Mechanisms of action of NIP71 on N-myristoyltransferase activity.
N-Myristoyl-CoA:protein N-myristoyltransferase (NMT) is the enzyme that catalyses the transfer of myristate from myristoyl-CoA to the N-terminal glycine of protein substrates. NMT was highly purified from bovine brain by procedures involving sequential column chromatography on DEAE-Sepharose CL-6B, phosphocellulose, hydroxylapatite, and mono S and mono Q f.p.l.c.. The highly purified NMT (termed NMT.II) possessed high specific activity with peptide substrates derived from the N-terminal sequences of the cAMP-dependent protein kinase and pp60src (29,800 and 47,600 pmol N-myristoylpeptide formed/min/mg, respectively), intermediate activity with a peptide based on the N-terminal sequence of a viral structural protein (microliter) (M2; 17,300 pmol N-myristoylpeptide formed/min/mg) and very low activity with a peptide derived from the N-terminal sequence of myristoylated alanine-rich C-kinase substrate (MARCKS; 1500 pmol myristoylpeptide formed/min/mg). An NMT protein inhibitor (NIP71) isolated from the particulate fraction of bovine brain (King MJ and Sharma RK: Biochem J 291:635-639, 1993) potently inhibited highly purified NMT activity (IC50 23.7 nM). A minor NMT activity (NMT.PU; 30% total NMT activity), which failed to bind to phosphocellulose, was insensitive to NIP71 inhibition. Inhibition of NMT was observed to be via mixed inhibition with respect to both the myristoyl-CoA and peptide substrates with NIP71 having an apparent higher affinity for NMT than the NMT.myristoyl.CoA complex. Inhibition by NIP71 at subsaturating concentrations of myristoyl-CoA and peptide resulted in a sigmoidal pattern of inhibition indicating that bovine brain possesses a potent and delicate on/off switch to control NMT activity.
King
M J
MJ
Department of Pathology, Royal University Hospital, University of Saskatchewan, Saskatoon, Canada.
Sharma
R K
RK
D000217:Acyltransferases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology; D002417:Cattle; D003600:Cytosol; D008565:Membrane Proteins / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000494:pharmacology*; D013379:Substrate Specificity
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase