Photosynthesis by isolated chloroplasts. Reversal of orthophosphate inhibition by Calvin-cycle intermediates

1. The orthophosphate inhibition of photosynthesis by isolated spinach chloroplasts can be reversed by 3-phosphoglycerate, dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, fructose 6-phosphate and fructose 1,6-diphosphate. 2. Metabolically related compounds such as ribulose 1,5-diphosphate, glucose 6-phosphate, 6-phosphogluconate and phosphoenolpyruvate are ineffective. 3. The kinetics of reversal are characteristic of the intermediate used, but, in each instance, the onset of oxygen evolution is accompanied by a carbon dioxide fixation and except with 3-phosphoglycerate the stoicheiometry is close to unity. 4. The nature of orthophosphate inhibition and its reversal is discussed in relation to metabolic control of photosynthesis.     

Inhibition of the phosphate transporter during isolation of intact chloroplasts from leaves of sunflower

Chloroplasts isolated from spinach leaves by the mechanical method were intact and exhibited high rates of CO₂-dependent oxygen evolution whereas chloroplasts isolated from sunflower leaves by the same technique were also intact but showed only low rates of oxygen evolution. The rate of uptake of orthophosphate (Pi) from the suspending medium with sunflower chloroplasts was less than 20% of that in spinach chloroplasts. The apparent Km for Pi transport was lower in sunflower chloroplasts but uptake was competitively inhibited by 3-phosphoglycerate in chloroplasts from both species. Uptake of malate (via the dicarboxylate transporter) and of ATP (via the adenine nucleotide transporter) was also reduced in sunflower chloroplasts compared to spinach chloroplasts. The endogenous Pi content and total exchangeable phosphate pool of sunflower chloroplasts were less than half that in spinach chloroplasts.Addition of a number of possible protective agents to the grinding medium failed to prevent the loss of photosynthetic activity during mechanical isolation of sunflower chloroplasts. Grinding mixtures of spinach and sunflower leaves together indicated that spinach chloroplasts were not inhibited by the sunflower leaf extract. Chloroplasts isolated from sunflower leaves via protoplasts had high rates of CO₂-dependent oxygen evolution. The Vmax and Km for Pi uptake, endogenous Pi content and total exchangeable phosphate pool of chloroplasts isolated from sunflower protoplasts were all similar to spinach chloroplasts. It is concluded that inner envelope membrane proteins are damaged during mechanical isolation of sunflower chloroplasts. The decrease in activity of the phosphate transporter and loss of endogenous phosphate may contribute to the low rates of photosynthesis observed in chloroplasts isolated by the mechanical method from leaves of sunflower and possibly other species.Abbreviations PGA 3-phosphoglyceric acid     

Regulation of sedoheptulose-1,7-bisphosphatase by sedoheptulose-7-phosphate and glycerate,and of fructose-1,6-bisphosphatase by glycerate in spinach chloroplasts

Using partially purified sedoheptulose-1,7-bisphosphatase from spinach (Spinacia oleracea L.) chloroplasts the effects of metabolites on the dithiothreitoland Mg²⁺-activated enzyme were investigated. A screening of most of the intermediates of the Calvin cycle and the photorespiratory pathway showed that physiological concentrations of sedoheptulose-7-phosphate and glycerate specifically inhibited the enzyme by decreasing its maximal velocity. An inhibition by ribulose-1,5-bisphosphate was also found. The inhibitory effect of sedoheptulose-7-phosphate on the enzyme is discussed in terms of allowing a control of sedoheptulose-1,7-bisphosphate hydrolysis by the demand of the product of this reaction. Subsequent studies with partially purified fructose-1,6-bisphosphatase from spinach chloroplasts showed that glycerate also inhibited this enzyme. With isolated chloroplasts, glycerate was found to inhibit CO₂ fixation by blocking the stromal fructose-1,6-bisphosphatase. It is therefore possible that the inhibition of the two phosphatases by glycerate is an important regulatory factor for adjusting the activity of the Calvin cycle to the ATP supply by the light reaction.Abbreviations DTT dithiothreitol - FBPase fructose-1,6-bisphosphatase - Fru-1,6-P₂ fructose-1,6-bisphosphate - Fru-6-P fructose-6-phosphate - 3-PGA 3-phosphoglycerate - Ru-1,5-P₂ ribulose-1,5-bisphosphate - Ru-5-P ribulose-5-phosphate - SBPase sedoheptulose-1,7-bisphosphatase - Sed-1,7-P₂ sedoheptulose-1,7-bisphosphate - Sed-7-P sedoheptulose-7-phosphate This work was supported by the Deutsche Forschungsgemein-schaft.     

