Properties of a multimeric protein complex from chloroplasts possessing potential activities of NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase

Abstract

A homogeneous multimeric protein isolated from the green alga, Scenedesmus ohliquus, 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 NADPHdependent 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 M , 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 M,-39 000 corresponds to that of NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase.The multimeric enzyme had a szo,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 (4 G) and active phosphoribulokinase (2 R). 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.There is now considerable evidence that the activities of several enzymes of the Calvin cycle are regulated by light [l]. The first enzyme demonstrated to be activated in vivo by light was NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase [2]. The effect of light was shown to be reversed by a subsequent period of darkness. The stimulatory effect of light was also observed in isolated spinach chloroplasts [3 -41. Incubation of the isolated chloroplast enzyme with a number of effector molecules (e.g. NADPH and ATP) stimulated the NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase activity [5 -71. The effect o...

A functional five‐enzyme complex of chloroplasts involved in the Calvin cycle

“…The results of the hydroxyapatite chromatography are reminiscent of the findings of Nicholson et al [24], which showed the existence of two fractions of phosphoribulokinase activity extracted from the green alga Scenedesmus obliquus. Table 1 shows the respective amounts of the various enzyme activities present in the fractions of the two peaks A and B.…”

CP12 provides a new mode of light regulation of Calvin cycle activity in higher plants

“…Powls and coworkers have described 560-kDa protein complexes in the chloroplast stroma of the green alga, Scenedesmus obliquus (25,26) and spinach (8), both having latent PRK and GAPDH activities. Incubation of these complexes with DTT and NADPH induced depolymerization and marked stimulation of the latent enzyme activities.…”

Regulation of Photosynthetic GAPDH Dissected by Mutants

“…Such archaic photosynthetic groups as cyanobacteria, red algae, and green microalgae contain a simple tetrameric NADP-GAPDH only constituted by A-type subunits (Figge et al, 1999). In some of these organisms including Synechocystis PCC6803 (Wedel and Soll, 1998), Chlamydomonas reinhardtii (Graciet et al, 2003), and Scenedesmus obliquus (Nicholson et al, 1987), the regulation of GAPDH appears to involve the reversible formation of a supramolecular complex between GAPDH and phosphoribulokinase (PRK), mediated by the small photosynthetic protein CP12.…”