The purification and properties of monoacylglycerol kinase from bovine brain.
Monoacylglycerol kinase (MGK) has been purified from bovine brain by six steps: isolation of cytosol, DEAE-cellulose chromatography, ammonium sulfate fractionation (0-40%), Bio-Gel A-1.5m, hydroxylapatite, and ATP-agarose column chromatography. The overall purification was 938 times with a 4.8% yield. The column separations (particularly Bio-Gel A-1.5m) and SDS- and nondenaturing-polyacrylamide gel electrophoresis of enzyme purified from ATP-agarose indicated that MGK exists as a complex (approximately 350 kilodaltons) that is stabilized by 0.5 M NaCl and, on complete dissociation, yields a major protein of 72 kilodaltons. Dithiothreitol, EDTA, and ATP helped to stabilize MGK during purification. The protein peak eluted from hydroxylapatite by 25 mM phosphate activated and stabilized MGK activity. Phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin inhibited MGK. These phospholipids and others activated MGK synergistically with the above protein peak. MGK copurified with diacylglycerol kinase (DGK) throughout giving MGK to DGK ratios of 0.05-0.36. Optimal activity required 0.5 mM 2-monoolein and 10 mM MgCl2. Strong inhibition by p-chloromercuriphenyl sulfonic acid, N-ethyl-maleimide, and 5,5'-dithio-bis(2-nitrobenzoic acid), and prevention of this inhibition by dithiothreitol indicated the involvement of intact SH groups in the action of MGK. Purified MGK showed preference for substrates with unsaturated fatty acids except for 1- or 2-monostearin. Overall the preference favored the selective generation of 1-stearoyl- and 2-arachidonoyl-lysophosphatidic acid.
Shim
Y H
YH
Department of Biochemistry, University of Western Ontario, London, Canada.
Lin
C H
CH
Strickland
K P
KP
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002413:Cations, Divalent; D002417:Cattle; D002850:Chromatography, Gel; D003600:Cytosol / Q000201:enzymology; D019852:Diacylglycerol Kinase; D004952:Esters / Q000032:analysis; D005227:Fatty Acids / Q000032:analysis; D008244:Lysophosphatidylcholines / Q000096:biosynthesis; D010770:Phosphotransferases / Q000032:analysis / Q000302:isolation & purification*; D017853:Phosphotransferases (Alcohol Group Acceptor); D013439:Sulfhydryl Reagents
D002413:Cations, Divalent; D004952:Esters; D005227:Fatty Acids; D008244:Lysophosphatidylcholines; D013439:Sulfhydryl Reagents; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); D019852:Diacylglycerol Kinase; C025944:acylglycerol kinase