The metabolism of glycine and glycollate by pea leaves in relation to photosynthesis

266

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 4 5 3 3 1

T H E METABOLISM OF G L Y C I N E AND GLYCOLLATE BY PEA LEAVES IN R E L A T I O N TO P H O T O S Y N T H E S I S B. J. M I F L I N * , A. F. H. M A R K E R

AND C. P. W H I T T I N G H A M

Botany Department, Imperial College, London (Great Britain) (Received N o v e m b e r I l t h , 1965)

SUMMARY

I. The metabolism of 14C-labelled glycollate and glycine both to sucrose and to a polyglucan via serine has been demonstrated in excised pea leaves. The effect of light, DCMU, isonicotinyl hydrazide and the partial pressure of CO 2 on the pathway has been studied. 2. The metabolism of [14C]glycine and serine formed during a preceding period of photosynthesis has been followed during a subsequent period of photosynthesis in ~*CO2. The radioactivity in the glycine and serine declined whilst that in sucrose rose, suggesting that in photosynthesis some sucrose is formed via glycine and serine. The effect of changes in partial pressure of carbon dioxide indicates that the photosynthetic pathway m a y be spatially separated from that which metabolises exogenous glyeine and glycollate.

INTRODUCTION

Glycollate formation in photosynthesis has been reported in tobacco 1 and isolated pea chloroplasts ~ but it has not been found in all higher plants a. The formation of glycine in sugar beeO and both glycollate and glycine in Chlorella 5 decreases as the partial pressure of CO 2 is raised. By feeding radioactive intermediates the existence of a pathway from glycollate to glycine to serine and then to sucrose has been shown in wheat leaves6, 7. TOLBERT3 has suggested that this pathway is the one taken by the C 2 compounds formed in photosynthesis. However, BASSHAM et al. 8 have found that the size of the photosynthetic amino acid pools in Chlorella are extremely small in relation to the total amount of amino acids in the cells. HELLEBURSTAND BIDWELL9 have postulated the existence of at least two amino acid pools in higher plant cells only one of which is connected with photosynthesis. It is therefore possible that the radioactive intermediates fed externally to the plant m a y enter a non-photosynthetic pool and their subsequent metabolism would reflect the metabolism of that pool and not of the photosynthetically produced intermediates. This paper attempts to relate the metabolism of externally fed radioactive glycollate and glycine to the further metabolism of these compounds produced endogenously in photosynthesis in pea leaves. A b b r e v i a t i o n : I N H , isonicotinyl hydrazide. * P r e s e n t a d d r e s s : D e p a r t m e n t of P l a n t Science, T h e University, Newcastle u p o n T y n e , i (Great Britain).

Biochim. Biophys. Acta, 12o (I966) 266--273

METABOLISM OF C 2 COMPOUNDS BY PEA LEAVES

267

METHODS

Peas (Pisum sativum var. Meteor) were soaked overnight in water and planted in compost in flat trays. They were grown in a greenhouse under natural light. The leaves of plants 3-4 weeks old were excised and radioactive intermediates fed via the petiole in the light unless otherwise stated. Inhibitors were fed prior to radioactive intermediates either via the petiole in the transpiration stream or b y vacuum infiltration. After the period of feeding the leaves were washed clean and plunged into 40 ml hot 80 % alcohol. The leaves were extracted b y boiling in the 80 % alcohol for 2 min. The alcohol was then poured off and the leaves extracted again in 2o ml boiling 80 % alcohol for a further 2 min. The next extraction was in 5 ml boiling water and the leaves finally extracted with 5 ml cold 50% alcohol. The extracts were combined, evaporated and chromatographed as previously described 1°. Residual activity in the leaves was determined after drying by placing them under an end-window Geiger Miiller tube. The methods employed in the degradation of the radioactive glucose will be described in a later publication. Essentially the method is that of GIBBS AND GUNSALUSn who described a bacteriological fermentation of glucose to carbon dioxide, ethanol and lactic acid by Leuconostoc mesenteroides. The subsequent degradation of ethanol and lactic acid was carried out b y some slight modifications of the method o f SHIBKO 12.

