Purification and properties of bovine spleen N-myristoyl-CoA protein:N-myristoyltransferase.
Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes the addition of myristate to the amino-terminal glycine residue of a number of eukaryotic proteins. In this report, a simple and rapid purification as well as the properties of this enzyme from bovine spleen is described. Using combination of ammonium sulfate precipitation, chromatography on SP-Sepharose fast flow, phenyl-Sepharose CL-4B, DEAE-Sepharose CL-6B, and Superose 12 (HR/30) gel filtration fast protein liquid chromatography, the enzyme was purified 1475-fold with a high yield. Under native conditions, the enzyme exhibited an apparent molecular mass of 58 kDa, whereas under denaturing conditions the enzyme represented an apparent molecular mass of 50 kDa, suggesting that spleen NMT is a monomeric protein. The NMT activity could be greatly activated to severalfold with the use of Tris-HCl buffer. Kinetic properties indicated that spleen NMT had an apparent low Km for pp60src and myristoylated alanine-rich C kinase substrate as compared with cAMP-dependent protein kinase and the M2 gene segment of reovirus type 3-derived peptides. Bovine spleen NMT was potently inhibited in a concentration-dependent manner by NIP71 (a bovine brain NMT inhibitory protein) with a half-maximal inhibition of 0.816 microgram/ml. Results of this study along with the existing knowledge on NMT indicate that the activity of enzyme resides in a single polypeptide chain of molecular mass between 50 and 68 kDa.
Raju
R V
RV
Department of Pathology, College of Medicine, University of Saskatchewan, Royal University Hospital, Saskatoon, Canada.
Kalra
J
J
Sharma
R K
RK
D000214:Acyl Coenzyme A / Q000138:chemical synthesis / Q000378:metabolism; D000217:Acyltransferases / Q000302:isolation & purification* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010171:Palmitoyl Coenzyme A / Q000138:chemical synthesis / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D013154:Spleen / Q000201:enzymology*; D013379:Substrate Specificity; D014316:Tritium
D000214:Acyl Coenzyme A; D014316:Tritium; D010171:Palmitoyl Coenzyme A; C040349:S-tetradecanoyl-coenzyme A; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase