Assay of N-myristoyl transferase by selective adsorption of myristoyl-coenzyme A on acidic alumina.
We have developed a simple and rapid method for detecting the enzyme myristoyl-CoA:protein N-myristoyl transferase. The enzyme catalyzes the transfer of the myristoyl moiety of myristoyl-CoA to the amino-terminal glycine residue of a peptide (protein). Incorporation of the [14C]myristate into the peptide is quantified after separation of the [14C]myristoyl-peptide from unreacted [14C]myristoyl-CoA by selective adsorption of [14C]myristoyl-CoA on acidic alumina. Optimal assay concentrations were 200 microM synthetic peptide, 1 microM [14C]myristoyl-CoA, 10 mM Tris-HCl/1 mM dithiothreitol/0.1 mM ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid/aprotinin (10 micrograms/ml) buffer, pH 7.4, and 1-10 micrograms protein.
Wagner
A P
AP
University of Karlsruhe, Institute of Organic Chemistry and Biochemistry, Federal Republic of Germany.
Retey
J
J
D000214:Acyl Coenzyme A / Q000378:metabolism*; D000217:Acyltransferases / Q000378:metabolism*; D000327:Adsorption; D000537:Aluminum Oxide; D006863:Hydrogen-Ion Concentration; D007700:Kinetics
D000214:Acyl Coenzyme A; C040349:S-tetradecanoyl-coenzyme A; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase; D000537:Aluminum Oxide