For measurements of photosynthesis the detached leaves were placed in glass chambers IO cm long × 2 cm internal diameter and illuminated from above at a light intensity of 9.1o 4 ergs/cmS.sec. 14C02 was generated by injecting Na214COs at a constant rate into acid phosphate buffer through which a stream of air at constant pressure was bubbled. The gas containing the 1'C0 s (estimated CO s content 0.045 %) was then passed over the leaves. When a rapid change from ~4CO2 to ~sC02 was required it was achieved b y using a cylinder of compressed air (or 4 % COs) to displace rapidly the ~4CO2 out of the chambers. At the end of the period of photosynthesis leaves were killed b y placing in boiling 80 % alcohol and then treated as above. RESULTS

Glycine and glycollate were readily metabolised b y excised pea leaves (Table I~. In the light these compounds were metabolised faster with the formation of a wider variety of products. Both glycollate and glycine gave rise to sucrose, aspartate, malate, alanine, glycerate, phosphoglyceric acid, sugar phosphates, serine and alcohol-insoluble compounds in the light but only the latter two compounds were formed in the dark. The presence of DCMU inhibited the formation of sucrose and other products but not of serine and alcohol-insoluble compounds. The metabolism of the C2 compounds appeared to be relatively independent of the partial pressure of carbon dioxide in the surrounding atmosphere (Table II). Addition of I N H has been shown to result in the accumulation of glycoliate in Chlorella and is believed to inhibit the conversion of glycine to serine 5. The results in Table I I I show that in peas in the presence of I N H glycollate was only converted to glycine although the amount of glycollate metabolised was nearly the same in the presence as in the absence of INH. At a lower concentration of I N H there was Biochim. Biophys. Acta, 12o (1966) 266-273

268

B. J . M I F L I N ,

A. F. H . M A R K E R ,

C. P. W H I T T I N G H A M

only from their than

partial inhibition of the foimation of sucrose and alcohol-insoluble compounds glycine. The formation of flee keto acids was measured by trapping them as dinitrophenylhydrazone derivatives but this fraction was found to contain less 1% of the total activity. When the alcohol-insoluble fraction was hydrolysed with acid the hydrolysate consisted chiefly of glucose. This glucose and the glucose moiety of sucrose were

TABLE THE

I

METABOLISM

OF

GLYCINE

AND

GLYCOLLATE

BY

EXCISED

PEA

LEAVES

D a t a f o r i n d i v i d u a l c o m p o u n d s g i v e n a s O//o t o t a l m e t a b o l i s e d a n d t h a t f o r t o t a l m e t a b o l i s e d a s lO 3 c o u n t s / m i n . E x p t . i . I /zC [2-14C~ g l y c i n e o r [ I - l a C ] g l y c o l l a t e f e d t o 6 l e a v e s p e r t r e a t m e n t . M e t a b o l i s m c o n t i n u e d f o r 35 r a i n i n t h e l i g h t o r I h i n t h e d a r k . P r e t r e a t m e n t s : DCMU, leaves v a c u u m i n f i l t r a t e d w i t h IO - 5 M D C M U ; c o n t r o l , l e a v e s v a c u u m i n f i l t r a t e d w i t h w a t e r . E x p t . 2. i # C [ I - 1 4 C ] g l y c o l l a t e f e d t o 6 l e a v e s f o r 15 m i n . P r e t r e a t m e n t s : DCMU, Io -s M DCMU vacuum infiltrated; control, water.

Compound

Expt. •

Expt. 2

Glycine feeding (% activity) Control Glycollate Glycine Serine Sucrose Aspartate Malate Alanine S u g a r p h o s p h a t e s plus phosphoglycerie acid Glycerate Insolubles Total metabolised c o u n t s / m i n × lO 3

TABLE THE

DCMU

Dark

Control

Control

2.9

48.5 12.2 3.2 3.1 2.7

58.5 5.2 2.8 1.9 3.2

67. 9 0.9 --

Trace 1.4 9 .2

-22.5

i.o 3-9 5.4

18.8

11.2

89.4

20.8

.

