474 BIOCHIMICA ET BIOPHYSICA AGTA

F A C T O R S A F F E C T I N G CO 2 F I X A T I O N BY C H L O R O P L A S T S

M A R T I N GIB B S , C L A N T O N C. B L A C K AND B E S S E L K O K

Department o/Biochemistry, Cornell University, Ithaca, N . Y . ( U.S.A .) ; Rias, Inc., Baltimore, Md. (U.S.A )

(Received March 2oth, 1961)

S U M M A R Y

The low rate of CO s uptake by spinach chloroplasts may be due to the loss of photo- synthetic pyridine nucleotide reductase during isolation of the particle. The addition of orthophosphate, ADP and Mg *+ accelerates TPN reduction, which may account for an earlier observation tha t small amounts of phosphate st imulate CO s reduction by chloroplasts. Aging destroys the ability of chloroplasts to assimilate CO, but does not affect TPN reduction.

INTRODUCTION

The maximum rate of photosynthesis calculated as ~moles of CO s ass imila ted/h/mg of chlorophyll is approx. 18o for intact leaves of higher plants 1 and 24 ° for intact Chore l la ~. In contrast, CO s fixation by chloroplasts is in the order of 3 or a rate of about I O/o of intact material3, 4. The question can be then raised as to what is limiting the ability of the isolated chloroplast to assimilate CO,.

The low rate of CO s fixation has generally been assumed to be due to a fault of the COs reduction cycle. Using pea chloroplasts, SMILLIE AND FULLER 5 have shown that the COs fixation rate parallels the amount of ribulose 1,5-diphosphate carboxylase. Without singling out a specific enzyme, ARNON et al. 6 have also concluded that the reduction of CO s is limited by the activity of the carbon cycle.

The photochemical act, which gives rise to the reductant, may also be a factor for CO s reduction. Little at tention has been given this function of the chloroplast apparently due to extremely high rates of ATP formationT, s and TPN reduction 9 recorded by various workers. These rates are sufficient to account for the requirements of the carbon cycle proposed by the CALVYN group 10.

I t is misleading, perhaps, to compare the observed rates of COs reduction with T P N H and ATP formation by chloroplast preparations since each is determined under a different set of conditions. While phosphate esterification and T P N H formation are assayed in the presence of various additives, phenazine methosulfate, riboflavin 5'-phosphate or PPNR, CO s uptake by intact chloroplasts is measured without similar cofactors. The cofactors are omitted since they either inhibit or have no effect on assimilation of CO, by the intact chloroplast11,1..

Abbrev ia t ion : P P N R , p h o t o s y n t h e t i c pyr id ine nucleot ide reductase .

Biochim. Biophys. Acta, 52 (1961) 474-477

FACTORS AFFECTING CO 2 FIXATION BY CHLOROPLASTS 475

For this reason, it became one purpose of this study to compare the TPN reduc- tion rate of the isolated chloroplast without additives with that of the parent intact chloroplast's ability to assimilate CO v to determine whether the photochemical act may be limiting the uptake of CO S . This approach was deemed advisable since it is known from the studies of SAN PIETRO AlqD LANG TM and DAVENPORT 9 that the re- duction of TPN requires an easily extracted enzyme, PPNR. The stability of endoge- nous TPN and the effect of Pt on the photochemical act (as assayed by TPNH forma- tion) were also studied as they may affect the rate of CO 2 assimilation.

MATERIALS AND METHODS

Reagents

TPN and ADP were products of the Sigma Chemical Company. P P N R was kindly provided by Dr. A. SAN PIETRO.

Reaction conditions

The detailed method for preparing spinach chloroplasts is described elsewherO, 14. Chlorophyll content was determined by the method of ARNON 15. The reactions were carried out aerobically in silica cells of I cm light path and 3 ml capacity. A light intensity of approx. 6o00 foot candles was used. This was provided by a 2oo-W tung- sten lamp suspended in a 2-1 beaker containing I ~/o (w/v) cupric sulfate. Water flowing through a copper coil suspended in the beaker maintained an even temperature. The temperature in the reaction cell was 18 ° to 20 °.