The phosphate translocator of the chloroplast envelope. Isolation of the carrier protein and reconstitution of transport

This report describes the solubilization and purification of the phosphate translocator of spinach chloroplasts and the reconstitution of its activity by incorporation into liposomes. (1) Prior to the isolation, the carrier is specifically labelled by treatment with 2,4,6-trinitrobenzenesulfonic acid and NaB[³H]H₄. (2) After preextraction of purified envelope membranes with Brij 58 for removing other loosely bound membrane proteins, the phosphate translocator is extracted with Triton X-100. After passing the resulting extract over a DEAE-Sepharose column followed by sucrose density gradient ultracentrifugation, the translocator protein is purified to apparent homogeneity. The 5–6-fold purification thus obtained concurs with earlier findings that the phosphate translocator protein represents 15–20% of the envelope membrane protein. This highly purified protein is suitable for studies of the hydrodynamic parameters of the translocator. (3) Since the exposure to detergents affects the activity of the translocator protein, alternatively, a rapid batch procedure for the purification of the translocator protein employing hydroxyapatite is used, yielding within 15 min the phosphate translocator protein of about 70% purity. (4) After incorporation of this protein fraction into liposomes, a specific transport of phosphate into these liposomes is observed, which van be terminated by inhibitor stop with pyridoxal 5′-phosphate. This uptake is only observed when the liposomes have been preloaded with phosphate or 3-phosphoglycerate, but not with 2-phosphoglycerate. Thus, like in intact chloroplasts, also the reconstituted transport facilitates an obligatory and specific counter exchange of anions. The apparent K_m for the transport of phosphate by this reconstituted system is about 0.8 mM, which is comparable to the corresponding value in intact chloroplasts. The calculated turnover of 150–300 min⁻¹ (20°C) accounts for 3–6% of the original activity.     

The reduction of 3-phosphoglycerate by reconstituted chloroplasts and by chloroplast extracts

1. The rate of 3-phosphoglycerate reduction in extracts from spinach chloroplasts, assayed by spectrophotometric measurement of 3-phosphoglycerate-dependent NADPH oxidation, was strongly inhibited by ADP. AMP was much less inhibitory.

2. Oxygen evolution by reconstituted chloroplasts with 3-phosphoglycerate as substrate was also inhibited by the addition of ADP or following uncoupling by added NH₄Cl.

3. In all cases the inhibitory effects of ADP were reversed by addition of phosphocreatine and creatine phosphokinase activity.

4. The stoichiometry of 3-phosphoglycerate reduction to NADPH oxidation in chloroplast extracts was 1:1 and there was negligible turnover of the Benson-Calvin cycle in either chloroplast extracts or in reconstituted chloroplasts under the particular conditions employed.

5. The maximum rate of 3-phosphoglycerate-dependent O₂ evolution by reconstituted chloroplasts was ultimately limited by NADP reduction and photo-phosphorylation, and was similar to the maximum rate of oxygen evolution under optimal conditions by intact chloroplasts. In the presence of sufficient ADP phosphorylating activity, the rate of enzymic 3-phosphoglycerate reduction was relatively high. The inhibition of this reaction by ADP may represent a control mechanism in photosynthesis.     

