Lecture Slides on Cell Biology

Kedir W. (M.Sc. Molecular Biology and

Biotechnology)Department of Biology

Hawassa University

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Cell Biology Cell is the basic, structural and functional unit of life. The science that deals with cells is known as cell

biology(cytology)How cells were discovered? 1665- English Scientist, Robert Hooke, discovered cells while

looking at a thin slice of cork using his own microscope.He described the cells as tiny boxes or a honeycombHe thought that cells only existed in plants and fungi

1673- Anton van Leuwenhoek: Used a handmade microscope to observe pond scum & discovered single-celled organisms

He called them “animalcules”q `

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Development of Cell Theory1838- German Botanist, Matthias Schleiden, concluded that all

plant parts are made of cells1839- German physiologist, Theodor Schwann, who was a close

friend of Schleiden, stated that all animal tissues are composed of cells.

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Cont. 1858- Rudolf Virchow, German physician, after

extensive study of cellular pathology, concluded that cells must arise from preexisting cells.

The 3 Basic Components of the Cell Theory All organisms are composed of one or

more cells. (Schleiden & Schwann)(1838-39) The cell is the basic unit of life in all living things. (Schleiden & Schwann)(1838-39) All cells are produced by the division of

preexisting cells. (Virchow)(1858)

BUT HOW DID THE FIRST CELL AROSE?

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Evolution of the cellCells are divided into two main classes, initially defined by

whether they contain a nucleus. • Prokaryotic cells (e.g. bacteria) lack a nuclear envelope• Eukaryotic Cells have a nucleus in which the genetic

material is separated from the cytoplasm. Prokaryotic cells are

• generally smaller and simpler than eukaryotic cells; • in addition to the absence of a nucleus, their genomes

are less complex and • they do not contain cytoplasmic organelles or a

cytoskeleton. 5

Cont. In spite of these differences, the same basic molecular

mechanisms govern the lives of both prokaryotes and eukaryotes, indicating that all present-day cells are descended from a single primordial ancestor. How did this first cell develop? And how did the

complexity and diversity exhibited by present-day cells evolve?

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1. Evolution from Molecules to the First Cell Living cells probably arose on earth about 3.5 billion years

ago by spontaneous reactions between molecules in pre-biotic condition

Pre-biotic conditions the earth was a violent place with volcanic eruptions,

lightning, and torrential rains the atmosphere contain small molecules such as

ammonia, carbon dioxide, methane. There was no free oxygen and no layer of ozone to absorb

the ultraviolet radiation from the sun. These conditions may have helped to keep the atmosphere

rich in reactive molecules that produced simple organic molecules such as amino acids, sugars, purines and pyrimidines

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Evidence The best evidence came from laboratory experiments by Stanley Miller.

If mixtures of gases such as CO2, CH4, NH3, and H2 are heated with water and energized by electrical discharge or by ultraviolet radiation, they react to form small organic molecules.

Among these products are compounds, such as hydrogen cyanide (HCN) and formaldehyde

(HCHO) that readily undergo further reactions in aqueous solution.

Most important, representatives of most of the major classes of small organic molecules found in cells are generated, including amino acids sugars the purines and pyrimidines

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Cont. Simple organic molecules such as amino acids and

nucleotides can associate to form polymers known as polypeptides and polynucleotides respectively.

Catalysts for early polymerizationThe earliest polymers may have formed in any of several

ways - for example, by the heating of dry organic compounds or by the catalytic activity of high concentrations of

inorganic polyphosphates or By crude mineral catalysts.

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What are the critical characteristic required of the macromolecule from which life evolved ?

Origin of life requires molecules that have the property of: • autocatalysis (the ability to catalyze reactions that lead to

production of more molecules of the catalyst itself) and • Self-replicating (Molecules that are Capable of Directing Their

Own Synthesis) Which molecules do have these properties?1. Polypeptides: In present-day living cells, the most versatile catalysts

are polypeptides. Although polypeptides are versatile as catalysts, there is no known way in which one such molecule can reproduce itself by directing their own synthesis

2. Polynucleotides: have properties that contrast with those of polypeptides. Both DNA and RNA directly guide the formation of exact copies of their own sequence. In addition to self replicating RNA molecules have catalytic property (e.g. Ribozymes).

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Cont. An RNA molecule therefore has these two special characteristics:

1. it carries information encoded in its nucleotide sequence that it can pass on by the process of replication and

2. the catalytic properties. • There are strong suggestions, therefore, that between 3.5 and 4 billion years

ago, somewhere on earth, self-replicating systems of RNA molecules, mixed with other organic molecules including simple polypeptides, began the process of evolution- a period of evolution known as the RNA world

• The first cell is presumed to have arisen by the enclosure of self-replicating RNA in a membrane composed of phospholipids

The enclosure of self replicating RNA and associated molecules in phospholipids membrane would thus have maintained them as a unit, capable of self reproduction and further evolution.

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Evolution of metabolism• Cells were originated in a sea of organic molecules, they were able to

obtain food and energy directly from their environment.• As resources get depleted cells needed to evolve their own mechanism

for generating energy. All present cells use ATP as their source of metabolic energy. The

mechanisms used by cells for generation of ATP are thought to have evolved in three stages corresponding to the evolution of:

GlycolysisPhotosynthesisOxidative metabolism

In the initially anaerobic atmosphere of earth, the first energy generating reaction involved the breakdown of organic molecules in the absence of oxygen. These reactions are likely to have been a form of present day Glycolisis C6H12O6 _____ 2C3H6O3 + 2ATP

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Cont.The development of photosynthesis

is the next major evolutionary step, which allowed the cell to harness energy from sunlight

provided independence from the utilization of preformed organic molecules.

The first photosynthetic bacteria probably used H2S as source of electron and hydrogen to convert CO2 to organic

molecules. • The use of H2O as a donor of electron and hydrogen for conversion of CO2

to organic molecules evolved later had important consequence of changing the earths atmosphere

by producing free O2 as a byproduct 6CO2 + 6H2O ______C6H12O6 + 6O2

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Cont.The release of O2 changed the environment in which cells

evolved have led to the development of oxidative metabolism

Oxidative metabolism utilizing O2 provided a mechanism for generating energy from organic

molecules that is more efficient than anaerobic glycolisis.

C6H12O6 + 6O2 ______ 6CO2 + 6H2O + 36-38ATP

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Evolution from prokaryotes to Eukaryotes

all organisms living now on earth were derived from a single primordial cell born more than 3.5 billion years ago.This first cell was prokaryotic, unicellular and anaerobic

prokaryotes are divided into two groups1. The archaebacteria and 2. The eubacteria

Indication: Archaebacteria

live in extreme environments, which are unusual today but may have been prevalent in primitive Earth.

For example, thermoacidophiles live in hot sulfur springs with temperatures as high as 80°C and pH values as low as 2.

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Cont.The eubacteria

Also live in a wide range of environments, including soil, water, and other organisms (e.g., human pathogens).

Photosynthesis was first evolved in prokaryotes known as Cyanobacteria

Consequences of accumulation of molecular oxygen in the atmosphere i. some anaerobic prokaryotes become extinctii. others may have evolved the capacity for aerobic respiration iii.others found niches in which oxygen was largely absent, where

they could continue an anaerobic way of life. iv.Others became predators or parasites on aerobic cells.

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Evolution of Eukaryotic Cells

A critical step in the evolution of eukaryotic cells was the acquisition of membrane-enclosed sub cellular organelles,

allowing the development of the complexity characteristic of these cells.

The organelles are thought to have been acquired as a result of the association of prokaryotic cells with the ancestor of eukaryotes.

The hypothesis that eukaryotic cells evolved from a symbiotic association of prokaryotes—endosymbiosis—is particularly well supported by studies of mitochondria and chloroplasts, which are thought to have evolved from bacteria living in large cells.

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EvidenceBoth mitochondria and chloroplasts are similar to bacteria in

size, and like bacteria, They reproduce by dividing in two(Binary Fission). Most important, both mitochondria and chloroplasts contain

their own DNA, which encodes some of their components. Like bacteria they contain 70s ribosome

• An endosymbiotic origin for these organelles is now generally accepted,

with mitochondria thought to have evolved from aerobic bacteria and

chloroplasts from photosynthetic bacteria, such as the cyanobacteria.

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Features of prokaryotes which make them different from eukaryotic Cells

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The Development of Multicellular OrganismsMulticellular organisms evolved from unicellular eukaryotes at least

1.7 billion years ago. Some unicellular eukaryotes form multicellular aggregates that

appear to represent an evolutionary transition from single cells to multicellular organisms. For instance, the cells of many algae (e.g., the green alga Volvox)

associate with each other to form multicellular colonies, which are thought to have been the evolutionary precursors of present-day plants.

Increasing cell specialization then led to the transition from colonial aggregates to truly multicellular organisms.

Continuing cell specialization and division of labor among the cells of an organism have led to the complexity and diversity observed in the many types of cells that make up present-day plants and animals, including human beings.

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Cont.

Figure . Four closely related genera of green algae, showing a progression from unicellular to colonial and multicellular organization.

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Figure Evolution of cells: Present-day cells evolved from a common prokaryotic ancestor along three lines of descent, giving rise to archaebacteria, eubacteria, and eukaryotes. Mitochondria and chloroplasts originated from the endosymbiotic association of aerobic bacteria and cyanobacteria, respectively, with the ancestors of eukaryotes.

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Cell size Cells vary greatly in size which ranges from less than1 micrometer - 200

micrometers. The size of a cell is directly related to its level of activity and the rate that

molecules move across its membranes. In order to stay alive, a cell must have a constant supply of nutrients, oxygen, and other molecules. It must also be able to get rid of carbon dioxide and other waste products that are harmful to it. The larger a cell becomes, the more difficult it is to satisfy these requirements; consequently, most cells are very small.

Advantages of being small: Small cells have large surface to volume ratio, so things can be moved in and out efficiently

There is a mathematical relationship between the surface area and volume of a cell referred to as the surface area-to-volume ratio.

As cells grow, the amount of surface area increases by the square (X2) but volume increases by the cube(X3). The surface area increases at a slower rate than the volume. Thus, the surface area-to-volume ratio changes as the cell grows

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There is a mathematical relationship between the surface area and volume of a cell referred to as the surface area-to-volume ratio.

As cells grow, the amount of surface area increases by the square (X2) but volume increases by the cube(X3). The surface area increases at a slower rate than the volume. Thus, the surface area-to-volume ratio changes as the cell grows.

As a cell gets larger, cells have a problem with transporting materials across the plasma membrane. For example, diffusion of molecules is quite rapid over a short distance, but becomes slower over a longer distance. If a cell were to get too large, the center of the cell would die because transport mechanisms such as diffusion would not be rapid enough to allow for the exchange of materials. When the surface area is not large enough to permit sufficient exchange between the cell volume and the outside environment, cell growth stops.

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Cell shape Cells show great variation in their shape . In human beings for example, there are about 200 types of cells in that vary greatly in

shape. Examples Squamous cells: are thin and flat cells. Such cells line the esophagus and cover the

skin. Polygonalcells : have irregularly angular shapes with four, five, or more sides. Stellate cells: Some nerve cells have multiple extensions that give them a star like, or

stellate, shape. Cuboidal cells: are squarish and approximately as tall as they are wide; liver cells are a

good examples. Columnar cells: such as those lining the intestines, are markedly taller than wide. Spheroid cells: Egg cells and fat cells are spheroid too void(round to oval). Discoid cells: Red blood cells are discoid (disc-shaped). Fusiform cells: Smooth muscle cells are fusiform - thick in the middle and tapered

toward the ends. Fibrous cells: Skeletal muscle cells are described as fibrous because of their threadlike

shape

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Tools of Cell Biology As in all experimental sciences, research in cell biology depends on

the laboratory methods that can be used to study cell structure and function.

