Ferredoxin-activated Fructose Diphosphatase of Spinach Chloroplasts

Abstract

Abstract The hydrolytic cleavage of Pi from fructose diphosphate by the fructose diphosphatase (FDPase) system of spinach chloroplasts was found to require (a) an alkaline FDPase component insensitive to inhibition by AMP or fructose diphosphate; (b) reduced ferredoxin; (c) a protein factor; and (d) Mg++. Reduced spinach ferredoxin could be replaced by reduced methyl viologen or dithiothreitol but not by reduced bacterial ferredoxin. Activation of the alkaline FDPase component by the nonphysiological substitutes required the protein factor. The alkaline FDPase component in isolated intact chloroplasts was also activated by light. The protein factor component of the FDPase system has been partly purified and found to have a molecular weight of 40,000. The alkaline FDPase component, purified to homogeneity, had a molecular weight of 145,000 and contained 2% carbohydrate. Except for a remarkably high half-cystine content (16% by weight as compared to 2% for mammalian FDPases) which reflects an apparent abundance of cystine, the amino acid composition of the chloroplast alkaline FDPase component resembled that of the mammalian FDPases. In the absence of reduced ferredoxin and protein factor, the chloroplast alkaline FDPase component can be activated by high concentrations of Mg++. Like liver alkaline FDPase, the chloroplast enzyme component is also activated by cystamine or 5,5'-dithiobis(2-nitrobenozic acid) (DTNB). Ferredoxin-activated FDPase was inhibited completely by 5 x 10-4 m EDTA but was insensitive to KCN, cuprizone, and diethyldithiocarbamate (all at 1 x 10-3 m). The activity of chloroplast FDPase during photosynthesis appears to be controlled photochemically through reduced ferredoxin. What, if any, physiological role can be ascribed to the activation of the enzyme by high concentrations of Mg++ and disulfide reagents, independently of reduced ferredoxin, is not clear.