Soluble guanylate cyclase activation by nitric oxide and its reversal. Involvement of sulfhydryl group oxidation and reduction.
Pre-incubation of either crude or purified nitric oxide-stimulated soluble lung guanylate cyclase resulted in a temperature-dependent decay of enzyme activity. The decay of nitric oxide-stimulated activity during pre-incubation was associated with a reduced responsiveness of the enzyme to reactivation by a second exposure to nitric oxide. This loss of enzyme responsiveness to reactivation by nitric oxide was greater with purified guanylate cyclase than with the crude enzyme and was highly dependent upon the nitric oxide dose. The addition of dithiothreitol or other thiols to nitric oxide-stimulated enzyme markedly accelerated the decay of activity in a dose-dependent manner. In addition, thiols prevented the loss of responsiveness of guanylate cyclase to reactivation by nitric oxide. Nitric oxide-stimulated enzyme activity was, therefore, reversed by the addition of thiol reducing agents. The addition of the thiol oxidizing agents, diamide or oxidized glutathione, to nitric oxide-stimulated guanylate cyclase caused a rapid and irreversible loss of activity. The effects of diamide or oxidized glutathione on the crude enzyme were prevented by excess dithiothreitol. Dithiothreitol did not prevent the destruction of purified nitric oxide-stimulated guanylate cyclase activity by diamide or oxidized glutathione, however. The results suggest that nitric oxide activation and its reversal are linked to the reversible oxidation and reduction, respectively, of sulfhydryl groups on guanylate cyclase which are involved in enzyme activation. The results further suggest the existence of a second class of sulfhydryl groups involved in the maintenance of enzyme activity.
D000818:Animals; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006162:Guanylate Cyclase / Q000378:metabolism*; D008168:Lung / Q000201:enzymology*; D008297:Male; D009569:Nitric Oxide / Q000494:pharmacology*; D010084:Oxidation-Reduction / Q000187:drug effects; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D013438:Sulfhydryl Compounds; D009569:Nitric Oxide; D006162:Guanylate Cyclase; D004229:Dithiothreitol