Triacylglycerol synthesis by an sn-1,2(2,3)-diacylglycerol transacylase from rat intestinal microsomes.
A membrane-associated sn-1,2(2,3)-diacylglycerol transacylase activity was purified 550-fold from the microsomes of rat intestinal villus cells. The enzyme was solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and was isolated and purified by sequential chromatography on hydroxylapatite, an anion exchanger, Affi-Gel heparin and Cibacron blue A-agarose. SDS-polyacrylamide gel electrophoresis revealed two major closely migrating polypeptides of apparent molecular mass of 50 and 52 kDa. Incubation of the isolated enzyme with sn-1,2(2,3)dioleoyl-[2-3H]glycerol yielded trioleoyl[2-3H]glycerol and 2-oleoyl[2-3H]glycerol. The synthesis of triacylglycerols was independent of acyl-CoA, as indicated by the absence of incorporation of radioactivity from [1-14C]oleoyl-CoA into mono-, di-, or triacylglycerols under these conditions. The transacylase did not hydrolyze triacylglycerols readily and was not affected by the acyltransferase inhibitors, N-ethylmaleimide and 4,4'-diisothiocyanostilbene-2,2'-disulfonate, while the lipase/hydrolase inhibitors NaF, phenylmethylsulfonyl fluoride, and diethyl p-nitrophenyl phosphate caused partial inactivation. The enzyme was specific toward 1(3)-positions of rac 1,2-diacylglycerols but did not utilize the -1,3-diacylglycerols or other neutral lipid esters effectively and was not capable of removing fatty acids from phosphatidylcholine or of transfer of fatty acids from X (unspecified enantiomer)-1,2-diacylglycerol to lysophosphatidylcholine. Since the diacylglycerol transacylation did not lead to accumulation of either sn-1,2- or sn-2,3-diacylglycerols during incubation with the racemates, it is concluded that both sn-1,2- and sn-2,3-s are utilized at similar rates.
Lehner
R
R
Banting and Best Department of Medical Research, C. H. Best Institute, University of Toronto, Ontario, Canada.
Kuksis
A
A
D000217:Acyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003067:Coenzymes / Q000378:metabolism; D051048:Diacylglycerol O-Acyltransferase; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007583:Jejunum / Q000201:enzymology*; D007700:Kinetics; D008297:Male; D008861:Microsomes / Q000201:enzymology*; D051381:Rats; D013379:Substrate Specificity; D014280:Triglycerides / Q000096:biosynthesis*
D003067:Coenzymes; D014280:Triglycerides; D000217:Acyltransferases; D051048:Diacylglycerol O-Acyltransferase