An appreciation of the experimental tools available to the cell biologist is thus critical to understanding both the current status and future directions of this rapidly moving area of science.

Some of the important general methods of cell biology are: Light Microscopy: Electron Microscopy: Sub-cellular Fractionation: Cell Culture: Viruses …..etc

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Light Microscopy Because most cells are too small to be seen by the naked eye,

the study of cells has depended heavily on the use of microscopes.

Robert Hooke first coined the term "cell" following his observations of a piece of cork with a simple light microscope in 1665

Antony van Leeuwenhoek, in 1673, was able to observe a variety of different types of cells, including sperm cells, red blood cells, and bacteria.

Microscopic studies of plant and animal cell by Matthias Schleiden and Theodor Schwann respectively led to the proposal of the cell theory.

Thus, the cell achieved its current recognition as fundamental unit of all living organisms because of observations made with the light microscope. 30

Since most cells are between 1 µm and 100 µm in diameter, they can be observed by light. However, the light microscope is not sufficiently powerful to reveal fine details of cell structure, for which resolution—the ability of a microscope to distinguish objects separated by small distances—is even more important than magnification.

The limit of resolution of the light microscope is approximately 0.2 µm Two objects separated by less than 0.2 µm appear as a single image,

rather than being distinguished from one another. • Theoretical limitation of light microscopy is determined by two factors

The wavelength (λ) of visible light and The light-gathering power of the microscope lens (numerical aperture,

NA)—according to the following equation:

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The wavelength of visible light is 0.4 to 0.7 µm, so the value of A is fixed at approximately 0.5 µm for the light microscope.

The numerical aperture can be envisioned as the size of the cone of light that enters the microscope lens after passing through the specimen. It is given by the equation

Where I is the refractive index of the medium through which light travels between the specimen and the lens. The value of for air is 1.0, but it can be increased to a maximum of approximately 1.4 by using an oil-immersion lens to view the specimen through a drop of oil.

The angle α corresponds to half the width of the cone of light collected by the lens. The maximum value of α is 90°, at which sin α = 1, so the highest possible value for the numerical aperture is 1.4.

The theoretical limit of resolution of the light microscope can therefore be calculated as follows:

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Types of light MicroscopeBright-field microscopy

Light passes directly through the cell and the ability to distinguish different parts of the cell depends on contrast resulting from the absorption of visible light by cell components.

cells are stained with dyes that react with proteins or nucleic acids in order to enhance the contrast between different parts of the cell.

Staining procedures kill the cellsnot suitable for many experiments in which the observation of living

cells is desired. Phase-contrast microscopy

Use optical systems that convert variations in density or thickness between different parts of the cell.

Enhances contrast in unstained cells by amplifying variations in density within specimen

useful for examining living, unsyatined cells.

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Fluorescence microscopy A fluorescent dye is used to label the molecule of interest within either

fixed or living cells. The fluorescent dye is a molecule that absorbs light at one wavelength

and emits light at a second wavelength. used to study a variety of molecules within cells

Differential-interference-contrast (Nomarski). Like phase-contrast microscopy, it uses optical modifications to

exaggerate differences in density, making the image appear almost 3D.Confocal Microscopy

Uses lasers and special optics for “optical sectioning” of fluorescently-stained specimens.

Uses computer.

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Electron MicroscopyBecause of the limited resolution of the light microscope, analysis of

the details of cell structure has required the use of more powerful microscopic techniques—namely electron microscopy,

The electron microscope can achieve a much greater resolution and magnification than that obtained with the light microscope because the wavelength of electrons is shorter than that of light.

The wavelength of electrons in an electron microscope can be as short as 0.004 nm: about 100,000 times shorter than the wavelength of visible light. Theoretically, this wavelength could yield a resolution of 0.002 nm, but such a resolution cannot be obtained in practice, because resolution is determined not only by wavelength, but also by the numerical aperture of the microscope lens.

Modern electron microscopes have a practical resolving power of about 2 nm.

Enhanced resolution and magnification allowed researchers to clearly identify sub-cellular organelles and to study cell ultra-structure.

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Types of electron microscopes The transmission electron microscopy (TEM)

Thin section of specimen is stained with metals to absorb electrons and enhance contrast.

Electrons transmitted through the specimen are focused and the image is magnified by using electromagnetic lenses (rather than glass lenses)

Image is focused onto a viewing screen or film. It is used to study internal cellular ultra-structure.

Scanning electron microscopy used to provide a three-dimensional image of cells. In scanning electron microscopy the electron beam does not pass

through the specimen. Instead, the surface of the cell is coated with a heavy metal, and a beam of electrons is used to scan across the specimen.

Electrons that are scattered or emitted from the sample surface are collected to generate a three-dimensional image as the electron beam moves across the cell.

Can usually only view dead cells because of the elaborate preparation required. May also introduce structural artifacts.37

Sub cellular Fractionation Although the electron microscope has allowed detailed visualization of cell

structure, microscopy alone is not sufficient to define the functions of the various components of eukaryotic cells.

Used to isolate the organelles of eukaryotic cells in a form that can be used for biochemical studies.

accomplished by differential centrifugation—a method used to separate the components of cells on the basis of their size and density.

The process of cell fractionation involves • Homogenization of tissue: sonication, grinding in a mechanical

homogenizer, or treatment with a high-speed blender. All these procedures break the plasma membrane and the endoplasmic reticulum into small fragments while leaving other components of the cell (such as nuclei, lysosomes, peroxisomes, mitochondria, and chloroplasts) intact.

• Centrifugation of the resulting homogenate at a slow speed. Nuclei and other larger particles settle at the bottom of the tube, forming a pellet. The unpelleted fluid or supernatant is decanted into another tube and centrifuged at a faster speed, separating out smaller organelles.

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Tissuecells

Homogenization

Homogenate1000 g

(1000 times theforce of gravity)

10 minDifferential centrifugation

Supernatant pouredinto next tube

20,000 g20 min

Pellet rich innuclei andcellular debris

Pellet rich inmitochondria(and chloro-plasts if cellsare from a plant)

Pellet rich in“microsomes”(pieces of plasma mem-branes andcells’ internalmembranes)

Pellet rich inribosomes

150,000 g3 hr

80,000 g60 min

Organelles and Protein Sorting Each eukaryotic cell is subdivided into functionally distinct, membrane-

bound compartments called organelles Each of the various organelles within cells is specialized for one or more

tasks and therefore needs a specialized set of proteins to carry out its function.

These proteins (with the exception of those produced in mitochondria and chloroplast) are synthesized on ribosomes in the cytosol and have to be destined to their proper locationsFor example

Receptors – functions on plasma membrane DNA polymerase – functions in the nucleus Catalase – functions in the peroxisomes Insulin – functions outside the cell

The delivery of specialized set of proteins to their proper cellular destinations is referred to as protein targeting or protein sorting or protein translocation

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Protein targeting can follow two distinct pathways:1. Post-translational targeting

Involve protein targeting after polypeptide synthesis is completed on cytosolic ribosome

Proteins that are going to nucleus, mitochondria, chloroplasts and peroxisomes are targeted post-translationally

2. Co-translational targeting The synthesis of proteins begin on cytosolic ribosomes. The

ribosome with the nascent peptide is targeted to the endoplasmic reticulum(ER) and sorting is done during translation (co-translationally)

This pathway is utilized by proteins destined for the ER, Golgi, lysosome, the plasma membrane and protein that is going to be secreted from the cell

How do proteins know where to go?

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Targeting Sequences Targeting of proteins to their proper destination is done by short amino acid

sequences called targeting sequences or signal sequence or signal peptide or sorting signals

Targeting sequences function like cellular Postal Codes targeting proteins towards their destination in the cell.

Upon the delivery of the protein, the signal sequence may be removed by the resident protein called SIGNAL PEPTIDASE or may remain as permanent part of protein

Retention signal Another class of sorting signal that give a signal to the cell that the protein has

reached its final destination and should not be movedOther requirements for protein targeting

Specific receptor that recognize the signal sequence A channel (translocon) or two (for double membrane organelles) to

translocate (cross the organelle’s membrane) ATP to provide energy Chaperones proteins for proper folding of proteins

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The Nucleus found in all the eukaryotic cells. certain eukaryotic cells such as the mature

sieve tubes of higher plants and mammalian erythrocytes contain no nucleus

the number of the nucleus may vary from cell to cell. According to the number of the nuclei following types of cells have been recognized :Mononucleate cells. E.g. Most plant and animal cellsBinucleate cells. E.g. Paramecium and cells of cartilage and liver.Polynucleate cells. The cells which contain many (from 3 to 100) nuclei .

The polynucleate cells of the animals are termed as syncytial cells, while the polynucleate cells of the plants are known as coenocytes. The most common example of the syncytial cells are the osteoblast (polykaryocytes of the bone morrow) which contain about 100 nuclei per cell and striated muscle fibers each of which contains many hundred nuclei.

Structure of the nucleusis the largest organelle surrounded by two concentric membranes called

the inner and outer nuclear membranes separated by an intermembrane space44

• The outer nuclear membrane is continuous with the endoplasmic reticulum

• The nuclear envelop contains small channels or holes that allow regulated exchange of molecules between nucleus and cytoplasm. These pores are called nuclear pore complexes(NPC).

• An NPC is made up of multiple copies of some 50 different proteins called nucleoporins.

The spherical inner nuclear membrane contains specific proteins that act as binding sites for the supporting fibrous sheath of intermediate filaments (IF), called nuclear lamina.

Nuclear lamina maintains the shape of the nucleus. These lamins also serve as sites of chromatin attachment and organize other proteins into functional nuclear bodies.

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Internal Organization of the Nucleus NucleoplasmThe cell nucleus contains the fluid filled space known as nucleoplasm or

karyolymph. The majority of the cell's genetic material (DNA) is located in nucleoplasm

Nucleolus The nucleolus is a discrete densely stained structure found in the nucleus Main roles of the nucleolus are to synthesize rRNA and assemble rRNA and

proteins to ribosome

Protein targeting to the nucleus All proteins found in the nucleus are synthesized in the cytoplasm and imported

into the nucleus through nuclear pore complexes. Such proteins contain a targeting sequences called nuclear-localization signal

(NLS) NLS directs the selective transport of proteins into the nucleus. Examples: Histones, Ribosomal proteins, DNA and RNA polymerases, and

Transcription factors are guided into nucleus with the help of NLS. NLS consist of four to eight amino acid residues and include several

consecutive basic aminoacids (Arg or Lys). In addition to NLS, Nuclear import requires

Nuclear import receptor: Importin α and β GTP as source of energy Ran GTPase Fully folded cargo protein: protein to be transported into nucleus

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Mechanism of Nuclear Import Importin α and β bind to the cargo protein at its NLS, forming cargo-

importin complexThe complex is translocated through the nuclear pore complex(NPC)

into the nucleus In the nucleoplasm, interaction of Ran·GTP with the importin causes a

conformational change that decreases its affinity for the NLS, releasing the cargo in the nucleoplasm.

To support another cycle of import the importin-Ran·GTP complex diffuses back through the NPC.

