title: The involvement of catalytic site thiol groups in the activation of soluble guanylate cyclase by sodium nitroprusside.
abstract: Sodium nitroprusside, a potent activator of soluble guanylate cyclase, potentiated mixed disulfide formation between cystine, a potent inhibitor of the cyclase, and enzyme purified from rat lung. Incubation of soluble guanylate cyclase with nitroprusside and [35S]cystine resulted in a twofold increase in protein-bound radioactivity compared to incubations in the absence of nitroprusside. Purified enzyme preincubated with nitroprusside and then gel filtered (activated enzyme) was activated 10- to 20-fold compared to guanylate cyclase preincubated in the absence of nitroprusside and similarly processed (nonactivated enzyme). This activation was completely reversed by subsequent incubation at 37 degrees C (activation-reversed enzyme). Incorporation of [35S]cystine into guanylate cyclase was increased twofold with activated enzyme, while no difference was observed with activation-reversed enzyme, compared to nonactivated enzyme. Cystine decreased the activity of nonactivated and activation-reversed enzyme about 40% while it completely inhibited activated guanylate cyclase. Mg+2- or Mn+2-GTP inhibited the incorporation of [35S]cystine into nonactivated or activated guanylate cyclase. Also, diamide, a potent thiol oxidant that converts juxtaposed sulfhydryls to disulfides, completely blocked incorporation of [35S]cystine into nonactivated or activated guanylate cyclase. These data indicate that activation of soluble guanylate cyclase by nitroprusside results in an increased availability of protein sulfhydryl groups for mixed disulfide formation with cystine. Protection against mixed disulfide formation with diamide or substrate suggests that these groups exist as two or more juxtaposed sulfhydryl groups at the active site or a site on the enzyme that regulates catalytic activity. Differential inhibition by mixed disulfide formation of nonactivated and activated enzyme suggests a mechanism for amplification of the on-off signal for soluble guanylate cyclase within cells.
mesh_terms: D000818:Animals; D001665:Binding Sites; D003553:Cystine / Q000378:metabolism / Q000494:pharmacology; D003958:Diamide / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005292:Ferricyanides / Q000494:pharmacology*; D006162:Guanylate Cyclase / Q000378:metabolism*; D008168:Lung / Q000201:enzymology; D009599:Nitroprusside / Q000494:pharmacology*; D051381:Rats; D012995:Solubility; D013438:Sulfhydryl Compounds / Q000378:metabolism*
chemical_list: D005292:Ferricyanides; D013438:Sulfhydryl Compounds; D003958:Diamide; D009599:Nitroprusside; D003553:Cystine; D006162:Guanylate Cyclase