Glycollate feeding (% activity)

3.2

8.6 5.8 8.7

.

Glycollate feeding (% activity)

. 6.6 22. 5 36.8 3.7 4.2 2.9

Dark .

. 7.1 72.1 0.3 Trace Trace Trace

DCMU

. 21.2 17.6 21.5 1.3 2.8 1.8

38.2 37.2 3.2 Trace i.o Trace

--Trace 9 .6

15.6 3.7 3 .6

3.7 2. I 1-9

46.0

34 .0

35.5

11

EFFECT

OF THE

EXTERNAL

PARTIAL

PRESSURE

OF CO 2 ON THE

METABOLISM

OF GLYCINE

AND

GLYCOLLATE

D a t a f o r i n d i v i d u a l c o m p o u n d s a s % t o t a l m e t a b o l i s e d a n d t h a t f o r t o t a l m e t a b o l i s e d a s lO 3 c o u n t s / m i n . E x p t . I. [ i - l i C l G l y c i n e f e d f o r IO m i n t o 6 l e a v e s i n a n a t m o s p h e r e of a i r o r C O 2 f r e e a i r a s i n d i c a t e d . E x p t . 2. [2-14C~Glycine f e d f o r 4 ° m i n t o 6 l e a v e s i n a n a t m o s p h e r e o f a i r o r of C O 2 - a i r ( 4 : 9 6 ) - E x p t . 3. [ I - 1 4 C ] G l y c o l l a t e f e d t o 6 l e a v e s f o r 4 ° m i n i n a n a t m o s p h e r e o f a i r o r CO2-air (4:96).

Compound

Glycollate Glycine Serine Sucrose Total metabolised c o u n t s / r a i n × lO 3

Expt. x

Expt. 2

Air (% activity)

C02free (% activity)

1.4 . . 39 .8 3 i. 8

i .2 . 43-5 29.4

72.2

.

75-4

Biochim. Biophys. Acta, 12o (1966) 2 6 6 - 2 7 3

.

Air (% activity)

.

1.6 . 17.8 44 .o 92.5

Expt. 3 C02 (% activity)

4%

o.9

Air (% activity)

C02 (% activity)

4%

18.0 41.5

-2.8 8. 4 5° .I

3.8 IO. I 4o.4

94.4

65-7

64.3

269

METABOLISM OF C 2 COMPOUNDS BY PEA LEAVES

degraded and the distribution of radioactivity in the glucose molecules derived from [I-14C!glycollate and [2-I4C~glycine feeding are shown in Table IV. [2-14ClGlycine produced C-I, C-2, C-5 and C-6 labelled glucose both in sucrose and the polyglucan but the glucose formed from the [I-14Clglycollate feeding was approximately equally labelled. Similar results have been reported for wheat leavese,is. Prior infiltration of pea leaves with non-radioactive serine decreased the percentage of glycollate and glycine metabolised to sucrose, sugar phosphates and glycerate but had little effect on the formation of aspartate, malate and alanine TABLE

III

GLYCOLLATE AND GLYCINE FEEDING IN THE PRESENCE OF I N H D a t a g i v e n a s lO s c o u n t s / m i n f o r t o t a l f i x a t i o n . I n d i v i d u a l c o m p o u n d s a s p e r c e n t a g e f i x a t i o n . E x p t . i . i . o / t C [2-14C~glycine f e d t o 6 p e a l e a v e s f o r i h. P r e t r e a t m e n t s : I N H , l e a v e s a l l o w e d t o t a k e u p I N H (lO m g / m l ) f o r I h ; c o n t r o l , l e a v e s a l l o w e d t o t a k e u p w a t e r f o r I h. E x p t . 2. i . o / , C [2-14C~ G l y c i n e f e d t o 6 p e a l e a v e s f o r i h. P r e t r e a t m e n t s : INH, leaves vacuum infiltrated with INH (5 ° m g / m l ) ; c o n t r o l , l e a v e s v a c u u m i n f i l t r a t e d w i t h w a t e r . E x p t . 3 . 0 . 2 5 / ~ C E I - 1 4 C l g l y c o l l a t e f e d t o 6 p e a l e a v e s f o r 1 h. P r e t r e a t m e n t s : I N H , l e a v e s v a c u u m i n f i l t r a t e d w i t h I N H (IO m g / m l ) . Control, leaves vacuum infiltrated with water.

