J. Mol. Biol. (1970) 52, 221-226

Electron Microscopy of Proteolipid Macromolecules from Cerebral Cortex

C. V~SQUEZ, F. J. BARRANTES, J. L. LA TORRE AND E. DE ROBERTIS

Institute de An&n&a General y Embrioloqia Facuihd de Medic& an&

Departamento de Biolqba, Facultad de Farmacia y Bioquimicu, Universidad de Buenos Aires, Argentina

Dedicated to the memory of Nicole Granboulan

(Received 18 December 1969, and in revised form 14 April 1970)

Electron micrographs have been obtained of proteolipid macromolecules from cerebral cortex during the successive steps of extraction and purification. These macromolecules were seen as short filaments having a width of about 15 A and a minimum length of 150 li and showed a high tendency to aggregate. This study was mainly concentrated on the “receptor proteolipid” which, by previous studies, was shown to have high affinity binding for different drugs active in nerve transmission. The binding of atropine sulphate to the proteolipid macromolecules was followed and different patterns of organization were observed. Ordered arrays of fibres, forming tubule-like structures were found with low concentrations of the drug. Then, as the concentration of atropine sulphate increased a drastic change occurred consisting of aggregation and the formation of amorphous clusters of proteolipid macromolecules.

1. Introduction Proteolipids constitute a special type of lipoprotein macromolecule found in biological membranes. They were first isolated from brain white matter (Folch-Pi & Lees, 1951) and have since been separated from a variety of sources such as mitochondria (Joel, Karnovsky, Ball & Cooper, 1958), chloroplasts (Zill & Harmon, 1961; Duffy, Katoh 6 San Pietro, 1966), bovine heart muscle (Murakami, Sekine & Funahashi, 1962), bovine heart microsomes (Shichi, Sugimura t Funahashi, 1965) and various subcellular mem- branes of the cerebral cortex (Lapetina, Soto & De Robertis, 1968). According to Zand (1968) proteolipids from brain white matter contain at least 25% of protein, high amounts of phospholipids and upon extensive purification may yield an essentially lipid- free protein, called the proteolipid apoprotein. The most remarkable characteristics of these lipoproteins are their insolubility in water and solubility in organic solvents (Folch- Pi & Lees, 1951). In the central nervous system, proteolipids are most abundant in white matter where they constitute the main protein component of myelin. The concentra- tion of proteolipid in white matter is about five times higher than that of grey matter (Amaducci, 1962). In our laboratory it was shown that proteolipid fractions prepared from nerve-ending membranes had a high affinity binding for transmitters and drugs acting on synaptic transmission. These so-called ‘receptor proteolipids’ were also characterized by their chromatographio properties and their reaction to light- scattering with atropine sulphate (De Robertis, Fiszer & Soto, 1967; Fiszer I% De

221

222 C. VASQUEZ ET AL.

Robertis, 1968,1.969; De Robertis, Fiszer, Pasquini & Soto, 1969; De Robertis, Gonzalez-Rodriguez & Teller, 1969). However, so far t,hey have not been chemically charact.erized as discrete molecular entities.

The present study was started to throw light upon the morphological characteristics of this macromolecule and on the nature of the interaction with drugs. Electron microscope studies were designed to observe the proteolipid of grey matter during the successive stages of extraction and purification. The proteolipid was seen as short filaments having a width of about 15 A; these filaments showed a high tendency to aggregate or to form net-like arrangements. The atropine sulphate-proteolipid inter- action was manifested by a progressive aggregation of these macromolecules.

