The modification of onc-proteins with the fatty acid myristate is an early step associated with the transformation of normal to neoplastic cells and mammalian retroviral reproduction and it is thought to be part of the mechanism by which cytoplasmic oncogene protein kinases or viral qaq structural proteins are localized to the inner plasma membrane surface. Since the transforming activity of onc-kinases and viral replication is dependent upon this membrane binding, this project will investigate the role of myristoylation as it relates to the mechanism of this subcellular localization. N-Myristoyl transferase has been partially purified from bovine brain. Several potential inhibitors of the enzyme have been synthesized and are being tested for their in vitro effects on the purified enzyme. N-Myristoyl and nonmyristoyl peptides homologous to the N-terminus of nave been used to identify high affinity protein acceptors in red cell membrane vesicles. The results suggest the existence of src-specific acceptors in the plasma membrane which might be involved in the normal cellular regulation and transforming activity of c-src and v-src. respectively. A new method for identifying N-myristoyl-proteins has been developed. The N-myristoylglycine from myristoylated proteins is released by mild acid hydrolysis, derivitized to p- nitrobenzylazlactone, and identified at pmole levels by reversed phase HPLC. This procedure has been used to identify the alpha subunit of the GTP-binding protein, Go, as a myristoylated protein. Information on the enzymology of myristoylation and the role of myristic acid in membrane binding will be used to design and synthesize specific inhibitors of myristoylation and membrane association with the goal of developing chemotherapeutic agents specific for critical early steps of tyrosine kinase mediated malignant transformation and mammalian retroviral reproduction.
