title: Partial purification of a diacylglycerol lipase from bovine aorta.
abstract: A diacylglycerol (DG) lipase has been purified from a soluble subcellular fraction of bovine aorta by (NH4)2SO4 precipitation in the presence of 5.0% (w/v) Triton X-100, followed by chromatography on DEAE-Sephacel, heparin-Sepharose and octyl-Sepharose in the presence of either CHAPS or Triton X-100 detergents. Under basal conditions, the hydrolysis of a short-chain [3H]dioctanoylglycerol ([3H]diC8) substrate was much greater than that of a long-chain 1-[1-14C]palmitoyl-2-oleoyl-sn-glycerol (1-[14C]POG) substrate. Lipase activity measured with 1-[14C]POG was markedly enhanced by Triton X-100. In the presence of 0.1% Triton X-100, specific enzyme activities in the octyl-Sepharose fraction determined with 1-[14C]POG or 1-stearoyl-2-[1-14C]-arachidonoyl-sn-glycerol as substrates were the same as that measured with [3H]diC8. MgCl2 (5mM) or CaCl2 (2 mM) also selectively stimulated lipase activity (up to 10-13-fold) measured with the long-chain (1-[14C]POG) substrate only. The increase in relative specific activity in the octyl-Sepharose fraction was 60-fold and 155-fold, based on hydrolysis of [3H]diC8 and 1-[14C]POG (+ Triton X-100), respectively. Unlabelled diC8 was a competitive inhibitor of 1-[14C]POG hydrolysis, suggesting that a single lipase hydrolyses both the short-chain and long-chain DG substrates; selective stimulatory effects of non-ionic detergents and bivalent cations on the hydrolysis of 1-[14C]POG may be due to effects on the physical properties of the substrate preparation. Monoacylglycerol lipase, DG kinase and cholesterol esterase activities could not be detected in the partially purified lipase preparation.
authors.affiliation: MRC Signal Transduction Group, Faculty of Medicine, University of Calgary, Alberta, Canada.
authors.forename: M W
authors.initials: MW
authors.lastname: Lee
authors.forename: D L
authors.initials: DL
authors.lastname: Severson
mesh_terms: D000818:Animals; D001011:Aorta / Q000201:enzymology*; D002417:Cattle; D002852:Chromatography, Ion Exchange; D003902:Detergents; D004075:Diglycerides / Q000378:metabolism; D007700:Kinetics; D008071:Lipoprotein Lipase / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D003902:Detergents; D004075:Diglycerides; D008071:Lipoprotein Lipase