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

Eur J Biochem. 2002 Nov;269(22):5617-24. doi: 10.1046/j.1432-1033.2002.03269.x.

Abstract

Light/dark modulation of the higher plant Calvin-cycle enzymes phosphoribulokinase (PRK) and NADP-dependent 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blotting, Western
  • Chloroplasts / enzymology*
  • Chromatography, Gel
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / metabolism
  • Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / chemistry*
  • Kinetics
  • Light
  • Oxygen / metabolism
  • Phosphotransferases (Alcohol Group Acceptor) / chemistry*
  • Precipitin Tests
  • Recombinant Proteins / chemistry
  • Spinacia oleracea / enzymology

Substances

  • Recombinant Proteins
  • Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)
  • Phosphotransferases (Alcohol Group Acceptor)
  • phosphoribulokinase
  • Oxygen