MOLECULAR-CLONING AND BIOCHEMICAL-CHARACTERIZATION OF BOVINE SPLEEN MYRISTOYL COA-PROTEIN N-MYRISTOYLTRANSFERASE

Myristoyl-CoA:protein N-myristoyltransferase (NMT) is an essential euk aryotic enzyme that catalyzes the cotranslational transfer of myristat e to the NH2-terminal glycine residue of a number of important protein s of diverse function. We have isolated full-length cDNA encoding bovi ne spleen NMT (sNMT). The single long open reading frame of 1248 bp of sNMT specifies a protein of 416 amino acids with a predicted mass of 46,686 Da. The protein coding sequence was expressed in Escherichia co li resulting in the production of functionally active 50-kDa NMT. Dele tion mutagenesis showed that the C-terminus is essential for activity whereas up to 52 amino acids can be deleted from the N-terminus withou t affecting the function. One of the N-terminal deletions resulted in threefold higher NMT activity. Genomic Southern analysis indicated the presence of two strong hybridizing bands with three different restric tion enzyme digests suggesting the possibility of two copies of the NM T gene in the bovine genome. RNA blot hybridization analysis of total cellular RNA prepared from bovine brain, heart, spleen, lung, liver, k idney, and skeletal muscle probed with bovine sNMT cDNA revealed a sin gle 1.7-kb mRNA. Western blot analysis of various bovine tissues with human NMT peptide antibody indicated a common prominent immunoreactive band with an apparent molecular mass of 48.5-50 kDa in all tissues. A dditional immunoreactive bands were observed in brain (84 and 50 kDa), lung (58 kDa), and skeletal muscle (58 kDa). Activity measurements de monstrated that brain contained the highest NMT activity followed by s pleen, lung, kidney, heart, skeletal muscle, pancreas, and liver. It a ppears therefore that mRNA and protein expression do not correlate wit h MMT activity, suggesting the presence of regulators of the enzyme ac tivity. (C) 1997 Academic Press.