Ribosomes Ribosomes are non membrane bounded cytoplasmic organelle found in all

organisms. are complexes of rRNA and protein constructed in the nucleolus in eukaryotic

cells Cells with high rates of protein synthesis have prominent nucleoli and many

ribosomes (e.g., human liver cell has a few million). It is the site for protein synthesis 48

Ribosomes function either free in the cytosol or bound to endoplasmic reticulum.

Most proteins made by free ribosomes will function in the cytosol. Bound ribosomes generally make proteins that are destined for membrane inclusion or export.

Types of ribosomesAccording to the size and the sedimentation coefficient (S) two types of

ribosomes have been recognized70S Ribosomes.

comparatively smaller in size and have sedimentation coefficient 70S and the molecular weight 2.7× 106 daltons.

occurs in the prokaryotic cells of the blue green algae and bacteria and also in mitochondria and chloroplasts of eukaryotic cells.

consists of two subunits, viz., 50S and 30S. The 50S ribosomal subunit is larger in size and the 30S ribosomal subunit is smaller in size

80S Ribosomes. have the sedimentation coefficient of 80S and the molecular weight 40 ×

106 daltons. 49

The 80S ribosomes occur in eukaryotic cells. The 80S ribosome also consists of two subunits, viz., 60S and 40S.

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MitochondriaSurrounded by a double membrane, the inner and outer mitochondrial

membranes are separated by intermembrane space.The inner membrane forms numerous folds called cristae that surround

the inner space, matrixFound in almost all eukaryotic cellsMitochondria plays a critical role in generation of metabolic energy

(ATP): power house of cellsThe matrix contains the enzymes for TCA cycle, the inner membrane

contains protein complexes involved in ETS and oxidative metabolismMitochondria have their own DNA and manufacture a small number of

their own proteins.

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Import of proteins to Mitochondria The majority of mitochondrial proteins are coded for by nuclear genes. These are

synthesized on free ribosomes and only imported into the mitochondrion post-translationally.

Only unfolded proteins can be imported into the mitochondria in order for the proteins to be pulled through small pores of mitochondrial translocons.

The unfolded state of such proteins is maintained by proteins called cytosolic chaperones

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Proteins targeting to mitochondria requires Mitochondrial targeting sequences called pre-sequences One TOM complex (translocase of outer mitochondrial membrane)-

for targeting to outer mitochondrial membrane and Two TIM complexes (Translocase of inner mitochondrial membrane).

One TIM complex for targeting to mitochondrial matrix and one TIM complex for inner mitochondrial membrane

• After protein import, the uptake-targeting sequence is removed by proteases called signal peptidase within the matrix and the proteins are folded into their correct shape

Plastids:Plastids are groups of plant and algal membrane-bound organelles that

include Leucoplasts, chromoplasts and chloroplasts. Leucoplasts:

The leucoplasts are the colorless plastids which are found in embryonic and germ cells.

They store the food materials as carbohydrates, lipids and proteins

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Chromoplastsare the colored plastids containing carotenoids and other pigments. They impart color (e.g., yellow, orange and red) to certain portions of

plants such as flower petals, fruits and some roots. Chloroplast

are chlorophyll-containing plastids which are the sites of photosynthesis

Found in eukaryotic algae, leaves and other green plant organs.Like the mitochondrion, the chloroplast is surrounded by an outer and

an inner membrane and posses their own genetic materialChloroplasts contain an extensive internal system of interconnected

membrane called thylakoids which serve as the site of electron transport system for ATP synthesis

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Thylakoids are arranged in stacks called grana embedded in a matrix, the stroma.

The thylakoid membranes contain green pigments (chlorophylls) and other pigments that absorb light and generate ATP during photosynthesis

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Protein targeting to Chlorolast Most chloroplast proteins are synthesized on free ribosomes in the cytosol and

targeted into chloroplast in unfolded state Protein import into the chloroplast requires

Targeting sequences called transit sequences that target chloroplast proteins to the import machinery in the chloroplast membranes

TOC complex (Translocase at outer membrane of chloroplast) and TIC complexes (Translocase at inner membrane of chloroplast.

After targeting, the transit sequence is cleaved by stromal processing peptidase (SPP), and the proteins are folded into their correct shape by chaperon proteins

Peroxisomes Peroxisomes are small single membrane bounded organelles that contain

enzymes involved in variety of metabolic reactions Functions include:

Decomposing harmful chemicals such as H2O2.

Fatty acid oxidation: into acetyl group that provide source of enery Biosynthesis of lipids such as cholestrol etc56

Protein import to Peroxisomes All proteins destined for peroxisomes are also translated on free cytosolic

ribosomes and then transported as completed polypeptide chain fully folded in cytosol

The Import requires Two peroxisome targeting signals: PTS1 and PTS2 Peroxins - peroxisome transport receptors that recognize PTS1 and PTS2 Translocation channel

During the transport: Peroxins bind to cargo proteins with PTS1 or PTS2 and dock to the

translocation channel The complex is transported through the membrane into peroxisome, protein

is released and Peroxin is recycled Unlike transport to mitochondria and chloroplast ,the targeting signals are not

usually cleaved during the import of proteins into peroxisomes

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Endoplasmic Reticulum The endoplasmic reticulum is a network of membrane enclosed channels

that run throughout the cell It forms a continuous network whose lumen (inside) is separated from the

cytosol by a single membrane. The membrane of the endoplasmic reticulum is continuous with the outer

nuclear membrane There are two regions of ER

1. Smooth endoplasmic reticulum 2. Rough endoplasmic reticulum

The basic difference is that, unlike the smooth endoplasmic reticulum, rough endoplasmic reticulum is covered in ribosomes, which give it its rough appearance in the electron microscope.

Smooth endoplasmic reticulum Used for the synthesis of fatty acids and phospholipids in the liver it is the site of detoxication of foreign chemicals including drugs.

It is also the storage site of calcium ions.Rough endoplasmic reticulum

Ribosome bound to the rough ER synthesize proteins58

Protein import to ER Synthesis of proteins destined for import into the endoplasmic reticulum

starts on free ribosome in cytosol.

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Requirements for the import to ER Signal peptide of about 20 amino acids long Signal recognition particle (SRP): recognize the signal sequence of protein to

be targeted to endoplasmic reticulum is made up of a small RNA molecule and proteins

The signal recognition particle receptor(SRPR): Receptor for SRP Protein translocator

Mechanism of targeting After synthesis of a protein begins on free ribosomes in the cytosol, the

endoplasmic reticulum signal sequence is recognized by a signal recognition particle

The signal recognition particle brings the ribosome to the endoplasmic reticulum membrane where it interacts with a signal recognition particle receptor

The SRP and SRP receptor then mediate insertion of the nascent protein into the translocon (channel within the membrane)

As the ribosome attached to the translocon continues translation, the unfolded protein chain is extruded into the ER lumen

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Once the polypeptide chain has entered the lumen of the endoplasmic reticulum, the signal sequences may be cleaved off by an enzyme called signal peptidase.

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Post translational modification of proteins Membrane proteins and soluble proteins synthesized on the rough ER

undergo a number of modifications before they reach their final destinations: Glycosylation: Most polypeptides synthesized on the rough endoplasmic

reticulum are glycosylated, that is, they have sugar residues added to them

Formation of disulfide bonds: disulfide bonds (–S–S–) help stabilize the tertiary and quaternary structure of many proteins

Folding: Only properly folded proteins and assembled subunits are transported from the rough ER to the Golgi complex in vesicles. Unassembled or misfolded proteins in the ER often are transported back through the translocon to the cytosol, where they are degraded.

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Golgi Apparatus Golgi, is a distinctive stack of flattened sacks called cisternae. The Golgi apparatus is the distribution point of the cell where proteins made

within the rough endoplasmic reticulum are further processed and then directed to their final destination.

The stack of flattened Golgi sacs has three defined regions — the cis, the medial, and the trans.

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Transport from the ER through the Golgi Apparatus(VESICULAR TRAFFICKING)

In vesicular transport, newly synthesized proteins are transported from the ER to the Golgi apparatus and from the Golgi apparatus to the cell surface and elsewhere,

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Vesicular transport Transport mediated by membrane bound vesicles It involve formation and budding of a vesicle from one organelle, followed by

fusion with a second membrane to transport proteins to the new compartment.

Vesicle Formation Vesicle formation is the process during which cargo is captured and the lipid

membrane is shaped with the help of cytosolic proteins into a bud, which is then pinched off in a process called fission.

There are two types of coats that serve this function: 1. Coatomer coats (COP)2. Clathrin coats.

Coatomer-Coated Vesicles used in trafficking between the endoplasmic reticulum and Golgi

i. COPII-coated vesicles: bud from the ER and transport proteins to Golgi complex (Anterograde Transport)

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ii. COPI-coated vesicles: bud from pre-Golgi compartments and transport mis-targeted proteins back to ER(Retrograde transport). if resident proteins of ER lumen containing a retention signal KDEL (Lys-Asp-

Glu-Leu) are mis-targeted to Golgi, they targeted back to ER, coated by COPI coated vesicle

Clathrin-Coated Vesicles Clathrin-coated vesicles mediate selective transport from

the Golgi to lysosome, plasmamembrane and secretary proteins to the surface of plasma membrane

proteins and lipids from the plasma membrane to the endosome, and operate in other places where selective transport is required.

Vesicle fusion with target organelle vesicle fusion is the process by which a vesicle membrane incorporates its

components into the target membrane and releases its cargo into the lumen of the organelle or, in the case of secretion, into the extracellular medium

Vesicle fusion is highly specific. vesicle must only dock with and fuse with the correct target membrane in order to avoid mixing of proteins

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Specificity in targeting is ensured because all transport vesicles and target membranes display unique surface markers known as SNAREs

SNARE proteins have a central role both in providing specificity and in catalyzing the fusion of vesicles with the target membrane

SNARES located on the vesicles are known as v-SNARES and SNAREs on the target membranes are known as t-SNARES

Each v-SNARE in a vesicular membrane specifically binds to a t-SNARE proteins in the target membrane, inducing fusion of the two membranes and release cargo protein.

After fusion is completed, the SNARE complex is disassembled in an energy dependent reaction

GTP hydrolysis by proteins Rabs is thought to provide energy for membrane fusion.

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Lysosome Lysosomes are a single membrane bounded acidic organelles that contain a

battery of degradative enzymes All the lysosomal enzymes work most efficiently at acid pH values and

collectively are termed acid hydrolasesImport to lysosomes Proteins that are destined for the lysosome are synthesized on the rough

endoplasmic reticulum. Because they do not have an endoplasmic reticulum retention signal such as KDEL, they are transported to the Golgi apparatus

In the Golgi apparatus the proteins are modified by addition of Mannose containing oligosaccharides and mannose is phosphorylated at carbon number 6 ( form Mannose-6-phosphate)

Mannose-6-phosphate is used as a surface marker distinguishing proteins destined to lysosome from others

Proteins containing mannose-6-phosphate are coated with a clathrin coat at transgolgi network and targeted to lysosome

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The plasma membraneo The plasma membrane also called plasmalemma or cell membrane is

the structure that encloses the cell & defines cell boundariesFunctions1. The plasma membrane encloses the cell & defines cell boundaries2. Maintains the essential differences between the cytosol and the extra

cellular environment. 3. Inside eukaryotic cells, maintain the characteristic differences

between the contents of each organelle and the cytosol. 4. Ion gradients across membranes is established by the activities of

specialized membrane proteins5. Can be used to synthesize ATP6. Selective transport of solutes 7. in nerve and muscle cells, to produce and transmit electrical signals.8. In all cells, the plasma membrane also contains proteins that act as

sensors of external signals

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Membrane structureDespite their differing functions, all biological membranes

have a common general structure: each is a very thin film of lipid bilayer and protein

molecules, held together mainly by non-covalent interactions.

