Arachidonoyl-diacylglycerol kinase from bovine testis. Purification and properties.
Previous work in our laboratory demonstrated the existence of a membrane-bound diacylglycerol kinase highly selective for diacylglycerols containing arachidonate as the sn-2 fatty acyl moiety (MacDonald, M. L., Mack K. F., Richardson, C. N., and Glomset, J. A. (1988) J. Biol. Chem. 263, 1575-1583). We now report the purification of arachidonoyl-diacylglycerol kinase 34,400-fold to apparent homogeneity from bovine testis. High concentrations of both salt and detergent were required to extract the enzyme from membranes and stabilize its activity, suggesting that in vivo the enzyme is part of a complex with other membrane or cytoskeletal proteins. Arachidonoyl-diacylglycerol kinase had an apparent M(r) of 58,000 both on SDS-polyacrylamide gels and by size exclusion chromatography. The enzyme appeared to be an integral membrane protein. In a mixed micellar assay, arachidonoyl-diacylglycerol kinase followed surface dilution kinetics with respect to diacylglycerol. The purified enzyme retained the arachidonate selectivity observed previously in membranes. Kinetic analyses indicated a Km for sn-1-stearoyl-2-arachidonoylglycerol of 2.4 mol %, as compared to 43 mol % for sn-1-palmitoyl-2-oleoylglycerol. Calcium, an activator of some other diacylglycerol kinases, had no apparent effect on the arachidonate-specific enzyme. Guanosine triphosphate could effectively substitute for ATP as the phosphoryl donor and Mg2+ could be replaced by Mn2+ or Ca2+. Phosphatidylserine and, to a lesser extent, phosphatidylinositol inhibited the purified enzyme. Phosphatidylcholine and phosphatidylethanolamine had only small effects.
Walsh
J P
JP
Howard Hughes Medical Institute Research Laboratory, University of Washington, Seattle 98195.
Suen
R
R
Lemaitre
R N
RN
Glomset
J A
JA
D016475:3T3 Cells; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002413:Cations, Divalent; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D019852:Diacylglycerol Kinase; D004075:Diglycerides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D010743:Phospholipids / Q000494:pharmacology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000302:isolation & purification* / Q000378:metabolism; D013737:Testis / Q000201:enzymology*; D014018:Tissue Distribution
D002413:Cations, Divalent; D004075:Diglycerides; D008565:Membrane Proteins; D010743:Phospholipids; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); D019852:Diacylglycerol Kinase