S-ADENOSYL-L-METHIONINE-MEDIATED ENZYMATIC METHYLATIONS IN THE RAT RETINAL MEMBRANES

Enzymatic step-wise methylation of membrane phosphatidylethanolamine ( PE) to phosphatidyl-N-methylethanolamine (PME) and then phosphatidyl-c holine (PC) has been known to alter membrane properties and responsive ness of cells for activation of receptors by chemical transmitters. Co nversion of PE to PME and PME to PC in the presence of S-adenosyl-L-me thionine (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 wa s not studied for PMT activity. Therefore, we studied the rat retina f or PMT I activity. Methylation of PE in the rat retinal sonicates was assayed using H-3-SAM (2 mu M) at 37 degrees C in Tris-glycylglycine b uffer (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 c hromatography 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 me thylated phospholipids were (M +/- SE, N=5) 19.90 +/- 4.03 fmol/mg pro tein/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 am ounted 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 mu M). These obser vations indicate that rat retina contains both PMTs and FACM.