Effect of photosynthetic intermediates on the magnesium inhibition of oxygen evolution by barley chloroplasts

Millimolar concentrations of Mg²⁺ inhibited CO₂-dependent O₂ evolution by barley (Hordeum vulgare L.) chloroplasts and also prevented the activation of NADP-glyceraldehyde-3-phosphate dehydrogenase, ribulose-5-phosphate kinase, and fructose-1,6-diphosphatase by light in intact chloroplasts. When added in the dark, 3-phosphoglycerate prevented the inhibition of O₂ evolution by Mg²⁺ and reduced the Mg²⁺ inhibition of enzyme activation by light. Fructose 1,6-diphosphate and ribulose 5-phosphate also prevented the inhibition of O₂ evolution by Mg²⁺ whereas glucose 1-phosphate, glucose 6-phosphate, ribulose 1,5-diphosphate, and citrate had no effect. Phosphoenolpyruvate gave an intermediate response. Metabolites that prevented the Mg²⁺ inhibition of O₂ evolution shortened the lag phase of CO₂-dependent O₂ evolution in the absence of M²⁺. Loading chloroplasts in the dark with 3-phosphoglycerate reduced both the lag phase of O₂ evolution and the inhibition of O₂ evolution by Mg²⁺. The results suggested that Mg²⁺ inhibition was lessened either by external metabolites that compete with inorganic phosphate for transport into the chloroplast or by a high concentration of internal metabolites.     

Photosynthesis in a reconstituted chloroplast system from spinach. Some factors affecting CO2-dependent oxygen evolution with fructose-1, 6-bisphosphate as substrate

When envelope-free spinach chloroplasts are incubated with stromal protein, catalytic NADP, catalytic ADP, radioactive bicarbonate and fructose 1,6-bisphosphate, ¹⁴CO₂ fixation starts immediately upon illumination but oxygen evolution is delayed. The delay is increased by the addition of fructose 6-phosphate and by a variety of factors known (or believed) to increase fructose bisphosphatase activity (such as dithiothreitol, more alkaline pH, higher [Mg] and antimycin A). Conversely, the lag can be decreased or eliminated by the addition of an ATP-generating system. Bearing in mind the known inhibition, by ADP, of sn-phospho-3-glycerate (3-phosphoglycerate) reduction it is concluded that the lag in O₂ evolution results from the production of ribulose 5-phosphate from fructose bisphosphate and that this in turn inhibits the reoxidation of NADPH by adversely affecting the ADP/ATP ratio. The results are discussed in their relation to the mode of action of antimycin A and to regulation of the reductive pentose phosphate pathway.     

Isolation of intact chloroplasts with high CO2 fixation capacity from sugarbeet leaves containing calcium oxalate

Intact chloroplasts were isolated from sugarbeet leaves by the mechanical disruption technique normally used for spinach. The chloroplast pellet contained a ring of white irregularly shaped crystals which were identified as calcium oxalate. The chloroplasts were greater than 90% intact yet good rates of CO₂ fixation were only obtained when inorganic pyrophosphate or 3-phosphoglycerate were added to the assay medium. Chloroplasts free of calcium oxalate were prepared by purification on a three step Percoll gradient. These purified chloroplasts were highly intact and showed high rates of CO₂ fixation without adding inorganic pyrophosphate or 3-phosphoglycerate. With optimal assay conditions (0.2 mM orthophosphate and pH 8.0) rates of 110–130 mole per milligram chlorophyll per hour were routinely obtained. It is concluded that intact chloroplasts capable of high rates of CO₂ fixation can be prepared from sugarbeet leaves using a simple three step Percoll gradient.Abbreviations BSA bovine serum albumin - Chl chlorophyll - Pi inorganic orthophosphate - PPi inorganic pyrophosphate - PGA 3-phosphoglycerate - EDTA ethylenediamine tetraacetic acid - EGTA ethyleneglycol-bis-(aminoethyl ether) - N,N tetraacetic acid     

Uptake of ATP analogs by isolated pea chloroplasts and their effect on CO2 fixation and electron transport

