Effect of Adenosine Monophosphate, Adenosine Diphosphate, and Reduced Nicotinamide Adenine Dinucleotide on Adenosine Triphosphate-dependent Carbon Dioxide Fixation in the Autotroph Thiobacillus neapolitanus

The observation that adenosine triphosphate (ATP)-dependent CO(2) fixation in extracts of chemosynthetic and photosynthetic autotrophs may be regulated in part by adenosine monophosphate (AMP) was extended to the strict autotroph Thiobacillus neapolitanus (X). In addition, this report presents data which include adenosine diphosphate (ADP) in the regulatory role. When the primary CO(2) acceptor, ribose-5-phosphate, was replaced by ribulose-1,5-diphosphate, no inhibition of CO(2) fixation occurred unless the Mg(++) concentration was limiting. A molar ratio of 5:1 AMP or ADP to ATP reduced the specific activity (micromoles of CO(2) fixed per milligram of protein per minute) of the extracts from 0.22 to 0.12 and 0.11, respectively. The reported stimulation of the carboxylative phase of ATP-dependent CO(2) fixation by reduced nicotinamide adenine dinucleotide (NADH(2)) was investigated. Adding NADH(2) to the extracts did not stimulate CO(2) fixation, even at carbonate levels from 0.05 to 30 mumoles, except in the absence of ribose-5-phosphate. Slight increases in CO(2) fixation were noted when the assay system was incubated in air instead of the usual helium atmosphere.     

Evidence for the presence of phosphoriboisomerase and ribulose-1,5-diphosphate carboxylase in extracts of Desulfovibrio vulgaris.

Cell extracts of Desulfovibrio vulgaris were found to incorporate 14CO2 into acid-stable products when ribose-5-phosphate or ribulose-1,5-diphosphate was used as a substrate. This CO2 fixation required adenosine triphosphate and produced 3-phosphoglyceric acid as one of the products. The assimilation of CO2 by pentose phosphates was unrelated to the pyruvate-CO2 exchange reaction. The pyruvate-CO2 exchange did not require adenosine triphosphate, did not produce phosphorylated compounds, and, unlike the pentose phosphate system, required an acidic protein fraction for activity.     

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).     

Kinetics of light-dark CO2 fixation and glucose assimilation by Aphanocapsa 6714.

Cells of Aphanocapsa 6714 were subjected to alternating ligh-dark periods (flashing-light experiments). The corresponding activation (in the light) and inactivation (in the dark) of the reductive pentose cycle was measured, in vivo, from initial rates of 14CO2 incorporation and also by changes in the total concentration of 14C and 32P in soluble metabolites. Two principle sites of metabolic regulation were detected: (i) CO2 fixation was inactivated 15 to 20 s after removal of the light source, but reactivated rapidly on reentering the light; (ii) hydrolysis of fructose-1,6-diphosphate (FDP) and sedoheptulose-1,7-diphosphate (SDP) by their respective phosphatase(s) (FDP + SDPase) was rapidly inhibited in the dark but only slowly reactivated in the light. The time required for reactivation of FDP + SDPase, in the light, was on the order of 20 to 30 s. As a consequence of the timing of these inactivation-reactivation reactions, newly fixed CO2 accumulated in the FDP and SDP pools during the flashing-light experiments. Changes in the concentrations of the adenylate pools (mainly in the levels of adenosine 5'-triphosphate and adenosine diphosphate) were fast in comparison to the inactivation-reactivation reactions in the reductive pentose cycle. Thus, these regulatory effects may not be under the control of the adenylates in this organism. The activation of CO2 fixation in the light is at least in part due to activation of phosphoribulokinase, which is required for formation of ribulose-1,5-diphoshate, the carboxylation substrate. Phosphoribulokinase activity in crude extracts was found to be dependent on the presence of strong reducing agents such as dithiothreitol, but not significantly dependent on adenylate levels, although adenosine 5'-triphosphate is a substrate.     

