The co-translational amide linkage of myristic acid to an N-terminal glycine in certain tyrosine kinase onc- and retroviral gag-proteins (i.e. N-myristoylation) has been shown to play an essential role in the targeting of cytoplasmic onc- kinases and retroviral gag structural proteins to various subcellular membrane compartments. Site directed mutagenesis designed to block N-myristoylation prevents membrane binding and inhibits both cellular transformation and viral replication. We have purified N-myristoyltransferase (NMT) from cow brain approximately 7,500-fold and shown that the enzyme is made up of at least 2 different similar sized subunits and that the active enzyme appears to exist in a reversible equilibrium of multimeric complexes consisting of 1, 2, and/or 6 subunits. The HIV pl7(gag) proteins was expressed and purified from E. coli and a method for in vitro N-myristoylation was developed. The pl7(gag) was shown to be phosphorylated in vivo and in vitro by protein kinase C consistent with the presence of a single obvious consensus PKC phosphorylation site which is highly conserved in HIV gag and could represent a new target for anti-HIV drug development. A new hetero-bifunctional photo cross-linking reagent was synthesized and coupled to N-myristoylated and non-myristoylated peptides homologous to the N-terminus of p6O(src) for use in the characterization of N-myristoyl-src """"""""acceptors"""""""".