Early observations:The first glimpse of the cell membrane's structure came in

1925 when Gorter & Grendell found a way to release the contents of red blood cells leaving only the membranes, called erythrocyte ghosts.

Analysis of these ghosts revealed the presence of lipids. Thus, Gorter & Grendell demonstrated that the plasma

membrane consisted of two layers of lipid --it was a bimolecular layer.

A little later in the mid-1930s, Schmidt based on observation of the rotation of polarized light by myelin sheaths (membranes around neurons) suggested the presence of protein. 72

Membrane Models1. The Davson- Danielli Sandwich Model of Membranes

In part, the work just cited lead Davson and Danielli to develop a model of membrane structure known as sandwich model

They defined membrane as a bimolecular lipid layer capped on the inside and outside by protein.

also postulated the presence of protein-lined pores for movement small molecules through membranes •The Davson-Danielli

model can be thought of as a sandwich where the bread represents the outer and inner layers of protein and the sandwich filling the lipid component (however, this sandwich model is inadequate since it has no protein pores

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2. Robertson's Unit Membrane Model Electron micrographs of tissue sections fixed in osmium tetroxide (a

fixative) revealed membranes as solid dark lines about 75 Å thick. In 1959 Robertson used a different fixative and stained his specimens

with potassium permanganate. This gave much better resolution. He found that membranes consisted of a light area about 35 Å thick ( lipid) surrounded by dark lines each 20 Å in thickness (proteins)..

The membranes in Robertson's electron micrograph were interpreted as consisting of a protein-lipid-protein sandwich, consistent with the Davson-Danielli model.

Furthermore, all cellular membranes had the same structure that led Robertson to conclude that they were identical. This led him to his "unit membrane hypothesis" which stated simply that all membranes had essentially the same sandwich like structure

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Conflict – Problems with the Davson-Danielli Sandwich and Unit Membrane models Despite the unifying effect of Robertson's unit

membrane hypothesis, problems developed when it was found that each membrane-bound organelle had its own particular function. How could membranes vary in function if

they all had the same structure? Variations in membrane thickness were also

observed which cast further doubt on Robertson's generalization. Furthermore, the Davson-Danielli model seemed inadequate to explain some membrane properties, e.g. how could lipid soluble material pass

through membranes regardless of size if a protein cap protected the lipid?

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3. The Fluid Mosaic ModelProposed by Singer & Nicholson proposed in 1972 This model, called the fluid mosaic model, emphasized the

dynamic nature of membranes in sharp contrast to the static Davson-Danielli model.

According to Singer & Nicholson: the molecular structure of the membrane is not rigid and

fixed, but rather fluid. The lipids are not capped with a solid protein coating. Instead,

protein molecules are dispersed throughout the membrane leaving many portions of the lipid bare and exposed to the extra-and intra-cellular environments.

It is through these bare areas that lipid soluble molecules pass.

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In this model, the phospholipids bilayer is a fluid matrix, in essence, a two-dimensional solvent for proteins.

Both lipids and proteins are capable of rotational and lateral movement.

Singer and Nicolson also pointed out that proteins can be associated with the surface of this bilayer or embedded in the bilayer to varying degrees

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The lipid bilayerThe lipid bilayer:

provides the basic fluid structure of the membrane serves as a relatively impermeable barrier to the

passage of most water-soluble molecules.all of the other functions of the membrane, transporting

specific molecules across it, or catalyzing membrane-associated reactions, such as ATP synthesis are provided by membrane proteins

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Properties of lipid bilayer1. All of the lipid molecules in cell membranes are amphipathic

(or amphiphilic)—that is, they have a hydrophilic (“water-loving”) or polar end and a hydrophobic (“water-fearing”) or nonpolar end.

The hydrophobic ends are composed of fatty acids FA differ in length (they normally contain between

14 and 24 carbon atoms). One of the tails is unsaturated while the other tail

is saturated. The hydrophilic head group is composed of alcohol

attached with phosphate

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Cont.2. Self sealing property:

Lipid molecules spontaneously aggregate to bury their hydrophobic tails in the interior and expose their hydrophilic heads to water.

Depending on their shape, they can aggregate in either of two ways:

1. They can form spherical micelles, with the tails inward or

2. They can form bimolecular sheets, or bilayers, with the hydrophobic tails sandwiched between the hydrophilic head groups.

Micelles Liposome Bilayer

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Cont.3. The lipid component of a biological membrane is a two-

dimensional liquid in which the constituent molecules are free to move laterally

4. Biological membranes are asymmetric structures.Composition of lipid bilayerThe lipid bilayer of many cell membranes is composed of

PhospholipidsCholesterol Glycolipids.

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Phospholipids: Composed of four components:

2 fatty acid chainsa phosphate, an alcohol (Glycerol or

sphingosine) attached to the phosphate

Head group(choline, serine, ethanolamine….. etc)

The fatty acid components provide a hydrophobic barrier

A phosphate , head group and an alcohol molecules has hydrophilic properties to

enable interaction with the environment.

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Cont.Phospholipids derived from glycerol

are called phosphoglycerides (glycerophospholipids). Phospholipids derived from sphingosine are called spingophospholipids

The four major phospholipids are:

•Only phosphatidylserine carries a net negative charge, •the other three are electrically neutral at physiological pH, carrying one positive and one negative charge.

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Cholesterol It is a steroid, built from four linked hydrocarbon rings. Attached to the ring are a hydrocarbon tail and a hydroxyl group. In membranes,

the hydrophobic part interact with the fatty acid chains of the phospholipids

the hydroxyl group interacts with the nearby phospholipid head groups.

absent from prokaryotes but is found in all animal membranes.

constitutes almost 25% of the membrane lipids in certain nerve cells but is essentially absent from some intracellular membranes.

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Glycolipids.Glycolipids, as their name implies, are sugar-

containing lipids. found exclusively on the extracellular layer of the membrane

Constitute 2–10 percent of the total lipid in plasma membranes; they are most abundant in nervous tissue.

.

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Membrane fluidityPlasma membrane is a two dimensional fluid.It can change from a liquid state to a two-dimensional

rigid crystalline (or gel) state at a characteristic freezing point.

This change of state is called a phase transition The temperature at which the structure

undergoes the transition from ordered to disordered (ie, melts) is called the transition temperatures or melting temperature (Tm).

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Factors affecting membrane fluidity1. Fatty acid chain length

A shorter chain length reduces the tendency of the hydrocarbon tails to interact with one another thus increase membrane fluidity

2. Degree of saturation of the hydrocarbon chain Presence of cis-double bonds produce kinks in the

hydrocarbon chains that make them more difficult to pack together, so that the membrane remains fluid

3. Amount of Cholesterol because of its shape cholesterol prevents long-chain

fatty acids from packing close to each other at low temperature and increase membrane fluidity.

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Cont. Cholesterol at high temperature, decrease membrane

fluidity by making the lipid bilayer less deformable. In this way, it inhibits possible phase transitions

4. TemperatureFluidity increases with increased temperature

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Membrane proteins There are two classes of membrane proteins. 1. Peripheral proteins (or extrinsic proteins)2. Integral proteins (or intrinsic proteins or transmembrane

proteins)Peripheral proteins

do not penetrate the bilayer to any significant degree associated with the membrane by weak non-covalent

bonds at the surface of membrane o can be dissociated from the membrane by treatment

with salt solutions or by changes in pH (treatments that disrupt hydrogen bonds and ionic interactions).

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Cont. Integral proteinspossess hydrophobic surfaces that can readily

penetrate the lipid bilayer itself can insert into the membrane or extend all the way

across the membrane and expose themselves to the aqueous solvent on both sides.

Because of these intimate associations with membrane lipid, integral proteins can only be removed from the membrane by agents capable of breaking up the hydrophobic interactions within the lipid bilayer itself (such as detergents and organic solvents).

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Functions of membrane proteinsProteins of the plasma membrane provide 6 membrane functions:1. Transport Proteins: transport lipid insoluble molecules and ions across

the membrane2. Receptor Proteins: Bind to chemical messengers (Ex. hormones) which

sends a message into the cell 3. Enzymatic Proteins: Carry out enzymatic reactions right at the

membrane when a substrate binds to the active site4. Cell Recognition Proteins: Glycoproteins (and glycolipids) on

extracellular surface serve as ID tags (which species, type of cell, individual)

5. Attachment Proteins: Attach to cytoskeleton to maintain cell shape 6. Intercellular Junction Proteins: Bind cells together

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Distribution of membrane components in two layers

The lipid compositions of the two halves of the lipid bilayer are different. i.e asymetrical

For example, in the human red blood cell membrane Phosphatidylcholine and sphingomyelin are located

in the outer half of the lipid bilayer, phosphatidylethanolamine and phosphatidylserine

are located in the inner half. Because the negatively charged

phosphatidylserine is located in the inner monolayer the inner is slightly negative in charge than the outer

Glycolipids are found exclusively in the non-cytoplasmic half(outer) of the lipid bilayer

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TRANSPORT ACROSS BIOLOGICAL MEMBRANESThe internal composition of the cell is maintained because the

plasma membrane is selectively permeable to small molecules. Most biological molecules are unable to diffuse through the

phospholipids bilayer, so the plasma membrane forms a barrier that blocks the free exchange of molecules between the cytoplasm and the external environment of the cell.

Specific transport proteins (carrier proteins and channel proteins) then mediate the selective passage of small molecules across the membrane, allowing the cell to control the composition of its cytoplasm

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In generalSmall non-polar molecules, such as O2

and N2 readily dissolve in lipid bilayers and therefore diffuse rapidly across them.

Small uncharged polar molecules, such as water and urea, also diffuse across a bilayer, albeit much more slowly

By contrast, lipid bilayers are highly impermeable to charged molecules (ions) such as Na+ and K+ Cl-, HCO3

-, small hydrophilic molecules like glucose, macromolecules like proteins and RNA can not freely diffuse through the plasma membrane

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Types of Membrane TransportGenerally there are two membrane transport systems based on

energy requirements1. Passive transport

Simple Diffusion Facilitated Diffusion

2. Active transport Direct active transport Indirect Active transport

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Simple DiffusionThe simplest mechanism by which molecules can cross

the plasma membrane is simple diffusion.Diffusion is the movement of molecules from region of

high concentration to the region of low concentration. During simple diffusion,

A molecule dissolves in the phospholipid bilayer and diffuses across it

It does not need energy for the process. No membrane proteins are involved the direction of transport is determined simply by

the relative concentrations of the molecule inside and outside of the cell.

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The net flow of molecules is always down their concentration gradient—from a compartment with a high concentration to one with a lower concentration of the molecule

Molecules that can pass through plasma membrane by simple diffusion are

Gases (such as O2 and CO2) Hydrophobic molecules (such as benzene) Small polar but uncharged molecules (such as H2O and ethanol) are

able to diffuse across the plasma membrane. Other biological molecules, however, are unable to dissolve in the

hydrophobic interior of the phospholipid bilayer and can not diffuse freely through the plasma membrane. This includes:

Larger uncharged polar molecules such as glucose, proteins Charged molecules of any size (including small ions such as H+, Na+,

K+, and Cl-).

