A Labile C02-Fixing Enzyme Complex in Spinach Chloroplasts

Activity and activation of ribulose 5-phosphate kinase, ribulose diphosphate carboxylase, phosphoglycerate kinase, and the NADPand NAD-dependent glyceraldehyde phosphate dehydrogenases from isolated chloroplasts were traced in tbe course of differential ultracentrifugation. Activation of some of these enzymes was carried out by incubation with NADPH2 or ATP. Activity of ribulose diphosphate carboxylase could be increased 2,4-fold by incubation with NADPH2 while phosphoglycerate kinase could not be activated. The degree of activation of the NADP-specific glyceraldehyde phosphate dehydrogenase and ribulose phosphate kinase was correlated witb the velocity of sedimentation. Enzymes, which could be activated manifold, sedimented more quickly. Two fractions could be obtained after ultracentri fugation: the sedimented fraction could be highly activated while the fraction in the supernatant could not. It was shown by gel chromatography with Sephadex for the NADP-specific glyceraldehyde phosphate dehydrogenase and ribulose phosphate kinase that the enzyme fractions which could be greatly activated had molecular weights of at least 400 000. In contrast, the enzyme fraction which could not be activated had a molecular weight of 50 000 for the ribulose phosphate kinase and of 240 000 for the glyceraldehyde phosphate dehydrogenases. Results are discussed in the way that a reversible dissociation of a labile enzyme complex takes place between ribulose phosphate kinase, glyceraldehyde phosphate dehydrogenases, and ribulose diphosphate carboxylase. Enzyme activity changes with the state of aggregation. The dissociated enzymes have higher activity than the complexed ones. Dissociation can be performed by ATP and NADPH2, which are assumed to be the physiological regulators, and by unphysiological means as dilution and high salt concentrations.