CHLOROPLAST STRUCTURE AND FUNCTION IN ac-20, A MUTANT STRAIN OF CHLAMYDOMONAS REINHARDI : I. CO2 Fixation and Ribulose-1,5-Diphosphate Carboxylase Synthesis

A mutant strain of the green alga Chlamydomonas reinhardi, ac-20, is described in which both the rate of CO₂ fixation by whole cells and the rate of carboxylation of ribulose-1,5-diphosphate in cell-free extracts are reduced, particularly when sodium acetate is present in the growth medium. Of the enzymes of the reductive pentose phosphate cycle tested, only ribulose-1,5-diphosphate carboxylase activity is reduced in the mutant strain, and it appears that the low carboxylase activity limits the strain's rate of photosynthetic carbon metabolism. Evidence is presented to show that the fluctuation in the level of the enzyme activity in the presence or absence of acetate results from the fluctuation in the level of some factor(s) limiting the rate of synthesis of the protein.     

Phosphoribulokinase from Nitrobacter winogradskyi: activation by reduced nicotinamide adenine dinucleotide and inhibition by pyridoxal phosphate.

CO2 fixation by particle-free extracts from Nitrobacter winogradskyi increased by addition of reduced nicotinamide adenine dinucleotide (NADH). Ribulose-1,5-diphosphate, however, increased CO2 fixation, even in the absence of NADH. Phosphoribulokinase (EC 2.7.1.19) was the enzyme of Nitrobacter extracts that was activated specifically by NADH. Pyridoxal-5-phosphate inhibited both CO2 fixation and NADH-activated phosphoribulokinase from Nitrobacter. However, it did not affect phosphoribulokinase from spinach leaves. Since the spinach enzyme had also no requirement for reduced pyridine nucleotides, it appears that pyridoxal phosphate interferes only with the binding of NADH and not with the binding of ribulose-5-phosphate and adenosine-5'-triphosphate. The regulation of phosphoribulokinase activity by NADH provided Nitrobacter with an energy-dependent control mechanism of CO2 assimilation.     

14CO2 fixation and glycolate metabolism in the dark in isolated maize (Zea mays L.) bundle sheath strands

Bundle sheath strands capable of assimilating up to 68 μmoles CO₂ per mg chlorophyll per hr in the dark have been isolated from fully expanded leaves of Zea mays L. This dark CO₂-fixing system is dependent on exogenous ribose-5-phosphate, ADP or ATP, and Mg²⁺ for maximum activity. The principal product of dark fixation in this system is 3-phosphoglycerate, indicating that the CO₂-fixing reaction is mediated by ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39). The rate of dark CO₂ uptake in the strands in the presence of saturating levels of ribose-5-phosphate plus ADP is inhibited by oxygen. The inhibitory effect of oxygen is rapidly and completely reversible, and is relieved by increased levels of CO₂. Glycolate is synthesized in this dark system in the presence of [U-¹⁴C]ribose-5-phosphate, ADP, oxygen, and an inhibitor of glycolate oxidase (EC 1.1.3.1). Glycolate formation is completely abolished by heating the strands, and the rate of glycolate synthesis is markedly reduced by either lowering the oxygen tension or increasing the level of CO₂.These results, obtained with intact cells in the absence of light, indicate that the direct inhibitory effect of oxygen on photosynthesis is associated with photosynthetic carbon metabolism, probably at the level of ribulose-1,5-bisphosphate carboxylase, and not with photophosphorylation or photosynthetic electron transport. Furthermore, the findings indicate that the synthesis of glycolate from exogenous substrate can readily occur in the absence of photosynthetic electron transport, an observation consistent with the ribulose-1, 5-bisphosphate “oxygenase” scheme for glycolate formation during photosynthesis.     

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.     

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.     

The Association of d-Ribulose- 1,5-Bisphosphate Carboxylase/Oxygenase with Phosphoribulokinase

