Chloroplasts possess an ATP-Pa-exchange activity, which is induced by a transition from acid to base. The reaction is dependant on the presence of Mg 2 ® and a reducing agent, DTT. The exchange reaction induced by acid-base transition has properties similar to the light-triggered ATPPa-exchange and the ATP-synthesis induced by acid-base transition.Die Austauschreaktion zwischen ATP * und anorganischem Phosphat, wie auch die Reaktion der ATP-ase, gelten nach heutigen Erkenntnissen allgemein als Teilreaktionen der mit einem Elektronentransport (Atmungskette oder photosynthetischer Elektronentransport) gekoppelten ATP-Synthese. Während ATP-ase-und die ATP -Pa-Austauschreaktion an Mitochondrien und Mitochondrienfragmenten schon vor längerer Zeit nachgewiesen werden konnten (vgl. die zusammenfassende Darstellung von RACKER 1 ), glaubte man lange, daß isolierte Chloroplasten zwar bei Belichtung zur ATP-Bildung befähigt sind, aber weder ATP-ase-Reaktion noch ATP -Pa-Austausch katalysieren können (AVRON und JAGENDORF 2 , AVRON 3 ). Vor wenigen Jahren gelang es jedoch, in Chloroplasten Enzyme nachzuweisen, die unter besonderen Bedingungen ATP spalten: eine durch Ca 2 ® und eine durch Mg 2 ® und ein Thiolreagens aktivierte ATP-ase. Erstere funktioniert nur im Licht (BENNUN und AVRON 4 ), während die durch die zweite ATP-ase katalysierte Reaktion durch eine kurze Belichtungszeit induziert wird und anschließend im Dunkeln abläuft (PETRACK und LIP-MANN 5 , HOCH und MARTIN 6 , MARCHANT und PACKER 7 ).Falls ATP-ase und ATP -Pa-Austausch-Reaktion Umkehrungen der Endstufen der Photophosphorylierung sind, sollte unter den oben genannten Bedingungen für die ATP-ase-Aktivität auch ein ATP -* Verwendete Abkürzungen: ATP = Adenosintriphosphat, Pa = anorganisches Phosphat, PMS = Phenazinmethosulfat, DTT = Dithiothreit. 1 E. RACKER, Mechanisms in bioenergetics, New York 1965. 2 M. AVRON U. A. T. JAGENDORF, J. biol. Chemistry 234, 967 [1959]. 3 M. AVRON, Biochim. biophys. Acta [Amsterdam] 40, 257 [1960]. 4 A. BENNUN U. M. AVRON, Biochim. biophys. Acta [Amsterdam] 79, 646 [1964]. 5 B.Pa-Austausch induziert werden können. Dies gelang kürzlich CARMELI und AVRON 8 und MCCARTHY und RACKER 9 . Es erwies sich auch für die Induktion dieser Austauschreaktion eine kurzzeitige Belichtung als notwendig bei gleichzeitiger Anwesenheit eines Kofaktors der Photophosphorylierung (z.B. PMS); ferner benötigte die Reaktion Mg 2 ® und DTT.In den letzen Jahren fand die chemiosmotische Hypothese der Phosphorylierung von MITCHELL (zusammenfassender Artikel siehe MITCHELL 10 ) immer stärker Beachtung, besonders seit es gelang, sie experimentell zu belegen (JAGENDORF und URIBE 11 ). In der vorliegenden Arbeit wird gezeigt, daß in der ATP-Pa-Austauschreaktion die Lichtinduktion durch den für die chemiosmotische ATP-Bildung notwendigen Säure-Base-Ubergang vollständig ersetzt werden kann. MethodenChloroplastenfragmente wurden aus Handelsspinat nach WHATLEY und ARNON 12 hergestellt; die Chlorophyllbestimmung erfolgte nach ARNON 13 . Vor der Durchführung der Austausc...
1. Spinach was harvested once a week, beginning five weeks after sowing. The leaves were divided into three categories, according to their height of insertion. For every category, Hill-activity and manganese content (by atomic absorption) were measured. The remaining leaf material was homogenised in distilled water or in a mixture of 0.6 M sucrose and 0.05 M Tris/maleate buffer, pH 7.8, and the homogenate was separated by differential centrifugation into two fractions: a green precipitate consisting mainly of chloroplast fragments and a yellow supernatant. Chloroplast fragments, isolated in sucrose medium, were washed with distilled water. Manganese and nitrogen content of the precipitate and the supernatant were determined, as was the chlorophyll concentration of the precipitate. 2. The manganese content in the old leaves and their fractions increased; nitrogen concentration, chlorophyll concentration, chlorophyll/nitrogen and chlorophyll/manganese ratios decreased. 3. The lowest inserted leaves and their fractions had a different chemical composition from the rest: they had a higher manganese and a lower nitrogen content. The chlorophyll/nitrogen ratio of the lamellae was also higher. 4. At the beginning, the lowest inserted leaves had the same manganese content as the seeds. Then the manganese concentration decreased slightly and increased again when leaf decomposition began. 5. We found a chlorophyll/manganese ratio of 104 molecules chlorophyll to 1 atom manganese. 6. Hill activity decreased with increasing age of the shoot. It seemed to be a function of the shoot age rather than the leaf age. 7. The manner of preparation had an effect on the chlorophyll/dry weight ratio, but not on the manganese/dry weight and nitrogen/dry weight ratios. Therefore the manganese/chlorophyll and the chlorophyll/nitrogen ratios depended on the manner of preparation.
Dinactin, an antibiotic forming complexes with K(+) ions, uncouples phosphorylation in chloroplasts without requiring the presence of a substance increasing the permeability of the membrane for protons. To inhibit photophosphorylation, less Dinactin is necessary in the absence than in the presence of K(+).When added before the light phase, Dinactin affects the light-triggered ATP-Pi exchange reaction in the same way as it does the complete photophosphorylation. Addition of the antibiotic after the activation by light inhibits the exchange reaction independently of the presence of K(+), possibly by blocking the energy transfer to ATP.The inhibition of the light-induced proton transport by Dinactin is more pronounced in the presence of K(+) than of Na(+) ions. The manner in which changes in the permeability of the chloroplast membrane for K(+) ions caused by Dinactin may influence photophosphorylation and reactions coupled with it is discussed.
Light induced the formation of an energy pool in chloroplast fragments which was emptied within 20-30 min in the dark, while an ATP-Pi exchange was going on.The exchange reaction was dependent on Mg(2+) and was inhibited by NH 4 (+) but not by ADP. The optimum pH in the light and in the dark stage of the reaction lay between pH 7.5 and 8.The high energy intermediate was formed only in the presence of DTT in the light stage; phosphate had no influence on the formation of the intermediate in that period, but increased its stability in the dark.The relation between both high energy intermediates, that of the light-induced ATP-Pi exchange reaction and that of the light-induced ATP synthesis, is discussed in its meaning for the hypothesis of chemiosmotic phosphorylation.
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