Properties of a multimeric protein complex from chloroplasts possessing potential activities of NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase
Article first published online: 3 MAR 2005
European Journal of Biochemistry
Volume 162, Issue 2, pages 423–431, January 1987
How to Cite
NICHOLSON, S., EASTERBY, J. S. and POWLS, R. (1987), Properties of a multimeric protein complex from chloroplasts possessing potential activities of NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase. European Journal of Biochemistry, 162: 423–431. doi: 10.1111/j.1432-1033.1987.tb10619.x
- Issue published online: 3 MAR 2005
- Article first published online: 3 MAR 2005
- (Received August 4/October 8, 1986) – EJB 86 0841
A homogeneous multimeric protein isolated from the green alga, Scenedesmus obliquus, has both latent phosphoribulokinase activity and glyceraldehyde-3-phosphate dehydrogenase activity. The glyceraldehyde-3-phosphate dehydrogenase was active with both NADPH and NADH, but predominantly with NADH. Incubation with 20 mM dithiothreitol and 1 mM NADPH promoted the coactivation of phosphoribulokinase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase, accompanied by a decrease in the glyceraldehyde-3-phosphate dehydrogenase activity linked to NADH. The multimeric enzyme had a Mr of 560000 and was of apparent subunit composition 8G6R. R represents a subunit of Mr 42000 conferring phosphoribulokinase activity and G a subunit of 39000 responsible for the glyceraldehyde-3-phosphate dehydrogenase activity. On SDS-PAGE the Mr-42000 subunit comigrates with the subunit of the active form of phosphoribulokinase whereas that of Mr-39000 corresponds to that of NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase.
The multimeric enzyme had a S20,W of 14.2 S. Following activation with dithiothreitol and NADPH, sedimenting boundaries of 7.4 S and 4.4 S were formed due to the depolymerization of the multimeric protein to NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (4G) and active phosphoribulokinase (2R). It has been possible to isolate these two enzymes from the activated preparation by DEAE-cellulose chromatography.
Prolonged activation of the multimeric protein by dithiothreitol in the absence of nucleotide produced a single sedimenting boundary of 4.6 S, representing a mixture of the active form of phosphoribulokinase and an inactive dimeric form of glyceraldehyde-3-phosphate dehydrogenase.
Algal thioredoxin, in the presence of 1 mM dithiothreitol and 1 mM NADPH, stimulated the depolymerization of the multimeric protein with resulting coactivation of phosphoribulokinase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase. Light-induced depolymerization of the multimeric protein, mediated by reduced thioredoxin, is postulated as the mechanism of light activation in vivo. Consistent with such a postulate is the presence of high concentrations of the active forms of phosphoribulokinase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase in extracts from photoheterotrophically grown algae. By contrast, in extracts from the dark-grown algae the multimeric enzyme predominates.