BIOCHIMICA ET BIOPHYSICA ACTA 299

D Y N A M I C S OF T H E P H O T O S Y N T H E S I S OF C A R B O N COMPOUNDS

II . AMINO ACID SYNTHESIS

DAVID C. SMITH, J. A. BASSHAM AND MARTHA K I R K

Lawrence Rad~at,on Laboratory, Umversdy o[ Cah~orma, Berkeley, Cal~/ (U S .4 I

(Received August I3th, I96O)

SUMMARY

Further kinetic studies have been made of the rates of appearance of 14C in individual compounds formed by Chlorella pyrenoidosa during steady state photossnathesis with t4CO2. Total and "act ive" pools of several amino acids have been determined. The effects of adding unlabeled acetate and of turning off the hght have been studied m this system.

From these experiments it is concluded that synthesis and utilization of alanme, serine, aspartlc acid, glutamic acid and several other amino acids are most active within the chloroplasts during photosynthesis and that these ammo acids are formed rather directly from intermediates of the carbon reduction cycle. The major portion of the carbon utilized for amino acid synthesis is accounted for in the synthesis of these compounds. Evidence for the presence cf at least two separated pools of these amino acids is given, and the effect of light and dark and of the addition of unlabeled acetate upon the synthesis of these amino acids is discussed.

INTRODUCTION

Methods have been developed which permit comparison of externally measured rates of photosynthesis in Chlorella pyrenoidosa with the rates of flow through individual compounds in the biosynthetic pathways. These methods depend on the observation of appearance of radiocarbon in indivldual compounds as a function of t ime of exposure of the plant to 14C02 during steady s tate photosynthesis 1. I t was found that the carbon reduction cycle 2,3 and carboxylation of phosphoenolpyruvic acid account for at least 73 °o and probably much more of the total carbon assimilation. In the present report we will describe measurements of the flow of carbon into several amino acids which are among the more important secondary intermediates in the photosynthesis of carbon compounds.

In the early studies of photosynthesis in algae with 14C0~, alanine, aspartic acid, and several other amino acids were identified as being among the first compounds to become labeled 4. These compounds, as well as malic acid, were slowly labeled even in the dark when the algae were exposed to 14C02. They were labeled much more

Abbreviations' PGA or 3-PGA, 3-phosphoglycenc acid, PEPA, phosphoenolpyruvlc acid, ATP, adenosine tnphosphate , TPNH, reduced tr lphosphopyndlne nucleotlde

Bzochzm B~ophys. Acta, 48 (I96I) 299-313

300 D. C. SMITH, J. A. BASSHAM, M. KIRK

rapidly if the algae were photosynthesizing or were in the hght until the moment of addition of radiocarbon 5. I t was recognized that these compounds were products of photosynthetic reduction of 14CO2, even though they could also be slowly labeled by reversible decarboxylation reactions of respiration

Accelerated incorporation of 14C02 into the amino acids of higher plants during photosynthesis has been noted in this laboratorve,: and in many others s-l~. Some workers have suggested that certain amino acids such as glutamlc acid 12 and aspartlc acid 11 are pr imary products of CO 2 incorporation during photosynthesis.

NICHIPOROVITCH s has presented and reviewed evidence that the synthesis of proteins in the chloroplasts of higher plants is greatly accelerated during photo- synthesis. This accelerated protein synthesis appears to utilize intermediates of photo- synthetic carbon reduction since the proteins were labeled when 14C02 was ad- ministered but not when [~4C]carbohydrates were supplied. $ISSAKIAN 13 has reported and reviewed experiments that show that protein can be synthesized in isolated chloroplasts from non-protein nitrogen, Including peptldes

HOLM-HANSEN 14 found that the incorporation of ~4C02 into amino acids was considerably increased in algae when they were supplied with ammonia. This increase was very marked in the case of glutamlc acid. I t had previously been found that algae photosynthesizing in distilled water made very little labeled glutamic acid until the light was turned off ~'~, ~6, whereupon there was a large Increase in the labeling of both glutamlc acid and citric acid, but no notable increase in labeled alanine

In the s tudy reported here, we have sought answers to the following questions: (a) What are the rates of flow of carbon through amino acids during photosynthesis and what portion of the total carbon uptake can be ascribed to amino acid synthesis? (b) Is there more than one reservoir of each amino acid? If so, what are the concen- trations of the reservoirs directly involved in photosynthesis? (c) What are the metabolic pathways leading from pr imary products of photosynthesis to these amino acids, and are any of these amino acids themselves pr imary products of carbon reduction? (d) What is the explanation of light-dark transient effects on the rates of synthesis of various amino acids?

E X P E R I M E N T A L

The experimental methods relating to maintenance of the algae (ChIorella pyrenoidosa) under steady s tate photosynthesis and the analysis of the labehng of compounds with 14C has been described in this journal 1. The same methods have been used in these studies except as noted.

Nutrient solution

The nutrient solutions used earher, while adequate for the purposes of those experiments, were insufficient to maintain the levels of amino acids and their rates of formation constant over several hours. We have therefore experimented with various nutrient solutions, testing for the maintenance of steady state photosynthesis as well as for the effect on paper chromatography. We have now found two solutions which appear to satisfy our requirements. One of these is a "s tar t ing" medium (Table I) which is used for suspension of the algae at the s tar t of the experiment. The other is

Bzoch,m. Biophys Acta, 48 (19611 299-313

PHOTOSYNTHESIS O1 ~ AMINO ACIDS 301

the "adding" medium (Table II) which is added automatically to the algal vessel as the pH of the suspension changes in response to mineral uptake by the algae.