Compound

Expt. x

Expt. 2

Control (% activity) Glycollate 1.2 Glycine 3.7 Serine 15.o Sucrose 36.0 Aspartate o.5 Malate 0. 7 Alanine I. 3 Sugar phosphates 1. 5 Glycerate o.9 Insolubles 2. I Total c o u n t s / m i n × lO 3 6 2 . 9

TABLE

INH (% activity) 1.4 26.0 4-4 24.1 Trace -Trace

Control (% activity)

Expt. 3 INH (% activity)

Control (% activity)

INH (% activity)

Trace o.5

0.8 8. 5 9.8 21.8 7.o 8.0 6. i 4.2 1.2 9.8

0. 7 56.2 i. i 0.8 ------o.7

7-7 2.8 4.6 7.0 o.4 0. 3 Trace 1. 3 o.2 3.7

14.5 9.7 1.3 0. 7 ----Trace o.3

56.4

77.2

59.5

28.0

26. 5

-

-

IV

THE INTRAMOLECULAR DISTRIBUTION OF RADIOACTIVITY IN GLUCOSE MOLECULES DERIVED FROM GLYCINE AND GLYCOLLATE METABOLISM Data given as percentage of the total radioactivity in the molecule. In each case feeding of the radioactive intermediates was continued for 4° min.

Carbon atom [2-14C~Glycine feeding of glucose From From sucrose insolubles

From sucrose

From insolubles

i 2 3 4 5 6

19.2 15. 3 18.3 19.9 13.2 14.1

15. 3 16.2 15.9 20.2 18.7 13.8

(%)

(%)

23. 4 22. 4 4-5 5-5 19.7 24.6

18.o 23.o 4 .o 7-4 25.7 22.0

Ez-14 C]GlycoUate feeding

(%)

(%)

Biochim. Biophys. Acta, l a O (1966) 2 6 6 - 2 7 3

270

B . J . MIFLIN, A. F. H. MARKER, C. P. WHITTINGHAM

TABLE V GLYCOLLATE

AND

GLYCINE

FEEDING

IN

THE

PRESENCE

OF

NON-RADIOACTIVE

INTERMEDIATES

D a t a for i n d i v i d u a l c o m p o u n d s g i v e n as % t o t a l m e t a b o l i s e d a n d t h a t for t o t a l m e t a b o l i s m as io 3 c o u n t s / m i n . E x p t . i. 1.5/~C [I-14C]glycollate fed t o 6 l e a v e s pe r t r e a t m e n t for i h i n l i ght . P r e t r e a t m e n t s : control, v a c u u m i n f i l t r a t e d w i t h w a t e r ; serine, v a c u u m i n f i l t r a t e d w i t h serine (i m g / m l ) ; g l y o x y l a t e , v a c u u m i n f i l t r a t e d w i t h g l y o x y l a t e (I m g / m l ) . E x p t . 2. 1. 5/~C [I-14C]glycine fed to 6 l e a v e s per t r e a t m e n t for 20 min. E x p t . 3. 0.5/*C[2-14C]glycine fed to 6 l e a v e s pe r t r e a t m e n t for 20 min. P r e t r e a t m e n t s as above.

Compound

Glycollate Glycine Serine Sucrose Aspartate Malate Alanine

S u g a r p h o s p h a t e s plus p h o s p h o g l y c e r i c acid Glycerate Insolubles Total metabolised c o u n t s / m i n × IOa

Expt. r GlyeoUate feeding (°/o activity)

Expt. 2 Glycine feeding (% activity)

Expt. 3 Glycine feeding (% activity)

Control

Control

Control

Serine

Glyoxylate

I3. 5 15.5 13. 3 1.8 2.3

19.8 16.4 8.1 2.6 2.4

I2.9 12.4 12. 4 2. t o.9

1.8

2.6

3.5

1.5

2.1

1. 3

i.o

2.5 2.0 35.9

1. 5 0.9 35. i

Trace Trace 42.2

15. 3 4.1 8. I

4 .6 2.9 5.6

7.5 i.o 14. 4

3 .1 0. 4 t 1.5

90.5

02.4

4 °.2

47.7

26-3

34 .1

4 °.0

3.3 . 4o.7 18. 7 2. I 1.6

.