When Ribulose- 1,5-bisphosphate carboxylase/oxygenase was purified from spinach leaves (Spinacia oleracea) using precipitation with polyethylene glycol and MgCl₂ followed by DEAE cellulose chromatography, 75% of phosphoribulokinase and 7% of phosphoriboisomerase activities copurified with ribulose- 1,5-bisphosphate carboxylase/oxygenase. This enzyme preparation showed ribose-5-phosphate and ribulose-5-phosphate dependent carboxylase and oxygenase activities which were nearly equivalent to its corresponding ribulose- 1,5-bisphosphate dependent activity. The ribose-5-phosphate and ribulose-5-phosphate dependent reaction rates were stable and linear for much longer time periods than the ribulose- 1,5-bisphosphate dependent rates. When sucrose gradients were used to purify ribulose- 1,5-bisphosphate carboxylase/oxygenase from crude stromal extracts, phosphoribulokinase was found to cosediment with ribulose- 1,5-bisphosphate carboxylase. Under these conditions most of the phosphoriboisomerase activity remained with the slower sedimenting proteins. Ammonium sulfate precipitation resulted in separation of the ribulose- 1,5-bisphosphate carboxylase peak from phosphoribulokinase peak. Crude extracts of peas Pisum sativum and spinach contained 0.725 to 0.730 milligram of phosphoribulokinase per milligram of chlorophyll, respectively, based on an enzyme-linked immunosorbent assay.     

Early effects of illumination on the activity of some photosynthetic enzymes

Leaves of dark-grown corn (Zea mays) were illuminated for periods ranging from 3 minutes to 12 hours. The changes in the activities of ribose-5-phosphate isomerase, ribulose-5-phosphate kinase, and ribulose-1,5-diphosphate carboxylase were followed.

The activity of ribose-5-phosphate isomerase did not change significantly until between 12 and 24 hours of illumination. An increase in ribulose-5-phosphate kinase activity occurred after a lag of about 6 hours. The increase in carboxylase activity began after 3 minutes of illumination and increased until after 3 to 6 hours in the light, after which it began to decline. The increases in these enzymes appear to be the result of protein synthesis.

     

Reversible inhibition of the calvin cycle and activation of oxidative pentose phosphate cycle in isolated intact chloroplasts by hydrogen peroxide

Hydrogen peroxide (6x10^-4 M) causes a 90% inhibition of CO₂-fixation in isolated intact chloroplasts. The inhibition is reversed by adding catalase (2500 U/ml) or DTT (10 mM). If hydrogen peroxide is added to a suspension of intact chloroplasts in the light, the incorporation of carbon into hexose- and heptulose bisphosphates and into pentose monophosphates is significantly increased, whereas; carbon incorporation into hexose monophosphates and ribulose 1,5-bisphosphate is decreased. At the same time formation of 6-phosphogluconate is dramatically stimulated, and the level of ATP is increased. All these changes induced by hydrogen peroxide are reversed by addition of catalase or DTT. Additionally, the conversion of [¹⁴C]glucose-6-phosphate into different metabolites by lysed chloroplasts in the dark has been studied. In presence of hydrogen peroxide, formation of ribulose-1,5-bisphosphate is inhibited, whereas formation of other bisphosphates,of triose phosphates, and pentose monophosphates is stimulated. Again, DTT has the opposite effect. The release of ¹⁴CO₂ from added [¹⁴C]glucose-6-phosphate by the soluble fraction of lysed chloroplasts via the reactions of oxidative pentose phosphate cycle is completely inhibited by DTT (0.5 mM) and re-activated by comparable concentrations of hydrogen peroxide. These results indicate that hydrogen peroxide interacts with reduced sulfhydryl groups which are involved in the light activation of enzymes of the Calvin cycle at the site of fructose- and sedoheptulose bisphophatase, of phosphoribulokinase, as well as in light-inactivation of oxidative pentose phosphate cycle at the site of glucose-6-phosphate dehydrogenase.Abbreviations ADPG ADP-glucose - DHAP dihydroxyacetone phosphate - DTT dithiothreitol - FBP fructose-1,6-bisphosphate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - HMP hexose monophosphates (fructose-6-phosphate, glucose-6-phosphate, glucose-1-phosphate) - 6-PGI 6-phosphogluconate - PMP pentose monophosphates (xylulose-5-phosphate, ribose-5-phosphate, ribulose-5-phosphate) - RuBP ribulose-1,5-bisphosphate - S7P sedoheptulose-7-phosphate - SBP sedoheptulose-1,7-bisphosphate Dedicated to Prof. Dr. W. Simonis on the occasion of his 70th birthday     

Ribose-5-phosphate biosynthesis in Methanocaldococcus jannaschii occurs in the absence of a pentose-phosphate pathway

Recent work has raised a question as to the involvement of erythrose-4-phosphate, a product of the pentose phosphate pathway, in the metabolism of the methanogenic archaea (R. H. White, Biochemistry 43:7618-7627, 2004). To address the possible absence of erythrose-4-phosphate in Methanocaldococcus jannaschii, we have assayed cell extracts of this methanogen for the presence of this and other intermediates in the pentose phosphate pathway and have determined and compared the labeling patterns of sugar phosphates derived metabolically from [6,6-2H2]- and [U-13C]-labeled glucose-6-phosphate incubated with cell extracts. The results of this work have established the absence of pentose phosphate pathway intermediates erythrose-4-phosphate, xylose-5-phosphate, and sedoheptulose-7-phosphate in these cells and the presence of D-arabino-3-hexulose-6-phosphate, an intermediate in the ribulose monophosphate pathway. The labeling of the D-ara-bino-3-hexulose-6-phosphate, as well as the other sugar-Ps, indicates that this hexose-6-phosphate was the precursor to ribulose-5-phosphate that in turn was converted into ribose-5-phosphate by ribose-5-phosphate isomerase. Additional work has demonstrated that ribulose-5-phosphate is derived by the loss of formaldehyde from D-arabino-3-hexulose-6-phosphate, catalyzed by the protein product of the MJ1447 gene.     

Transient inhibition by ribose 5-phosphate of photosynthetic O2 evolution in a reconstituted chloroplast system.

Photosynthetic oxygen evolution by a reconstituted chloroplast system utilising sn-phospho-3-glycerol (3-phosphoglycerate) ceases upon the addition of ribose 5-phosphate even though the presence of this metabolite permits a rapid and immediate CO2 fixation. The period of cessation is appreciable at 0.1 mM ribose 5-phosphate. It is lengthened as the amount of added ribose 5-phosphate is increased and by the addition of dithiothreitol, a known activator of ribulose-5-phosphate kinase. Ribulose 1,5-bisphosphate is without effect. A similar interruption of O2 evolution may also be brought about by the addition of ADP or by ADP-generating systems such as glucose plus hexokinase. Spectrophotometric experiments indicate that the reoxidation of NADPH in the presence of sn-phospho-3-glycerol is similarly affected. The transient inhibition by ribose 5-phosphate is not observed in the presence of an active ATP-generating system or in the presence of sufficient DL-glyceraldehyde to inhibit ribulose-5-phosphate kinase activity. It is concluded that ribose 5-phosphate inhibits photosynthetic O2 evolution by adversely affecting the steady-state ATP/ADP ratio and consequently the reduction of sn-phospho-3-glycerol to glyceraldehyde 3-phosphate. The results are discussed in their relation to ADP regulation of photosynthetic carbon assimilation and metabolite transport.     

Enzymatic Evidence for a Complete Oxidative Pentose Phosphate Pathway in Chloroplasts and an Incomplete Pathway in the Cytosol of Spinach Leaves

The intracellular localization of transaldolase, transketolase, ribose-5-phosphate isomerase, and ribulose-5-phosphate epimerase was reexamined in spinach (Spinacia oleracea L.) leaves. We found highly predominant if not exclusive localization of these enzyme activities in chloroplasts isolated by isopyknic centrifugation in sucrose gradients. Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glucose phosphate isomerase, and triose phosphate isomerase activity was present in the chloroplast fraction but showed additional activity in the cytosol (supernatant) fraction attributable to the cytosol-specific isoforms known to exist for these enzymes. Anion-exchange chromatography of proteins of crude extracts on diethylaminoethyl-Fractogel revealed only a single enzyme each for transaldolase, transketolase, ribose-5-phosphate isomerase, and ribulose-5-phosphate epimerase. The data indicate that chloroplasts of spinach leaf cells possess the complete complement of enzymes of the oxidative pentose phosphate path-way (OPPP), whereas the cytosol contains only the first two reactions, contrary to the widely held view that plants generally possess a cytosolic OPPP capable of cyclic function. The chloroplast enzymes transketolase, ribose-5-phosphate isomerase, and ribulose-5-phosphate epimerase appear to be amphibolic for the Calvin cycle and OPPP.     