1. The ATP analog, adenylyl-imidodiphosphate rapidly inhibited CO₂-dependent oxygen evolution by isolated pea chloroplasts. Both α, β- and β, γ-methylene adenosine triphosphate also inhibited oxygen evolution. The inhibition was relieved by ATP but only partially relieved by 3-phosphoglycerate. Oxygen evolution with 3-phosphoglycerate as substrate was inhibited by adenylyl-imidodiphosphate to a lesser extent than CO₂-dependent oxygen evolution. The concentration of adenylyl-imidodiphosphate required for 50% inhibition of CO₂-dependent oxygen evolution was 50 μM.2. Although non-cyclic photophosphorylation by broken chloroplasts was not significantly affected by adenylyl-imidodiphosphate, electron transport in the absence of ADP was inhibited by adenylyl-imidodiphosphate to the same extent as by ATP, suggesting binding of the ATP analog to the coupling factor of phosphorylation.3. The endogenous adenine nucleotides of a chloroplast suspension were labelled by incubation with [¹⁴C]ATP and subsequent washing. Addition of adenylyl-imidodiphosphate to the labelled chloroplasts resulted in a rapid efflux of adenine nucleotides suggesting that the ATP analog was transported into the chloroplasts via the adenine nucleotide translocator.4. It was concluded that uptake of ATP analogs in exchange for endogenous adenine nucleotides decreased the internal ATP concentration and thus inhibited CO₂ fixation. Oxygen evolution was inhibited to a lesser extent in spinach chloroplasts which apparently have lower rates of adenine nucleotide transport than pea chloroplasts.     

Isolation of intact chloroplasts with high CO2 fixation capacity from sugarbeet leaves containing calcium oxalate

Intact chloroplasts were isolated from sugarbeet leaves by the mechanical disruption technique normally used for spinach. The chloroplast pellet contained a ring of white irregularly shaped crystals which were identified as calcium oxalate. The chloroplasts were greater than 90% intact yet good rates of CO₂ fixation were only obtained when inorganic pyrophosphate or 3-phosphoglycerate were added to the assay medium. Chloroplasts free of calcium oxalate were prepared by purification on a three step Percoll gradient. These purified chloroplasts were highly intact and showed high rates of CO₂ fixation without adding inorganic pyrophosphate or 3-phosphoglycerate. With optimal assay conditions (0.2 mM orthophosphate and pH 8.0) rates of 110–130 μ mole per milligram chlorophyll per hour were routinely obtained. It is concluded that intact chloroplasts capable of high rates of CO₂ fixation can be prepared from sugarbeet leaves using a simple three step Percoll gradient.     

Photosynthetic induction phenomena in spinach chloroplasts in relation to the nature of the isolating medium

1. Measurements were made of photosynthetic CO₂ fixation and O₂ evolution by spinach chloroplasts isolated in sorbitol media containing 2-(N-morpholino)ethanesulphonate (MES).

2. The chloroplasts isolated in MES-sorbitol media exhibited induction phenomena which were similar to those shown by chloroplasts isolated in orthophosphate-sugar mixtures. Added ribose 5-phosphate shortened the lags which preceded the attainment of maximal rates of CO₂ fixation and O₂ evolution. O₂ evolution reached its maximum rate almost immediately in the presence of 3-phosphoglycerate. Induction periods were shortened by pre-illumination of the parent tissue prior to separation of the chloroplasts.

3. In the absence of added substrate (other than CO₂) lags exhibited by chloroplasts isolated in MES-sorbitol were shorter than those observed with chloroplasts prepared in orthophosphate-sorbitol. These shorter lags could be extended by briefly exposing the chloroplasts to sugar media containing orthophosphate, malate or acetate or to Tris-NaCl.

4. The results are discussed in relation to photosynthetic induction phenomena and current methods of chloroplast isolation.     