T A B L E I

EFFECT OF AGING ON STABILITY OF CO 2 FIXATION AND T P N RI~DUCTION BY SPINACH CHLOROPLASTS

Chloroplasts were prepared in 0.35 M NaCl, o.o2 M Tris, p H 7.5, divided in two groups and suspended either in the ext rac t ing solution and used to assay CO s fixation or in o.o35 M NaCI, o.oo2 M Tris, p H 7.5, and used for T P N reduction. Both assays were carried out for io min in a to ta l volume of 1.5 ml containing chloroplasts equivalent to 2oo/~g chlorophyll. The reaction mix- ture for isotope fixation contained the following components in ;umoles : Tris, p H 7.5, xoo; MnCI~, i ; sodium ascorbate, p H 7.5, i ; po tass ium phosphate , p H 7.5, o.3; NaCl, 38o and sodium bicarbo- nate, 0.29 containing 3.55/~C. The CO s fixation rate is corrected for dark fixation by exposing the complete mix ture to darkness for IO rain. Fu r t he r details are given elsewhere 4. For the T P N re- duct ion studies, the following components were added in / ,moles: Tris, p H 7.5, 80; MgCI v 4; po tass ium phosphate , p H 7.5, 4; TPN, 1.5, and ADP, 2. 5. A Carey Model x4 recording spectro- pho tomete r was used to determine the a m o u n t of T P N H formed. This was done by scanning the difference in absorbancy in the l ight-exposed mixture against a dark-exposed mixture f rom 4oo m/, to 3o0 m/~. The increase in ext inct ion (o.i is equivalent to the reduction of o.o48/~mole TPN) at

34 ° m/~ was used for calculation.

Expt. Time after TPN reduced grinding ( ra in ) Itm°les/h/mg 14C0, fixed

chlorophyll counts/rain/aliquot

i 3 ° 22. 4 4800 265 22.9 216

2 I i o 19.1 2200 320 18.1 IOO 420 16.4 7 °

Bioct~im. Biophys . Acta. 52 (I96I) 474-477

476 M. GIBBS, C. C. BLACK, B. KOK

RESULTS

Stability of CO 2 fixation and T P N reduction

Table I~ shows the effect of aging the spinach chloroplast on CO s fixation and TPN reduction. In comparison to endogenous TPN reduction, chloroplasts prepared and stored in o.35 M NaC1 at o ° lose their ability to assimilate COy

Rate of endogenous T P N reduction

In Table II it can be seen that chloroplast fragments reduce approx. IO ~moles TPN/h/mg chlorophyll. The addition of Mg 2+, ADP and Pt resulted in a stimulation of the reduction rate. I t should be noted that this stimulation varied considerably. An increase in the reduction rate was always observed, but, on occasion, the stimulation was only 25 %. The major cause is apparently due to variation in the age, manner of growth, and storage conditions of the store-bought spinach. The rate was further increased by the addition of PPNR. Here again, Mg 2+, PI and ADP brought about a stimulation in the reduction of TPN.

T A B L E II

RATE OF TPN REDUCTION

Chloroplas t s were p repa red in 0. 4 M sucrose, 0.02 M Tris, p H 7.8, and o.oI M KC1 a n d r e suspended in th i s solut ion. Assa ys were carr ied ou t in a to ta l v o l u m e of 1.5 m l con t a in ing ch loroplas t s equiva l - en t to I85 # g chlorophyl l . An exposure t i m e of 3 m i n ( + P P N R ) or 9 rain ( - - P P N R ) was used. T h e reac t ion m i x t u r e con ta ined t he following c o m p o n e n t s i n / z m o l e s : Tris, p H 7.5, 12; T P N , 1.8 a n d when ind ica ted MgCI~, 4; ADP , 2.5 ; p o t a s s i u m phospha t e , p H 7.5, 4 ; and 4.7 un i t s of P P N R ,

4o-fold purified. For fu r the r detai ls , see Table I.