In order to assure solution of all the elements in the "adding" medium it is necessary to dissolve the ammonium carbonate in about 80 % of the water and to

TABLE I

STARTING MEDIUM

KH,POI 0.20 m;V/ MgSO 4" 7H~O o 20 m_M MgCIz 0.20 m M K2CO a o. io m M Ca(NOa) 2 o.o2 m~ / KNO a o 05 m~V/ A solution of trace elements plus CoC12. 6H20 (4 ° mg/l) and

M o O a (15 m g / 1 ) I ml/1 NH4VO a (o.o2 3 g/l) i ml/1 (NH,),CO 3 o.15 m.~/

Saturate with 2 % CO 2 by bubbl ing mixture of 2 % CO2-in-air through solution unti l pH reaches a constant level

Add FeC1 a 0.02 m/V/

TABLE I I

A D D I N G MEDIUM

(NH4)2CO s 6 75 m M NH4H2PO 4 i . i o m M KH~PO 4 o 4 ° m M MgSO 4. 7H~O 1.5 m2~l MgClz 1.5 m M KNO 3 o o 5 m M Ca(NO3) 2 o o5 m M A solution of trace elements plus CoC1,.6H20 (4 ° mg/l) and

MoO a (15 mg/l) I ml/1 NH4VO a (o 023 g/l) I roll1 FeC1 a o.I m M

bubble a mixture of 2 % C02-in-air through it until a constant pH is obtained. All the other elements, except the FeC13, are dissolved in the remaining 20 °/o of water. These two parts are then mixed and the proper amount of a concentrated solution of FeC13 is added. Even so, when the FeC13 solution is added to the rest of the "adding" medium, some cloudiness occurs. However, this precipitate does not settle out during the course of the experiment and it is presumed that the chemicals contained in the fine suspension are available to the algae once it is added to the more acidic algal s o l u t i o n .

W h e n t h e a lgae a re f i r s t s u s p e n d e d in t h e " s t a r t i n g " m e d i u m t h e p H is a b o v e 6.

A f t e r 2 ° o C 0 2 - i n - a l r h a s b e e n p a s s i n g t h r o u g h t h e a lga l s u s p e n s i o n for s e v e r a l

m i n u t e s , t h e p H d r o p s t o a b o u t 5.8. T h e l i g h t s a re t h e n t u r n e d o n t o I l l u m i n a t e t h e

a lgae cel l a n d p h o t o s y n t h e s i s beg i n s . F o r a few m i n u t e s t h e p H r i ses to a b o u t 6.2.

W h e n t h e p H b e g i n s t o fa l l d u e t o N H ~ + u p t a k e , t h e p H c o n t r o l u n i t is se t a t p H = 6

B,ochzm B,ophys Acta. 48 (1961) 299-313

3o2 D . C . SM1TH, J . A. BASSHAM, M. K I R K

and is then tu rned on. Occasional ly i t is necessary to InJect a smal l amoun t of o. I .\; HCI to br ing the p H down to 6.o. The ra te of add i t i on of " a d d i n g " med ium is then found to be fa i r ly cons tan t for several hours The dens i ty of the algae suspension also remains cons tan t .

Table I I I (see R E S U L T S ) shows the ra tes of u p t a k e of a m m o n i a and CO 2 along with the ra te of oxygen evolu t ion when the above med ia are used over a per iod of t ime to ma in t a in the cont inuous g rowth of a r ° 0 suspension of algae

The ra te of NH4+ up t ake is ca lcu la ted from the ra te of add i t i on of the " a d d i n g " med ium and the known concen t ra t ion of NH4 ÷ in the " a d d i n g " medium. Since some NH4+ Ion is r emoved when samples of algae suspension are wi thdrawn, th is calculat ion gives an upper l imi t to the ac tua l NH~+ u p t a k e rate .

T A B L E I I I

RATES OF N H 4 + AND CO 2 UPTAKE AND 0 2 EVOLUTION OF Chlorella

Erpt No

Grown *n descr*bed medm

Rate

T~me Hours after hghts on

pmoles/mzn/g wet-packed alaae

NH4 + O: CO, (upper hind)

3 ° o 5 20. 4 22.2 I . o 2.4 o 2 o 19 8 21.1 3 .26 2 75 20.8 19.3

31 o 5 16 6 19 4 2 3 I o 18 2 18 i 2.8 1 5 2 6 2 o 18 o 14 8

32 o 5 19 o 19 3 I .O 3 0 2 0 2 6

2.5 I9 6 15 7 2 5 3 0 2 5

0 - 8 m l n d a r k 4 3

Administration of x'C0 2 and H14COa- Administration of 14CO2 and H 1 4 C 0 3 - w e r e made by the same method as described

in the previous paper 1. Specific radioactivlties of from 6 to 7/~C .'/,mole of carbon were used in each experiment.

Analysis of samples The analyses were carried out as previously reported I. Approximately i ml

samples were taken into weighed tubes containing 4 ml of methanol. Samples were then extracted and analyzed by two dimensional paper chromatography. Radio- autographs of the chromatograms were prepared and radioactivity of each compound from each sample was determined.

B~och~m. B z o p h y s Ac ta , 48 (1961) 299 -313

PHOTOSYNTHESIS OF AMINO ACIDS 303

Amino acid determination

Carbon labeling: As indicated in the earlier work 1, the concentration of carbon in a pool which is rapidly turning over may be measured by allowing it to reach a constant level of labeling with 14C. At this t ime the specific radioactivity of this pool of the given compound is the same as that of the carbon dioxide which is administered to the plant. Such a pool will be referred to as the "ac t ive" pool. We desired to measure the concentrations of amino acids in the "active" pools and to compare them with the total amino acid concentrations. As before, we shall express as "/zmoles of 14C" the amount of 12C and 1'C in/ ,moles which corresponds to the measured amount of radio- activity, using the specific radioactivity of the administered ~*CO2 + ~2COv In other words, if S = specific radioactivity of the CO2 (In/*C/~moles) and A =rad ioac t iv i ty (In /*C) found in a given compound from I ml of wet packed algae, then A/S -~ "/*moles of 14C" in I ml of algae.

