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 ...
Evidence is provided that amyloplasts from pea roots contain a translocator which transports, in a counter exchange mode, phosphate, glucose 6-phosphate, dihydroxyacetone phosphate and 3-phosphoglycerate. The translocator has a low affinity for 2-phosphoglycerate and glucose 1-phosphate. Metabolite transport was measured by silicone oil filtering centrifugation either directly by uptake of radioactive labelled compounds or indirectly by back exchange.
Extracts have been rapidly prepared from spinach leaves, and the activity of fructose-6-phosphate, 2-kinase (Fru6P,2kinase) and fructose-2,6-bisphosphatase (Fru2.6P2ase) measured. The enzyme activities do not change during light-dark transitions, but there is an increase of Fru6P,2- kinase and decrease of Fru2,6P2ase activity over several hours in the light. This increase of the Fru6P,2kinase: Fru2,6P2:ase ratio shows that a previously unrecognized mechanism, which may include protein modification, controls Fru2,6P2 levels in leaves. It operates as sucrose accumulates in the leaf, and will be involved in regulating the partitioning of photosynthate.
Ralstonia eutropha JMP 134 was continuously grown on phenol and 2,4-dichlorophenoxyacetate under nutristatic conditions at elevated stationary concentrations of 90±650 mg phenol/l and 25±100 mg 2,4-D/l, respectively, in order to study the response of the bacterial population to long-term exposure to these potentially toxic substrates. The course of the cells' response over time was observed by determining distinctive growth parameters and by the on-line measurement of¯uorescence spectra of intracellular and extracellular¯uorophores. The latter were monitored using a modi®ed¯uorescence spectrophotometer. The results of the nutristat experiments indicate that the adaptation of the culture to long-term exposure to phenol and 2,4-D exhibited dynamic characteristics of the growth pattern determined by the individual substrates and their concentration, including enforced and reduced levels of substrate conversion. This growth pattern is interpreted as an expression of superimposing cellular events in order to withstand unfavorable environmental conditions. Finally, the growth rate attained retarded levels under stationary conditions, slowing down to almost zero for example in the case of about 100 mg 2,4-D/l.The growth rate pro®le within the various phases of adaptation was well re¯ected by the¯uorescence signals. The NAD(P)H¯uorescence was almost exclusively emitted by the cellular pool of NADPH and behaved inversely to the growth rate. A similar relationship was obtained for the cellular¯uorescence of a¯avin-containing compound. Sharply reduced growth was additionally accompanied by a rapid rise of the background¯uorescence. These data indicate that¯uorescence-derived signals provide a useful re¯ection of cellular events in inhibited growth situations.
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