Cyclic GMP concentrations can be markedly increased (up to 400-fold) by tobacco smoke and many known chemical carcinogens, by various vasodilators such as nitroprusside, and by hormones and neurotransmitters, but the mechanism(s) of guanylate cyclase regulation and the fuction of cyclic GMP have not been established. We have purified to apparent homogeneity, both the soluble and particulate forms of the enzyme, and now intend to study the mechanisms of regulation using these pure enzymes. We intend to produce monoclonal antibodies to both the particulate and soluble forms of guanylate cyclase and to characterize such antibodies with respect to affinity, specificity and sub-type. Monoclonal antibody to the particulate enzyme from sea urchin spermatozoa will be used in attempts to purify the particulate enzyme from mammalian letina. Radioimmunoassays for both forms of the enzyme will be developed, and the amounts of both forms will be traced in cultured cells under various growth conditions, as well as in regenerating liver. An epidermal growth factor-stimulated phosphorylation of the particulate form of guanylate cyclase will be examined in detail, with special emphasis on the chemical nature of the phosphoamino acid formed and on the effects of such phosphorylation on enzyme activity. The effects of the EGF-stimulated kinase on the soluble form of the enzyme also will be determined. The amino acid sequence of the catalytic site and of the phosphorylation site(s) of both the particulate and soluble enzymes will be examined; the phosphorylation site(s) will be compared to that of pp60v-src and of pp 60c-src. The mechanism of regulation of the soluble enzyme will be studied in detail with emphasis on the functions of the heme group on the question of whether or not copper is an integral enzyme component, and on the effect of total-SH content on enzyme activity. These experiments will examine spectral and enzyme activity data as a function of enzyme activators (NO, nitroprusside, fatty acid hydro peroxides) and potential antagonists (cyanide, azide, carbon monoxide). Reconstitution of the pure, apo-heme form of the enzyme will represent a major part of this work. This proposed research has potential direct applications to health problems as diverse as vision defects, heart disease and cancer.
