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 approximately 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 approximately 391 kDa. Subsequent treatment of the approximately 391 kDa activity peak with an NMT peptide reaction product (i.e. N-myristoyl-peptide) results in approximately 75% of the activity re-eluting as a approximately 126-kDa peak in 1 M NaCl. Rechromatography also yields small amounts of a approximately 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 125I-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 approximately 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 NMT 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.