Comparison of the kinetic properties,inhibition and labelling of the phosphate translocators from maize and spinach mesophyll chloroplasts

The kinetic properties of the phosphate translocator from maize (Zea mays L.) mesophyll chloroplasts have been determined. We have used a double silicone-oil-layer centrifugation system in order to obtain true initial uptake rates in forward-reaction experiments. In addition, it was possible to perform back-exchange experiments and to study the effects of illumination and of preloading the chloroplasts with different substrates on transport. It is shown that the phosphate translocator from mesophyll chloroplasts of maize, a C₄ plant, transports inorganic phosphate and phosphorylated C₃ compounds in which the phosphate group is linked to the C₃ atom (e.g. 3-phosphoglycerate and triose phosphate). The affinities of the transported metabolites towards the translocator protein are about one order of magnitude higher than in mesophyll chloroplasts from the C₃ plant, spinach. In contrast to the phosphate translocator from C₃-mesophyll chloroplasts, that of C₄-mesophyll chloroplasts catalyzes in addition the transport of C₃ compounds where the phosphate group is attached to the C₂ atom (e.g. 2-phosphoglycerate, phosphoenolpyruvate). The phosphate translocator from both chloroplast types is strongly inhibited by pyridoxal-5-phosphate (PLP), 2,4,6-trinitrobenzenesulfonic acid and 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS). In the case of the spinach translocator protein these inhibitors were shown to react with the same amino-acid residue at the substrate binding site, and one molecule of either DIDS or PLP is obviously required per substrate binding site for the inactivation of the translocation process. In the functionally active dimeric translocator protein only one substrate-binding site appears to be accessible at a particular time, indicating that the site might be exposed to each side of the membrane in turn. Using [³H]-H₂DIDS for the labelling of maize mesophyll envelopes the radioactivity was found to be associated with two polypeptides of about 29 and 30 kDa. Since Western-blot analysis showed that only the 30 kDa polypeptide reacted with an antiserum directed against the spinach phosphate translocator protein it is suggested that this polypeptide presumably represents the phosphate translocator from maize mesophyll chloroplasts.Abbreviations DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid - PEP phosphoenolpyruvate - 2-,3-PGA 2-,3-phosphoglycerate - PLP pyridoxal-5-phosphate - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TNBS 2,4,6-trinitrobenzenesulfonic acid - triose P triose phosphate This work was supported by the Deutsche Forschungsgemeinschaft     

Properties of a Membrane-bound Phosphatase from the Thylakoids of Spinach Chloroplasts

A 3-phosphoglycerate phosphatase activity of about 2 micromoles per minute per milligram chlorophyll is associated with the thylakoid membranes of spinach chloroplasts. The K_m for 3-phosphoglycerate is 3 millimolar. The enzyme can be solubilized from thylakoid membranes by treatment with 0.33 molar MgCl₂ or sodium deoxycholate. The activity is not stimulated by sulfhydryl reagents or the addition of 10 millimolar MgCl₂. The enzymic activity is insensitive to ethylenediaminetetraacetate. The pH optimum is broad, between 5.5 to 7.5. Although the substrate specificity is broad, 3-phosphoglycerate is the best substrate of those tested at neutral pH. However, p-nitrophenyl phosphate was a more effective substrate at pH 5.5. The enzyme exhibits the general characteristics of an acid phosphatase.     

Control of CO2 fixation. Regulation of spinach ribulose-5-phosphate kinase by stromal metabolite levels

The effect of stromal metabolites on the light-activated form of ribulose-5-phosphate kinase was studied with the enzyme rapidly extracted from illuminated spinach chlorplasts. In some instances, the effect of metabolites on the dark-inactivated enzyme extracted from darkened chloroplasts was also investigated. (1) The light-activated form of the enzyme is competitively inhibited with respect to ribulose 5-phosphate by 6-phosphogluconate, ribulose 1,5-bisphosphate, 3-phosphoglycerate and phosphate. Also, fructose 1,6-bisphosphate is inhibitory. All these compounds, except ribulose 1,5-bisphosphate, show an increasing inhibitory effect at lower pH values. Therefore, in the presence of these inhibitors, ribulose-5-phosphate kinase becomes strongly pH dependent. These compounds also exert an inhibitory effect on the dark-inactivated enzyme. (2) The assay of stromal levels of 6-phosphogluconate showed that this compound increased dramatically during a light-dark transient. (3) The dark-inactivated form of ribulose-5-phosphate kinase is strongly inhibited by ADP, the inhibition being competitive with respect to ATP. (4) A simulation of stromal metabolite levels in the enzyme activity assay indicates that in illuminated chloroplasts ribulose-5-phosphate kinase attains only about 4% of its maximal activity. When the fully light-activated enzyme is assayed under conditions occurring in the stroma in the dark, the activity is further decreased by a factor of 20. The same assay with the dark-inactivated enzyme yields an activity of virtually zero. (5) These results demonstrate that in the chloroplasts ribulose-5-phosphate kinase can not only be very efficiently switched off in the dark, but also be subjected to fine control during the illuminated state through the action of stromal metabolites.     

