Nitric oxide regulation of glyceraldehyde-3-phosphate dehydrogenase activity in Dictyostelium discoideum cells and lysates.

The ability of compounds releasing nitric oxide (NO) to regulate glyceraldehyde-3-phosphate dehydrogenase (GraPDH) activity was analysed both in cell homogenates and in intact Dictyostelium discoideum. The time course of GraPDH inactivation in cell lysates by NO-releasing compounds suggests that two processes may be involved, one of which accounts for the majority of the inactivation and shows a close correlation with GraPDH ADP-ribosylation. Maximal ADP-ribosylation under these conditions exhibited a stoichiometry of about 0.4 mol ADP-ribose/mol enzyme tetramer. NO-mediated inhibition of GraPDH activity was attenuated if specific substrates, cofactors, or cysteine were added to cytosol preparations. Under such conditions, ADP-ribosylation of the enzyme was correspondingly reduced or negligible. Intact cells treated with NO-releasing compounds were shown to respond by rapidly decreasing their GraPDH activity. This inhibition was transient and, after a 10-min incubation, enzyme activity returned to the level seen in control cells. The time course of these in vivo changes correlated well with those of the NO-stimulated ADP-ribosylation of GraPDH also seen in intact cells. The basis underlying the NO-stimulated inhibition of GraPDH activity was investigated and found to reflect a decreased Vmax. No changes in either the Km of the enzyme for its substrates or its state of polymerization were observed.