Partial purification and characterization of phospholipid N-methyltransferases from murine thymocyte microsomes

Abstract

Significant amounts of phospholipid N-methyltransferase activity in murine thymocytes were found to be distributed on the plasma membrane. The enzyme activity had an optimum pH of 9. The presence of divalent cations, Mg 2+ (10 m m) or Ca 2+ (1 m m), and EGTA separately in the assay had only a small effect on the enzyme activity. However, addition of both 10 m m Mg 2+ and 1 m m Ca 2+ increased the enzyme activity. The presence of two enzymes for each conversion of phosphatidylethanolamine (PE) to phosphatidylmonomethylethanolamine (PME) and PME to phosphatidylcholine (PC) was suggested by the result of the determination of the incorporated radioactivity into PME, phosphatidyldimethylethanolamine (PDE) and PC; the apparent K m values for S-adenosyl- l-methionine were 20 and 400–500 μ m for the conversion of PE to PME and for the conversion of PME to PC they were 5 μ m and 40 μ m. S-Adenosyl- l-homocysteine (AdoHcy), a known inhibitor of enzymatic methylation, competitively inhibited [ 14C]methyl incorporation into total lipid. The apparent K i value for AdoHcy was 44.7 μ m. Two phospholipid N-methyltransferases were partially purified by extraction with sodium deoxycholate, gel filtration on Sephadex G-75, and affinity column chromatography on AdoHcy-Sepharose. One enzyme, mainly catalyzing the formation of PME, was purified approximately 1548-fold and the other catalyzing the formation of PDE and PC, was purified approxiamtely 629- to 703-fold. However, the former still contained a little activity for PDE and PC formation and the latter contained a little activity for PME formation. In these partially purified phospholipid N-methyltransferase preparations, little contaminating protein O-carboxylmethyltransferase activity was observed; however, significant PC-phospholipase A 2 activity was detected. This result may suggest that phospholipid N-methyltransferases associate with phospholipase A 2 in the thymocyte plasma membrane.