Identification, purification and characterization of a membrane-associated N-myristoyltransferase inhibitor protein from bovine brain.
N-Myristoyl-CoA: protein N-myristoyltransferase (NMT) is the enzyme that catalyses the covalent transfer of myristic acid from myristoyl-CoA to the N-terminal glycine residue of a protein substrate. Subcellular fractionation of bovine brain indicates that NMT activity was located in both the cytosolic and the particulate fraction of the cell. Removal of the particulate fraction resulted in a 2-fold enhancement of NMT activity. Reconstitution of the particulate fraction and cytosolic fraction resulted in inhibition of the elevated cytosolic NMT activity. These results indicated the existence of putative inhibitor(s) activity of NMT located in the particulate fraction of bovine brain. The inhibitor was stable to heat and was identified as a protein, on the basis of its susceptibility to the proteases trypsin and chymotrypsin. Protease degradation first required the delipidation of the particulate fraction. The inhibitor was purified to near-homogeneity by heat treatment, solvent extraction and Sephacryl S-300 gelfiltration column chromatography. The inhibitor was purified 630-fold from the particulate fraction with a 20% yield. The protein inhibitor had an apparent molecular mass of 92 kDa by gel filtration and 71 kDa by SDS/PAGE, indicating the protein is monomeric. The inhibitor did not interact directly with myristoyl-CoA and possessed no protease, thioesterase or demyristoylase activity. Purified inhibitor protein inhibited the formation of 1167 pmol of myristoyl-peptide/min per mg of protein.
King
M J
MJ
Department of Pathology, University of Saskatchewan, Royal University Hospital and Saskatoon Cancer Centre, Canada.
Sharma
R K
RK
D000214:Acyl Coenzyme A / Q000378:metabolism; D000217:Acyltransferases / Q000037:antagonists & inhibitors*; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000648:ultrastructure; D001923:Brain Chemistry; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry; D003600:Cytosol / Q000737:chemistry; D004355:Drug Stability; D006358:Hot Temperature; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology
D000214:Acyl Coenzyme A; D009227:Myristic Acids; D009419:Nerve Tissue Proteins; D019814:Myristic Acid; C040349:S-tetradecanoyl-coenzyme A; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase