Redox Transfer across the Inner Chloroplast Envelope Membrane

Abstract

In leaves of spinach plants (Spinacia oleracea L.) grown in ambient CO2 the subcellular contents of adenylates, pyridine nucleotides, 3-phosphoglycerate, dihydroxyacetone phosphate, malate, glutamate, 2-oxoglutarate, and aspartate were assayed in the light and in the dark by nonaqueous fractionation technique. From the concentrations of NADP and NADPH determined in the chloroplast fraction of illuminated leaves the stromal NADPH to NADP ratio is calculated to be 0.5. For the cytosol a NADH to NAD ratio of 10-3 is calculated from the assay of the concentrations of NAD, malate, glutamate, aspartate, and 2-oxoglutarate on the assumption that the reactions catalyzed by the cytosolic glutamate oxaloacetate transaminase and malate dehydrogenase are not far away from equilibrium. For the transfer of redox equivalents from the chloroplastic NADPH to the cytosolic NAD two metabolite shuttles are operating across the inner envelope membrane: the triosephosphate-3-phosphoglycerate shuttle and the malate-oxaloacetate shuttle. Although both shuttles would have the capacity to level the redox state of the stromal and cytosolic compartment, this apparently does not occur. To gain an insight into the regulatory processes we calculated the free energy of the enzymic reactions and of the translocation steps involved.From the results it is concluded that the triosephosphate-3-phosphoglycerate shuttle is mainly controlled by the chloroplastic reaction of 3-phosphoglycerate reduction and of the cytosolic reaction of triosephosphate oxidation. The malate-oxaloacetate shuttle is found to be regulated by the chloroplastic NADP-malate dehydrogenase and also by the translocating step across the envelope membrane.The metabolism of a leaf cell is distributed between various compartments, e.g. the cytosol, chloroplast stroma, and mitochondrial matrix. Each of the metabolic compartments has its specific function and hence also its special milieu. Specific translocators catalyze the transfer of metabolites between these compartments (16 transferred from the chloroplast stroma to the cytosol by two different metabolite shuttles, the triosephosphate-3-phosphoglycerate shuttle ( 14) catalyzed by the Pi-trioseP-3-PGA3 translocator (6) and the malate-OAA shuttle (1) facilitated by specific transport of malate and OAA ( 12). As both metabolite shuttles would have the capacity to level the redox state of the stromal and cytosolic compartment, a regulation of these processes is required to maintain the specific redox states of the two metabolic compartments.To gain an insight into such regulatory processes, we attempted in the present publication to analyze the redox state of pyridine nucleotides by the measurement of their concentrations and the concentrations of substrates of pyridine nucleotide-linked reactions in subcellular compartments of spinach leaves. These measurements were carried out mainly by nonaqueous fractionation of frozen leaves carried out by Heber (13) earlier and later refined in our laboratory (8). It will be shown that ...

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