When the radioactivity of a given metabolic pool of a compound no longer increases as a function of the time of exposure of the photosynthesizing algae to 14COv the pool is said to be "sa tura ted" with 14C, and the/ ,moles of "14C" as defined above is considered to be equal to the steady state concentration of the metabohc pool. A given metabolic pool is usually only one of several pools of the same compound in the plant. Therefore the total cellular concentration of a given compound is usually greater than the measured "act ive" pool.

In the case of the sugar phosphates of the carbon reduction cycle, and of PGA, the "ac t ive" pools saturate in 3 to 5 min after the introduction of x4CO2, and there is seldom any problem m deciding what the "ac t ive" pool concentration is 1, ~e. In the present s tudy we found tha t the "ac t ive" pools of some amino acids saturated quickly and thus the levels could be easily determined. In the case of other amino acids, slowness of the "act ive" pools to saturate, coupled with appreciable rates of labeling of other pools of the same compound, have made measurement of the "ac t ive" pool size difficult or at times Impossible. Thus some "act ive" pool sizes are given as approximations or as a range of values.

Ninhydrin estimation: In order to provide a comparison between "ac t ive" pool sizes and total concentrations a method was developed to est imate the concentrations of amino acids in spots on the chromatograms. In these studies it was necessary to chromatograph the extract from no more than 5 mg of algae, due to the necessarily high salt content of the extracts. The methods of colorlmetrlc estimation of amino acids previously reported from this laboratory 17 did not afford sufficient sensitivity and accuracy for this purpose.

Methods involving ninhydrin are generally the most satisfactory for estimating small amounts of amino acids, but the major difficulty of using these methods on chromatograms or their eluates is that the paper may give a high and variable back- ground color. This is presumably due mainly to traces of ammonia absorbed from the atmosphere or contained in the chromatographic solvents I t is particularly trouble- some when the nmhydrm is in the reduced and more sensitive form necessary for the estimation of small amounts of amino acids.

I t was found that this background color could be reduced to a low and constant value if the eluates are made alkaline and then scrubbed as described below. Since accurate estimates with reduced mnhydrin must be made under buffered conditions,

Bzoch,m. B~ophys Acta, 48 (1961) 299-3i 3

304 D . c . SMITH, J. A. BASSHAM, M. KIRK

it is possible to use the alkaline component of the buffer system for scrubbmg of the eluates, and then the acid component can be added afterwards

After location of the amino acid spots on the chromatograms by radioautography, they are cut out and each eluted with about 0.3 ml of water into graduated centrifuge tubes. Then o.I ml of o 5 N NaOH is added and the solution is scrubbed with NHs- free air under partial vacuum for I5 mln at room temperature. Next o.I ml of a 0.8 N citric acid solution is added to bring the pH to 5.0, followed by the addition of 0. 4 ml of o 83 % ninhydrin and o.15 mM KCN in methoxyethanol. The total volume is made up to 0.9 ml with water and after mixing well, the solution is heated for 15 min in a boiling water bath (the mouths of the reaction tubes being stoppered with glass marbles to prevent loss of volume). After cooling on ice, I.O ml 60 °'o ethanol is added and the absorbancy at 57 ° m/, is determined in a Beckman spectrophotometer using cells of 2.0 ml capacity. Under these conditions, for example, I /zg of glutamic acld l~ eqmva- lent to an absorbancv change of o 075. There is a linear relationship between ab- sorbancy and amount of amino acid over the range o.i to 1.5/~g amino nitrogen.

The background values for blank areas of chromatograms are determined and appropriate corrections applied to the amino acid values, depending upon the areas of the spots. (A typical area of about 30 cm 2 of paper weighing 300 mg would have a blank of about o.I absorbancy units.) The radioactlvlty of the papers after elution is also checked. Usually 97 °o to 98.5 °o of the activity has been eluted. Since finger- prints leave ninhydrm positive marks on chromatogram papers, rubber gloves should be worn at all stages of handling the paper chromatograms.

The ninhydrln method described above was modified from that described by YEMM AND COCKING 18 bv changing the volumes and concentrations of the reagents in order to increase the sensitivity and by including the method of scrubbing the eluates.

Acetate feeding experiments In Expt. SS3I, after the algae had been photosynthesizing for 60 min In 14CO2

under steady state conditions, 80 ~1 unlabeled I.O N ammonium acetate was injected into the algal suspension (volume 80 ml) to give an initial concentration of Io -3 M acetate After IO rain and again after another IO mln, similar additions were made. All other environmental factors were maintained constant, including pH.

Light-dark experiments

In Expts. SS3 o and SS32, after the algae had been photosynthesizing in 14CO2 under steady state conditions for 60 and lO4 rain, respectively, the light was turned off, all other conditions being maintained constant. Samples were taken for another IO mln After analysis by chromatography, the radioactlvitles of certain compounds were measured to study tile effect of the hght-dark transient period. During this time the rate of addition of nutrient was still followed.

RESULTS AND DISCUSSION

"Active" pool s,zes and rates of photosynthesis

In Table I II , the rates of C02 and NH4 + uptake and of 02 evolution during the course of three steady state experiments are shown. The times given are from the

B,ochzm B~ophys Acta, 48 (1961) 299-313

PHOTOSYNTHESIS OF AMINO ACIDS 305

40

35

30

~25 :t_

2o

15

lO

beginning of illumination of algae in the apparatus, the administration of z4C02 being about 1-I .5 h after the s tar t of the experiment. The rate of oxygen evolution is the best maintained of the three. The calculated rate of N H , + uptake is also reasonably constant in the last two experiments. There is a tendency for the CO 2 uptake rate to decrease about 25 °o after a time. Since the algae in the steady state apparatus are exposed to greater illumination than in their gro~ang tubes, this decrease in CO 2 fixation rate may inchcate some quanti ta t ive change in the overall metabolism. For example, the amount of fa t ty acid synthesis may have Increased. Thus, a perfect "s teady s ta te" has not been achieved in these experiments. Nonetheless, the approach to the true steady state in these experiments is sufficiently close to permit us to measure a number of properties of the system.

