Identification and properties of two methyltransferases in conversion of phosphatidylethanolamine to phosphatidylcholine.

Abstract

Two methyltransferases involved in the methylation of phosphatidylethanolamine to form phosphatidytcholine were demonstrated in a microsomal fraction of bovine adrenal medulla. The first methyltransferase catalyzes the methylation of phoshatidylethanol'amine to form phosphatidyl-N-monomethylethanolamine. This enzyme has an optimum pH of 6.5, a low Km for S-adenosyl-L-methionine (1.4 ;M), and an absolute requirement for Mg2+. The second methyltransferase catalyzes the two successive methylations of phosphatidyl-N-monomethylethanolamine to phosphatidyl-N,N-dimethylethanolamine and phosphatidylcholine. In contrast to the first methyltransferase, it has an optimum pH of 10 and a high Km for S-adenosyl-L-methionine (0.1 mM) and does not require MgS+.Several investigations have shown that enzymatic methylations can occur on the amino group of phospholipids to form phosphatidyicholine (1-4). The enzyme(s) catalyzing this sequence of methylation were shown to reside in the microsomes of rat liver and Neurospora. A preparation of rat liver microsomes has been described that catalyzed the stepwise methylation of phosphatidyl-N-monomethylethanolamine to phosphatidylcholine but not of phosphatidylethanolamine (1; 4). The enzyme catalyzing the first methylation step has been suggested to be rate-limiting (1), but its properties have not yet been described. Recently, our laboratory reported on the ability of the enzyme, protein carboxymethylase, to transfer a methyl group from S-adenosyl-L-methionine to carboxy groups of membrane proteins of chromaffin granules in the adrenal medulla (5-7). In studies to examine the effects of cations on this enzyme activity with various membrane fractions, it was observed that methylation of lipids also occurred which depended upon the presence of Mg2+. Since the methylation of phospholipids has not been shown to require Mg2+ (1-4), this led us to search for and characterize the Mg2+ dependent enzyme that methylates lipids. This communication presents evidence that this Mg2+-dependent enzyme is involved in the conversion of phosphatidylethanolamine to phosphatidyl-N-monomethylethanolamine and that a second methyltransferase converts the latter compound to phosphatidylcholine. METHODS AND MATERIALSAssay of Phosphatide Methyltransferases. The methylation of phosphatidylethanolamine to phosphatidyl-N-monomethylethanolamine was assayed by measuring incorporation of the methyl group from S-adenosyl-L-[methyl-3H]methionine into phospholipids. The assay medium, in a 6-ml stoppered polyethylene tube, contained 4 ,uM S-adenosyl-L-[methyl-3H]-The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "'advertisement"9 in accordance with 18 U. S. C. §1734 solely to indicate this fact. 1718 methionine (2 ,uCi), 10 mM MgCl2, 0.1 mM sodium EDTA, 50 mM sodium acetate buffer (pH 6.5), and tissue extract (0.1 mg of protein) in a total volume of 50 1l. The reaction was started by the addition of radioactive S-adenosyl-L...

Phospholipid methylation stimulates lactogenic binding in mouse mammary gland membranes.

“…The methyltransferases present in membranes prepared from several animal tissues catalyze the synthesis of phosphatidylcholine from phosphatidylethanolamine (2,3). This, in turn, results in rapid translocation of the phosphatidylcholine to the external surface of the membranes (3, 4) and alteration in membrane microviscosity and charge (4, il).…”

“…Membranes prepared from either rat erythrocytes or bovine adrenal medulla contain two enzymes that synthesize phosphatidylcholine from phosphatidylethanolamine by using Sadenosyl-L-methionine (AdoMet) as the methyl donor (2,3). These two enzymes, their substrates, and products are localized within the membranes in such a way as to be implicated in maintaining the asymmetric environment of the phospholipids in the membranes (3).…”

Concanavalin A stimulates phospholipid methylation and phosphatidylserine decarboxylation in rat mast cells.

“…The overall yield of '4C-labeled phosphatidylserine was approximately 5% of the initial total radioactivity. The final product was free of phosphatidylcholine and was >92% pure as shown by thin-layer chromatography with several solvent systems (11 [methyl-3Hlmethionine, and the incorporation of the [3H]-methyl group into the lipid fraction in the presence of Con A was measured. In the early stage of the reaction, there was a marked increase in the incorporation of the [3H]methyl group into the lipid fraction ( Fig.…”

“…Histamine in the supernatant was measured fluorometrically (10). The precipitate was washed with 1 ml of 5% trichloroacetic acid containing 10 mM methionine and then extracted with 3 ml of chloroform/ methanol, 2:1 (vol/vol), as described (11). Because 4 hr, the reaction was stopped by the addition of 0.2 ml of 1 M HCl, and the lipids were extracted with 4 ml of ethyl ether/ethanol, 4:1 (vol/vol).…”

“…The first involves activation of the membrane cell surface and the second, fusion of histamine-containing granules with plasma membranes. Ca2+ is required for the second stage but not for the first (2), and Mg2+ is necessary for the methylation of phosphatidylethanolamine (11). To clarify further the role of phospholipid methylation in histamine release, the effect of Ca2+ and Mg2+ was examined.…”