Raju R V, Datla R S, Kakkar R, Sharma R K
Department of Pathology and Saskatoon Cancer Centre, College of Medicine, University of Saskatchewan, Canada.
Mol Cell Biochem. 1998 Dec;189(1-2):91-7. doi: 10.1023/a:1006861417562.
Myristoyl-CoA:protein N-myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the co-translational transfer of myristate to the NH2-terminal glycine residue of a number of important proteins of diverse function. Recently, we have isolated full length cDNA encoding bovine spleen NMT [27] the full length cDNA was cloned and expressed in E. coli, resulting in the expression of functionally active 50 kDa NMT. Using the combination of SP-Sepharose fast flow and Mono S fast protein liquid chromatography, the enzyme was purified 20-fold with a high yield. The spleen NMT (sNMT) fusion protein exhibited an apparent molecular weight of 53 kDa on SDS-PAGE. Upon cleavage by the Enterokinase the sNMT exhibited an apparent molecular weight of 50 kDa without loss of catalytic activity. The two synthetic peptide substrates based on the N-terminal sequence of pp60src (GSSKSKMR) and cAMP dependent protein kinase (GNAAAKKRR) have different kinetic parameters of Km values of 40 and 200 microM. Recombinant sNMT was also potently inhibited by Ni2+ (histidine binder) in a concentration dependent manner with a half maximal inhibition of 280 microM. The E. coli expressed sNMT was homogenous and showed enzyme activity.
肉豆蔻酰辅酶A：蛋白质N-肉豆蔻酰转移酶（NMT）是一种必需的真核酶，它催化肉豆蔻酸共翻译转移至多种功能各异的重要蛋白质的NH2末端甘氨酸残基上。最近，我们分离出了编码牛脾NMT的全长cDNA[27]，该全长cDNA被克隆并在大肠杆菌中表达，从而表达出具有功能活性的50 kDa NMT。通过使用SP-琼脂糖凝胶快速流和Mono S快速蛋白质液相色谱相结合的方法，该酶以高产率被纯化了20倍。脾NMT（sNMT）融合蛋白在SDS-PAGE上显示出表观分子量为53 kDa。经肠激酶切割后，sNMT显示出表观分子量为50 kDa，且催化活性未丧失。基于pp60src（GSSKSKMR）和cAMP依赖性蛋白激酶（GNAAAKKRR）的N端序列的两种合成肽底物具有不同的动力学参数，Km值分别为40和200 microM。重组sNMT也被Ni2+（组氨酸结合剂）以浓度依赖性方式强烈抑制，半数最大抑制浓度为280 microM。大肠杆菌表达的sNMT是均一的，并显示出酶活性。
