Abstract
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 (μl) (M2; 17,300 pmol N-myristoylpeptide formed/min/mg) and very low activity with a peptide derived from the N-terminal sequence ofmyristoylatedalanine-richC-kinasesubstrate (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 myristolyl-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.
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Abbreviations
- NMT:
-
N-myristoyl-CoA:protein N-myristoyltransferase
- NMT·I:
-
mono Q N-myristoyl-CoA:protein N-myristoyltransferase peak I
- NMT·II:
-
mono Q N-myristoyl-CoA:protein N-myristoyltransferase peak II
- NMT·III:
-
mono Q N-myristoyl-CoA:protein N-myristoyltransferase peak III
- NIP71 :
-
71 kDa heat-stable N-myristoyltransferase inhibitor protein
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King, M.J., Sharma, R.K. Mechanisms of action of NIP71 on N-myristoyltransferase activity. Mol Cell Biochem 141, 79–86 (1994). https://doi.org/10.1007/BF00926170
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DOI: https://doi.org/10.1007/BF00926170