Antimycin A Stimulation of Rate-limiting Steps of Photosynthesis in Isolated Spinach Chloroplasts

Changes in levels of metabolites in isolated spinach (Spinacia oleracea) chloroplasts seen upon addition of antimycin A suggest that the activities of enzymes mediating several regulated reactions are affected. Apparently, the presence of added antimycin A does not increase the level of CO₂ in the chloroplasts, nor does it stimulate CO₂ fixation by increasing the level of the carboxylation substrate, ribulose-1,5-diphosphate. Rather, it appears that antimycin A increases CO₂ fixation rate by indirectly stimulating the enzyme, ribulose-1,5-diphosphate carboxylase (E.C. 4.1.1.39), which mediates the carboxylation of ribulose-1,5-diphosphate to give 3-phosphoglycerate. Another rate-limiting enzyme of the reductive pentose phosphate cycle, hexose diphosphatase (E.C. 3.1.3.11), seems also to be stimulated. The synthesis of polysaccharides (mostly starch) seems also to be stimulated. These results are interpreted as indicating that antimycin A addition enhances the general activation of those enzymes which already are activated during photosynthesis but are inactive in the dark. The ratio of adenosine triphosphate-adenosine diphosphate under conditions of photosynthesis was only moderately decreased in the presence of antimycin A, perhaps accounting in part for an observed increase in accumulation of 3-phosphoglycerate as compared with dihydroxyacetone phosphate. No significant effect on movement of metabolites from the chloroplast to the medium was seen.     

Effect of antimycin a on photosynthesis of intact spinach chloroplasts

Low concentrations (0.5-10 μm) of antimycin A were shown to increase the rate of CO₂ fixation, O₂ evolution and inorganic phosphate esterification in intact spinach (Spinacia oleracea) chloroplasts. The increase was highest when the light intensity was saturating. Stimulation was independent of the bicarbonate concentration and was accompanied by an enhancement in the synthesis of glycerate 3-phosphate with a decrease in dihydroxyacetone phosphate. The antibiotic decreased the Michaelis constant of the chloroplast but not of ribulose 1,5-diphosphate carboxylase for bicarbonate. It was suggested that antimycin A is affecting that portion (outer envelope) of the intact chloroplast which contains the enzyme mechanism for controlling the pace of CO₂ fixation.     

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.     

Regulation of Soybean Net Photosynthetic CO(2) Fixation by the Interaction of CO(2), O(2), and Ribulose 1,5-Diphosphate Carboxylase

Kinetic properties of soybean net photosynthetic CO₂ fixation and of the carboxylase and oxygenase activities of purified soybean (Glycine max [L.] Merr.) ribulose 1, 5-diphosphate carboxylase (EC 4.1.1.39) were examined as functions of temperature, CO₂ concentration, and O₂ concentration. With leaves, O₂ inhibition of net photosynthetic CO₂ fixation increased when the ambient leaf temperature was increased. The increased inhibition of CO₂ fixation at higher temperatures was caused by a reduced affinity of the leaf for CO₂ and an increased affinity of the leaf for O₂. With purified ribulose 1,5-diphosphate carboxylase, O₂ inhibition of CO₂ incorporation and the ratio of oxygenase activity to carboxylase activity increased with increased temperature. The increased O₂ sensitivity of the enzyme at higher temperature was caused by a reduced affinity of the enzyme for CO₂ and a slightly increased affinity of the enzyme for O₂. The similarity of the effect of temperature on the affinity of intact leaves and of ribulose 1,5-diphosphate carboxylase for CO₂ and O₂ provides further evidence that the carboxylase regulates the O₂ response of photosynthetic CO₂ fixation in soybean leaves. Based on results reported here and in the literature, a scheme outlining the stoichiometry between CO₂ and O₂ fixation in vivo is proposed.     

On the Participation of Phosphoribulokinase in the Light Regulation of CO(2) Fixation

CO₂ fixation by a suspension of isolated spinach chloroplasts was terminated by turning off the light, and changes of metabolite levels in the chloroplast stroma and the surrounding medium were assayed. Whereas CO₂ fixation comes to a total stop within 15 seconds, a conversion of triose phosphates to heptose, hexose, and pentose monophosphates is found to occur for 1 to 2 minutes afterwards. It seems from these data that an inactivation of fructose and sedoheptulose bisphosphatases proceeds with a lag period. In contrast, the conversion of pentose monophosphates to ribulose 1,5-bisphosphate is inhibited immediately after the stop of illumination. As the stromal level of freely available ATP was not depleted under this condition, these data demonstrate that ribulose 5-phosphate kinase was very rapidly inactivated after darkening of the chloroplasts. Essentially, the same effect is also observed when CO₂ fixation is partially inhibited by addition of moderate concentrations of m-chlorocarbonyl phenylhydrazone, partially uncoupling photophosphorylation. It appears from these results, that the activity of ribulose 5-phosphate kinase is not only regulated by light through the mediation of reduced carriers like thioredoxin but also by alternative parameters, e.g. stromal metabolite levels.     

Microenvironmental manipulation of the observed michaelis constant of ribulose diphosphate carboxylase

Urease and ribulose 1, 5-diphosphate carboxylase can be bound to Sepharose to give an immobilized two-enzyme system which catalyzes the reaction urea → H₂CO₃ → phosphoglyceric acid. The observed Km of the system for urea approaches the lower value for urease when carboxylase levels on the gel exceed urease levels. If a similar system operates in the chloroplast, the high Km (H₂CO₃) of ribulose 1,5-diphosphate carboxylase may not be metabolically significant.     

Carbon dioxide fixation in the light and in the dark by isolated spinach chloroplasts

Factors affecting CO₂ fixation in the spinach (Spinacia oleracea) chloroplast were investigated. Free magnesium ions are shown to be highly inhibitory for photosynthetic CO₂ fixation in isolated intact spinach chloroplasts. The pH optimum for CO₂ fixation is about 8.5 but is dependent upon the reaction medium. Conditions are defined under which chloroplasts illuminated in the absence of CO₂ accumulate ribulose 1,5-diphosphate, and fix CO₂ in a subsequent dark period when high magnesium ion concentrations are provided. The regulation of photosynthetic CO₂ assimilation by these factors is discussed.     

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.     

Level of photosynthetic intermediates in isolated spinach chloroplasts

The level of intermediates of the photosynthetic carbon cycle was measured in intact spinach chloroplasts in an attempt to determine the cause of the induction lag in CO₂ assimilation. In addition, transient changes in the level of the intermediates were determined as affected by a light-dark period and by the addition of an excess amount of bicarbonate during a period of steady photosynthesis. Assayed enzymically were: ribulose 1,5-diphosphate, pentose monophosphates (mixture of ribose 5-phosphate, ribulose 5-phosphate and xylulose 5-phosphate, hexose monophosphates (mixture of glucose 6-phosphate, glucose 1-phosphate, and fructose 6-phosphate), glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, glycerate acid 3-phosphate, a mixture of fructose 1,6-diphosphate and sedoheptulose 1,7-diphosphate, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP).     

Relative Contribution of Ribulose 1,5-Diphosphate Carboxylase and Phosphoenolpyruvate Carboxylase to CO2 Fixation Activity in Roots of Dark-grown or Light-grown Lens culinaris Seedlings

The pathway of carbon assimilation in greening roots was compared to the pathway in leaves of Lens culinaris seedlings by means of labelling distribution analysis among the products of ¹⁴CO₂ fixation in vivo, and in vitro with ribulose 1,5-diphosphate as the substrate. In green leaves, CO₂ fixation via ribulose 1,5-diphosphate carboxylase predominated largely while, in green roots, this carboxylase activity and the phosphoenolpyruvate carboxylase contributed almost equally to the whole in vivo CO₂ fixation. A participation of the activities of both carboxylases according to the double carboxylation pathway in the synthesis of dicarboxylic acids (malate and aspartate) was demonstrated in vitro after 48 h of greening in roots but seemed to be absent in in vivo experiments.     

Evidence for Cyclic Photophosphorylation during CO(2) Fixation in Intact Chloroplasts: Studies with Antimycin A, Nitrite, and Oxaloacetate

This study examines the effect of antimycin A and nitrite on ¹⁴CO₂ fixation in intact chloroplasts isolated from spinach (Spinacia oleracea L.) leaves. Antimycin A (2 micromolar) strongly inhibited CO₂ fixation but did not appear to inhibit or uncouple linear electron transport in intact chloroplasts. The addition of small quantities (40-100 micromolar) of nitrite or oxaloacetate, but not NH₄Cl, in the presence of antimycin A restored photosynthesis. Antimycin A inhibition, and the subsequent restoration of photosynthetic activities by nitrite or oxaloacetate, was observed over a wide range of CO₂ concentration, light intensity, and temperature. High O₂ concentration (up to 240 micromolar) did not appear to influence the extent of the inhibition by antimycin A, nor the subsequent restoration of photosynthetic activity by nitrite or oxaloacetate. Studies of O₂ exchanges during photosynthesis in cells and chloroplasts indicated that 2 micromolar antimycin A stimulated O₂ uptake by about 25% while net O₂ evolution was inhibited by 76%. O₂ uptake in chloroplasts in the presence of 2 micromolar antimycin A was 67% of total O₂ evolution. These results suggest that only a small proportion of the O₂ uptake measured was directly linked to ATP generation. The above evidence indicates that cyclic photophosphorylation is the predominant energy-balancing reaction during photosynthesis in intact chloroplasts. On the other hand, pseudocyclic O₂ uptake appears to play only a minimal role.     

Studies on the mechanism of glycerate 3-phosphate synthesis in tomato and maize leaves

The net carbon incorporation in maize (Zea mays) and tomato (Lycopersicum esculentum) leaves was mainly the result of the carboxylation of ribulose 1,5-diphosphate. In both of these organisms synthesis of glycerate 3-phosphate was studied during short chase experiments (2 or 3 seconds in ¹⁴CO₂ then 8 to 27 seconds in unlabeled CO₂). Changes in the radioactivity in the individual carbon atoms of glycerate 3-phosphate, malate, and aspartate are consistent with the formation, in both leaves, of 2 molecules of glycerate 3-phosphate for each CO₂ molecule incorporated. The CO₂, before reacting with ribulose 1,5-diphosphate, is first incorporated in an intracellular CO₂ pool which has a different composition according to the species. This pool is constituted in tomato by volatile compounds (50 nanomoles per gram of fresh weight) more or less in equilibrium with atmospheric CO₂. In maize the pool consists of carbon atoms 4 of malate and aspartate (for at least 80% of the pool) and volatile compounds which correspond, in all, to 540 nanomoles per gram of fresh weight where atmospheric CO₂ enters through an irreversible reaction.     

Chlorophyll Accumulation, CO2 Fixation Enzymes, and Hill Activity, in Greening Roots of Lens culinaris

The carboxylation of ribulose-1,5-diphosphate was demonstrated in vitro with extracts of ctiolated seedling roots. The presence of ribulose-1,5-diphosphate carboxylase was characterized in the subcellular fraction enriched in amyloplasts. Synthesis of chlorophyll, development of CO² fixation capacities and of Hill activity upon illumination have been studied with roots of Lens culinaris seedlings. The marked increases in CO² fixation with ribulose-1,5-diphosphate as the substrate and in Hill activity that occur after a lag phase seem to be related to cytological changes during the greening of roots.     

The Reducing Power and the CO2 Fixation Activity of Isolated Choroplasts

Antimycin A, which stimulates the Co₂ fixation activity of isolated intact spinach (Spinacia oleracea L) chloroplasts, was shown to stimulate also the oxygen evolution in the presence of oxaloacetate, as do the uncouplers. Some uncoupers (tetramethlethylenediamine, Nh₄CL) were shown to stimulate the CO₂ fixation activity, and to decrease the intrachloroplastic ATP levels, like antimycin A. The intrachloroplastic NADPH levels were not affected by the inhibitors studied.     

Enzyme activities of the carbon reduction cycle in some photosynthetic organisms

Profile analyses of the enzymes comprising the photosynthetic carbon reduction cycle have been performed in extracts of dark grown and greening Euglena gracilis var. bacillaris. Chlorella pyrenoidosa grown photoautotrophically, in the light with glucose or in the dark with glucose, Tolypothrix tenuis, Chromatium and leaves of spinach. Amounts of activity are compared with the level of photosynthetic CO₂ fixation. Only in Chromatium were all enzyme activities sufficient to support the in vivo rate of CO₂ fixation. In organisms other than Chromatium, some enzymes and particularly fructose 1,6-phosphatase and ribulose 1.5-diphosphate carboxylase appeared to be present in insufficient amounts to support the photosynthetic rate of the intact cell. Developmental studies with Euglena and growth studies with Chlorella led to the conclusion that these enzymes were associated with the cycle. Suppression of CO₂ fixation in heterotrophically grown Chlorella was accompanied by a striking decrease in the same enzymes whose activities increased in greening Euglena.     

Mesophyll Resistance and Carboxylase Activity: A Comparison under Water Stress Conditions

The response of several leaf gas exchange parameters were monitored with decreasing leaf water potential in Phaseolus vulgaris L. leaflets. These included photosynthesis, transpiration, CO₂ compensation point, ribulose 1,5-diphosphate carboxylase activity, boundary layer plus stomatal, and mesophyll resistance to diffusion of CO₂. Mesophyll resistance was calculated under two assumptions: (a) the CO₂ concentration at the chloroplast was zero, and (b) it was equal to the CO₂ compensation point.     

CO(2) Fixation Rate and RuBisCO Content Increase in the Halotolerant Cyanobacterium, Aphanothece halophytica, Grown in High Salinities

The growth of the halotolerant cyanobacterium Aphanothece halophytica, previously adapted to 0.5 molar NaCl, was optimal when NaCl concentration in culture medium was in the range 0.5 to 1.0 molar. The growth was delayed at either too low or too high salinities with lag time of ca. 0.5 day in 0.25 molar NaCl and ca. 2 days in 2 molar NaCl under the experimental conditions. However, the growth rates at the logarithmic phase were similar in the culture media containing NaCl in the range 0.25 to 2.0 molar. The capacity of photosynthetic CO₂ fixation increased 3.7-fold in the cells at the logarithmic phase as NaCl concentration in the culture medium increased from 0.25 to 2.0 molar. The protein level of ribulose 1,5-bisphosphate carboxylase/oxygenase was also found to increase with increasing salinity using both an immunoblotting method and protein A-gold immunoelectron microscopy. These results indicate that high photosynthetic capacity and high ribulose 1,5-bisphosphate carboxylase/oxygenase content may entail an important role in betaine synthesis and adaptation of the A. halophytica cells to high NaCl level.     