Serine

3-5 . 59.1 9.2 3-8 3-7

.

2.o . 34.8 32.5 3.2 i .8

Glyoxylate o.7 33.5 48.5 o.4 o.2

(Table V). It increased the percentage incorporated into glycine from glycollate and into serine from glycine. In contrast infiltration with non-radioactive glyoxylate increased the metabolism of glycine to sucrose and had little effect on the formation of serine and insolubles. Glyoxylate decreased the uptake of glycollate (but not of glycine) but had little effect on the distribution of such glycollate as was metabolised, except that the percentage radioactivity in the sugar phosphates was decreased. These results are in agreement with a sequence of reactions from glycollate to glycine to serine and thence to sucrose. The question remains whether glycine and serine formed during photosynthesis may also be metabolised to sucrose. After 20 min photosynthesis in 14C02 the distribution of the 14C during a subsequent period of photosynthesis in a2C02 whs investigated. The change in the radioactivity of the major compounds (expressed as the change in percentage of the total fixed) during the "flushing" period is plotted in Fig. I. In air there was an increase in the radioactivity in sucrose and a decrease in the glycine plus serine fraction of approximately equal magnitude. In the presence of I N H there was a delayed loss of activity from glycine, a small increase in serine, and a delayed increase in sucrose. If at the time the gas stream was changed the cell suspension was darkened, the changes in the absence of I N H in glycine plus serine were small and the increase in sucrose was slower and smaller than in the light. In the presence of I N H there was a steady decline in the 14C in glycine and an increase in serine and other amino acids but little change in sucrose. In a shorter term experiment (Fig. 2) the same general results were observed in the controls. Increasing the Biochim. Biophys. Acta, 12o (1966) 266-273

METABOLISM OF C 2 COMPOUNDS BY PEA LEAVES

271

partial pressure of CO~ in the flushing gas to 4 % resulted in a much more rapid loss in radioactivity from the sugar phosphates but there was no loss of activity from the glycine and serine fractions and no increase in the sucrose. In this case radioactivity is lost from sugar phosphates and appears in amino acids other than glycine and serine and in the alcohol-insoluble fraction. Ca)

.c E

._8 '°

._o 4

0

x

r::~-.--..~........

-IC

r

.

- ""-o

L

~c 1(

(c)

-1C

e-

$

,~

-2o,

Time (mir

"

6

,'~

!

~


6

0

2

4

6

Time (rnin)

Fig. i. Left. The further metabolism of 14C fixed in p h o t o s y n t h e s i s a and b. Leaves were allowed to photosynthesize in 14COs for 2o min. The gas s t r e a m was t h e n rapidly changed to lsCOs (in air) a n d p h o t o s y n t h e s i s allowed to continue for a further 12 min. Samples were t a k e n a t o, 3, 7 a n d 12 m i n after the gas change. The leaves were pretreated b y allowing t h e m to take up a, water, b, I N H (io mg/ml) for I h; c, a n d d, comparable to a a n d b except t h a t the leaves were placed in the d a r k w h e n the gas was changed. The ordinate is the % fixation in the c o m p o u n d at t h e sample time m i n u s the corresponding % in the sample at zero time. a, O - - O , glycine + serine; • - - A , sucrose; m - - m , other amino acids; 0 - - 0 , insolubles, b. 0 - - 0 , glycine ; A - - A , sucrose; ,--., other amino acids; 0 - - 0 , serine, c, 0 - - 0 , glycine + serine; A - - A , sucrose; ~ - - ~ , other amino acids; 0 - - 0 , insolubles, d, 0 - - 0 , glycine; A - - A , sucrose; n - - m , other amino acids; 0 - - 0 , serine. Fig. 2. Right. Leaves were allowed to photosynthesize for 20 min in l l C O 2 and the gas s t r e a m t h e n changed to either 0.o 3 % 12COs or 4.o % lsCOs, a, O - - 0 , total fixed control; A - - A , total fixed 4 % COs. b, 0 - - 0 , insolubles control; A - - A , insolubles 4 % COs; A - - A , glycerate 4 % COs; O - - O , glycerate control, c, 0 - - 0 , glycine + serine control; A - - ~ k , glycine + serine 4 % COs ; A - / ~ , other amino acids 4 % COs; 0 - O, other amino acids control, d, 0 - - 0 , sucrose control; A - - ~ k , sucrose 4 % COs; A - - • , sugar phosphates 4 % COs; O - - O , sugar phosphates control.