If one takes the average rate of NH4+ uptake to be 2.5 Fmoles/min/ml of algae, then amino acid synthesis appears to account for about half of the total synthesis of carbon compounds (protein being assumed to be composed of 3.2 atoms of carbon/atom of nitrogen)•

The appearance of llC in alanine and in glutamic acid in SS32 is shown in Fig. I. These curves illustrate two extreme cases observed with the rapidly labeled amino acids during photosynthesis. In contrast to the glutamic acid pool, the "act ive" pool of alanlne ~s clearly saturated after 3 ° min and its size can be determined to be 20 Fmoles of carbon/ml of algae Glutamlc acid continues to increase In labeling for the entire IOO mln in Expt SS 32 (Fig. I) I t is impossible from these data to determine accurately the size of its most "ac t ive" pool. Nonetheless, if one fits an exponential curve to the experimental labehng curve, an approximate "act ive" pool size of about 35 ~moles can be determined. Since the total pool of glutamic acid in Expt. SS32 was found to be about 77 Fmoles, it is apparent that there is more than one pool of glutamlc acid.

The labeling curve of glutamm acid is further complicated by the mdmation that a less "ac t tve" or secondary pool of glutamlc acid is being slowly labeled from 30 min onward. This can be seen more clearly in Fig. 2 (SS3o) where the curve for the specific radioactivity of the total glutamic acid pool has followed an exponentml course until after 2o min and then begins to rise more steeply again.

601

g I m ! o 50 l

40!

30 u ,

u 2 o l

1o I i

I~) ~ / 3[0 40 510 60 70 80 90 1~) II0 T~rne (m inu tes ) a f t e r adrnlss=on of 14C02

Fzg. z. L a b e h n g of g l u t ami c aczd a n d a l a n m e m s t e a d y s t a t e E x p t . 32.

• ' ° /o14C m totol corbon

~o Total carbon

..-

...-'" o 14 C

• . - ; ' " " " o

/ o

10 20 30 40 50 60 Time(minutes ) a f ter odm;ss;on of I4CO2

Flg. e. To ta l carbon, labeled carbon a n d overal l specific a c h v l t y of g lu t amlc acid m s t e a d y s t a t e

E x p t 3o.

B*ochim. B~ophys Acta, 48 (1961) 299-313

306 D. C. SMITH, J. A. BASSHAM, M. KIRK

Aspartic acid (Fig 3) becomes labeled In a manner kmetlcally similar to alanlne labeling and its "active" pool is nearly saturated after I h Serlne (not shown) is intermediate between alanlne and glutamlc acid in the shape of its labehng curve, its secondary pool(s) apparently being slowly labeled during photosynthesis O/ o o 4 S o °°~

,~3s

/

2I 1 0

I i t I t I ~ I I I 0 tO 20 30 40 50 60 70 80 90 100

Minu tes o f Ler i n t r o d u c t i o n 14C0 2

Ftg. 3 Appearance of 1~C in aspartm acid m steady state Exp t 32.

10C

oo 9c {8c -~7c u

,r 6 c

5C

3C

2C

10 O5

0

Fig

/

,,// °

G l u t a m m e

~o 2o 3b io ~'o 6b ~ ~ 9'o ~o M i n u t e s a f t e r i n t roduc t ion 14C02

4. Appearance of I~C in glutamine in steady state E x p t 32

The carbon labeling curve of glutamlne (Fig. 4) is similar to that of glutamlc acid. In fact the curves for these two compounds, if divided by their respective labels at IOO mln, give curves that are essentially superlmposable. From this fact, we can say that no major pool of glutamlc acid provides the precursor for glutamine synthesis Rather, glutamine and glutamic acid must come from a common precursor. Of course, all such kinetic arguments must be qualified by the fact that the common precursor could be a complex, such as an enzyme complex, of one of the two compounds. There is no reason to assume that the precursor is such a complex, and we are inclined to think that the precursor is a blochemlcally identifiable compound Earlier work from this laboratory, in which lnhibitors of amino acid formation were employedlY, 19 indicated the possibility that glutamine could be formed m photosynthesizing Chlordla pyrenoidosa without the prior formation of glutamm acid. For these reasons we shall regard the Incorporation of NH~ + and carbon into glutamine as not part of that Incorporated into glutamic acid.

In Table IV, the sizes of such "active" pools and of the totals of all pools are given for the four most rapidly labeled amino acids In Expts SS3o, SS3I, and SS32 the "active" pool size is taken as the saturation level of an exponential curve fitted to the first 2o min of the experimental curve for glutamlc acid and serlne The differences in total concentrations of amino acids between the experiments shown in Table IX; presumably reflect changes in the stock algal cultures during the six month period of the experiments The sizes of the "active" pool~ are generally less than half of those of the total pools. In each case there are pools other than the one whmh is so rapidly labeled during photosynthesis

In order to calculate the rates of flow of carbon through the "active" pools of several primary amino acids, we determined the maximum slope of the labeling curves as shown in Figs 3 and 4 by the thin hne Th~s slope, N, is the net rate of

