Partial purification and characterization of phospholipid N-methyltransferases from murine thymocyte microsomes |
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| Authors: | F Makishima S Toyoshima T Osawa |
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| Affiliation: | 1. Department of Agricultural, Food and Environmental Science, University of Perugia, Borgo XX Giugno 74, Perugia 06100, Italy;2. Faculty of Agricultural and Environmental Sciences, Kaposvár University, 40, Guba S. str., H-7400 Kaposvár, Hungary;3. Department of Animal Medicine, Production and Health, University of Padova, Agripolis, Viale dell''Università 16, 35020 Legnaro, Padova, Italy;1. Levine Cancer Institute, Carolinas HealthCare System, Charlotte, NC, United States;2. Charing Cross Trophoblastic Disease Centre, Department of Medical Oncology, Charing Cross Campus of Imperial College London, London, United Kingdom;3. Spectrum Health Medical Group, Department of Obstetrics, Gynecology, and Womens Health Group, Grand Rapids, MI, United States;1. China CDC Key Laboratory of Environment and Population Health, National Institute of Environmental Health, Chinese Center for Disease Control and Prevention, Beijing 100050, China;2. The Key Laboratory of Trace Element Nutrition of National Health Commission of the People''s Republic of China, National Institute for Nutrition and Health, Chinese Center for Disease Control and Prevention, No. 29 Nan Wei Road, Xicheng District, Beijing 100050, PR China;3. Enshi Center for Disease Control and Prevention, Enshi 445000, Hubei, China;4. Department of Biostatistics, Indiana University School of Medicine, Indianapolis, IN 46202-2872, USA |
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| 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, Mg2+ (10 mM) or Ca2+ (1 mM), and EGTA separately in the assay had only a small effect on the enzyme activity. However, addition of both 10 mM Mg2+ and 1 mM Ca2+ 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 Km values for S-adenosyl-L-methionine were 20 and 400-500 microM for the conversion of PE to PME and for the conversion of PME to PC they were 5 microM and 40 microM. S-Adenosyl-L-homocysteine (AdoHcy), a known inhibitor of enzymatic methylation, competitively inhibited [14C]methyl incorporation into total lipid. The apparent Ki value for AdoHcy was 44.7 microM. 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 approximately 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 A2 activity was detected. This result may suggest that phospholipid N-methyltransferases associate with phospholipase A2 in the thymocyte plasma membrane. |
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