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.
1. The distribution -before, during and after photosynthesis -of different phosphate esters in chloroplasts and cytoplasm of leaf cells of spinach and Elodea has been investigated. In steady state experiments intact leaves were fed, during illumination, with 14 C02 . The kinetics of the distribution of labelled phosphate esters between chloroplasts and cytoplasm were determined. In further experiments with intact leaves fluctuations in the pool sizes of phosphate esters in chloroplasts and in the cytoplasm were recorded in the dark/light and the light/dark transient. Independent fluctuations served as an indication that little or no exchange between chloroplasts and cytoplasm takes place. Concomitant fluctuations suggest rapid exchange.2. Although labelling takes place in the chloroplasts, a number of labelled phosphorylated intermediates appear rapidly in the cytoplasm during steady state photosynthesis of intact leaves in the presence of 14 C02 . This is particularly true for phosphoglyceric acid, glucose-6-phosphate, fructose-6-phosphate and fructose-l,6-diphosphate. On the other hand, labelled ribulosediphosphate, sedoheptulosediphosphate and sedoheptulosemonophosphate remain largely in the chloroplasts. This agrees with earlier work on the behaviour of phosphorylated intermediates during the induction period of photosynthesis.3. In the dark the level of the bulk of the sugar diphosphates is lower in the chloroplasts than in the cytoplasm of intact leaves. On illumination, a large accumulation occurs only in the chloroplasts. This behaviour suggests impermeability of the chloroplast membrane towards at least some of the sugar diphosphates. In contrast, concomitant large fluctuations in the levels of dihydroxyacetonephosphate and fructose-l,6-diphosphate have been observed during the transients from dark to light and vice versa in chloroplasts and cytoplasm alike indicating that at least one of these compounds functions as a transport metabolite. Changes in the concentrations of glucose-6-phosphate and fructose-6-phosphate were much smaller under the influence of light than those of other sugar phosphates.4. The results demonstrate the role of phosphorylated transport metabolites in carbon metabolism in chloroplasts and cytoplasm. Implications of these findings in relation to photosynthesis, respiration and the regulation of metabolism are discussed. In vorangegangenen Arbeiten haben wir mit Hilfe von 14 C02 und 32 P04 39 die intrazelluläre Lokalisation und die Wanderung von Zwischenprodukten der Photosynthese im Verlauf der Induktionsphase näher untersucht 2 . Dabei ergab sich, daß bei 14 C02-Gabe im Dunkeln und anschließender Belichtung einige Intermediärprodukte der Photosynthese wie RuDP**, SuDP und SuMP auch über längere Versuchszeiten (4 Min.) 14 C-markiert ausschließlich oder fast ausschließlich in den Chloroplasten vorkommen, während andere Verbindungen wie PGS, * Gegenwärtige Adresse: University of Bristol, Dept. Agr.-and Horticulture, Res. St., Long Ashton, Bristol. ** Benutzte Abkürzungen: ADP, ATP ...
Abstract. The photochemical activities of various species of unicellular algae (Anacystis nidulans, Chlorella pyrenoidosa, and Porphyridium cruentum) were studied following chemical fixation. Fixation with formaldehyde and glutaraldehyde yielded cells which retained their ability to perform photosystem I and photosystem II reactions. The photochemical efficiencies of some fixed algae are as great as those of unfixed spinach chloroplasts. Fixed algae containing accessory pigments appear to be useful models for further studies of the light reactions of photosynthesis.In 1966 Park et al. (14) reported that glutaraldehyde fixed Chlorella cells and chloroplasts isolated from glutaraldehyde fixed spinach leaves performed light dependent O2 production in 'the presence of suitajble electron acceptors. The quantum yields for photosystem II reactions of spinach chloroplasts from fixed leaves were about 25 % those of chloroplasts from unfixed leaves. At that time we fores!aw that this method might be a useful technique for studying photosystems I and II in algae containing accessory pigments. Such algal cells are generally impermeable to the components of reaction mixtures used to study photosystems I and II. We predicted that aldehyde fixation should remove this permeability barrier and circumvent many of the problems caused by cell breakage in these algae. Cell breakage is usually -accompanied by considerable loss of accessory pigment from thylakoid membranes (4,17).However in our initial efforts we failed to demonstrate Hill reaction either in glutaraldehyde fixed algae which contained accessory pigments or in spinach chloroplasts which were fixed after isolation.Both these obstacles are now removed. In this paper we report retention of both photosystems I and II
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