Effects of thiol inhibitors on hepatic guanylate cylase activity

Several thiol blocking agents inhibit basal guanylate cyclase activity of 100 000 X g hepatic supernatant fractions and the stimulation of enzyme activity by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), NaN3, NaNO2 and nitroprusside. The relative potency of the thiol blockers as inhibitors was CdCl2 greater than p-hydroxymercuribenzoate greater than N-ethylmaleimide greater than arsenite greater than iodoacetamide. Inhibition of basal and MNNG-responsive soluble guanylate cyclase activities by arsenite was markedly potentiated by an equimolar concentration of 2,3-dimercaprol, but not by mercaptoethanol. Inhibition of soluble guanylate cyclase by either arsenite or CdCl2 was completely reversed by excess 2,3-dimercaprol. Qualitatively similar effects were observed with DE-52 cellulose purified soluble hepatic guanylate cyclase, and suggested an involvement of closely juxtaposed thiol groups in the regulation of enzyme activity. For several reasons inhibition by thiol blockers appeared to be mediated through multiple mechanisms and/or sites of interaction: (1) Concentrations of the thiol inhibitors which had no effect on basal activity strikingly inhibited the responsiveness of the enzyme to a submaximal concentration of MNNG. (2) CdCl2 abolished the action of excess MnCl2 to stimulate purified guanylate cyclase, but was a relatively ineffective inhibitor when MnCl2 and GTP were present in equimolar concentrations. By contrast, arsenite-2,3-dimercaprol was uniformly effective in inhibiting guanylate cyclase activity in the presence or absence of excess MnCl2. (3) Arsenite-2,3-dimercaprol increased the Km for MnGTP (control, 0.13 +/- 0.02 mM; 0.2 mM arsenite-2,3-dimercaprol, 0.31 +/- 0.03 mM), whereas CdCl2 had no effect on this parameter. (4) Hepatic particulate guanylate cyclase activity was significantly inhibited by arsenite 2,3-dimercaprol but not by CdCl2. Thus, the data not only indicate that vicinal dithiol groups are required for expression of basal guanylate cyclase activity and enzyme responses to agonists, but strongly suggest the involvement of more than one interacting site containing free thiol residues.