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Facilitated Diffusion Facilitated diffusion like simple diffusion, involves

No external source of energy The direction of the transport is down hill

Facilitated diffusion differs from simple diffusion in that The transported molecules do not dissolve in the phospholipid

bilayer. Instead, their passage is mediated by proteins that enable the transported molecules to cross the membrane without directly interacting with its hydrophobic interior.

It allows the movement of polar and charged molecules, such as carbohydrates, amino acids, nucleosides, and ions, to cross the plasma membrane.

The direction of their transport is determined both by the concentration and voltage(electrical) differences- Electrochemical gradient

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Types of proteins that mediate facilitated diffusion

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Uniporters: transport a single type of molecule down its concentration gradient via facilitated diffusion

Symporters are also known as Co-transporters They transport two different

solutes simultaneously in the same direction

Antiporters: are also known as counter transporters They also transport two

different solutes simultaneously but in opposite direction

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Mechanism of transport by carrier proteins Carrier proteins transport molecules by binding to specific molecules to

be transported on one side of the membrane. They then undergo conformational changes that allow the molecule to

pass through the membrane and be released on the other side. Are responsible for the facilitated diffusion of sugars, amino acids, and

nucleotides across the plasma membranes of most cells.

E.g. Glucose transporter - functions by alternating between two conformational state In the first conformation, a glucose-binding site faces the outside of the cell. The binding of glucose to this exterior site induces a conformational change in the transporter, such that the glucose-binding site now faces the interior of the cell. Glucose can then be released into the cytosol, followed by the return of the transporter to its original conformation s

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Channel proteinsthey form a hydrophilic passageway across the membrane

through which polar molecules or ions move In contrast to carrier proteins, Channel proteins simply form open

pores in the membrane, allowing small molecules of the appropriate size and charge to pass freely through the lipid bilayer. E.g.

Porins: permit the free passage of ions and small polar molecules through the outer membranes of bacteria.

aquaporins: water channel proteins through which water molecules are able to cross the membrane much more rapidly than they can diffuse through the phospholipid bilayer.

Ion channels: are the best-characterized channel proteins which mediate the passage of ions across plasma membrane

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Properties of ion channels Transport through ion channels is extremely rapid. More than a

million ions per second flow through open channels—a flow rate approximately a thousand times greater than the rate of transport by carrier proteins.

Ion channels are highly selective because narrow pores in the channel restrict passage of ions of the appropriate size and charge. Thus, specific channel proteins allow the passage of Na+, K+, Ca2+, and Cl- across the membrane.

Some ion channels are open much of the time; these are referred to as non-gated channels. Most ion channels, however, open only in response to specific chemical or electrical signals; these are referred to as gated channels.

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Examples of gated channels1. Ligand-gated channels: open in response to the binding of

neurotransmitters or other signaling molecules; Example The binding of the neurotransmitter acetylcholine at certain synapses opens channels that admit Na+ and initiate a nerve impulse or muscle contraction.

2. Voltage-gated channels: open in response to changes in electric potential across the plasma membrane.

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OsmosisOsmosis: is the diffusion of water through a

selectively permeable membrane Water moves from high water area to low

water area or from dilute(weak) solution to concentrated(strong) solution

If two solutions have unequal solute concentration, the solution with higher solute concentration is known as Hypertonic (hyper= more than) and the one with low solute concentartion is known as hypotonic(Hypo= less than).

If the solute concentration of two solutions are equal the solutions are said to be Isotonic ( Iso= same)

•In cells, plasma membrane separates two aqueous solutions, one inside the cell(cytoplasm) and one outside(extracellular fluid).•The direction of net diffusion of water is determined by solute concentration of either side

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Direction of OsmosisThe net direction of osmosis depends on the relative

concentration of solutes on the two sides of the membrane. When the concentration of solute molecules outside the cell is

lower than the concentration in the cytosol, the solution outside is hypotonic to the cytosol. In this situation, water diffuses into the cell until equilibrium is reached.

When the concentration of solute molecules outside the cell is higher than the concentration in the cytosol, the solution outside is hypertonic to the cytosol. The water will diffuse out of the cell until equilibrium is established.

When the concentration of solutes outside and inside the cell are equal, the outside solution is said to be isotonic to the cytosol. The water diffuses into and out of the cell at equal rates, so there is no net movement of water.

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Water balance of cellsAnimal cells

1. If placed in Hypotonic solution: Cells swell and finally burst as water enters them by

osmotic flow. Rupture of the plasma membrane by a flow of water into

the cytosol is termed osmotic lysis. 2. Hypertonic solution

causes them to shrink as water leaves them by osmotic flow a condition called crenation.

3. Isotonic solution No net flow of water into or out of the cell. This maintains

normal animal cell volume

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Plant, algal, fungal, and bacterial cells 1. Hypotonic solution

Unlike animal cells they are surrounded by a rigid cell wallBecause of the cell wall, the osmotic influx of water that

occurs when such cells are placed in a hypotonic solution (even pure water) leads to an increase in intracellular pressure called osmotic pressure

The osmotic pressure also called turgor pressure, generated from the entry of water into the cytosol pushes the cytosol and the plasma membrane against the resistant cell wall. In this case the cell get turgid and turgidity maintains normal cell volume.

2. Hypertonic solutionThe water will diffuse out of the cell and the protoplasm of

the cell shrink. The condition known as plasmolysis. 109

What will happen to plant and animal cells if placed in the following solutions?

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Active transport

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Direct Active Transport: ExampleThe Na+/K+ ATPase

The cytosol of animal cells contains a concentration of potassium ions (K+) as much as 20 times higher than that in the extracellular fluid. Conversely, the extracellular fluid contains a concentration of sodium ions (Na+) as much as 10 times greater than that within the cell.

These concentration gradients are established by the active transport of both ions. And, in fact, the same transporter, called the Na+/K+ ATPase(Pump), does both jobs.

It uses the energy from the hydrolysis of ATP to actively transport 3 Na+ ions out of the cell for each 2 K+ ions pumped into the cell.

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The process involve ATP-driven conformational changes in the pump.

1. Na+ ions bind to sites inside the cell. This binding stimulates the hydrolysis of ATP and phosphorylation of the pump,

2. Phosphorylation induce a conformational change that exposes the Na+-binding sites to the outside of the cell and reduces their affinity for Na+. Consequently, the bound Na+ is released into the extracellular fluids.

3. At the same time, K+-binding sites are exposed on the cell surface. The binding of extracellular K+ to these sites then stimulates hydrolysis of the phosphate group bound to the pump

4. Hydrolysis of phosphate induces a second conformational change, exposing the K+-binding sites to the cytosol and lowering their binding affinity so that K+ is released inside the cell.

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Secondary active transport: Active Transport driven by Ion Gradients

function as coupled carriers, in which the transfer of one solute strictly depends on the transport of a second.

Coupled transport involves either the simultaneous transfer of a second solute in the same direction, performed by symporters or the transfer of a second solute in the opposite direction, performed by antiporters

In this transport system, the free energy released during the movement of an inorganic ion down an electrochemical gradient is used as the driving force to pump other solutes uphill, against their electrochemical gradient.

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Example of Indirect active transport The Na+/glucose transporter(Co-transporter or symporter)

the Na+/glucose transporter protein allows transport of sodium ions and glucose to enter the cell together.

Glucose molecules are pumped actively into the cell using the energy released from the flow of sodium ions down their concentration gradient

Later the sodium is pumped back out of the cell by the Na+/K+ ATPase.

The Na+-H+ exchange protein(counter transporter or antiporter)The Na+-H+ antiporter couples the transport of Na+ into the cell

with the export of H+, thereby removing excess H+ produced by metabolic reactions and preventing acidification of the cytoplasm.

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Endocytosis and ExocytosisEndocytosisThe carrier and channel proteins discussed in the preceding

section transport small molecules through the phospholipid bilayer.

Eukaryotic cells are also able to take up macromolecules and particles from the surrounding medium by a distinct process called endocytosis using ATP energy.

In endocytosis, the material to be internalized is surrounded by an area of plasma membrane, which then buds off inside the cell to form a vesicle containing the ingested material.

There are three types of Endocytosis1. Phagocytosis2. Pinocytosis3. Receptor mediated endocytosis116

Phagocytosis (cell eating) Phagocytosis is the process by which cells engulf large particles such as

bacteria, cell debris, or even intact cells.Mechanism of Phagocytosis

The particle to be transported binds to receptors on the surface of the phagocytic cell and triggers the extension of pseudopodia.

The pseudopodia eventually surround the particle and their membranes fuse to form a large intracellular vesicle called a phagosome.

The phagosomes then fuse with lysosomes, producing phagolysosomes in which the ingested material is digested by the action of lysosomal acid hydrolases.

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Role of Phagocytosisamoebas use phagocytosis to capture food particles provide a defense against invading microorganisms and to

eliminate aged or damaged cells from the body e.g Macrophages

Pinocytosis or “cell drinking,” is the process of taking in droplets of ECF containing molecules of some use to the cell. The process begins as the plasma membrane becomes dimpled, or caved in, at points. These pits soon separate from the surface membrane and form small membrane-bounded vesicles in the cytoplasm.

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Receptor-Mediated EndocytosisMovement of very specific molecules

into the cell with the use of vesicles coated with the protein clathrin.

Coated pits are specific locations coated with clathrin and receptors. When specific molecules (ligands) bind to the receptors, then this stimulates the molecules to be engulfed into a coated vesicle.

Ex. Uptake of cholesterol (LDL) by animal cells

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ExocytosisMovement of large molecules

bound in vesicles out of the cell with the aid of ATP energy.

Vesicle fuses with the plasma membrane to eject macromolecules.

Ex. Proteins, polysaccharides, polynucleotides, whole cells, hormones, mucus, neurotransmitters, waste

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Cell Walls and The Extracellular MatrixAlthough cell boundaries are defined by the plasma membrane,

Cells of bacteria, fungi, algae, and higher plants are surrounded by rigid cell walls

Although animal cells are not surrounded by cell walls, most of the cells in animal tissues are embedded in an extracellular matrix consisting of secreted proteins and polysaccharides.

Cell Walls The rigid cell walls that surround bacteria and many types of

eukaryotic cells (fungi, algae, and higher plants) determine cell shape and prevent cells from swelling and bursting as a result of osmotic pressure. Despite their common functions, the cell walls of bacteria and eukaryotes are structurally very different.

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Bacterial Cell Walls determine cell shape and prevent the cell from bursting as a

result of osmotic pressure. The structure of their cell walls divides bacteria into two

broad classes that can be distinguished by a staining procedure known as the Gram stain, developed by Christian Gram in 1884.

1. Gram-negative bacteria (Example: E. coli) have a two membrane system, the plasma membrane and

the outer lipopolysaccharide membrane. These bacteria have thin cell walls located between, the two

membrane systems, their inner and outer membranes.

1. Gram-positive bacteria (Example: Staphylococcus aureus) have only a single membrane, which is surrounded by a much

thicker cell wall.

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The unique structure of their cell walls also makes bacteria vulnerable to some antibiotics. Penicillin, for example, inhibits the enzyme responsible for forming cross-links between different strands of the peptidoglycan, thereby interfering with cell wall synthesis and blocking bacterial growth.