B~och,m B~ophys .4cla, 48 (19611 299-313

P H O T O S Y N T H E S I S OF AMINO ACIDS 3 0 7

T A B L E I V

AMINO ACID LEVELS IN STEADY STATE, EXPERIMENTS

A s u m m a r y of a m i n o a c i d c o n c e n t r a t i o n s ( exp re s sed in / , m o l e s c a r b o n / g w e t p a c k e d a lgae) in 5 s t e a d y s t a t e e x p e r i m e n t s I4C poo l s m e a s u r e d a t t i m e w h e n " a c t i v e " poo l s of a m i n o ac id s

b e c o m e s a t u r a t e d (for e x p l a n a t i o n see t ex t )

Glutam,t at zd ,4lamne 4spartw actd Serme T~me at u,kwh

I~moles "4etwe" ttmoles "Actwe" itmoles "Actwe" itmoles "Actwe" values mtasured total pool total pool total pool total pool for i4C content

carbon t,moles t*C carbon t, moles t~C carbon ltmoles t~C carbon t~moles t4C

SS26 44 7 I8 I2 3 5 5 20 m m S S D S I 41.2 17 9.5 5 3 2o m l n SS3 o 47-5 15-2o 41 o 15 6 4 3 .0 6 3 3 . 8 - 4 .2 * SS3I 43 4 1 5 - 2 o 32 o 7 i 2 6 7 * SS32 77.5 2 5 - 4 0 42.3 20 o 19.5 4 2 2o 2 3 2 -4 -0 *

* V a l u e e s t i m a t e d on bas i s of e x t r a p o l a t i o n of e x p o n e n t i a l c u r v e s for g l u t a m m ac id a n d s e r m e , l e v e l i n g o f f of e x p e r i m e n t a l c u r v e fo r a l a n l n e a n d a s p a r t l c a c i d

T A B L E V

RATES OF FLOW OF CARBON THROUGH "ACTIVE" POOLS OF AMINO ACIDS

Steady stale Expt 32

Calculated rate Equwalents NH4 + Compound Net *ale (N) *n Degree of of syntheses (R) uptake

l~moles o f t x* salural~on l~moles of carbon ltmoles of N H t +

A l a n l n e 2 oo o 25 2 67 S e n n e o 42 o 15 0 .49 A s p a r t l c ac id o 67 o 25 o 89 G l u t a m l c ac id o 73 o 25 o 98 G l u t a m l n e o 23 o 25 o 32 G l y c l n e * o 034 o i 0 .04 C l t r u l h n e * * o 09 - - o 09 T h r e o n m e * o . i o o 5 o 20

T o t a l 5 44 E x t e r n a l l y m e a s u r e d u p t a k e 17 o P e r c e n t of t o t a l t h r o u g h t h e s e pools 32 °5

o 89 o .16 o 22 o 2o o 1 3 o 02 o 09 o 05

i 69 2 6

65 o;

* N o t i n c l u d e d in t o t a l s ** F i g u r e s a r e for c a r b a m y l c a r b o n o n l y

increase in 1~C at a given time after the introduction of 1'C0,. If the actual rate of flow of carbon of both isotopes through the pool is R, and if the precursor is saturated with 14C, the rate of flow of ~4C into the pool is also R. If the degree of saturation of the pool is x, at the time where N is measured, the rate of flow of I~C out of the pool i s Rx , and R = N / ( I - - x) . The calculated rates are given in Table V. A range of pool sizes of glutamic acid (the most uncertain case) from 25/~moles to 50/~moles would cause a range in calculated synthesis rates from 1.o4-o.92/~moles/min/ml algae; that is, ~ 7 % of the reported value. The slopes are measured at times from 5 to 16 min from the introduction of 14C0~, by which time the known precursors (PGA and phosphoenolpyruvic acid) are saturated 1. The slower achievement of maximum

B w c h ~ m . B w p h y s A c t a , 48 ( I 9 6 I ) 2 9 9 - 3 1 3

308 D. C. SMITH, J. A. BASSHAM, M. KIRK

labeling rates (measured at 16 rain) in glutamic acid and glutamine indicates that these amino acids have additional, unidentified precursors. It is possible that unstable, volatile, or non-extractable precursors (which we would not see) are not saturated by 16 rain and that the measured and calculated synthetic rates are therefore too low. In any event, no large pools of unknown radioactive compounds are seen during thL- period

The total of the carbon dioxide and ammonium ion used in the synthesis of these principal amino acids as calculated in Table V represents about 65 °'o of all the amino acid synthesis as calculated from the externally measured ammonium ion uptake rate and about 32 o o of the total carbon dioxide uptake Other amino acids are labeled at appreciable rates and no doubt account for some of the remaining ammonium ion uptake. In addition, amination of some amino acid moieties of proteins may not take place until the carbon chain has been incorporated into the protein or peptlde chain. In particular the very small rate of labelling of glycine as compared with the abundance of glycine moieties m plant protein may indicate that free glycine is not an important intermediate in protein synthesis in these organisms. Amination of the two-carbon skeleton to give glycme moieties probably would account for at least IO ° o of the NH4 + fixation.

The results and the conclusions so far may be summarized as follows' I The "active" pools of several amino acids have been measured by the laC

saturation technique, and are found to represent only 20 to 50 °o of the total pools of these amino acids in the cases of alamne, aspartic acid, glutamic acid and serlne Therefore two or more pools of each of these amino acids exist in the algal cells.

2. The rates of synthesis of these amino acids from carbon dioxide have been calculated, and are found to account for at least 32 °'o of all carbon fixation during steady state photosynthesis under the chosen environmental conditions. If certain assumptions are correct, the synthesis of these amino acids also accounts for about 62 °,o of all the uptake of ammonium Ion, which IS the sole source of nitrogen in these experiments

3. The net increase of i4C m alanine, serlne, and aspartlc acid reaches maximum rates by 5 mln after the introduction of 14CO2, at the same time that intermediates of the carbon reduction cycle of photosynthesis have become saturated with a4C, but long before such secondary products of carbon reduction as sucrose and these amino acids themselves have become saturated with ~4C. This indicates that the immediate precursors of these amino acids are Intermediates m the carbon reduction cycle, or are in isotopic equilibrium with intermediates of the cycle (e.g phosphoenol- pyruvic acid).

