Phospholipid synthesis in isolated fat cells. Studies of microsomal diacylglycerol cholinephosphotransferase and diacylglycerol ethanolaminephosphotransferase activities.
Diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) activities were investigated in microsomes from isolated rat fat cells. Assays based on the conversion of CDP-[14C]choline of CDP-[14C]ethanolamine to phosphatidylcholine or phosphatidylethanolamine utilized ethanol-dispersed diacylglycerols and 1 to 5 microng of protein. Cholinephosphotransferase and ethanolaminephosphotransferase activities had similar dependences on MgCl2 and pH, and were inhibited similarly by CaCl2, organic solvents, Triton X-100, Tween 20, and dithiothreitol. Ethylene glycol bis(beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid stimulated both activities similarly. With 1,2-dioleoyl-sn-glycerol, the cholinephosphotransferase activity had an apparent Km for CDP-choline of 23.9 micronM and a V max of 8.54 nmol/min/mg. CDP-ethanolamine and CDP were competitive inhibitors of the cholinephosphotransferase activity (apparent Kl values of 227 micronM and 360 micronM, respectively). With 1,2-dioleoyl-sn-glycerol, the ethanolaminephosphotransferase activity had an apparent Km of 18.3 micronM for CDP-ethanolamine and a V max of 1.14 nmol/min/mg. CDP-choline appeared to be a noncompetitive inhibitor of the ethanolaminephosphotransferase activity (apparent Kl of 1620 micronM). Inhibition of the ethanolaminephosphotransferase activity by CDP appeared to be of a mixed type. The dependences on diacylglycerols containing fatty acids 6 to 18 carbons in length were investigated...
D000273:Adipose Tissue / Q000166:cytology / Q000201:enzymology*; D000818:Animals; D001667:Binding, Competitive; D003566:Cytidine Diphosphate Choline / Q000494:pharmacology; D002798:Diacylglycerol Cholinephosphotransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004075:Diglycerides / Q000494:pharmacology; D004355:Drug Stability; D004983:Ethanolamines / Q000494:pharmacology; D005260:Female; D006358:Hot Temperature; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D010743:Phospholipids / Q000096:biosynthesis*; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D013329:Structure-Activity Relationship
D004075:Diglycerides; D004983:Ethanolamines; D010743:Phospholipids; D003566:Cytidine Diphosphate Choline; D010770:Phosphotransferases; D002798:Diacylglycerol Cholinephosphotransferase