Composition of Bacterial cell wallThe cell wall of both gram positive and gram negative bacteria are

composed of a polysaccharide known as peptidoglycanPeptidoglycan is made up of two monomeric subunits-

N-acetylglucoseamine (NAG) and N-acetylmuramic acid (NAM)

These subunits are cross linked with a short peptide that provide strength to the bacterial cell wall

Eukaryotic Cell Walls In contrast to bacteria, the cell walls of eukaryotes (including fungi, algae,

and higher plants) are composed principally of polysaccharides The basic structural polysaccharide of fungal cell wall is chitin (a polymer of

N-acetylglucosamine residues), which also forms the exoskeleton of arthropods (e.g., the shells of crabs).

The cell walls of most algae and higher plants are composed principally of cellulose (a polymer of β-glucose).

Cellulose is the most abundant polymer on earth. One of the critical functions of plant cell wall is to prevent cell swelling as a

result of osmotic pressure.

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The Extracellular Matrix of Animal cellsAlthough animal cells are not surrounded by cell walls, many of the cells

in tissues of multi-cellular organisms are embedded in an extracellular matrix consisting of secreted proteins and polysaccharides. It fills the spaces between cells and binds cells and tissues together

Extracellular matrices are composed of Tough fibrous proteins(collagen) – provide structural supportA stretchable protein (elastin)- provide flexibilityPolysaccharide: glycosaminoglycans(GAGs) such as N-

acetylglucosamine or N-acetylgalactosamine. GAGs are negatively charged modified sugars and they bind

positively charged ions and trap water molecules to form hydrated gels, thereby providing mechanical support to the extracellular matrix.

Adhesion proteins: are responsible for linking the components of the matrix to one another and to the surfaces of cells. Examples: Fibronectins, entactin and Laminins

The major cell surface receptors responsible for the attachment of cells to the extracellular matrix are the integrins. In addition to attaching cells to the extracellular matrix the integrins serve as anchors for the cytoskeleton

Cell-Cell InteractionsDirect interactions between cells, as well as between cells and the

extracellular matrix, are critical to the development and function of multicellular organisms.

Some cell-cell interactions are transient, such as the interactions between cells of the immune system and the interactions that direct white blood cells to sites of tissue inflammation. In other cases, stable cell-cell junctions play a key role in the organization of cells in tissues. For example, several different types of stable cell-cell junctions are critical to the maintenance and function of epithelial cell sheets.

The connections between one cell and another are called intercellular junctions. These attachments enable the cells to resist stress and communicate with each other.

The principal types of intercellular junctions are tight junctions, gap junctions, adhesion junctions.

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Tight Junctions A tight junction joins two neighboring cells

tightly to each other by proteins that hold two adjacent cells by forming a zipperlike pattern.

This seals off the intercellular space and provide a tight barrier for some substances to pass between the cells.

For example, In the stomach and intestines, tight junctions prevent digestive juices from seeping between epithelial cells and digesting the underlying connective tissue.

They also help to prevent intestinal bacteria from invading the tissues, and they ensure that most digested nutrients pass through the epithelial cells and not between them.

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Gap (Communicating) Junctions A gap junction is formed by a ringlike

proteins known as connexin, which consists of six transmembrane proteins surrounding a water-filled channel.

Gap junctions provides direct connections between the cytoplasms of adjacent cells. Gap junctions are open channels through the plasma membrane, allowing ions and small molecules (less than approximately a thousand daltons) to diffuse freely between neighboring cells, but preventing the passage of proteins and nucleic acids.

Plasmodesmata are the only intercellular junctions in plants; they function like gap junctions

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3. Adhesion junction (desmosome). In an adhesion junction

(desmosome), the adjacent plasma membranes do not touch but are held together by intercellular filaments or adhesion proteins which can be divided into four major groups: the selectins, the integrins, the immunoglobulin (Ig) super family and the cadherins

The cell-cell interactions mediated by the selectins, integrins, and most members of the Ig superfamily are transient

Stable adhesion junctions are based largely on cadherins.

The neighboring cells are separated by a small gap, which is spanned by dhesion proteins that hold the cells together.

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The CytoskeletonThe cytoplasm of eukaryotic cells is crisscrossed by a network of

protein fibers called cytoskeleton.There are three types of cytoskeleton that can be distinguished

on the bases of their diameter, type of their subunit, and subunit arrangment1. Actin filaments2. Intermediate filaments3. Microtubules

1. Actin Filaments Are thin, flexible fibers approximately 8nm in diameter They are the thinnest of the cytoskeletons and also called

microfilaments Formed from monomeric subunits called actin proteins.

Actin proteins polymerize to form actin filaments. Each filament is composed of two protein chains that form a

twisted two-stranded structure.132

Some functions of Actin filaments: Provides mechanical strength to the cell Links transmembrane proteins to cytoplasmic proteins anchors the centrosomes at opposite poles of the cell during

mitosis Pinches dividing animal cells apart during cytokinesisGenerate cytoplasmic streaming in some cells and generate

locomotion in cells such as white blood cells and the amoeba Interact with myosin ("thick") filaments in skeletal muscle fibers

to provide the force of muscular contraction

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2. Intermediate filaments Diameter – about 10nm- intermediate in diameter between actin

filaments(8nm) and microtubules(25nm) Whereas actin filaments and microtubules are polymers of single types of

proteins (actin and tubulin, respectively), intermediate filaments are composed of a variety of proteins that are expressed in different types of cells.

More than 65 different intermediate filament proteins have been identified and classified into six groups based on similarities between their amino acid sequences Intermediate filaments found in the nucleus are composed of Lamins. epithelial cells is composed of Keratin mesenchymal cells are composed of Vimentin muscle cells are composed of Desmin long axons of neurons are composed of Neurofilaments-

Functions They provides mechanical support to cells and organelles. They mediate cell–cell adhesion and cell–matrix adhesion

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3. Microtubules Are hollow tubes of about 25nm in diameter Composed of a ring of 13 protein protofilaments formed by the polymerization of a molecule that consists of two

subunits, called alpha and beta tubulin Polymerization starts from the centrosome, which is the primary

microtubule-organizing center (MTOC) in animal cells.Functions Used as a “railroad tracks" for movement of organelles and

molecules with in the cell.The motion is provided by protein "motors" that use the

energy of ATP to move along the microtubule.The two major motor proteins that drag themselves along

microtubules and move molecules are kinesins and dyneins The migration of chromosomes in cell division takes place on

microtubules that make up the spindle fibers.

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Cilia and Flagella Cilia and flagella are microtubule-based projections of the plasma

membrane responsible for movement of a variety of eukaryotic cells. Bacteria flagellaare protein filaments projecting from the cell surface, rather than

projections of the plasma membrane supported by microtubules. Eukaryotic cilia and flagella are very similar in structures, each with a diameter of approximately

0.25µmCilia are responsible both for cell motility and for sweeping food

organisms over the cell surface and into the oral cavity. In animals, an important function of cilia is to move fluid or mucus over the surface of epithelial cell sheets. A good example is provided by the ciliated cells lining

Flagella differ from cilia in their length (they can be as long as 200 µm) and in their wavelike pattern of beating. Cells usually have only one or two flagella, which are responsible for the locomotion of a variety of protozoans and of sperm.

Structure of cilia and flagella The fundamental structure of both cilia and flagella is the axoneme Axoneme is composed of microtubules and their associated proteins In their structures the microtubules are arranged in a characteristic "9 + 2"

pattern in which a central pair of microtubules is surrounded by nine outer microtubule doublets.

The two fused microtubules of each outer doublet are distinct: One (called the A tubule) is a complete microtubule consisting of 13 protofilaments; the other (the B tubule) is incomplete, containing only 10 or 11 protofilaments fused to the A tubule.

The outer microtubule doublets are connected to each other by links of a protein called nexin. In addition, two arms of dynein are attached to each A tubule, and it is the motor activity of these axonemal dyneins that drives the beating of cilia and flagella.

Cell MetabolismMetabolism: refers to the sum of all the enzyme-catalyzed reactions in

a living organismThere are two types metabolic reactions in the body1. Catabolism: The energy releasing (Exergonic rxn) process in which a

chemical or food is used (broken down) by degradation or decomposition, into smaller pieces.

2. Anabolism: is just the opposite of catabolism. In this portion of metabolism, the cell consumes energy (Endegonic rxn) to produce larger molecules from smaller ones. Anabolism is driven by the energy that catabolism releases, so endergonic and exergonic processes, anabolism and catabolism, are inseparably linked.

All the chemical reactions that take place in the cell require catalysis.The agents that carryout catalysis in living organisms are called enzymes

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Enzyme properties Catalysts for biological reactions Most are proteins Are highly specific Work by lowering the activation energy Increase the rate of reaction without being changed themselves Their activity is lost if denatured May be simple proteins or may contain cofactors such as metal ions or

organic (vitamins)Enzyme Nomenclature and classification The name of an enzyme has two parts. The first part indicates name of

its substrate and second part ending in ‘ase’ Example:

sucrase – reacts sucrose lipase - reacts lipid

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Systematic NameThe International Union of Biochemistry (IUB) assigned a name called a

systematic name In this scheme an enzyme is assigned a four-number (Enzyme

commission number or E.C number) classification and a two-part name In each enzyme code (EC) number.

The first digit indicates major classsecond digit indicates sub classthird digit denotes sub-sub class andfinal digit indicates specific enzyme

For example, the enzyme commonly called “hexokinase” is designated “ATP: D-hexose-6-phospho-transferase E.C. 2.7.1.1.” This identifies hexokinase as a member of class 2 (transferases), subclass 7

(transfer of a phosphoryl group), sub-subclass 1 (alcohol is the phosphoryl acceptor). Finally, the term “hexose-6” indicates that the alcohol phosphorylated is that of carbon six of a hexose.

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Enzyme classificationAccording to the IUB there are six major classes of enzymes 1. Oxidoreductases.

Oxidoreductases catalyze oxidation-reduction reactions. Subclasses of this group include the dehydrogenases, oxidases,

oxygenases, reductases, peroxidases, and hydroxylases. 2. Transferases.

Transferases catalyze reactions that involve the transfer of groups from one molecule to another. Examples of such groups include amino, carboxyl, carbonyl, methyl, phosphoryl, and acyl (RC=O).

Examples include the transcarboxylases, transmethylases, and transaminases.

3. Hydrolases. Hydrolases catalyze reactions in which the cleavage of bonds is

accomplished by adding water. The hydrolases include the esterases, phosphatases, and peptidases.

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4. Lyases Lyases catalyze reactions in which groups (e.g., H2O, CO2, and NH3)

are removed to form a double bond or are added to a double bond. Decarboxylases, hydratases, dehydratases, deaminases, and

synthases are examples of lyases. 5. Isomerases

They catalyze inter conversion of optical, functional and geometrical isomers.

6. Ligases Ligases catalyze bond formation between two substrate molecules. The energy for these reactions is always supplied by ATP hydrolysis. The names of many ligases include the term synthetase.

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How enzymes workA catalyst does not change the chemical reaction but it accelerates the

reaction. They are not consumed in overall reaction. But they undergo chemical or physical change during reaction and returns to original state at the end of reaction.

For a chemical reaction A → B to occur, energy is required. When enough energy is supplied. A undergoes to transition state which is an unstable state. So, it gets converted to product B which is more stable.

The amount of energy needed to convert a substance from ground state to transition state is called activation energy.

In presence of catalyst, A undergoes to transition state very fast and requires less energy. Hence, a catalyst accelerates the rate of reaction by decreasing the energy of activation.

Likewise enzymes also speed up reaction by lowering energy of activation. Further, the activation energy is very much less for a reaction in presence of enzyme than non-enzyme catalyst. Therefore enzymes are more efficient than non-enzyme catalyst.