Properties of phosphoribulokinase from Thiobacillus neapolitanus

Partially purified preparations of ribulose-5-phosphate kinase (specific activity, 50 to 125 mumoles per min per mg of protein) were employed in a series of kinetic experiments in the presence of several concentrations of H(+), Mg(2+), adenosine triphosphate (ATP), and phosphoenolpyruvate (PEP). The pH optimum of the enzyme was found to be 7.9; at this pH and above, response of the enzyme to variations in ATP concentration was hyperbolic, exhibiting a K(m) of 7 x 10(-4)m ATP. At pH values below the optimum the response to ATP was sigmoidal, as it was throughout the entire pH range in the presence of PEP at a concentration greater than 5 x 10(-4)m. In the presence of PEP the pH optimum shifted to pH 8.4. In contrast, phosphoribulokinase from spinach exhibited hyperbolic responses throughout its pH range with no inhibition caused by PEP. Thiobacillus neapolitanus phosphoribulokinase was inhibited by PEP in a sigmoidal manner; however, in the presence of suboptimal concentrations of Mg(2+) the addition of PEP caused significant stimulation of activity. It is postulated that the enzyme consists of interacting subunits with several sites on the enzyme for binding ATP and with several separate sites binding PEP. It is suggested that PEP functions as a regulator of CO(2) fixation when the organism is under conditions of unlimited concentrations of substrate and CO(2).     

Activation of CO2 fixation in isolated spinach chloroplasts

1. 1. The effect of the Mg²⁺ concentration on the CO₂ fixation activity in situ in isolated and intact spinach chloroplasts upon suspension in hypotonic medium was examined. CO₂ fixation in the dark was activated 25–100 fold by 20 mM Mg²⁺ in the presence of added ATP plus either ribulose 5-phosphate or ribose 5-phosphate. 20 mM Mg²⁺-stimulated fixation only 2–3 fold in the presence of the substrate of fixation, ribulose 1,5-diphosphate. The highest Mg²⁺-stimulated rate of fixation in the dark observed with chloroplasts was 480 μmoles CO₂ fixed per mg chlorophyll per h.

2. 2. The concentration of bicarbonate at half of the maximal velocity (apparent K_m) during the Mg²⁺-stimulated fixation of CO₂ was 0.4 mM in the presence of ATP plus ribose 5-phosphate and 0.6 mM with ribulose 1,5-diphosphate.

3. 3. Dithioerythritol or light enhanced Mg²⁺-stimulated CO₂ fixation 1–3 fold in the presence of ATP plus ribose 5-phosphate but not ribulose 1,5-diphosphate.

4. 4. These results indicate that Mg²⁺ fluxes in the stroma of the chloroplast could control the activity of the phosphoribulokinase with a lesser effect on the ribulosediphosphate carboxylase. An increase in Mg²⁺ of 6–10 mM in the stroma region of the chloroplast would be enough to activate CO₂ fixation during photosynthesis.

Glucose-6-phosphate dehydrogenase of Anabaena sp.

The kinetic and molecular properties of cyanobacterial glucose-6-phosphate dehydrogenase, partly purified from Anabaena sp. ATCC 27893, show that it undergoes relatively slow, reversible transitions between different aggregation states which differ in catalytic activity. Sucrose gradient centrifugation and polyacrylamide gel electrophoresis reveal three principal forms, with approximate molecular weights of 120 000 (M ₁), 240 000 (M ₂) and 345 000 (M ₃). The relative catalytic activities are: M ₁M ₂<M ₃. In concentrated solutions of the enzyme, the equilibrium favors the more active, oligomeric forms. Dilution in the absence of effectors shifts the equilibrium in favor of the M ₁ form, with a marked diminution of catalytic activity. This transition is prevented by a substrate, glucose-6-phosphate, and also by glutamine. The other substrate, nicotinamide adenine dinucleotide phosphate (NADP⁺), and (in crude cell-free extracts) ribulose-1,5-diphosphate are negative effectors, which tend to maintain the enzyme in the M ₁ form. The equilibrium state between different forms of the enzyme is also strongly dependent on hydrogen ion concentration. Although the optimal pH for catalytic activity is 7.4, dissociation to the hypoactive M ₁ form is favored at pH values above 7; a pH of 6.5 is optimal for maintenace of the enzyme in the active state. Reduced nicotamide adenine dinucleotide phosphate (NADPH) and adenosine 5-triphosphate (ATP), inhibit catalytic activity, but do not significantly affect the equilibrium state. The relevance of these findings to the regulation of enzyme activity in vivo is discussed.Abbreviations G6PD glucose-6-phosphate dehydrogenase - 6PGD 6-phosphogluconate dehydrogenase - RUDP ribulose-1,5-diphosphate - G6P glucose-6-phosphate - 6PG 6-phosphogluconate     

Phosphofrucktokinase and glucose catabolism of Mucor and Penicillium species.