2. Materials and Methods (a) Puri&ztion

Proteolipid preparations having a high affinity binding were prepared using the tech- niques developed in this laboratory (Soto, Pasquini, Placid0 & La Terre, 1969). Lipid extracts of bovine and cat cerebral cortex were obtained by homogenization of the tissue in 20 vol. of chloroform-methanol (2 : 1 v/v) (Folch-Pi, Lees & Sloan-Stanley, 1957). This was washed according to the procedure of Folch-Pi et al. (1957) and the resulting lower phase was referred to as the total lipid extract. The addition of diethyl ether to the lower phase resulted in the precipitation of most of the proteolipid, along with some phospho- lipids and cerebrosides. The diethyl ether precipitate was further purified by chromato- graphy on Sephadex LH20 (Soto et al., 1969) resulting in the isolation of a single peak of proteolipid protein previously shown to have the high affinity binding (De Robertis, Fiszer, Pasquini & Soto, 1969). A proteolipid apoprotein was prepared from the peak of receptor proteolipid using the prolonged acid-dialysis procedure described by Folch-Pi & Sherman, 1969, Proc. 2nd Int. Meeting I&. Sot. Neurochem., p. 169.

(b) Electron microscopy

Carbon supports, reinforced with collodion were mounted on platinum grids (Siemens type). The samples, containing 25 pg of proteolipid protein/ml. were transferred onto them by means of micropipettes. About 2 ~1. of the proteolipid solution were allowed to stand for 30 set on the grid surface and the grids were then blotted with the edge of a filter paper. Different negative staining techniques were used, of which 1 y0 uranyl acetate in chloroform- methanol (2 : 1 v/v) rendered the best results. Proteolipid apoprotein was stained with aqueous 1% uranyl acetate using the same procedure as above. After staining, the grids were dried with filter paper and the collodion removed in an oven at 180°C for 10 min. Electron micrographs were taken at magnifications of 40,000 with an Elmiskop 1A. In some cases fixation of the material was performed by immersing a needle wetted with the proteolipid solution into 3% glutaraldehyde in 0.1 M-phosphate buffer, pH 7.3. The thin film which was formed on the surface of the fixative was touched with a carbon-mounted platinum grid and the subsequent steps were carried out as described above.

3. Results (a) Proteolipid puri&ation

Different samples of the extracted and ether-precipitated material were examined under the electron microscope. All preparations showed the same general features, i.e. amorphous clusters of small size and randomly distributed elongated structures. The over-all diameter of the clusters varied between 100 and 500 A and were composed of small aggregates forming more or less compact structures.

The elongated structures were variable in width and length but occasionally, short isolated filaments with an individual width of about 15 A were observed (Plate I (a)).

I’LATE I. Electron micrographs of proteolipid from bovine cerebral rort<ax rxtractrv( with c.hloroform-methanol and precipitated with diethyl ether. x 200,000.

(a) Fibrilar elements are scattered throughout. The arrows point to short and fine filanwnts which constitute the main component negatively stained tvith uranpl acrtatr in rhlorof’orm-- methanol (2 : 1 v/v).

(b) Similar material spread on the surface of 3% glataraldohyde and stained with aquwus uranyl awtate. The arrows point to about, 15 Ak wide filaments. Most filaments RI-P seen in parallel arrays.

! frcr’tny p, 221

PLATE II. Electron micrographs of a last peak of proteolipitl similar to that shown in Plate I, under the action of increasing concentrations of atropine mdphate. lTranyl wetate in chloroform- methanol (2 : 1 v/v). x 200,000.

(a) Final concentration 6 x 10m7 M-atropine sulphato. A few isolated filaments are obsorvod (arrows). Most, of the protrolipids appear as elongated structurrs resulting from the association of filaments. Some small clusters are also seen.

(b) Final concentration 10e6 iwatropine sulphate. Only clusters of aggregated and CvAlapsed macromolecules are observed.

(c) Final eoncontration lo-” iv-atropine sulphate. More compact clusters aggregated into chains are seen.

PLATE III. Electron micrographs of proteolipid macromolecules from a diethyl ether precipitatca of the last peak obtained from the Sephadex LH20 column as in Plate I. Atropine sulphate was addud to tho final concentration of lo-* M. Uranyl acctatr in chlor(,form-methanol (2 : I v/x-). x 200,000.

(a) Macromolecular assemblies with a high degree of order are observed. The dense rods of 40 .& in width correspond to the uranyl stain and the intervening clear lines to the organized proteolipid macwxnolecules. The arrows point to some end views of tubular structures penetrated by the stain.