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Enzymes Active site The active site is a specialized region of the enzyme where the enzyme

interacts with the substrate. The active site of an enzyme is generally a pocket or cleft that is specialized to

recognize specific substrates and catalyze chemical transformations. It is formed in the three-dimensional structure by a collection of different amino acids (active-site residues) that may or may not be adjacent in the primary sequence

There are two models that describe the binding interaction between the enzyme and substrate.

The “lock-and-key” Model The “induced-fit”model for substrate

Lock-and-key model for substrate bindingProposed by Emil Fischer in 1894The lock and key model for enzyme-substrate binding uses

complementarities between the enzyme active site (the lock) and the substrate (the key). Simply, the substrate must fit correctly into the active site—it must be the right size and shape.

The two shapes are considered as rigid and fixed148

In the induced-fit model Proposed by Daniel E. Koshland in 1958 In this model, the structure of the enzyme

is different depending on whether the substrate is bound or not.

The enzyme changes shape (undergoes a conformation change) on binding the substrate. This conformation change converts the enzyme into a new structure in which the substrate and catalytic groups on the enzyme are properly arranged to accelerate the reaction.

The induced fit model recognizes that the substrate binding site is not a rigid “lock” but rather a dynamic surface created by the flexible overall three-dimensional structure of the enzyme.

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Co-enzymes and prosthetic groupsMany enzymes requires the presence of small , non protein units

or cofactors to carryout their particular reaction Cofactors can be either inorganic ions such as Zn2+, Fe2+ or

complex organic molecules called coenzymes (e.g NAD+, NADP+ FAD+)

A metal or coenzyme that is covalently attached to enzyme is called prosthetic group.

A complete catalytically active enzyme together with its prosthetic group is called holoenzyme

A protein part of the enzyme on its own without its cofactor is termed apoenzyme

Many coenzymes are derived from vitamins

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Enzyme Kinetics The rate of an enzyme catalyzed reaction is often called its velocity. The velocity of all enzymes is dependent on Substrate and enzyme

concentration, temperature, and pH1. Substrate and enzyme concentrationThe normal pattern of dependence of enzyme rate on substrate

concentration ([S]) is that at low substrate concentrations a doubling of [S] will lead to a doubling of the initial velocity (V0).

However, at higher substrate concentrations the enzyme becomes saturated and further increases in [S] lead to very small changes in V0. This occurs because at saturating substrate concentrations effectively all of the enzyme molecules have bound substrate. The overall enzyme rate is now dependent on the rate at which the product can dissociate from the enzyme, and adding further substrate will not affect this. The shape of the resulting graph when V0 is plotted against [S] is called a hyperbolic curve

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In situations where the substrate concentration is saturating (i.e. all the enzyme molecules are bound to substrate), a doubling of the enzyme concentration will lead to a doubling of V0. This gives a straight line graph when V0 is plotted against enzyme concentration.

The dependence of enzyme rate (activity) on substrate concentration has been provided in a quantitative way. The simplest of these equations, the Michaelis-Menten equation, relates the initial velocity (V0) to the concentration of substrate [S] and the two parameters Km and Vmax.

Michaelis-Menten equation The Michaelis-Menten model of enzyme kinetics applies to a simple reaction in

which the enzyme and substrate form an enzyme–substrate complex (ES) that can dissociate back to the free enzyme and substrate.

The enzyme (E), combines with its substrate (S) to form an enzyme–substrate

complex (ES). The ES complex can dissociate again to form E S, or can proceed chemically to form E and the product P. The rate constants k1, k2 and k3 describe the rates associated with each step of the catalytic process.152

From the observation of the properties of many enzymes it was known that the initial velocity (V0) at low substrate concentrations is directly proportional to [S], while at high substrate concentrations the velocity ends towards a maximum value that is the rate becomes independent of [S]. This maximum velocity is called Vmax. Michaelis and Menten derived an equation to describe these observations

The equation describes a hyperbolic curve. In deriving the equation, Michaelis and Menten defined a new constant, Km, the Michaelis constant

For many enzymes k2 is much greater than k3. Under these circumstances Km becomes a measure of the affinity of an enzyme for its substrate since its value depends on the relative values of k1 and k2 for ES formation and dissociation, respectively.

A high Km indicates weak substrate binding (k2 predominant over k1), a low Km indicates strong substrate binding (k1 predominant over k2).

Km can be determined experimentally by the fact that its value is equivalent to the substrate concentration at which the velocity is equal to half of Vmax.153

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2. TemperatureEnzymes have little activity at low temperatureRate of enzyme catalyzed rxn increases with temperatureEnzymes are most active at optimum temperatures (usually

37°C in humans) Activity lost with denaturation at high temperatures

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3. pH Each enzyme has an optimum pH at which the rate of reaction that

it catalyzes is maximum Deviations from optimum pH value decrease activity Larger deviation in pH lead to denaturation

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Enzyme inhibitionMany types of molecules exist which are capable of interfering with

the activity of an individual enzymeAny molecule which act directly on an enzyme to lower its catalytic

rate is called inhibitorEnzyme inhibition may be of two types

1. Irreversible inhibition2. Reversible inhibition which in turn can be divided into two

Competitive Non-competitive

Irreversible inhibition Inhibitor binds tightly by covalent bond to amino acid residue of

enzyme active site and permanently inhibit the enzymeE.g. a nerve gas diisopropylphosphofluoride(DIPF) react with serine

residue in the active site of enzyme actylcholinesterase irreversibly and prevent transmition of nerve impulses

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Reversible inhibition can be1. Competitive Inhibitor

A competitive inhibitor has a structure similar to substrate and occupies active site

Inhibitor competes with substrate for active siteHas effect reversed by increasing substrate concentration

2. A noncompetitive inhibitorDoes not have a structure like substrateBinds to the allosteric site of enzyme (site other than active site) Changes the shape of enzyme and active site so that substrate

cannot fit altered active site, No reaction occursEffect is not reversed by adding substrate

Enzyme Regulation In biological systems the rates of many enzymes are altered by the

presence of other molecules such as activators and inhibitors (collectively known as effectors).

1. Feedback inhibition:The end product in a metabolic pathway inhibit steps earlier in

the same pathwayThe end product bind to the enzyme at the control point at site

other than active site. Such enzymes are called allosteric enzymes

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2. Reversible covalent modificationThe activity of many enzymes is altered by the reversible making and

breaking of a covalent bond between the enzyme and a small non-protein group.

The most common such modification is the addition and removal of a phosphate group; phosphorylation and dephosphorylation, respectively.

Phosphorylation is catalyzed by protein kinases, often using ATP as the phosphate donor, whereas dephosphorylation is catalyzed by protein phosphatases.

The addition and removal of the phosphate group causes changes in the tertiary structure of the enzyme that alter its catalytic activity.

For example, glycogen phosphorylase, an enzyme involved in glycogen breakdown, is active in its phosphorylated form, and glycogensynthase, involved in glycogen synthesis, is most active in its unphosphorylated form

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3. Proteolytic activationSeveral enzymes are synthesized as larger inactive precursor forms called

proenzymes or zymogens. Activation of zymogens involves irreversible hydrolysisof one or more peptide bonds.

Example: Pancreatic proteasesThe digestive enzymes trypsin, chymotrypsin and elastase are produced

as zymogens in the pancreas. They are then transported to the small intestine as their zymogen forms and activated there by cleavage of specific peptide bonds.

Trypsin is synthesized initially as the zymogen trypsinogen. It is cleaved (and hence activated) in the intestine by the enzyme enteropeptidase which is only produced in the intestine. Once activated, trypsin can cleave and activate further trypsinogen molecules as well as other zymogens, such as chymotrypsinogen and proelastase

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Many tasks that a cell must perform, such as movement and the synthesis of macromolecules, require energy. A large portion of the cell's activities is therefore devoted to obtaining energy from the environment and using that energy to drive energy-requiring reactions.

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Glycolysis Glycolysis is a series of reactions that takes place in the cytoplasm of all

prokaryotes and eukaryotes. Glycolysis converts one molecule of glucose into two molecules of

pyruvate [which are then converted to acetyl coenzyme A(CoA) ready for entry into the citric acid cycle].

Overall, glycolysis has a dual role. The first is to generate ATP. it also feeds substrates into the citric acid

cycle and oxidative phosphorylation, where most ATP is made. The second role is to produce intermediates that act as precursors for

a number of biosynthetic pathways. Thus acetyl CoA, for example, is the precursor for fatty acid synthesis

In the process of glycolysis, two ATP molecules are needed for early reactions in the glycolytic pathway but four ATPs are generated later, giving a net yield of two ATPs per molecule of glucose degraded.

In addition to producing ATP, glycolysis converts two molecules of the coenzyme NAD+ to NADH.

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Steps of glycolysis

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Fates of Pyruvate1. Entry into the citric acid cycle.

Glycolysis releases relatively little of the energy present in a glucose molecule; much more is released by the subsequent operation of the citric acid cycle and oxidative phosphorylation.

Following this route under aerobic conditions, pyruvate is converted to acetyl CoA by the enzyme pyruvate dehydrogenase and the acetyl CoA then enters the citric acid cycle.

2. Conversion to fatty acid or ketone bodies.When the cellular energy level is high(ATP in excess), the rate

of the citric acid cycle decreases and acetyl CoA begins to accumulate. Under these conditions, acetyl CoA can be used for fatty acid synthesis or the synthesis of ketone bodies

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3. Conversion to lactate.The NAD+ used during glycolysis must be regenerated if glycolysis is to

continue. Under aerobic conditions, NAD+ is regenerated by the re-oxidation of NADH

via the electron transport chain When oxygen is limiting, as in muscle during vigorous contraction, the re-

oxidation of NADH to NAD+ by the electron transport chain becomes insufficient to maintain glycolysis. Under these conditions, NAD+ is regenerated instead by conversion of the pyruvate to lactate by lactate dehydrogenase

4. Conversion to ethanol.In yeast and some other microorganisms under anaerobic conditions, the

NAD+ required for the continuation of glycolysis is regenerated by a process called alcoholic fermentation.

The pyruvate is converted to acetaldehyde (by pyruvate decarboxylase) and then to ethanol (by alcohol dehydrogenase), the latter reaction reoxidizing the NADH to NAD+

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Tricarboxylic Acid Cycle is also called the Krebs cycle or the citric acid cycle Occurs in the mitochondria of eukaryotes and in the cytosol of prokaryotes. is used to oxidize the pyruvate formed duringthe glycolytic breakdown of

glucose into CO2 and H2O. It also oxidizes acetylCoA arising from fatty acid degradation and amino acid

degradation products In addition, the cycle provides precursors for many biosynthetic pathways. Oxidative decarboxylation of pyruvate Pyruvate, the end-product of aerobic glycolysis, must be transported into the

mitochondrion before it can enter the TCA cycle. This is accomplished by a specific pyruvate transporter that helps pyruvate

cross the inner mitochondrial membrane. Once in the matrix, pyruvate is converted to acetyl CoA by the pyruvate dehydrogenase

During oxidative decarboxylation of pyruvate, one carbon of each pyruvate is released as C02, and the remaining two carbons are added to CoA to form acetyl CoA.