Coupling of Carbon Dioxide Fixation to the Oxyhydrogen Reaction in the Isolated Chloroplast of Chlamydomonas reinhardtii

The oxyhydrogen reaction (the reduction of O₂ to water by H₂) in the presence of CO₂ was studied in the isolated Chlamydomonas reinhardtii chloroplast by monitoring the rate of ¹⁴CO₂ incorporation into acid-stable products in the dark. The endogenous rate of CO₂ uptake (50-125 nmol/mg chlorophyll per h) was increased about 3- to 4-fold by ATP and additionally when combined with glucose, ribose-5-phosphate, and glycerate-3-phosphate. The rate was diminished 50 to 75%, respectively, when H₂ was replaced by N₂ or by air. Decrease in CO₂ uptake by dl-glyceraldehyde was taken to indicate that the regenerative phase and complete Calvin cycle turnover were involved. Diminution of CO₂ incorporation by rotenone, antimycin A, and 2,5-dibromo-3-methyl-6-isopropanol-p-benzoquinone was attributed to an inhibition of the oxyhydrogen reaction, resulting in an elevated NADPH/NADP ratio. If so, then the diminished CO₂ uptake could have been by “product inhibition” of the carbon metabolic network. Our data are consistent with the proposal (H. Gaffron [1942] J Gen Physiol 26: 241-267) that CO₂ fixation coupled to the oxyhydrogen reaction is dependent to some extent on exchloroplastic metabolism. This support is primarily ATP provided by mitochondrial respiration.     

Effects of glyoxylate on photosynthesis by intact chloroplasts

Because glyoxylate inhibits CO₂ fixation by intact chloroplasts and purified ribulose bisphosphate carboxylase/oxygenase, glyoxylate might be expected to exert some regulatory effect on photosynthesis. However, ribulose bisphosphate carboxylase activity and activation in intact chloroplasts from Spinacia oleracea L. leaves were not substantially inhibited by 10 millimolar glyoxylate. In the light, the ribulose bisphosphate pool decreased to half when 10 millimolar glyoxylate was present, whereas this pool doubled in the control. When 10 millimolar glyoxylate or formate was present during photosynthesis, the fructose bisphosphate pool in the chloroplasts doubled. Thus, glyoxylate appeared to inhibit the regeneration of ribulose bisphosphate, but not its utilization.

The fixation of CO₂ by intact chloroplasts was inhibited by salts of several weak acids, and the inhibition was more severe at pH 6.0 than at pH 8.0. At pH 6.0, glyoxylate inhibited CO₂ fixation by 50% at 50 micromolar, and glycolate caused 50% inhibition at 150 micromolar. This inhibition of CO₂ fixation seems to be a general effect of salts of weak acids.

Radioactive glyoxylate was reduced to glycolate by chloroplasts more rapidly in the light than in the dark. Glyoxylate reductase (NADP⁺) from intact chloroplast preparations had an apparent K_m (glyoxylate) of 140 micromolar and a V_max of 3 micromoles per minute per milligram chlorophyll.

     

Initial ATP Formation, NADP Reduction, CO(2) Fixation, and Chloroplast Flattening Upon Illuminating Pea Leaves

Chloroplasts in living cells of detached and sectioned leaves of Pisum sativum had a thickness of 2.68 ± 0.04 μ in the dark as determined from photographs made using a phase contrast microscope. Upon illumination with 4000 lux for 10 min, the chloroplasts flattened to 2.15 ± 0.04 μ. There was a short lag period of about 11 sec at 1000 lux and 2 sec at 4000 lux before appreciable light-induced flattening occurred. Both ATP and reduced nicotinamide adenine dinucleotide phosphate (NADPH) in detached pea leaves increased upon illumination and then fell during the initial 60 sec. The maximum ATP level was attained in 16 sec at 1000 lux and 10 sec at 4000 lux, while NADPH required about twice as long to reach a maximum. A sustained rate of carbon dioxide fixation occurred after a lag period coinciding in time with the drop in the NADPH level. ATP appeared to be involved not only with carbon dioxide fixation, but also with some reaction beginning sooner, perhaps the light-induced chloroplast flattening. Considering the initial photophosphorylation and the sustained CO₂ fixation rates, the ATP formation rate in vivo apparently increased after the leaves had been in the light for a few min.