II. Lokalisation von Enzymen des reduktiven und dem oxydativen Pentosephosphat-Zyklus in den Chloroplasten und Permeabilität der Chloroplasten-Membran gegenüber Metaboliten

Abstract

1. The interrelationship of metabolic activities in chloroplasts and cytoplasm of leaf cells of spinach, sugar beet and Elodea has been investigated. Different methods have been adopted to study the intracellular localization of enzymes and the flow of phosphorylated intermediates across the chloroplast membrane. The flow of substrates was investigated by determining the rates of the conversion of substrates added to aqueously isolated chloroplasts, prior to and after destruction of the outer chloroplast membrane. The observed differences yielded information as to whether a substrate could traverse the chloroplast membrane.Two methods mere used to investigate the localization of enzymes :a) The percentage distribution of photosynthetic and respiratory enzymes in chloroplasts and cytoplasm was calculated from data on enzyme activities in non-aqueous cell fractions.b) Low levels of enzymes in chloroplasts in the presence of high cytoplasmatic levels were detected by assaying enzyme activities in preparations of aqueously isolated chloroplasts prior to and after ultrasonic destruction of the outer chloroplast membrane.2. If chloroplasts are isolated in aqueous sucrose buffer, their outer membranes act as an efficient barrier against the penetration of NADP, RuDP, GAP and, in some but not all experiments, of FMP and GMP. PGA, DHAP and, probably to a lesser extent, aspartate, ɑ-ketoglutarate, oxaloacetate and FDP can traverse this membrane. Chloroplast membranes are significantly altered when isolated in NaCI-buffer systems and do not correspond to the in vivo situation.3. The conversion of Ri-5-P to RuDP occurs exclusively or nearly exclusively in the chloroplasts indicating that phosphoribulokinase and/or ribosephosphate isomerase are located only there.4. The conversion of Ri-5-P to GAP and SuMP, which is catalyzed by the enzymes ribosephosphate isomerase, xylulosephosphate epimerase and transketolase, proceeds likewise only or at least predominantly in the chloroplasts and not, or only to a small extent, in the cytoplasm.5. The major parts of glucose-6-phosphate dehydrogenase and of 6-phosphogluconate dehydrogenase reside in the cytoplasm. However, a small, but significant, level of these enzymes is to be found also in the chloroplasts. Hexokinase and transaldolase are also present there. Pyruvate kinase and phosphofructokinase appear to be absent from chloroplasts.6. Since, with the presence of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, hexokinase, transaldolase and enzymes of the Calvin cycle, the enzymic machinery of the oxidative pentose phosphate pathway is complete in the chloroplasts, the results suggest that chloroplasts are engaged in the oxidative decomposition of carbohydrates.7. In the dark the oxidative pentose phosphate pathway requires the control of NADPH formation and the transfer of hydrogen across the chloroplast membrane.8. The available data on the intracellular localization of enzymes and on the kinetics of the distribution of labelled intermediates show that the photosynthetic carbon cycle operates exclusively within the chloroplasts. There is nothing to suggest that enzymes of chloroplasts and cytoplasm cooperate in the cyclic regeneration of the carbon acceptor molecule. However, the existence of phosphorylated transport metabolites suggests that secondary reactions of photosynthesis such as sucrose and amino acid synthesis, which proceed, at least in part, outside the chloroplasts, are directly linked with chloroplastic reactions by activated (phosphorylated) intermediates.

Regulation of glucose-6-phosphate dehydrogenase in spinach chloroplasts by ribulose 1,5-diphosphate and NADPH/NADP+ ratios

“…1.1.1.43) are present in spinach chloroplasts [13], three assays were performed to show that the presence of 6-phosphogluconate dehydrogenase does not interfere with assaying glucose-6-phosphate dehydrogenase. One assay contained 1.0 mM glucose-6-phosphate and 1.0 mM 6-phosphogluconate, the other two contained all ingredients plus glucose-6-phosphate or 6-phosphogluconate, respectively.…”

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

“…Through cell fractionation, G6PDH and 6PGDH activities were detected in both chloroplasts and cytosol (Garnier-Dardart, 1965;Heber et al, 1967;Slack and Hatch, 1967;Latzko and Gibbs, 1968;Mukerji and Ting, 1968;Miflin and Beevers, 1974 , 1973;Simcox and Dennis, 1978;Nishimura and Beevers, 1981), it was widely assumed thereafter, yet never clearly demonstrated, that a11 other activities of the pathway also occur as such isoenzymes and are compartmentalized in the same manner.…”

“…Although many cell fractionation studies (Heber et al, 1967;Emes and Fowler, 1978;Feierabend and Gringel, 1983) indicated that most of the activities in the OPPP core segment may be confined to plastids, one notable report assumed that about 70% of these activities were cytosolic (Latzko and Gibbs, 1968) in spinach leaves. Attempts to distinguish plastid-and cytosol-specific isoenzymes of Rib-5-P isomerase and transketolase from various tissues (Rutner, 1970;Kawashima and Tanabe, 1976;Feierabend and Gringel, 1983) were mostly unsuccessful, and it was recognized (Feierabend and Gringel, 1983) that the cyclic mode of the cytosolic OPPP would be problematic in the absence of corresponding isoenzymes.…”

Development and Intracellular Distribution of Enzymes of the Oxidative Pentose Phosphate Cycle in Radish Cotyledons

“…When enzyme activities are present in both cell compartments, they are due to isoenzymes, one located in the chloroplasts and the other in the cytosol (1, 2, 4). This distribution also applies to the two key enzymes of the oxidative pentose phosphate cycle, glucose-6-P dehydrogenase and 6-P-gluconate dehydrogenase (11,25,26).…”

“…When enzyme activities are present in both cell compartments, they are due to isoenzymes, one located in the chloroplasts and the other in the cytosol (1, 2, 4). This distribution also applies to the two key enzymes of the oxidative pentose phosphate cycle, glucose-6-P dehydrogenase and 6-P-gluconate dehydrogenase (11,25,26).At present only little evidence is available about the intra-'This investigation was supported by the Deutsche Forschungsgemeinschaft. cellular distribution of enzymes in etiolated plant tissues, since most work has been performed using green leaves.…”