In the process, one molecule of NAD+ is reduced to NADH for each pyruvate. 170

Reactions of TCA cycle The citric acid cycle is composed of eight reactions The two-carbon acetyl group combines with oxaloacetate (four carbons) to

yield citrate (six carbons). Through eight further reactions, two carbons of citrate are completely oxidized

to C02 and oxaloacetate is regenerated. Output of TCA cycle for oxidation of one glucose molecule are

Two GTP Six molecules of NADH and Two FADH2

Total economy of complete oxidation of glucose Six molecules of C02. Four molecules of ATP - two from glycolysis and two from the citric acid

cycle Ten molecules of NADH (two from glycolysis, two from the conversion

of pyruvate to acetyl CoA, and six from the citric acid cycle) Two molecules of FADH2

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Electron transport chain In eukaryotes, electron transport and oxidative phosphorylation occur in

the inner membrane of mitochondria. These processes re-oxidize the NADH and FADH2 that arise from the citric acid cycle (located in the mitochondrial matrix) and NADH of glycolysis (located in the cytoplasm)

Oxidative phosphorylation is by far the major source of ATP in the cell. In prokaryotes, the components of electron transport and oxidative phosphorylation are located in the plasma membrane.

The oxidation of a molecule involves the loss of electrons. The reduction of a molecule involves the gain of electrons.

In electron transport system electrons are transferred from NADH and FADH2 to oxygen along the electron transport chain (also called the respiratory chain).

The components of electron transport chain are arranged according to their increasing redox potential (affinity to gain electron). Oxygen is the strongest oxidizing agent and has a tendency to accept electrons.

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The electron transport chain exist as four large protein complexes embedded in the inner mitochondrial membrane called:

NADH-Q reductase (Complex I)Succinate-Q reductase (Complex II)Q-cytochrome c reductase (Complex III)Cytochrome c oxidase (Complex IV)

In ETS, NADH passes its electrons to NADH-Q reductase (Complex I) and FADH2 to succinate-Q reductase (Complex II).

Energy liberated by electron transport in ETC is used to pump H ions out of the mitochondrion to create an electrochemical proton (H) gradient. The protons flow back into the mitochondrion through the ATP synthase located in the inner mitochondrial membrane, and this drives ATP synthesis.

Approximately three ATP molecules are synthesized per NADH oxidized and approximately two ATPs are synthesized per FADH2 oxidized.

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Cell cycle and cell cycle regulation All cells arise by the division of an existing cell. During cell cycle the cell doubles in mass, duplicates its genome and organelles,

and partitions these between two new progeny cells. Events in cell cycle have to be carried out with great precision and in the

correct order, and cells have established sophisticated control processes to ensure that the cell cycle proceeds with the required accuracy

To ensure the accuracy of cell cycle, cells contain genes known as “cell cycle control genes.”

The proper functioning of such genes not only determines how big we are, it also prevents cell division becoming “out of control” and leading to cancer

In humans the cells of some tissues, such as the skin, the lining of the gut, and the bone marrow continue to divide throughout life.

Others, such as the light-sensitive cells of the eye and skeletal muscle cells, show almost no replacement.

.

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The cell cycle The cell cycle is an ordered series of events leading to cell replication The cell cycle consists of four coordinated processes

Cell growth DNA replication Distribution of duplicated chromosomes to daughter cells and Cell division

In eukaryotes cell cycle consists of four major phases. G1 phase(gap1)

is the period between mitosis and phase of DNA replicationIt is the longest and most variable cell cycle phaseDuring G1 phase cells synthesize RNAs and proteins required for DNA

synthesis Most cells in multicellular organisms are differentiated to carryout specialized

functions and no longer divide Such cells are considered to be in special compartment of G1 phase called the

G0 phase. Example nerve cells177

S Phase(Synthesis)The period during which chromosomes duplicate in number

G2 Phase(gap2) During G2 phase cells proof

read the DNA structure andmake preparations for mitosis

The G1, S, and G2 phases are collectively referred to as interphase, the period between one mitosis and the next. The M phase( Mitosis)During mitosis and subsequent

cytokinesis, chromosomes and cytoplasm partitioned into two daughter cells

Divided into four phases Prophase, Metaphase, Anaphase and Telophase

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1. Prophase. Chromosome condensed and become visible under microscope as

distinct paired threads termed sister chromatids Chromosome condensation reduces the chance of long DNA

molecules becoming tangled and broken Nucleoli and nuclear envelop disappear Microtubules grow outward from spindle poles and attach on the

sister chromatids at specialized structures called Kinetochores at the centromere

2. Metaphase Chromosomes organized at equatorial plate(metaphase plate)

3. Anaphase Each chromatid is now considered a separate chromosome; there

are two complete and separate sets. The spindle fibers contract and pull the chromosomes, one set

toward each pole of the cell.

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5. TelophaseThe sets of chromosomes reach the poles of the cell and become

indistinct as their DNA uncoils to form chromatin. A nuclear membrane re-forms around each set of chromosomes.

Cytokinesis During the last stages of telophase, the cell cytoplasm itself divides in

to two. In animal cells, a cleavage furrow made of actin and its motor protein

myosin constricts the middle of the cell In plants, a structure called the phragmoplast forms at the equator of

the spindles where it directs the formation of a new cell wall.

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Cell cycle controlProgression of the cell cycle is regulated at several checkpoints, which

ensure that all cellular components are present and in good working order before the cell proceeds to the next stage.

The checkpoints are necessary to prevent cells with damaged or missing chromosomes from proliferating

the major control points are the G1/S boundary at which point the cell is committed to DNA

replication. the G2/M transition when it is committed to mitosis.

At G1/S, the cell must decide whether it is big enough andnutritional conditions are appropriate to begin the crucial process of

replicating its genome.

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• In G2 the primary concern is Whether its DNA is in perfect condition before entering mitosis. There are sensitive mechanisms for detecting the presence of unreplicated

or damaged DNA, and cells will not commit themselves to mitosis until any defects have been corrected.

Both the G1/S and the G2/M checkpoints are regulated by a mechanism in which two proteins interact. The Cyclin and cyclin dependent kinase (CDK)

1. the G2/M checkpoint. This checkpoint is regulated by cyclin B, which combines with CDK1 to form M-

phase promoting factor (MPF). After MPF is formed, it must be activated by the addition of a phosphate group

to one of the amino acids of CDK MPF senses unreplicated or damaged DNA and generate a signal that lead to

cell cycle arrest Operation of G2 check point therefore prevent initiation of M phase before

completion of S phase . This allow time for damage to be repaired rather than being passed on to daughter cells

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2. The G1/S checkpoint This check point is known as START in yeast and restriction point in

animalsAt this check point cells evaluate its internal as well as external

enviroment and asses if it is in a position to enter S phase If it find the condition suitable, it decide to enter the S phase. Otherwise it

arrest the cell cycle at G1 and enter to Go phaseentry into S phase is regulated by three cyclin-dependent kinases called

Cdk2, Cdk4, and Cdk6,which are activated by binding with cyclin D proteins

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Regulation of cell numbercell division occurs constantly through out the life of an individual

and yet all multicellular organisms have limited size. E.g. Humans can never be as big as elephant!

The reason that multicellular organisms do not become infinitely large is because the proliferation of cells is balanced by cell death.

Cells die for two quite different reasons. One is accidental, the result of mechanical trauma or exposure

to some kind of toxic agent, and often referred to as necrosis(pathological death). This is the only type of death seen in unicellular organisms.

The other type of death is deliberate, the result of an built-in suicide mechanism known as apoptosis or programmed cell death.

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Features of cell death by necrosis Cells that die by necrosis swell and then burst. The cell contents then leak out, causing the surrounding tissues to become

inflamed. Features of cell death by apoptosis

Cells that die by suicide shrink, and their cell contents are packaged into small membrane-bound packets called blebs.

The nuclear DNA becomes chopped up into small fragments, each of which becomes enclosed in a portion of the nuclear envelope.

The dying cell modifies its plasma membrane, signaling to macrophages, which respond by engulfing the blebs and the remaining cell fragments and by secreting cytokines that inhibit inflammation.

Apoptosis begin with the signal that can come from within cell(E.g. detection of radiation induced DNA break) or from outside (e.g. decrease

in the level of growth factors or hormones)These signals induce the cell to make a decision to commit suicide

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The changes that occur during apoptosis are the result of hydrolysis of cellular proteins by a family of proteases called caspases

All the cells of our body contain caspases, but they are normally locked in an inactive form by an integral inhibitory domain of the protein

Caspases can be activated by proteolytic removal of inhibitory domain releasing the active caspase

Cells are instructed to die when a ligand binds to one of a family of death domain receptors.

This occurs, for example, If a cell is infected by a virus, white blood cells recognize viral proteins on the cell surface and activate Fas, a death domain receptor on the surface of the unlucky cell.

On binding its ligand, a death domain receptor causes caspase 8 to activate. In turn, caspase 8 can hydrolyze and hence activate the effector caspases that begin the processes of cell destruction.

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Cancer and control of cell proliferationCell growth is a carefully regulated process that respond to specific

need of the bodyIn multicellular organisms, the process of cell birth and cell death

are balancedIf the control that regulate cell multiplication break down, cells

begin to grow and divide indefinitely and results in cancer.Cancer is unregulated cell proliferation often accompanied by

abnormal differentiation (neoplasia)Cancers are generally caused by the disruption of cell proliferation

mechanism either through the activity of virus or mutation of critical growth regulation genes.

There are two classes of such genes1. Oncogenes 2. Tumor suppressor genes

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OncogenesGenes that normally stimulate cell division are called proto-

oncogenesMutation that cause proto-oncogenes to be over expressed or

hyperactive convert them to oncogenes(onco=cancer) leading to excessive cell proliferation that is characteristic of cancer

Oncogenes can also be activated by retrovirusesTumor suppressor genes(TSG)In normal cells TSG control cell cycle by blocking passage through

the G1 check point by preventing cyclin from binding to CDK thus inhibiting cell division

Genes that normally inhibit cell division are called tumor supressor genes

When TSG genes are mutated, they can also lead to uncontrolled cell proliferation

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TumorTumor is any abnormal proliferation of cells which may be either a

benign or malignant tumor1. Benign tumor:

remains confined to its original location, neither invading surrounding tissue or spreading to distant body sites.

Such tumor can be removed surgically. E.g. skin wart2. Malignant tumor:

Tumor capable of both invading surrounding normal tissue or spreading throughout the body via circulatory systems( Metastasis)

Due to spreading ability their treatment is usually difficultOnly malignant tumors are properly referred to as cancer

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Types of cancerThere are more than 100 distinct types of cancersMost cancers fall into three main groups based on the type of cell

from which they arise1. Carcinomas: cancer of epithelial tissue. Include about 90% of

human cancers2. Sarcomas: tumors of connective tissues such as muscle, bone,

cartilage etc. it is rare in humans3. Leukemias (Lympomas): cancer that arise from blood forming

cells and cells of immune system. Account for about 7% of human cancers

Tumors are further classified according to tissue of origin. E.g lung cancer, breast cancer etc

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Causes of cancer Substances that cause cancer are called carcinogenes There are many carcinogens that cause cancer1. Radiation

Radiation act by damaging DNA and inducing mutation E.g Radiation of short wave length: UV radiation, gamma radiation

2. Chemicals: can act either by damaging DNA and inducing mutation or by stimulating cell proliferation E.g. chemicals in tobacco smoke( Lung cancer), aflatoxin from small

molds(Liver cancer) induce cancer though mutation Tumor promoters such as estrogen hormone cuase cancer through

stimulation of cell proliferation3. Tumor Viruses: E.g. Hepatitis B and C(liver cancer), Papillomavirus(cervical cancer),

Herpesvirus, Retroviruses( blood cancer)

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