ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 211, No. 2, October 15, pp. 762-773, 1981 Evidence for Two Methyltransferases Involved in the Conversion of Phosphatidylethanolamine to Phosphatidylcholine in the Rat Liver’ B. V. RAMA SASTRY,*P~‘~ CHARLES N. STATHAM,+ JULIUS AXELROD,t AND FUSAO HIRATAt *Department of Phnm, Vanderbilt University School of Medicine, Nash&, Tennessee 37232, and tsection on Pharmacology, Laboratory of Clinical Science, Naticmal Institutes of Mental Health, and *Molecular Toxicology Section, Clinical Pharmacology Branch, Clinical Oncology Program, Division of Cancer Treatment, National Cancer Institute, Bethesda, Margland 20205 Received January 5, 1981 The stepwise methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) occurs in the rat liver. The properties of two methyltransferases that are involved in this stepwise methylation of PE to PC in the rat liver are reported. Rat microsomal membranes were used as the source of the enzymes and S-adenosyl-L-[methyl- 3H]methionine as the methyl donor. The first methyltransferase converted PE to phos- phatidyl-N-methylethanolamine (PME), and the second methyltransferase converted PME to PC. The products were characterized by thin-layer chromatography and high- pressure liquid chromatography. After incubation of rat liver microsomes with the methyl donor, three peaks corresponding to PME, dimethyl-PME (PMME), and PC were found and quantified. The first methyltransferase had low K,,, (0.83 PM), pH optimum of 8, and was activated by Mg2f. The second methyltransferase had a high K, (-67 pM) and a pH optimum of 10. The proportion of the first methyltransferase in the microsomal mem- branes was increased by repeated washings in hypotonic medium containing EDTA. When the microsomal membranes were subjected to repeated mild sonication and centrifugation at 105,OOOg a fraction of the second methyltransferase was solubilized (i.e., appeared in the nonparticulate fraction). The solubilized enzyme utilized dipalmitoyl-PME and -PMME as substrates. Both enzymes were also present in mitochondrial and nuclear membranes with highest specific activities occurring in the microsomal membranes. Several investigations have indicated that phosphatidylethanolamine is meth- ylated by stepwise transmethylation with i Part of this investigation was presented at the Meeting of the Federation of American Societies for Experimental Biology and Medicine at Dallas, Tex., in April, 1979. z On sabbatical leave from the Department of Pharmacology, Vanderbilt University School of Med- icine, Nashville, Tenn. 37232. This author was par- tially supported by a Faculty Fellowship from the Vanderbilt University Research Council; United States Public Health Service Research Grants HD- 10607, AG-02077, HL-25358, and United States Public Health Service General Research Support Grant RR- 05424, and a grant from The Council for Tobacco Research U. S. A., Inc. The authors wish to thank Dr. Michael R. Boyd of the Molecular Toxicology Sec- tion, NCI, for consultations on HPLC and Professors John G. Coniglio and Erwin J. Landon of Vanderbilt University for critical evaluation of the manuscript. S-adenosylmethionine ( SAM)3 to form phosphatidylcholine in the mammalian liver (l-6). Bremer and Greenberg (1) as- sumed that there were three different en- zymes, each incorporating one methyl group. They found that incorporation of the first methyl group was the rate-lim- iting step in the overall reaction leading to phosphatidylcholine. Rehbinder and Greenberg (4) solubilized the microsomal methyltransferase and concluded that there was only one enzyme. In two other studies (5, 6) using solubilized methyl- transferase systems, no evidence was pre- sented for the involvement of more than z Abbreviations used: SAM, S-adenosylmethionine; TLC, thin-layer chromatography; HPLC, high-pres- sure liquid chromatography. 0003-9861/81/120762-12$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of repmduction in any form reserved. 762