T P N reduced (#moles~h/rag chlorophyll)

- - P P N R + P P N R

Control 7.8, 11.6 40.5 ADP, Pt, Mg ~+ 15.6, 18.4 93.0

DISCUSSION

The rate of TPN reduction catalyzed by chloroplast particles without additives is in the order of IO/~moles/h/mg chlorophyll (Table II). Assuming, as postulated by the CALVIN group 1°, that 2 moles or T P N H or its equivalent are consumed per mole of CO 2 reduced to the carbohydrate level, then maximum fixation rates in the order of 5 /~moles COJh/mg chlorophyll can be supported with preparations of this type. Values approaching this figure have been recorded by ARNON et al. 3 and in this labora- tory 4. Thus, it is possible that the factor limiting CO s fixation in chloroplasts prepared in isotonic solutions and used rapidly (Table I) may be the supply of T P N H or other substances produced by the photochemical act. The low rate of fixation by chloro- plasts compared with that of intact tissue may be due to the loss during extraction of PPNR, as the addition of this protein to the photochemical system resulted in an approximate 5-fold increase in the TPN reduction rate (Table II). I t should therefore be stressed that the data reported with chloroplasts and artificial acceptors resulting in extremely high rates of phosphorylation and TP N H formation indicate the unusual

B i o c h i m . B i o p h y s . A c t a , 52 (I96I) 474-477

FACTORS AFFECTING C 0 2 FIXATION BY CHLOROPLASTS 4 7 7

capabilities of the chloroplast but may have little value as a basis of comparison with CO, fixation rates. The question as to what is limiting the ability of the isolated chloro- plast to assimilate CO 2 at a rate approaching that of the intact cell can be best answered by stating that this may be due to removal of enzymes involved in the photochemical act and the carbon assimilation cycle.

Previously, GIBBS AND CALO 4 observed that the addition of small amounts of Pt to intact chloroplast preparations stimulates C02 fixation. Based on the data recorded in Table II which are in agreement with the observation of DAVENPORT 9 and SA~ PIETRO 16 that Pt together with Mg *÷ and ADP brings about a stimulation of the rate of TPN reduction, it would appear that PI affects CO, fixation by accelerating the formation of reductant. Phosphate would appear to have this function rather than one of giving rise to ATP since arsenate will replace PI in bringing about an increased CO~ uptake and increased TPNH synthesis.

ACKNOWLEDGEMENTS

This investigation was aided by grants from the National Science Foundation and from the United States Air Force through the Air Force Office of Scientific Research of the Air Research and Development Command, under contract No. AF 49 (638) 798. One of us (C.C.B.) is a postdoctoral fellow of the U.S. Public Health Service.

R E F E R E N C E S

1 E. I. RABINOWITCH, Pt~otosynthesis and Related Processes, Vol. II, P a r t 2, In te rsc ience Publ . Inc., New York, 1956.

2 0 . HOLM-NANSEN, N. G. PUN, K. NISHIDA, V. MOSES AND M. CALVIN, Physiol. Plantarum, 12

(1959) 475. 3 F. R. WHATLEY, M. B. ALLEN, A. V. TREBST AND D. I. ARNON, Plant Physiol., 35 (196o) 188. 4 M. GIBBS AND N. CALO, Plant Physiol., 34 (1959) 318. 5 R. M. SMILLIE AND R. C. FULLER, Plant Physiol., 34 (1959) 651. 6 D. I. ARNON, M. n . ALLEN AND F. R. WHATLEY, Biochim. Biophys. Acta, 20 (1956) 463. 7 M. AVROM, Biochim. Biophys. Acta, 4 ° (I96O) 257. 8 M. B. ALLEN, F. R. WHATLEY AND D. I. ARNON, Biochim. Biophys. Acta, 27 (1958) 16. 9 H. E. DAVENPORT, Biochem. J., 77 (196°) 471.

10 j . A. BASSHAM AND M. CALVIN, The Path o] Carbon in Photosynthesis, Prentice-Hall, Englewood Cliffs, New Jersey, 1957.

11 M. B. ALLEN, D. I. ARNON, J. B. CAPINDALE, F. R. WHATLEY AND L. J. DURHAM, J. Am. Chem. Soc., 77 (1955) 4149.

i2 M. GIBBS AND M. AL-NAEIDH, unpublished data. 13 A. SAN PIETRO AND H. M. LANG, J. Biol. Chem., 231 (1958) 211. 14 N. CALO AND M. GIBBS, Z. Naturforsch., i 5b (196o) 287. I5 D. I. ARNON, Plant Physiol., 24 (1949) I. IS A. SAN PIETRO, private communication.

Biochim. Biophys. Acta, 52 (1961) 474-477