Purification of soluble guanylate cyclase from rat liver

Abstract

Soluble guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] was purified from rat liver and exhibited a single protein band on polyacrylamide gels coincident with activity and indicative of MW of 150,000. The apparent specific activity of the purified enzyme was 276 nmol of cyclic[c]GMP formed/mg per min with Mn2+ as the cation of cofactor and 23.8 nmol of cGMP formed/mg per min with Mg2+. This represented 9200-fold and 7400-fold purifications of Mn2+ and Mg2+ activities, respectively. The specific activity of soluble guanylate cyclase was not constant with protein concentration. At all stages of purification, increasing the enzyme concentration in the guanylate cyclase assay increased the apparent specific activity of the preparation. The purified enzyme could be activiated by nitroprusside, nitric oxide, arachidonate, linoleate, oleate and superoxide dismutase. The degree of activation was dependent upon the concentration of enzyme protein assayed.