Co-existence of two regulatory NADP-glyceraldehyde 3-P dehydrogenase complexes in higher plant chloroplasts

Autor(en): Scheibe, R 
Wedel, N
Vetter, S
Emmerlich, V
Sauermann, SM
Stichwörter: ACTIVATION; B-SUBUNIT; Biochemistry & Molecular Biology; CALVIN CYCLE ACTIVITY; enzyme aggregation; ENZYMES; FRUCTOSE-1,6-BISPHOSPHATASE; LIGHT REGULATION; light/dark regulation; NADP-glyceraldehyde 3-P dehydrogenase; PHOSPHORIBULOKINASE; RIBULOSE-5-PHOSPHATE KINASE; SITE; spinach chloroplast; SPINACH-CHLOROPLASTS
Erscheinungsdatum: 2002
Herausgeber: BLACKWELL PUBLISHING LTD
Journal: EUROPEAN JOURNAL OF BIOCHEMISTRY
Volumen: 269
Ausgabe: 22
Startseite: 5617
Seitenende: 5624
Zusammenfassung: 
Light/dark modulation of the higher plant Calvin-cycle enzymes phosphoribulokinase (PRK) and NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (NADP-GAPDH-A(2)B(2)) 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 A(8)B(8) 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 A(8)B(8) complex immediately upon expression in E. coli.
ISSN: 00142956
DOI: 10.1046/j.1432-1033.2002.03269.x

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