Ridgway N D, Vance D E
Department of Biochemistry, University of Alberta, Edmonton, Canada.
J Biol Chem. 1987 Dec 15;262(35):17231-9.
Phosphatidylethanolamine (PE) N-methyltransferase catalyzes the synthesis of phosphatidylcholine by the stepwise transfer of methyl groups from S-adenosylmethionine to the amino head group of PE. PE N-methyltransferase was solubilized from a microsomal membrane fraction of rat liver using the nonionic detergent Triton X-100 and purified to apparent homogeneity. Specific activities of PE N-methyltransferase with PE, phosphatidyl-N-monomethylethanolamine (PMME), and phosphatidyl-N,N-dimethylethanolamine (PDME) as substrates were 0.63, 8.59, and 3.75 mumol/min/mg protein, respectively. The purified enzyme was composed of a single subunit with a molecular mass of 18.3 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Methylation activities dependent on the presence of PE, PMME, and PDME and the 18.3-kDa protein co-eluted when purified PE N-methyltransferase was subjected to gel filtration on Sephacryl S-300 in the presence of 0.1% Triton X-100. All three methylation activities eluted with a Stokes radius 2.1 A greater than that determined for pure Triton micelles (molecular mass difference of 27.4 kDa). Two-dimensional analysis of PE N-methyltransferase employing nonequilibrium pH gradient gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the enzyme is composed of a single isoform. Analysis of enzyme activity using PE, PMME, and PDME at various Triton X-100 concentrations indicated the enzyme follows the "surface dilution" model proposed for other enzymes that act at the surface of mixed micelle substrates. Initial velocity data for all three lipid substrates (at fixed concentrations of Triton X-100) were highly cooperative in nature. Hill numbers for PMME and PDME ranged from 3 at 0.5 mM Triton to 6 at 2.0 mM Triton. All three methylation activities had a pH optimum of 10. These results provide evidence that a single membrane-bound enzyme catalyzes all three methylation steps for the conversion of PE to phosphatidylcholine.
磷脂酰乙醇胺（PE）N - 甲基转移酶通过将甲基从S - 腺苷甲硫氨酸逐步转移到PE的氨基头部基团来催化磷脂酰胆碱的合成。使用非离子去污剂Triton X - 100从大鼠肝脏的微粒体膜部分中溶解PE N - 甲基转移酶，并纯化至表观均一性。以PE、磷脂酰 - N - 单甲基乙醇胺（PMME）和磷脂酰 - N，N - 二甲基乙醇胺（PDME）为底物时，PE N - 甲基转移酶的比活性分别为0.63、8.59和3.75 μmol/分钟/毫克蛋白质。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳测定，纯化的酶由单个亚基组成，分子量为18.3 kDa。当在0.1% Triton X - 100存在下对纯化的PE N - 甲基转移酶进行Sephacryl S - 300凝胶过滤时，依赖于PE、PMME和PDME以及18.3 kDa蛋白质存在的甲基化活性共同洗脱。所有三种甲基化活性的洗脱斯托克斯半径比纯Triton胶束的测定值大2.1 Å（分子量差异为27.4 kDa）。采用非平衡pH梯度凝胶电泳和十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳对PE N - 甲基转移酶进行二维分析表明，该酶由单一同工型组成。在不同Triton X - 100浓度下使用PE、PMME和PDME分析酶活性表明，该酶遵循为作用于混合胶束底物表面的其他酶提出的“表面稀释”模型。所有三种脂质底物（在固定的Triton X - 100浓度下）的初始速度数据本质上具有高度协同性。PMME和PDME的希尔系数范围从0.5 mM Triton时的3到2.0 mM Triton时的6。所有三种甲基化活性的最适pH为10。这些结果提供了证据，表明单一的膜结合酶催化将PE转化为磷脂酰胆碱的所有三个甲基化步骤。
