Activation of soluble guanylate cyclase by nitric oxide, nitroso compounds, NO-heme complexes and S-nitrosothiols was studied during three phases of enzyme purification. Comparisons between unpurified hepatic and coronary arterial enzymes, a partially purified hepatic enzyme (100-400 fold purified), and a highly purified apparently homogeneous hepatic enzyme (16,000 - 19,000 fold purified) revealed several of the intricacies involved in the activation of soluble guanylate cyclase. The partially purified and highly purified enzymes were deficient in heme. Reconstitution of heme-deficient soluble guanylate cyclase with heme or the direct addition of low concentrations (1 (mu)m) of hematin to enzyme reaction mixtures markedly enhanced soluble guanylate cyclase activation by nitric oxide, nitroso compounds and S-nitrosothiols. High concentrations (> 5 (mu)m) of hematin or hemoproteins inhibited the activation of soluble guanylate by these same agents but basal activity was unaltered. NO-heme and protoporphyrin IX were capable of activating heme-deficient soluble guanylate cyclase in the absence of added heme. Thiols enhanced the activation of soluble guanylate cyclase and partially reversed the hemoprotein inhibition of enzyme activation. Activation of soluble guanylate cyclase by nitric oxide and nitroso agents was much greater when Mg('2+), rather than Mn('2+), was employed as the required divalent cation. However, basal activity was greater when Mn('2+) was used. An activation enhancing factor was discovered and partially purified from rat liver. The activation enhancing factor was heat stable (75(DEGREES) for 15 min), did not contain heme and did not appear to be a thiol. The activation enhancing factor did not affect basal activity, however, activation by nitric oxide, nitroso compounds, NO-heme complexes, S-nitrosothiols and protoporphyrin IX was markedly enhanced. Furthermore, the activation enhancing factor has been the only agent shown, to date, that enhances the activation of soluble guanylate cyclase by protoporphyrin IX. The data in this dissertation support the view that soluble guanylate cyclase possesses sulfhydryl groups required for catalysis and that heme and/or other unknown factors are required for the full expression of enzyme activation by nitric oxide and nitroso compounds. In addition, NO-heme or protoporphyrin IX may be the ultimate factors involved in the mechanism of activation of soluble guanylate cyclase
