Sastry B V, Vidaver P S, Janson V E, Franks J J
Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, Tennessee.
J Ocul Pharmacol. 1994 Spring;10(1):253-63. doi: 10.1089/jop.1994.10.253.
Enzymatic step-wise methylation of membrane phosphatidylethanolamine (PE) to phosphatidyl-N-methylethanolamine (PME) and then phosphatidyl-choline (PC) has been known to alter membrane properties and responsiveness of cells for activation of receptors by chemical transmitters. Conversion of PE to PME and PME to PC in the presence of S-adenosyl-L-methionine (SAM) are catalyzed by two phospholipid N-methyltransferases, PMT I and PMT II, of which PMT I is the rate limiting enzyme. Retina is a good neuronal model for chemical transmission. However, retina was not studied for PMT activity. Therefore, we studied the rat retina for PMT I activity. Methylation of PE in the rat retinal sonicates was assayed using 3H-SAM (2 microM) at 37 degrees C in Tris-glycylglycine buffer (50 mM, pH 8.0) and methylated phospholipids were extracted with chloroform/methanol/HCl (2/1/0.02, v/v) and separated by thin layer chromatography on Silica Gel G plates. Chromatograms were developed in a solvent system of propionic acid/n-propyl alcohol/chloroform/water (2/2/1/1, v/v). This study gave the following results: (a) the total methylated phospholipids were (M +/- SE, N = 5) 19.90 +/- 4.03 fmol/mg protein/min; (b) the major methylated phospholipid was PME (4.21 +/- 0.68 fmol/mg protein/min; (c) the fatty acid methylesters formed by fatty acid carboxymethylase (FACM) which accumulated in the solvent front amounted to 18.82 +/- 2.84 fmol/mg protein/min. Both PMT I and FACM were inhibited by S-adenosyl-L-homocysteine (I50, 1.2-5 microM). These observations indicate that rat retina contains both PMTs and FACM.
已知膜磷脂酰乙醇胺（PE）逐步酶促甲基化生成磷脂酰-N-甲基乙醇胺（PME），然后再生成磷脂酰胆碱（PC），会改变膜的性质以及细胞对化学递质激活受体的反应性。在S-腺苷-L-甲硫氨酸（SAM）存在的情况下，PE向PME以及PME向PC的转化由两种磷脂N-甲基转移酶PMT I和PMT II催化，其中PMT I是限速酶。视网膜是化学传递的良好神经元模型。然而，尚未对视网膜的PMT活性进行研究。因此，我们研究了大鼠视网膜的PMT I活性。在37℃下，于Tris-甘氨酰甘氨酸缓冲液（50 mM，pH 8.0）中使用3H-SAM（2 μM）测定大鼠视网膜超声裂解物中PE的甲基化情况，并用氯仿/甲醇/HCl（2/1/0.02，v/v）提取甲基化的磷脂，然后在硅胶G板上通过薄层色谱法进行分离。色谱图在丙酸/正丙醇/氯仿/水（2/2/1/1，v/v）的溶剂系统中展开。本研究得出以下结果：（a）总甲基化磷脂为（M±SE，N = 5）19.90±4.03 fmol/mg蛋白/分钟；（b）主要的甲基化磷脂是PME（4.21±0.68 fmol/mg蛋白/分钟）；（c）在溶剂前沿积累的由脂肪酸羧甲基酶（FACM）形成的脂肪酸甲酯为18.82±2.84 fmol/mg蛋白/分钟。PMT I和FACM均被S-腺苷-L-高半胱氨酸抑制（I50，1.2 - 5 μM）。这些观察结果表明大鼠视网膜中同时含有PMT和FACM。