(1,) Another view of the orient&cd protoolipid macromoleroles forming tubular structures of variable length.

PLATE IV. Electron micrographs of proteoli~)itl ~,~ac.rc,rrlc,l~,r,~~l~,s frorr~ a tlic+h>~l ~~thc~r I)rc*c*il~it:tte of the last peak obtained from the Scphadrx LH2O rolllrnlr as in I’late I. Atrol)inct sulphat,cl \vas adtled to t’hr final conwnt,rat,ion of’ IO -R 11. I’ranyl ac~vtafc in c~hlor~)forrn--n~(~t,~l:~~~~~l (2 : I \./v). x 320,OUO.

(a) Paracrystallinc array of proteolipiri macromolecules forming tlrnsc~ly ~~a~lwcl tzllhular structures. The repeating distance between parallel lines is ahout 70 A.

(b) More dispersed tubular structures showing the filamcntous layer which envloscss thv lumen filled with uranyl arc obncrvrd.

apetatc. tllhlll~* stnwt 13res lying free in \vhirh some strands

BRAIN PROTEOLIPID MACROMOLECULES 223

Elongated components were also found when the material was spread on the surface of 3% glutaraldehyde; most of them appeared as filaments, presenting some degree of orientation (Plate I(b) ). In this case the amorphous clusters were barely seen on the background.

The supernatant of the ether precipitate was also examined. This fraction contains only 10% of the proteolipid but lOOo/o of the cholesterol, ‘75% of the lipid phosphorus and 50% of the cerebrosides of the total lipid extract. This preparation did not show the amorphous clusters previously described and no filaments were observed.

After column chromatography of the ether precipit’ate several fractions were generally obtained. The last peak which has a low lipid phosphorus content and a high binding affinity for n-[14C-dimethyl]tubocurarine (De Robertis, Fiszer, Pasquini & Soto, 1969), was thoroughly studied. Filamentous st,ructures were found, similar to those observed in Plate I(a); according to their concentration they were seen isolated or forming thread-like assemblies. The last peak was reprecipitated with diethyl ether, to reduce even further the remaining phospholipids, and in this ca,se large clusters wit’h a net-like filamentous arrangement were observed. These fibres were quite thick and were obviously composed by the aggregation of thin filaments.

The proteolipid protein moiety (apoprotein) obtained after prolonged dialysis was studied with 1% uranyl acetate either in chloroform-methanol or in aqueous media. Small thin filaments were observed, of about 15 d in width and about 150 A in length. These filaments were not very stable and showed a high hendency to coil. In t,his case, no clusters or bundle aggregation of filaments were found.

Control examinations were made of samples of cholesterol (Merck), phosphatidyl inositol (fraction I of Sigma) and phosphatidyl serine (brain extract III, Sigma) dissolved in chloroform-methanol (2 : 1 v/v). Mixtures of the above lipids were also examined as well as samples of the diethyl ether precipitate after repeated evaporations and dissolutions to denature the proteolipid protein moiety. The electron microscope appearance of these lipids showed a granular appearance with no resemblance to the filamentous structures described above.

(b) Changes with atropine sulphate

It has previously been established that the last peak of proteolipid eluted from the Sephadex LH20 column in the presence of increasing concentrations of atropine sulphate gives a typical light-scattering reaction. The curve starts at a concentration of 5 x lo- 7 M and follows a sigmoid shaped course, characteristic of a co-operative type of interaction, and with a Hill number of 3 (De Robertis, GonzBlez-Rodriguez & Teller, 1969). Studies by equilibrium dialysis with [3H]atropine showed that some binding had already occurred at lower concentrations (i.e. 1Om8 M) in which the light-scattering effect was not evident (GonzBlez-Rodriguez, La Torre 8: De Robertis, 1969). The electron microscope study of the drug-proteolipid interaction was carried out at various concentrations of the ligand. A solution of 5 x low7 M of atropine sulphate mixed with 25 pg of proteolipid protein/ml. produced the initiation of a series of events leading to the formation of macromolecular aggregates. As can be seen in Plate II(a), the first step in these series is an association of the filaments: small clusters of what appeared to be coiled and collapsed filaments were also observed in this preparation. A final concentration of at,ropine sulphate of 10m6 M produced a higher degree of aggregation and clusters of relatively small size were seen (Plat)e II(b)). With 10m5 M-&Opine

sulphate, the image is that of an aggregation of amorphous clusters into long chains of