In isolated chloroplasts from Swiss chard, TOLBERT 2° has shown that inter- mediates of the carbon reduction cycle and related compounds (phosphate esters of sugars and carboxylic acids) do not diffuse out of the chloroplast. He found, instead, that l~C labeled compounds in the supernatant fluid following 14CO2 fixation in the chloroplasts were glycollic acid and sucrose, and to a lesser extent, some free carboxylic acids, and serine and glycine. It is also noteworthy that alanme and aspartlc acid became labeled in the chloroplasts but did not diffuse to the super- natant fluid. If we may assume that chloroplasts from Chlorella and Swiss chard are similar with respect to formation and retention of these compounds, we are led to the conclusion that '

B~ochtm Btoph),s .4cta, 48 (1961) 299-313

PHOTOSYNTHESIS OF AM1NO ACIDS 309

4. The rapid s}mthesis of the amino acids found in the "act ive" pools in our experiments takes place within the chloroplasts.

5. Since carbon is not only going into these pools during steady s tate photo- synthesis but is also passing through them; and since the less "act ive" pools of the same amino acids (presumed to be outside the chloroplasts) are not becoming labeled at an appreciable rate, it follows that more than 60 °/o of the protein synthesis in Chlorella under these conditlons of photosynthesis takes place in the chloroplasts, via free amino acids.

6. The slowness of glycme synthesis in these experiments, coupled with the relative frequency of glycine moieties in most plant proteins suggests that the glycine moieties of the chloroplastic protein are either supplied by extra-chloroplastic synthesis from unlabeled substrates, or more likely, are incorporated into the protein chain as some other two carbon compound, such as glyoxyhc acid. This finding would seem to be consistent with the observations of SISSAKIAN 13 who found that [14Clglycine ad- mimstered to various fragments of tobacco leaf cells was incorporated much more readily into the mitochondrial protein than into the chloroplastlc protein. In either case, incorporation of glycine was inhibited by the supernatant solution. Perhaps the supernatant solution contains glyoxylate which is used preferentially for protein synthesis, especially in chloroplasts, or contains other factors favoring the formation of glyoxylate from endogenous substances.

10C A c e A t a t e ,, ,

9 C ,

: 1 l u

~,8c : i :

,~ 7¢ ' i i

6<: , o t a l c a r b o n

CO o . . . . . . . . . . . . 9 J - _ _ J ' , i 1 4 5C . . . . o p , t E ~ C a s °A u o o . , J "~ total

4C o,,,~ ~ # , w,, carbon

• ° ~ 1 4 C

.1~ r r t i i i i i , i i 10 20 30 40 50 60 70 8 0 9 0 100 110 120 130

T i m e in m i n u t e s

Fig. 5- To ta l pool size a n d rad*ocarbon in g l u t aml c acid d u n n g s t e a d y s t a t e E x p t . 32. Ace ta te addl taons were each 8 0 / , m o l e s to 8o ml a lgae suspens ion .

Feeding unlabeled acetate

Fig. 5 illustrates the results of the steady state experiment in which unlabeled acetate was supplied to the external medmm at intervals beginning 60 min after the introduction of 14C02 to the system. The first addition of acetate results in a large and immediate rise in the total amount of glutamic acid, the second addition causes a smaller rise and the third addition has no significant effect. However, none of the additions of acetate causes any significant change in the concentration of [14C]glutamic acid, so that its specific act ivi ty drops sharply after the first two additions. These results complement those of MOSES, HOLM-HANSEN, BASSHAM AND CALVIN 21, who found that when labeled acetate was supplied to Chlorella there was a much greater

Btochzm B,ophys. Acta, 48 ( i96t) 299--313

31o D. C SMITH, J A. BASSHAM, M. KIRK

incorporation of 14C into glutamic acid after 3 min than when the label was supplied as I~CO~. In the experiment Illustrated in Fig 5, addition of acetate caused no appreciable change in the concentration of either total or [I4C]alanine, this result again complements those of the above authors.

Two mare conclusions are suggested by the results of this experiment' (a) The rapidity with which externally supplied acetate is incorporated into glutamlc acid, and not into alanine, suggests that the latter may be synthesized entirely from the acetate, perhaps by the glyoxylate pathway described by H. L. KORNBERG22,~3; and (b) the fact that addition of acetate causes no appreciable change in the con- centration of [14Clglutamic acid it further evidence for the existence of separate pools of glutamic acid. The rapidity with which the externally supplied acetate enters the "inactive" pool suggests that it may be located outside the chloroplast, while the "active" pool is located in the chloroplast.

I t should be mentioned that acetate was added as ammonium acetate and that the results of addition could conceivably be caused by the increase in ammonium ion concentration. However, glutamic acid alone of the amino acids, and only the un- labeled glutamic acid, was significantly affected. Considering previously reported synthesis of glutamlc acid from labeled acetate in Chlorella, it seems likely that the observed results are caused by the added acetate, not the additional ammonium ion.

Light-dark transient experiments

If Scenedesmus in distilled water in the light is supplied with laCOz, very little labeled carbon appears in glutamlc acid, but if the light is turned off shortly after admission of 14CO2, then labeled carbon appears rapidly in this compound 15. When Scenedesm,ts (or Chlorella) is in an ammonia medium in the light, then appreciable amounts of labeled carbon will be incorporated into glutamlc acid from ~4CO~, (see ref. 14) but again, if the light is turned off shortly after admission of 14CO2, then there is a rapid rise in the amount of ~4C in glutamlc acid. In both these kinds of experiments, the hght-dark transient effects were observed before the pools of photosynthetic intermediates had become saturated with ~4C. It was therefore of Interest to see if these effects still occurred when the light was turned off some time after saturation of the pools of intermediates, and to determine if the\" were correlated with changes in the total amount of glutamlc acid.

