Molecular properties of chloroplastic thioredoxin f and the photoregulation of the activity of fructose 1,6-bisphosphatase.

Abstract

Thioredoxin f has been isolated and purified to homogeneity from spinach chloroplasts. The protein is a dimmer which dissociates in two apparently identical halves when ionic strength is raised. The dimmer has a molecular weight of 16000. In its oxidized state, chloroplastic thioredoxin f has no sulfhydryl group directly available, even after denaturation of the protein. When reduced by dithiothreitol, four -SH groups become available on the dimer: Taken together, these results show that there exists one disulfide bridge per monomer. Reduction by dithiothreitol results in the breaking of two disulfide bridges and the appearance of four sulfhydryl groups. Monomeric thioredoxin f does not result in a significant activation of fructose 1,6-bisphosphatase. Full activation is obtained in presence of the dimeric protein. This is understandable since activation of fructose bisphosphatase necessitates reduction of two disulfide bridges of the enzyme. Full activation of fructose bisphosphatase is obtained in the presence of either an excess of dimeric thioredoxin f, or in the presence of dithiothreitol, Moreover, a large excess of oxidized thioredoxin deactivates fructose bisphosphatase, These results suggest that there exists an equilibrium where rTH and oTh stand for reduced and oxidized thioredoxin, oFruP2ase and rFruP2ase for oxidized and reduced fructose bisphosphatase. Moreover, this equilibrium must be strongly shifted towards the left. NADPH in presence of NADP+-reductase may reduce thioredoxin, and conversely reduced thioredoxin may reduce NADP+if the reductase is present. These results together with the existence of the above equilibrium provides a tentative scheme of fructose bisphosphatase inactivation in vivo in the dark. When the light has been turned off, electrons are transferred from thioredoxin to NADP+via NADP+-reductase. Thioredoxin becomes oxidized and then deactivates fructose bisphosphatase.