Raju R V, Datla R S, Sharma R K
Department of Pathology and Saskatoon Cancer Centre, University of Saskatchewan, Canada.
Mol Cell Biochem. 1996 Feb 9;155(1):69-76. doi: 10.1007/BF00714335.
Myristoyl CoA:protein N-myristoyltransferase catalyzes the addition of myristate to the amino-terminal glycine residue of a number of eukaryotic proteins. Escherichia coli transformed with human NMT expression construct produced high levels of N-myristoyltransferase. Using the combination of ammonium sulfate precipitation, chromatography on SP-Sepharose fast flow and fast protein liquid chromatography on Mono-S, the enzyme was purified more than 100 fold with 40% yield. The hNMT fusion protein exhibited an apparent molecular weight of 53 kDa on SDS-polyacrylamide gel electrophoresis. Upon cleavage by the Enterokinase [(Asp)4-Lys], the hNMT exhibited an apparent molecular mass of 49 kDa without loss of catalytic activity. The hNMT activity could be greatly activated severalfold with the use of Tris, SDS, ethanol and acetonitrile. The catalytic activity of hNMT was potently inhibited in a concentration dependent manner by NIP71, a bovine brain NMT inhibitory protein with a half maximal inhibition of 31.0 nM. The E. coli expressed hNMT was homogeneous and showed enzyme activity.
肉豆蔻酰辅酶A：蛋白质N-肉豆蔻酰转移酶催化将肉豆蔻酸添加到许多真核蛋白质的氨基末端甘氨酸残基上。用人NMT表达构建体转化的大肠杆菌产生了高水平的N-肉豆蔻酰转移酶。通过硫酸铵沉淀、SP-琼脂糖快速流动柱色谱和Mono-S快速蛋白质液相色谱相结合的方法，该酶的纯化倍数超过100倍，产率为40%。hNMT融合蛋白在SDS-聚丙烯酰胺凝胶电泳上显示出约53 kDa的表观分子量。经肠激酶[（Asp）4-Lys]切割后，hNMT的表观分子量为49 kDa，且催化活性未丧失。使用Tris、SDS、乙醇和乙腈可使hNMT活性大幅激活几倍。hNMT的催化活性受到牛脑NMT抑制蛋白NIP71的浓度依赖性强力抑制，其半数最大抑制浓度为31.0 nM。大肠杆菌表达的hNMT是均一的且具有酶活性。