Figs 6 and 7 illustrate the results of this kind of ~teady state experiment in which the light was turned off some 60 or IO0 mm after the admission of ~4CO2. Several effects occur when the hght is turned off: the total amounts of glutamlc acid and alanlne immediately begin to rise, while the amount of 14C falls somewhat - -but rises again shortly afterwards The total amount of the acids then falls off, followed by a fall in the amount of 14C. During the first 8 mln of darkness, the rate of ammonia uptake increases (Table III).

The explanation of these effects is complex. The initial drop in ~4C during the first 30 see of darkness might result from the cessation of CO s fixation and reduction. The initial rise in the total amount of the amino acids, the subsequent rise in the amount of ~4C and the increased rate of ammonia uptake all imply that turning off the light has in some way increased the total rate of combination of ammoma into amino acids Light-dark transient effects have been previously explained in terms of a release of photoinhlbltlon of the decarboxylation of pyruvic acid 15-1~. This would

Brochure Bzophvs Acla, 48 (1961} 299-313

PHOTOSYNTHESIS OF AMINO ACIDS 311

not be a satisfactory explanation for the rise in the pool of " inact ive" amino acids observed here, since such an explanation would require that either the "react ive" pools are in the chloroplast, or that the photomhibition is operative in the cytoplasm.

7 O

~ 4o

g ao l : l L

E ~k ~ 0

Total glutamlc ,/ ",,, ~ carbon [ o ! ~ , /

, I %' " - , , \ a, . . . . . \ , Y ,/ c a r b o n

/ / %

/ {~ -------'~ "~ "'.,.,~ ""o'~ .[14C.] a lan me ~ t:._.._ ~ 0

~eg " [14C] ~[u t . . . .

L I i i [

T ime in m i n u t e s a f t e r t u r n , n g o f f h g h t

Fig 6 Changes in to ta l and [laC]glutamlc acid and a l a n m e when h g h t is tu rned off (SS32).

~3c tY~

§2c m

::&" 1C

O~

~ m ~ c (x02)

Alanlne (x03)

~,o o - o Ser lne

.~ ,.--.~ " ........... ,PGA

Malta acld %'~- ~- ~--I; .... ~--~__--~ Qtrm acid (x40)

) i i ~ i , , r i ,

1 2 3 4 S 6 7 8 9 10 T ime in m i n u t e s a f t e r t u r n i n g o u t h g h t

Fig. 7 Changes m I*C con ten t of several compounds when h g h t is tu rned off (SS3o)

More probably, switching off the light causes a change m the oxidation-reduction state of the cytoplasm, permit t ing increased oxidation of glucose. Such a change could be t ransmit ted from the chloroplast to the cytoplasm by the diffusion of some substance. For example, it is known tha t glycolic acid can diffuse out of chloroplasts 2°, and this may be involved in such a mechanism.

The small rise in 14C amino acids that begins about 60 sec after the light is turned off presumably corresponds to that previously reported. These transient increases in 14C-labehng may be due to a release of a photoinhibition. They could also be the result of the diffusion out of the chloroplast of labeled photosynthetic intermediates. For example, it is known that mltochondrlal membranes are permeable in the oxIdlsed state, but not in the reduced s tate 2~. Such a change in permeablhty might also occur in the chloroplast membrane when the hght is turned off.

Photosynthetic pathways to amino acids

If, as the evidence indicates, the principal site of amino acid photosynthesis is in the chloroplasts, one might expect the environment within the chloroplasts to influence the nature of the biochemical pathways of amino acid synthesis This environment Includes abundant supplies of reduced triphosphopyridine nucleotide, adenosine tr lphosphate and carbon incorporated into phosphoglyceric acid, phosphoenol- pyruvlc acid, and various sugar phosphates. I t would not be surprising if under these conditions there are pathways different from those commonly encountered In non- photosynthetic systems.

For example, alanme accounts for about half of all the synthesis from carbon of the amino acids studied, and its synthesis requires twice as much ammonium ion fixation as does the synthesis of glutamic acid and glutamlne combined. I t is clear that, however glutamlc acld is formed, it does not provide all the nitrogen, via trans- aminatlon, for the synthesis of alanine. I t seems more likely that alanine is synthesized

Blochzm B,ophys ,4cta, 48 ([96I) 299-313

312 1). C. SMITH, J. A. BASSHAM, M. KIRK

from Intermediates of the carbon reduction cycle directly bv reductlve aminatlon. While the light is on, no ItC is found in free pyruvlc acid upon subsequent

analysis by paper chromatography of the products of photosynthesis of 14CO2 until long after the "ac t ive" alanme pool is saturated (steady state Expt. 32) We have verified the fact that one can see labeled pyruxuc acid by radioautography of chromatograms by hydrolyzmg carbon-labeled phosphoenolpyruvlc amd from our chromatograms and rechromatographmg it Free pyruvlc acid does not seem to be an intermediate between the carbon reduction and alamne

If we tentat ively eliminate transamination and reductlve ammat lon of pyruvm acid as routes to the synthesis of alanlne, we are left with prevmusly undocumented, hypothetical routes. Of these, the one which seems to us the most probable is a reduction and ammation of phosphoenolpyruvlc acid to give alanine.

2-PGA P E P A

H,C--OH H C H

I li T P N H 3-PGA ~ H C - O P O a H ~ C - O P O s H - >

l I NH4+ CO,- CO 2-

NHa +

H 2 C - O H

1 H C - N H , + H O P O 3 H - + H +

I CO 2

Senne

CH 3 J

H C - N H 2 + H O P O a H - + H + I

C0~-

Alanme

Serine might be formed by a similar, but non-reductlve amlnation of 2-phospho- glyceric acid.

