Characterization of heart sarcolemmal phospholipid methylation.
The transmethylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) was studied in rat heart sarcolemmal membrane. Kinetically, three apparent Km values for S-adenosyl-L-methionine (AdoMet) were obtained when the total [3H]methyl groups incorporation into the phospholipids was examined in the presence of 0.01-250 microM AdoMet. A first methyltransferase active site having a very low Km (0.1 microM) for AdoMet showed a partial requirement for Mg2+ and an optimum pH of 8.0 with a major formation of phosphatidyl-N-monomethylethanolamine (PMME). Both Ca2+ and K+ were inhibitory to this site. A second active site with a Km of 3.6 microM showed an optimum pH of 7.0 with predominant formation of phosphatidyl-N,N-dimethylethanolamine (PDME) and no Mg2+ requirement; in addition, transmethylation activity was also observed over a broad alkaline pH range (9-11) with an optimum at pH 10.5. This site was insensitive to Ca2+ but was stimulated by Na+, while K+ had an inhibitory effect. A third active site with a Km of 119 microM showed an optimum pH of 10.5 with major formation of PC and no Mg2+ requirement. This site was also insensitive to Ca2+ but markedly inhibited by both K+ and Na+. Under optimal conditions, the activities of all three methyltransferase sites were linear for at least 30 min of incubation and the sensitivity to the inhibitory effect of S-adenosyl-L-homocysteine was different for each site. Addition of exogenous PMME and PDME as substrates enhanced the synthesis of the corresponding methylated products by 3-5-fold and 3-8-fold, respectively. In contrast, exogenous PE failed to increase methyltransferase activity. These results provide evidence for the existence of three distinct methyltransferase active sites in rat heart sarcolemma.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008780:Methyltransferases / Q000378:metabolism*; D009206:Myocardium / Q000201:enzymology*; D010713:Phosphatidylcholines / Q000378:metabolism*; D050918:Phosphatidylethanolamine N-Methyltransferase; D010714:Phosphatidylethanolamines / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D012436:S-Adenosylmethionine / Q000378:metabolism; D012508:Sarcolemma / Q000201:enzymology*; D012964:Sodium / Q000494:pharmacology
D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D012436:S-Adenosylmethionine; D012964:Sodium; D008780:Methyltransferases; D050918:Phosphatidylethanolamine N-Methyltransferase; D008274:Magnesium; D011188:Potassium; D002118:Calcium