TAILIEUCHUNG - Báo cáo Y học: Co-existence of two regulatory NADP-glyceraldehyde 3-P dehydrogenase complexes in higher plant chloroplasts

Light/dark modulation of the higher plant Calvin-cycle enzymes phosphoribulokinase (PRK) and NADP-depend-ent glyceraldehyde 3-phosphate dehydrogenase (NADP-GAPDH-A2B2) involves changes of their aggregation state inaddition to redox changes of regulatory demonstrate that plants possess two different complexes containing the inactive forms (a) of NADP-GAPDH and PRK and (b) of only NADP-GAPDH, respectively, in darkened chloroplasts. | Eur. J. Biochem. 269 5617-5624 2002 FEBS 2002 doi Co-existence of two regulatory NADP-glyceraldehyde 3-P dehydrogenase complexes in higher plant chloroplasts Renate Scheibe1 Norbert Wedel2 Susanne Vetter1 Vera Emmerlich3 and Sonja-Manuela Sauermann1 1 Plant Physiology University of Osnabrueck Germany 2Planton GmbH Kiel Germany 3Plant Physiology University of Kaiserslautern Kaiserslautern Germany Light dark modulation of the higher plant Calvin-cycle enzymes phosphoribulokinase PRK and NADP-depend-ent glyceraldehyde 3-phosphate dehydrogenase NADP-GAPDH-A2B2 involves changes of their aggregation state in addition to redox changes of regulatory cysteines. Here we demonstrate that plants possess two different complexes containing the inactive forms a of NADP-GAPDH and PRK and b of only NADP-GAPDH respectively in darkened chloroplasts. While the 550-kDa PRK GAPDH CP12 complex is dissociated and activated upon reduction alone activation and dissociation of the 600-kDa A8B8 complex of NADP-GAPDH requires incubation with dithiothreitol and the effector 1 3-bisphosphoglycerate. In the light PRK is therefore completely in its activated state under all conditions even in low light while GAPDH activation in the light is characterized by a two-step mechanism with 60-70 activation under most conditions in the light and the activation of the remaining 30-40 occurring only when 1 3-bisphosphoglycerate levels are strongly increasing. In vitro studies with the purified components and coprecipitation experiments from fresh stroma using polyclonal antisera confirm the existence of these two aggregates. Isolated oxidized PRK alone does not reaggregate after it has been purified in its reduced form only in the presence of both CP12 and purified NADP-GAPDH some of the PRK reaggregates. Recombinant GapA GapB constructs form the A8B8 complex immediately upon expression in E. coli. Keywords enzyme aggregation light dark regulation NADP-glyceraldehyde 3-P .

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