Inhibition of 3-Phosphoglycerate-Dependent O(2) Evolution by Phosphoenolpyruvate in C(4) Mesophyll Chloroplasts of Digitaria sanguinalis (L.) Scop

The effects of phosphoenolpyruvate (PEP), inorganic phosphate (Pi), and ATP on 3-phosphoglycerate (PGA)-dependent O₂ evolution by chloroplasts of Digitaria sanguinalis (L.) Scop. (crabgrass) were evaluated relative to possible mechanisms of PEP transport by the C₄ mesophyll chloroplast. Crude and Percoll purified chloroplast preparations exhibited rates of PGA-dependent O₂ evolution in the range of 90 to 135 micromoles O₂ per milligram chlorophyll per hour, and up to 180 micromoles O₂ per milligram chlorophyll per hour at optimal Pi concentrations (approximately 0.2 millimolar at 9 millimolar PGA). Higher concentrations of Pi were inhibitory. PEP inhibited O₂ evolution (up to 70%) in both chloroplast preparations when the PEP to PGA ratio was high (i.e. 9 millimolar PEP to 0.36 millimolar PGA). Usually no inhibition was seen when the PEP to PGA ratio was less than 2. PEP acted as a competitive inhibitor and, at a concentration of 9 millimolar, increased the apparent K_m (PGA) from 0.15 to 0.53 millimolar in Percoll purified chloroplasts. A low concentration of PGA and high ratio of PEP to PGA, which are considered unphysiological, were required to detect any inhibition of O₂ evolution by PEP. Similar results were obtained from crude versus Percoll purified preparations. Neither the addition of Pi nor ATP could overcome PEP inhibition. As PEP inhibition was competitive with respect to PGA concentration, and as addition of ATP or Pi could not prevent PEP inhibition of PGA-dependent O₂ evolution, the inhibition was not due to PEP exchange of adenylates or Pi out of the chloroplast. Analysis of the effect of Pi and PEP, separately and in combination, on PGA-dependent O₂ evolution suggests interactions between PEP, Pi, and PGA on the same translocator in the C₄ mesophyll chloroplast. C₃ spinach chloroplasts were also found to be sensitive to PEP, but to a lesser extent than crabgrass chloroplasts. The apparent K_i values (PEP) were 3 and 21 millimolar for crabgrass and spinach, respectively.     

Diversity of specificity and function of phosphate translocators in various plastids

This report gives a comparison of the specificity of phosphate translocators in various plastids. Whereas the phosphate translocator of the C₃ plant spinach mediates a counter exchange between inorganic phosphate, dihydroxyacetone phosphate, and 3-phosphoglycerate, the phosphate translocators in chloroplasts from C₄ and CAM plants transport phosphoenolpyruvate in addition to the above mentioned metabolites. In plastids from pea roots the phosphate translocator also transports glucose 6-phosphate. This diversity of phosphate translocators is discussed in view of the special functions of the various plastids.     

Enzyme regulation in c(4) photosynthesis : identification and localization of activities catalyzing the synthesis and hydrolysis of fructose-2,6-bisphosphate in corn leaves

