Purification and partial sequencing of myristoyl-CoA:protein N-myristoyltransferase from bovine brain.
The enzyme myristoyl-CoA:protein N-myristoyltransferase (NMT; EC 2.3.1.97) catalyses the transfer of myristic acid to the N-terminal glycine residue of cell and viral proteins. In this report the purification and partial sequencing of this enzyme from bovine brain is described. Using a combination of ammonium sulphate precipitation, chromatography on DEAE-Sepharose and affinity chromatography on CoA-agarose the enzyme was purified some 40-fold. Size-exclusion chromatography of this material in the presence of myristoyl-CoA yielded two peaks of enzyme activity with apparent molecular masses of 66 kDa and 43 kDa. Chromatography of the CoA-affinity-purified material on MONO-S followed by size-exclusion chromatography in the presence of myristoyl-CoA resulted in the isolation of the large form of the enzyme purified 3000-fold. Analysis by SDS/PAGE of this material showed a major 60 kDa silver-stained band. Similar analysis of the 43 kDa enzyme fraction from the same separation showed that this fraction contained several proteins including a major component with an apparent molecular mass of 49 kDa. Attempts at N-terminal sequencing of the 66 kDa form of the enzyme were unsuccessful and therefore this material was digested with trypsin and the resulting peptides separated by reverse-phase h.p.l.c. N-terminal protein sequencing of these peptides yielded sequences which show sequence similarity to those of yeast N-myristoyl-transferase.
McIlhinney
R A
RA
M.R.C. Anatomical Neuropharmacology Unit, Oxford, U.K.
McGlone
K
K
Willis
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AC
D000217:Acyltransferases / Q000737:chemistry / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D017386:Sequence Homology, Amino Acid
D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase