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  • 1
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Rapid changes of enzyme activity are obtained by post-translational modification of cysteine residues of some chloroplast enzymes. Individual fine-tuning is achieved by specific factors acting upon the redox cycle. In order to study the regulatory properties of these enzymes, they are purified from leaves or in a recombinant form from Escherichia coli. The various factors acting upon the enzyme in vivo can be simulated in vitro. However, in these studies, some subtle technical problems can be encountered. Two cases are presented in this article, and an attempt is made to explain some previous, seemingly contradictory results. The Calvin-cycle enzyme glyceraldehyde 3-phosphate dehydrogenase in its less active A8B8 form can be dissociated and thereby activated in vitro simply by diluting out the protein. On the other hand, activation requires the presence of reduced thioredoxin (Td) and an increase in ionic strength when performed at a high protein concentration, as present in vivo. Chloroplast fructose-1,6-bisphosphatase (FBPase) is purified from E. coli as an enzyme similar to that purified from leaves. However, using the standard protocol for lysis of the bacteria leads to a form with some unusual properties as changed isoelectric point, lack of Ca2+/fructose-1,6-bisphosphate (FBP) dependency of reductive activation, and lack of activity at high pH and high Mg2+ concentration. These observations are used in order to better understand the characteristics of the activation/inactivation process.
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  • 2
    ISSN: 1432-2048
    Keywords: Key words:Capsicum (fruit plastids) ; Dark metabolism ; Glyceraldehyde 3-phosphate dehydrogenase ; Malate dehydrogenase ; Malate valve ; Spinacia (chloroplasts)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract. Chloroplasts isolated from spinach (Spinacia oleracea L.) leaves and green sweet-pepper (Capsicum annuum L. var. grossum (L.) Sendt.) fruits contain NADP-dependent malate dehydrogenase (MDH; EC 1.1.1.82) and the bispecific NAD(P)-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; EC 1.2.1.13). The NADP-dependent MDH and GAPDH are activated in the light, and inactive in the dark. We found that chloroplasts possess additional NAD-dependent MDH activity which is, like the NAD-dependent GAPDH activity, not influenced by light. In heterotrophic chromoplasts from red sweet-pepper fruits, the NADP-dependent MDH and the NAD(P)-GAPDH isoenzymes disappear during the developmental transition and only NAD-specific isoforms are found. Spinach chloroplasts contain both NAD/H and NADP/H at significant concentrations. Measurements of the pyridine dinucleotide redox states, performed under dark and various light conditions, indicate that NAD(H) is not involved in electron flow in the light. To analyze the contribution of NAD(H)-dependent reactions during dark metabolism, plastids from spinach leaves or green and red sweet-pepper fruits were incubated with dihydroxyacetone phosphate (DHAP). Exogenously added DHAP was oxidized into 3-phosphoglycerate by all types of plastids only in the presence of oxaloacetate, but not with nitrite or in the absence of added electron acceptors. We conclude that the NAD-dependent activity of GAPDH is essential in the dark to produce the ATP required for starch metabolism; excess electrons produced during triose-phosphate oxidation can selectively be used by NAD-MDH to form malate. Thus NADPH produced independently in the oxidative pentose-phosphate pathway will remain available for reductive processes inside the plastids.
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  • 3
    ISSN: 1432-2048
    Keywords: Key words: Chloroplast ; Malate valve ; NADP-malate dehydrogenase ; Solanum ; Transgenic potato
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract. The contribution of the malate valve in the regulation of steady-state photosynthesis was studied in transgenic potato (Solanum tuberosum L. cv Désirée) plants with altered expression of plastidic NADP-dependent malate dehydrogenase (NADP-MDH; EC 1.1.1.82). Mutant plants were obtained after transformation with the homologous Nmdh gene in antisense orientation, or with the Nmdh gene from pea (Pisum sativum L.) in sense orientation. A total number of nine stable sense and antisense lines with 10% or 30%, and 400% of wild-type NADP-MDH capacity were selected. Intact chloroplasts were isolated from leaves of wild-type and mutant plants. In chloroplasts from sense transformants the increased enzyme amount was activated as in wild-type chloroplasts, but increased rates of oxaloacetate-dependent malate formation were only measured upon partial uncoupling. In contrast, chloroplasts from antisense transformants produced only little malate upon oxaloacetate addition. Measurements with intact leaves during steady-state photosynthesis yielded no differences in gas-exchange parameters and chlorophyll fluorescence. The leaf malate content was unchanged in NADP-MDH underexpressors, but twice as high in overexpressing plants. The altered NADP-MDH expression clearly influences the redox state of ferredoxin, especially in low light. Furthermore, the malate valve can successfully compete for electrons with cyclic electron flow, but the conditions under which this occurs are quite artificial.