224 C. Vk3QUEZ ET AL.

variable length (Plate II(c)). The visible coarse precipitation observed, at even higher concentrations, probably corresponds to the formation of large aggregates of prot,eo- lipid-atropine complexes.

In an attempt to find out which was the minimum concentration of atropine sulphate able to start the ultrastructural changes, a more purified preparation, i.e. the diethyl ether precipitate of a chromatographed proteolipid peak was assayed. Concentrations as low as 10e8 M-atropine sulpbate gave rise to molecular assemblies with a high degree of order. As shown on Plates III and IV the oriented filaments generally appear in a parallel two-dimensional array which may form multilayered three-dimensional complexes. In some preparations the degree of order was even higher, resulting in a paracrystalline pattern. The width of each filamentous layer, formed by the proteo- lipid macromolecules, was about 15 A and the distance between two successive pro- teolipid layers of the order of 40 A; this space was filled with the uranyl acetate stain. Thus, the over-all width of each “unit” was 70 A. In some places these “units” showed a rather extensive branching; others appeared isolated and it was possible to observe the presence of thin filamentous strands in the space occupied by the stain, in between the two external light layers.

The addition of atropine sulphate to the control lipid mixtures described previously did not result in any visible change in their appearance under the electron microscope.

4. Discussion There are no reports in the literature on the macromolecular structure of proteo-

lipids studied with the electron microscope. In the present work proteolipid macro- molecules were observed by negative staining during the successive stages of their isolation and purification from cerebral cortex. The diethyl ether precipitate of the total lipid extract we have examined is composed of 50 to 65% protein, 12 to 18% lipid phosphorus, 44 to 55% cerebrosides and traces of cholesterol, expressed as percentages of the amount present in tjhe original total lipid extract (Soto et al., 1969). Morphologically, this precipitate consisted of two main elements; amorphous clusters and filamentous material. In subsequent steps of purification, a diminution of the amount of the amorphous moiety was observed. The filaments were the most con- spicuous structures seen in this study and their relat’ive proportion markedly increased with purification of the proteolipid. In the precipitated total lipid extract, as well as in the chromatographed material, the proteolipid was observed as more or less straight filaments which showed a tendency to aggregate or to form net-like arrays. After prolonged dialysis, the filaments were seen as scattered elements, frequently coiled and collapsed. A possible explanation of this phenomenon might be the lack of the stabilizing properties exert,ed by phospholipids on the polUypeptide chain of the proteo- lipid. After prolonged acid dialysis, only some phosphoinositides and sulphatides remain attached to the myelin apoprotein (Folch-Pi & Sherman, 1969, Proc. Znd, Int. Meeting Int. Sot. Neurochem., p. 169. The phosphoinositides are the lipids most closely bound to brain proteolipids (Le Baron & Lees, 1962) and might act as a linkage between the apoprotein and the bulk of the lipid moiety (Le Baron, Hauser & Ruiz, 1962). Thus, the collapse of the apoprotein filaments may be indicative of a decreased stability of the proteolipid due to the loss of lipid mat,erial. Nevertheless, no striking differences in width were observed as t,he lipid moiety was removed after acid dialysis. The mean length of the filaments could not be accurately assessed because of their lateral

BRAIN PROTEOLIPID MACROMOLECULES 225

aggregation during the first steps of purification and their collapse after dialysis. Aggregation possibly occurred during blotting and dehydration of the samples on the grid surface. However, the minimum length which some filaments attain is approxi- mately 150 A. A 70% content in u-helix was reported for the myelin apoprotein in organic solvents using both circular dichroism and optical rotatory dispersion techniques (Folch-Pi & Sherman, 1969. Proc. 2nd Int. Meeting Int. Sot. Neurochem., p. 169). The filamentous nature of the macromolecules observed by us is compatible with such a finding.