The rapid labeling of aspartic acid and malic acid 1 is considered to be evidence for carboxylatlon of phosphoenolpyruvic acid

H C H

II CO 2 3-PGA ~ H C - O P O a H - >

I CO~- L

CO - I

C = O

I CH 2- I

CO S -

C0~- ]

or C - 0 P 0 a H -

CH

I CO 2-

T P N H , NH4+

CO~- I

H C - N H 2 I

H C H I

CO 2-

T P N H CO 2- l

H C - O H I

H C H ]

CO a

Mahc acid

Abpart lc acid

Bzochzm 13zophys Acta 48 (1961') 299-313

PHOTOSYNTHESIS OF AMINO ACIDS 313

Subsequent reduction of the carboxylation product would give malic acid, while reductive amination would give aspartic acid. An enzyme capable of bringing about the carboxylation of PEPA has been isolated from green leaves by BANDURSKI AND C-RIENER 25. Since the carboxylation product is not seen by our method of analysis, mahc acid and aspartic acid are, in this sense, the first stable, lsolable products of this carboxylation. With certain plants and under suitable physiological conditions, it is possible that either or both compounds might appear more prominently labeled than phosphoglyceric acid. Thus NEZGOVOROVA'S 11 observation of aspartic acid as a first stable carbon dioxide fixation product is not contradictory to our results.

We should like to suggest that glutamlc acid and glutamine are formed in the chloroplast independently from a common precursor which is m turn formed by condensation and reduction of two carbon and three carbon compounds derived directly from the carbon reduction cycle. It is likely that the two carbon compound would be acetate, glyoxylate or glycolate, while the three carbon compound would be phosphoenolpyruvate, phosphoglycerate, or even alanlne. Various possible reactions suggest themselves, but there is little evidence for any of them in green plants at the moment

ACKNOWLEDGEMENTS

The work described m this paper was sponsored by the United States Atomic Energy Commission, University of California, Berkeley, Calif. (U.S.A.).

The first Author is a Harkness Fellow of the Commonwealth Fund, New York, 1959-196o.

REFERENCES

1 j A. BASSHAM AND M KIRK, B*och*m B~ophys Acta, 43 (196o) 447- " J A. BASSHAM, A. A BENSON, L D KAY, A Z. HARRIS, A T WILSON AND • CALVIN, J Am.

Chem Soc., 76 (1954) 176o. 3 j . A. BASSHAM AND M CALVIN, The Path o] Carbon ~n Photosynthes,s, Prentice-Hall, Englewood

Chffs, New Jersey, 1957. 4 xV. STEPKA, A A. BENSON AI'iD M CALVIN, Science, lO8 (i948} 3o4 . 5 M. CALVIN, J. A BASSHAM, A. A. BENSON, V. LYNCH, C OUELLET, L SCHOU, W STEPKA AND

N. TOLBERT, Sympos*a Soc Exptl. B~ol , 5 (1951) 284- 6 S ARONOFF, A A. BENSON, W. Z HASSlD AND M. CALVIN, Sczence, lO5 (1947) 664. 7 A A BENSON, M. CALVIN, V. A. HAAS, S. ARONOFF, A G. HALL, J A BASSHAM AND J. W. VV'EIGL,

J FRANCK AND ~VV. E LOOMIS, Photosynthesis *n Plants, Iowa State College Press, 1949, p. 381. s A. A NICHIPOROVlCH, F,rst Geneva Con/erence on Peace/ul Uses otAtom*cEnergy, 1955, paper 697. 9 T F. ANDREYEVA, Dohlady Akad Nauk SSSR, 785 (I95 I) lO33.

10 N R VOSKRENSKAYA, Doklady Ahad. Nauh SSSR, 932 (1953) 9 I I . 11 L. A NEZOOVOROVA, F~zzol RastemL Akad. Nauk SSSR, 6 (1959) 45I lZ O. \VARBURG, Sc*ence, 128 (1958) 68 13 N M SISSAKIAN, Proceedzngs o/ the Second Umted Nations Con/erence on the Peace/ul Uses o/

Atomic Energy, Vol. 25, Pa r t 2, p. 159 14 O. HOLM-HANSEN, K NISHIDA, V. 3lOSES AND ~,I. CALVIN, J Exptl. Botany, IO (1959) lO 9 15 !VI CALVIN AND P MASSINI, Exper~entza, 8 (1952) 445- is j A BASSHAM, K SHIBATA, K STEENBERG, J. BOURDON AND M CALVIN, J . .4 m. Chem Soc.,

78 (1956 ) 412o 17 p ~r F. VAN DER MUELLEN AND J A ]~ASSHAM, dr A)n Chem. Sot., 81 (19.59) 2233 I8 E V~" YEMM AND E. C. COCKING, The .4nalyst, 80 (19.55) 209 19 H SIMON, Umvers~ty o/Cal*/orma Radzat~on Laboratory Report, UCRL-9o73 , March, 196o. 2o N. E. TOLBERT, Brookhaven Symposia sn Bzol, 2 (1958) 271 ~1 V MosEs, O. I-IOLM-HANSEN, J. A BASSHAM AND ~I CALVIN, J ~VIolecular B~ol, i (1959) 21. 22 H L KORNBERO AND N. B MADSEN, B~ochem., J , 68 (1958) 549- 2a H L KORNBERG, Fourth [nternat~onalCongress on B~ochem,stry, Vol XlI I , colloquia, p 251. 24 A. L LEHNINGER, Revs 21Iodern Phys , 31 (1959) 136 25 R. S BANDURSKI AND C M GREINER, J B~ol Chem., 204 (1953) 781

B,och~m. B,ophys Acta, 48 (1961) 299-313