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

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 M r of 560000 and was of apparent subunit composition 8G6R. R represents a subunit of M r 42000 conferring phosphoribulokinase activity and G a subunit of 39000 responsible for the glyceraldehyde‐3‐phosphate dehydrogenase activity. On SDS‐PAGE the M r ‐42000 subunit comigrates with the subunit of the active form of phosphoribulokinase whereas that of M r ‐39000 corresponds to that of NADPH‐dependent glyceraldehyde‐3‐phosphate dehydrogenase. The multimeric enzyme had a S 20,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.