Recombinant bovine spleen myristoyl CoA: Protein N-myristoyltransferase

Myristoyl-CoA:protein N-myristoyltransferase (NMT) is an essential eukaryot ic enzyme that catalyzes the co-translational transfer of myristate to the NH2-terminal glycine residue of a number of important proteins of diverse f unction. Recently, we have isolated full length cDNA encoding bovine spleen NMT [27] the full length cDNA was cloned and expressed in E. coli, resulti ng in the expression of functionally active 50 kDa NMT. Using the combinati on of SP-Sepharose fast flow and Mono S fast protein liquid chromatography, the enzyme was purified 20-fold with a high yield. The spleen NMT (sNMT) f usion protein exhibited an apparent molecular weight of 53 kDa on SDS-PAGE. Upon cleavage by the Enterokinase the sNMT exhibited an apparent molecular weight of 50 kDa without loss of catalytic activity. The two synthetic pep tide substrates based on the N-terminal sequence of pp60(src) (GSSKSKMR) an d cAMP dependent protein kinase (GNAAAKKRR) have different kinetic paramete rs of K-m values of 40 and 200 mu M. Recombinant sNMT was also potently inh ibited by Ni2+ (histidine binder) in a concentration dependent manner with a half maximal inhibition of 280 mu M. The E. coli expressed sNMT was homog enous and showed enzyme activity.