Activities catalyzing the synthesis of fructose-2,6-bisphosphate (fructose-6-phosphate,2-kinase or Fru-6-P,2K) and its breakdown (fructose-2,6-bisphosphatase or Fru-2,6-P₂ase) were identified in leaves of corn (Zea mays), a C₄ plant. Fru-6-P,2K and Fru-2,6-P₂ase were both localized mainly, if not entirely, in the leaf mesophyll cells. A partially purified preparation containing the two activities revealed that the kinase and phosphatase were regulated by metabolite effectors in a manner generally similar to their counterparts in C₃ species. Thus, corn Fru-6-P,2K was activated by inorganic phosphate (Pi) and fructose-6-phosphate, and was inhibited by 3-phosphoglycerate and dihydroxyacetone phosphate. Fru-2,6-P₂ase was inhibited by its products, fructose-6-phosphate and Pi. However, unlike its spinach equivalent, corn Fru-2,6-P₂ase was also inhibited by 3-phosphoglycerate and, less effectively, by dihydroxyacetone phosphate. The C₄ Fru-6-P,2K and Fru-2,6-P₂ase were also quite sensitive to inhibition by phosphoenolpyruvate, and each enzyme was also selectively inhibited by certain other metabolites.     

Some effects of inorganic phosphate on O2 evolution by isolated chloroplasts

1. After an initial lag, isolated spinach chloroplasts evolved O₂ in illuminated reaction mixtures containing bicarbonate but no added phosphate. This evolution soon ceased but could be restarted by the addition of phosphate.

2. The phosphate requirement could be met by orthophosphate, inorganic pyrophosphate, ATP or ADP but not by AMP. Approx. 3 molecules of O₂ were evolved for each molecule of orthophosphate added and approx. 6 for each molecule of pyrophosphate.

3. With CO₂ as the sole added substrate the extent of the initial lag in O₂ evolution was not greatly affected by small quantities of added orthophosphate but as the concentration of orthophosphate was increased there was a progressive increase in the lag and a progressive decrease in the maximum rate. Pyrophosphate failed to produce these effects at a 100 times the concentration and in the presence of pyrophosphate the orthophosphate inhibition was less severe. There was little or no orthophosphate inhibition in the presence of substrate quantities of 3-phosphoglycerate or ribose 5-phosphate and CO₂.

4. There was also a requirement for phosphate by chloroplasts evolving O₂ in the presence of 3-phosphoglycerate or ribose 5-phosphate plus CO₂. In the presence of endogenous phosphate only, added ribose 5-phosphate suppressed the O₂ evolution which normally followed the addition of 3-phosphoglycerate.

5. The results provide direct support for the proposed phosphate requirement of the photosynthetic carbon cycle and are discussed in this context. They also imply that orthophosphate, ribose 5-phosphate and 3-phosphoglycerate can penetrate the intact chloroplast envelope with considerable rapidity.     

Uptake of ATP analogs by isolated pea chloroplasts and their effect on CO2 fixation and electron transport.

1. The ATP analog, adenylyl-imidodiphosphate rapidly inhibited CO2-dependent oxygen evolution by isolated pea chloroplasts. Both alpha, beta- and beta, gamma-methylene adenosine triphosphate also inhibited oxygen evolution. The inhibition was relieved by ATP but only partially relieved by 3-phosphoglycerate. Oxygen evolution with 3-phosphoglycerate as substrate was inhibited by adenylyl-imidodiphosphate to a lesser extent than CO2-dependent oxygen evolution. The concentration of adenylylimidodiphosphate required for 50% inhibition of CO2-dependent oxygen evolution was 50 micronM. 2. Although non-cyclic photophosphorylation by broken chloroplasts was not significantly affected by adenylyl-imidodiphosphate, electron transport in the absence of ADP was inhibited by adenylyl-imidodiphosphate to the same extent as by ATP, suggesting binding of the ATP analog to the coupling factor of phosphorylation. 3. The endogenous adenine nucleotides of a chloroplast suspension were labelled by incubation with [14C]ATP and subsequent washing. Addition of adenylyl-imidodiphosphate to the labelled chloroplasts resulted in a rapid efflux of adenine nucleotides suggesting that the ATP analog was transported into the chloroplasts via the adenine nucleotide translocator. 4. It was concluded that uptake of ATP analogs in exchange for endogenous adenine nucleotides decreased the internal ATP concentration and thus inhibited CO2 fixation. Oxygen evolution was inhibited to a lesser extent in spinach chloroplasts which apparently have lower rates of adenine nucleotide transport than pea chloroplasts.