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  • 4
    ISSN: 1573-5079
    Keywords: CO2 assimilation ; cyclic electron flow ; malate valve ; nitrite reduction ; poising mechanisms ; redox state
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract For maximal rates of CO2 assimilation in isolated intact spinach chloroplasts the generation of the adequate NADPH/ATP ratio is achieved either by cyclic electron flow around photosystem I or by linear electron transport to oxaloacetate, nitrite or oxygen (Mehler-reaction). The interrelationships between these poising mechanisms turn out to be strictly hierarchical. In the presence of antimycin A, an inhibitor of ferredoxin-dependent cyclic electron transport, the reduction of both, oxaloacetate and nitrite, but not that of oxygen restores CO2 fixation. When oxaloacetate and nitrite are added at low concentrations simultaneously during steady-state CO2 fixation, the reduction of nitrite is clearly preferred over the reduction of oxaloacetate, but CO2 fixation is not influenced. Nitrite reduction is not decreased upon addition of oxaloacetate, but vice versa. This is due to the regulation of NADP-malate dehydrogenase activation by electron pressure via the ferredoxin/thioredoxin system on the one hand, and by the NADPH/(NADP+NADPH) ratio (anabolic reduction charge, ARC) on the other hand. Thus the closing of the ‘malate valve’ prevents drainage of reducing equivalents from the chloroplast (1) when a low ARC indicates a high demand for NADPH in the stroma and (2) when nitrite reduction reduces the electron pressure at ferredoxin. The ‘malate valve’ is opened when cyclic electron transport is inhibited by antimycin A. Under these conditions the rate of malate formation is higher than in the absence of the inhibitor even in the presence of oxaloacetate, thus indicating that the regulation of the ‘malate valve’ functions at various redox states of the acceptor side of Photosystem I.
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  • 5
    ISSN: 1573-5079
    Keywords: CO2 assimilation ; metabolic control analysis ; photosynthesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In order to explain the mechanisms of Calvin-cycle regulation, the general properties of metabolic systems under homeostatic flux control are analyzed. It is shown that the main characteristic point for an enzyme in such a system can be the value of a sharp transition from some constant homeostatic flux to a limitation by this enzyme. A special method for the quantitative treatment of the experimental dependence of a metabolic flux such as photosynthesis on enzyme content is developed. It is pointed out that reactions close to a thermodynamic equilibrium under normal conditions can considerably limit the homeostatic fluxes with a decrease of the enzyme content. Calvin-cycle enzymes are classified as non-limiting, near-limiting and limiting. The deduced rules for the regulation of a homeostatic metabolic pathway are used to explain the data obtained for transgenic plants with reduced activities of Calvin-cycle enzymes. The role of compensating mechanisms that maintain the photosynthesis rate constant upon the changes of enzyme contents is analyzed for the Calvin cycle. The developed analysis explains the sharp transitions between limiting and non-limiting conditions that can be seen in transgenic plants with reduced content of some Calvin-cycle enzymes, and the limiting role of such reversible enzymes as aldolase, transketolase and others. The attempt is made to predict the properties of plants with increased enzyme contents in the Calvin cycle.
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  • 6
    ISSN: 1573-5079
    Keywords: antimycin A ; chloroplasts ; hydrogen peroxide ; malate valve ; redox state
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Electron fluxes in isolated intact spinach chloroplasts were analyzed under saturating light and under optimal CO2 and Pi supply. When CO2 assimilation was the only ATP- and NADPH-consuming reaction, the ΔpH decreased and the chloroplasts showed clear evidence of over-reduction. This suggested that additional electron flow is required in order to maintain the ΔpH and the stromal NADPH/ATP ratio. The additional electron flow may be cyclic electron transport around Photosystem I and linear electron transport towards either oxaloacetate or O2. The contributions of, and the interrelationships between, these three electron transfer pathways were analyzed by following the reactions of chloroplasts in their presence or absence, and by monitoring to what extent they were able to compensate for each other. Inhibition of cyclic electron flow by antimycin A caused strong over-reduction and decreased the ΔpH. Only oxaloacetate, but not O2, was able to restore photosynthesis. In the presence of H2O2, there was a rapid build-up of a high ΔpH, and the reduction of any other electron acceptor was prevented. It is concluded that the different electron acceptors in the stroma are organized in a hierarchical manner; this allows electron flux towards CO2 and nitrite reduction to proceed without any competition for electrons, and any excess electrons to be taken by these additional non-assimilatory pathways. Hence, the ΔpH is maintained at the required level and over-reduction of the electron transport chain and the stromal redox components is avoided.
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