The primary catabolic pathways in the fungi Penicillium notatum and P. duponti, and Mucor rouxii and M. miehei were examined by measuring the relative rate of 14CO2 production from different carbon atoms of specifically labelled glucose. It was found that these organisms dissimilate glucose predominantly via the Embden--Meyerhof pathway in conjunction with the tricarboxylic acid cycle and to a lesser extent by the pentose phosphate pathway. Phosphofructokinase (EC 2.7.1.11) activity could not be detected initially in Penicillium species because of the interference from mannitol-1-phosphate dehydrogenase (EC 1.1.1.17) and NADH oxidase (EC 1.6.99.3). A combination of differential centrifuging and a heat treatment of Penicillium cell-free extracts in the presence of fructose-6-phosphate removed the interfering enzymes. The kinetic characteristics of phosphofructokinase from P. notatum and M. rouxii are described. The enzyme presents highly cooperative kinetics for fructose-6-phosphate. The kinetics for ATP show no cooperativity and inhibition by excess ATP is observed. The addition of AMP activated the P. notatum enzyme, relieving ATP inhibition; slight inhibition by AMP was observed with the M. rouxii enzyme. In contrast M. rouxii pyruvate kinase (EC 2.7.1.40) is activated 50-fold by fructose-1,6-diphosphate whereas pyruvate kinase from P. notatum and P. duponti were unaffected by fructose-1,6-diphosphate.     

Interactions between ribulose-1,5-bisphosphate carboxylase and stromal metabolites. II. Corroboration of the role of this enzyme as a metabolite buffer

The capacity of ribulose-1,5-bisphosphate carboxylase to bind reversibly chloroplast metabolites which are the substrates for both thylakoid and stromal enzymes was assessed using spinach chloroplasts and chloroplast extracts and with pure wheat ribulose-1,5-bisphosphate carboxylase. Measurements of the rate of coupled electron flow to methyl viologen in ‘leaky’ chloroplasts (which retained the chloroplast envelope and stromal enzymes but which were permeable to metabolites) and also with broken chloroplasts and washed thylakoids were used to study the effects of binding ADP and inorganic phopshate to ribulose-1,5-bisphosphate carboxylase. The presence of ribulose-1,5-bisphosphate carboxylase significantly altered the values obtained for apparent K_m for inorganic phosphate and ADP of coupled electron transport. The K_m (P_i) in washed thylakoids was 60–80 μM, in ‘leaky’ chloroplasts it was increased to 180–200 μM, while in ‘leaky’ chloroplasts preincubated with KCN and ribulose 1,5-bisphosphate the value was decreased to 40–50 μM. Similarly, the K_m (ADP) of coupled electron transport in washed thylakoids was 60–70 μM, in ‘leaky’ chloroplasts it was 130–150 μM and with ‘leaky’ chloroplasts incubated in the presence of KCN and ribulose 1,5-bisphosphate a value of 45–50 μM was obtained. The ability of ribulose 1,5-bisphosphate carboxylase to reduce the levels of free glycerate 3-phosphate in the absence of ribulose 1,5-bisphosphate was examined using a chloroplast extract system by varying the concentrations of stromal protein or purified ribulose 1,5-bisphosphate carboxylase. The effect of binding glycerate 3-phosphate to ribulose-1,5-bisphosphate carboxylase on glycerate 3-phosphate reduction was to reduce both the rate an the amount of NADPH oxidation for a given amount of glycerate 3-phosphate added. The addition of ribulose 1,5-bisphosphate reinitiated NADPH oxidation but ATP or NADPH did not. Incubation of purified ribulose-1,5-bisphosphate carboxylase with carboxyarabinitolbisphosphate completely inhibited the catalytic activity of the enzyme and decreased inhibition of glycerate-3-phosphate reduction. Two binding sites with different affinities for glycerate 3-phosphate were observed with pure ribulose-1,5-bisphosphate carboxylase.