IDENTIFICATION AND CHARACTERIZATION OF MULTIPLE FORMS OF BOVINE BRAIN N-MYRISTOYLTRANSFERASE

N-Myristoyltransferase (NMT) catalyzes the co-translational addition of myristic acid to the N-terminal glycine of many cellular, viral, and fungal proteins which are essential to normal cell functioning and/or are potential therapeutic targets. We have found that bovine brain NMT exists as a heterogeneous mixture of interconvertible high molecular mass multimers involving approximate to 60-kDa NMT subunit(s). Gel filtration chromatography of partially purified NMT at low to moderate ionic strength yields NMT activity eluting as 391 +/- 52 and 126 +/- 17 kDa peaks as well as activity which profiles the protein fractions and Likely results from NMT nonspecifically associating with background proteins and/or column matrix. Chromatography in 1 M NaCl causes 100% of this activity to elute as a single peak of approximate to 391 kDa. Subsequent treatment of the approximate to 391 kDa activity peak with an NMT peptide reaction product (i.e. N-myristoyl-peptide) results in approximate to 75% of the activity re-eluting as a approximate to 126-kDa peak in 1 M NaCl. Rechromatography also yields small amounts of a approximate to 50-kDa NMT monomer which increases with prior storage at 4 degrees C. Up to 5 NMT subunits were identified by SDS-polyacrylamide gel electrophoresis and specific immunoblotting with a human NMT peptide antibody and by cofactor-dependent chemical cross-linking with an I-125-peptide substrate of NMT. The prominent 60 kDa and minor 57-, 53-, 49-, and 47-kDa NMT immunoblotted subunits co-migrate with five of nine silver-stained proteins in an enzyme preparation purified >7,000-fold with approximate to 50% yield by selective elution from octyl-agarose with the myristoyl-CoA analog, S-(2-ketopentadecyl)-CoA. Storage at 4 degrees C also leads to conversion of the larger NMT subunit(s) into 49 and 47 kDa forms with no loss of NMT activity. These results identify two interconvertible forms of NR;IT in bovine brain that result from NMT subunit multimerization and/or complex formation with other cellular proteins. The data also identify a fully active NMT monomer which arises from subunit proteolysis. This study thus reveals a previously unappreciated level of NMT complexity which may have important mechanistic and/or regulatory significance for N-myristoylation in mammalian cells.