DISCUSSION

The conversion of glycollate to glycine and glycine to serine occurs in the dark but further metabolism to sucrose is light dependent. The addition of DCMU has a similar effect to placing the leaves in the dark suggesting t h a t the metabolism to sucrose is in some way related to photosynthesis. Since the metabolism of glycoUate and glycine is not affected b y the absence of CO s in the surrounding atmosphere, the Biochim. Biophys. Acta, 12o (I966) 266-273

272

B. J. MIFLIN, A. F. H. MARKER, C. P. WHITTINGHAM

need for light cannot be attributed to a requirement for specific carbon compounds. The inhibition of sucrose, phosphoglyceric acid, glycerate and sugar phosphate formation b y non-radioactive serine indicates that these compounds are formed from glycine and glycollate via serine and the inhibition by I N H is consistent with this. The pathway from serine to sucrose is probably via hydroxypyruvate, glycerate and phosphoglyceric acid. The formation of aspartate and malate from exogenous C 2 compounds is also light dependent but this is not affected by the presence of non-radioactive serine. This would suggest that aspartate and malate are derived from C 2 compounds but not via serine. Possible routes for the conversion of C 2 units to C 4 compounds have been found in bacteria. KORNBERG AND MORRIS14 have shown that glycine and glyoxylate can condense to give hydroxyaspartate and the synthesis of malate from glyoxylate and acetyl- CoA is also well established 15. Malate synthetase has been found in small amounts in the leaves of higher plants 16. The inhibition of aspartate and malate formation by I N H could be explained by the action of I N H as a general antagonist of vitamin Be-requiring enzymes 17, rather than specific for serine transhydroxymethylase. RABSON et al. 7 postulated the involvement of C1 units derived from glyoxylate in the conversion of glycine to serine. However, the fact that non-radioactive glyoxylate stimulates rather than inhibits the conversion of glycine to serine suggests that the reaction occurs by direct conversion of two glycine molecules to give one serine in which the C-2 of one glycine molecule becomes C-3 of serine. Such a reaction mechanism has been found in bacterialS, 19 and in avian livers 2°. In the latter tissue and in the present work the reaction is inhibited by INH. The labelling pattern in the sucrose molecule from [2-14Clglycine is consistent with the C-3 of serine being derived from the C-2 of glycine. The fixation pattern in peas at low partial pressures of carbon dioxide differs from that, for example, in Chlorella in the absence of any significant fixation in glycollate even in the presence of INH. However, the C 2 compound glycine accumulates instead of glycollate. The addition of I N H partially inhibits the production of sucrose which would be consistent if some of the sucrose formed in photosynthesis is derived via serine and glycine. This is also indicated b y the experiments when 14C0~ is rapidly replaced by 12C02, for at least part of the increase in the radioactivity in sucrose must have come from that originally in glycine and serine. Raising the partial pressures of carbon dioxide inhibited the transfer of radioactivity from glycine to sucrose presumably by greatly increasing the amount of non-radioactive phosphoglyceric acid and sugar phosphates. Thus the formation of sucrose from glycine and serine can be of importance in photosynthesis only at lower partial pressures of carbon dioxide (near that in air). The dependence on light of the metabolism of serine to sucrose, but not of glycine to serine, is the same whether these compounds are formed in photosynthesis or from exogenous feeding; however, whereas externally supplied glycine gives rise to alcohol-insoluble substances in the dark less of these compounds are formed in the dark from endogenous substrates. Also whilst the further metabolism of glycine and serine formed in photosynthesis is markedly dependent on the partial pressure of carbon dioxide this is not the case with exogenous feeding experiments. Hence although the metabolic path is similar for exogenous and endogenous substrates the Biochim. 13iophys. Acta, 12o (1966) 266--273