Preliminary observations on the “receptor proteolipid” from electroplaques of the electric eel (Torpedo) show the presence of similar types of filaments.

The interaction of the proteolipid with atropine sulphate has been studied using light-scattering (De Robertis, Gonzalez-Rodriguez & Teller, 1969) and polarization of fluorescence (Gonzalez-Rodriguez et aE., 1969). It was suggested that atropine sulphate is able to bind initially to the proteolipid at very low concentrations and to produce later on, at higher concentrations, the association of proteolipid macromolecules into larger assemblies. The co-operativity of this phenomenon was explained by a kind of chain reaction between the proteolipid molecules which have already bound to the ligand. This hypothesis is well supported by the ultrastructural observations presented here. The size of the macromolecular aggregates formed after addition of atropine sulphate was seen to increase with the concentration of the ligand. The lower limit of sensitivity of the light-scattering techniques used to reveal the proteolipid-atropine interaction was of the order of 5 x 10-l M-atropine sulphate. Our electron microscopic studies unveiled changes in the array of the proteolipid macromolecules with concentrations of atropine as low as lo-* M, at which there is no light-scattering effect. With this concentration the 15 A filaments showed a high degree of order and they were seen in parallel array, forming “units” having a width of 70 A and a variable length. The electron dense uranyl stain filled the 40 A space in between the two light outer layers of these units, where the filaments are located. It is difficult to ascertain whether these units are tubular structures or represent palisade-like arrangements. After considering these two alternative hypotheses, we concluded that the units observed could better be interpreted as tubular assemblies of filaments. Indeed, the presence of strands in the space between the walls of these tubular structures may be indicative of some kind of organization around a central axis. In some fields it was also possible to detect the end view of tubes penetrated by the stain (Plate III(a)). At the moment we were unable to explain satisfactorily how the proteolipid macromolecules assemble in order to form these tubules. The oriented tubular configuration observed at low atropine sulphate concentrations may represent a rather unstable, but unique structure, in which the filaments might be held at definite distances by an electrostatic interaction in the high dielectric medium. We suggest that the introduction of the polar molecule of atropine sulphate at the very low concentration of 10e8 M produces sufficient changes in the protein charges to promote the formation of ordered configurations. Then, as the concentration of atropine sulphate increases, the equilibrium point for the ordered arrays is passed and aggregates are produced.

The control studies using lipid mixtures having no protein constituent strongly suggest that the changes produced by atropine sulphate are a function of the proteo- lipid-protein moiety and not the result of unspecific interaction with the lipid components.

The visualization of the purified proteolipid macromolecules as filamentous struc-

226 C. VAc;QUEZ ET AL.

tures which may change their conformation upon tho addition of very low concentra- tions of physiological act’ive substances, may provide some enlightenment on bhc function of excitable membranes. It is possible that nithin the lipoprotein structure of the post’synaptic membrane, the rcueptor protoolipid may also adopt difTerentj configurations when it binds to the natural transmitter. Wallauh 85 Gordon (1969) have stressed the importance of penetrating protein segments having a hydrophobic face and probably lying perpendicular to the membranes’ surfaces. Because of its hydro- phobic character the receptor proteolipid could penetrate across the lipid layer and in this way contribute to the formation of pores in the membrane.

We are indebted to Dr G. G. Lunt for helpful suggestions in the preparation of the manu- script.

This work was supported by grants from the Consejo National de Investigaciones Cientificas y TBcnicas, Argentina, and the National Institutes of Health, U.S.A. (2 ROl NS 06953-04 NEUA).

Two of the authors (C.V. & E.D.R.) are members of the Carrere de1 Investigador, Consejo National de Investigacionos Cientificas y TBcnicas and another of the authors (F. J. B.) is in receipt of a Fellowship from the Consejo National de Investigaciones Cientificas y T&nicas, Argentina.

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