Binding and activation of pea chloroplast fructose-1,6-bisphosphatase by homologous thioredoxins m and f

Fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) binds its putative physiological activator thioredoxin f(Trx f) at pH 7.9, the pH in the stroma of the illuminated chloroplast. Since Trx m, described as specific in NADP + -malate dehydrogenase (NADP-MDH) activation, appears in pea (Pisum sativum L.) also to be functional in FBPase modulation, we have here analyzed the effect of pH and the redox status of the chloroplast stroma in the pea FBPase binding of homologous Trx f and m. Both pea Trx were strongly bound by purified FBPase when they were preincubated at pH 7.9 with 2.5 mM dithiothreitol (DTT), but not when the reductant was omitted. As occurs with Trx f, the Trx m/FBPase ratio of the complex was 4, but this was only observed with a Trx m/FBPase concentration ratio > 10 in the preincubation mixture. The FBPase-Trx m binding disappeared in the presence of 100 mM NaCl, even with 2.5 mM DTT at pH 7.9, with a concomitant appearance of different aggregation states of the FBPase subunit. A similar FBPase-Trx m complex was detected in the stromal solution when pea chloroplasts were lysed at pH 7.9 in the presence of DTT. No interaction was observed between NADP-MDH and Trx f or m, either in the presence or in the absence of DTT. Pea FBPase showed sigmoidal activation kinetics with pea Trx m, and an S 0.5 of 133 nM versus 6.6 nM with pea Trx f. About 10-fold higher concentration of the former than that of the latter was required for obtaining maximum activity; however, the V max with Trx f was only 2-fold higher than that with Trx m. We conclude that pea FBPase binds and is activated by the homologous Trx m, even though to a lesser extent than with Trx f. We also deduce that in the light the conditions in the chloroplast stroma are optimal for forming an FBPase-Trx complex.