Guanylate Cyclase From Bovine Lung

A kinetic characterization of the regulation of purified soluble guanylate cyclase from bovine lung by protoporphyrin M and hematin is reported. Purified guanylate cyclase was isolated with heme and had specific activities @mol of cGMP/min/mg of protein) of 0.1-0.2 and 0.3-0.6 in the presence of excess MgGTP and MnGTP, respectively, in the absence of added activators. Protoporphyrin IX, nitric oxide (NO), and NOheme increased the V,, up to 40-fold and decreased the K,,, for GTP (from 100 to 45-55 p ~ ) in the presence of excess Mg’. However, in the presence of excess Mn2+ the V,, was increased only slightly and the K, for GTP was unchanged. Protoporphyrin IX resembled NO and NO-heme also in lowering the K, and Ki for uncomplexed metal. This close similarity in the interactions of these activators with guanylate cyclase suggests that a common form of activated enzyme is generated. Hematin, in excess of 1.5 p ~ , inhibited guanylate cyclase activity. Smaller concentrations of hematin competitively inhibited protoporphyrin IX (KI = 0.35 p ~ ) , suggesting that both porphyrins compete for a common binding site on guanylate cyclase. The apparent K, for protoporphyrin IX (8-38 n ~ ) varied directly as a function of the guanylate cyclase concentration under the assay conditions employed. The equilibrium dissociation constant of the guanylate cyclase-protoporphyrin IX complex was estimated by Scatchard analysis to be 1.4 n ~ . The stoichiometry of binding was estimated to be 0.92 mol/mol of holoenzyme. Cyanide and certain oxidants inhibited guanylate cyclase activation by NO, NO-heme, and nitroso compounds without affecting activation by protoporphyrin IX or unactivated enzyme. These observations suggest that protoporphyrin IX, NO-heme, and perhaps other activators regulate guanylate cyclase by similar mechanisms. Moreover, protoporphyrin IX and heme may be important biological regulators of guanylate cyclase activity.