METABOLISM OF C 2 COMPOUNDS BY PEA LEAVES m e t a b o l i s m of t h e f o r m e r m a y t a k e p l a c e a t a s i t e s p a t i a l l y s e p a r a t e d c a r b o n p a t h w a y of p h o t o s y n t h e s i s , n a m e l y i n t h e c y t o p l a s m .

273 from the

ACKNOWLEDGEMENTS T h e a u t h o r s w i s h t o a c k n o w l e d g e t h e g i f t of D C M U f r o m D r . CALDERBANK of I C I O n e of u s ( B . J . M . ) w i s h e s t o t h a n k t h e A g r i c u l t u r a l R e s e a r c h C o u n c i l a n d a n o t h e r ( A . F . H . M . ) t h e D e p a r t m e n t of S c i e n t i f i c a n d I n d u s t r i a l R e s e a r c h f o r t h e g r a n t of a s t u d e n t s h i p d u r i n g t h e c o u r s e of t h i s w o r k .

REFERENCES i I. ZELITClt, J. Biol. Chem., 24o (1965) 1869. 2 D. A. WALKER, Biochem. J., 92 (I964) 22o. 3 N. E. TOLBERT, Photosynthetic Mechanisms in Green Plants, Natl. Res. Council, Washington, 1963, p. 648. 4 D. C. MORTIMER, Can. J. Botany, 37 (1959) I I 9 I . 5 C. P. WHITTINGHAM, R. G. HILLER AND M. BERMINGHAM, Photosynthetic Mechanisms in Green Plants, Natl. Res. Council, Washington, 1963, p. 675. 6 D. WANG AND E. R. WAYGOOD, Plant Physiol., 37 (1962) 829. 7 R. RABSON, N. E. TOLBERT AND P. C. KEARNEY, Arch. Bioehem. Biophys., 98 (1962) 154. 8 J. A. BASSIIAM, B. MORAWEICKA AND M. KIRK, Biochim. Biophys. ,4cta, 9o (1964) 542. 9 J. A. HELLEBURST AND R. G. S. BIDWELL, J. Exptl. Botany, 41 (1963) 968. IO G. G. FRITCHARD, W, J. GRIFFIN AND C. P. WHITTINGIIAM, J. Exptl. Botany, 13 (1962) 176. i i M. GIBBS AND I. C. GUNSALUS, J. Biol. Chem., 194 (1952) 871, 12 S. SHIBKO, P h . D . Thesis, University of London, 1958. 13 E. JIMINEZ, R. L. BALDWIN, N. E. TOLBERT AND W. A. WOOD, Arch. Biochem. Biophys., 98 (I962) 172. 14 H. L. KORNBERG AND J. G. MORRIS, Bioehem..]., 95 (1965) 577. 15 H. L. KORNBERG AND H. A. KREBS, Nature, 179 (1957) 988. I6 Y. YAMAMOTO AND H. BEEVERS, Plant Physiol., 35 (196o) lO2. 17 R. M. HICKS AND J. CYMERMAN-CRAIG, Biochem. J., 67 (1957) 353. 18 R. D. SAGERS AND I. C. GUNSALUS, J. Bacteriol., 81 (1961) 541. I9 R. D. SAGERS, Federation Proc., 24(1 ) (1965) 219. 20 D. A. RICHERT, R. AMBERG AND M. WILSON, J. Biol. Chem., 237 (1962) 99.

Biochim. Biophys. Acta, 12o (1966) 266-273