Arachidonoyl-diacylglycerol kinase from bovine testis. Purification and properties.

摘要: Previous work in our laboratory demonstrated the existence of a membrane-bound diacylglycerol kinase highly selective for diacylglycerols containing arachidonate as sn-2 fatty acyl moiety (MacDonald, M. L., Mack K. F., Richardson, C. N., and Glomset, J. A. (1988) Biol. Chem. 263, 1575-1583). We now report purification arachidonoyl-diacylglycerol 34,400-fold to apparent homogeneity from bovine testis. High concentrations both salt detergent were required extract enzyme membranes stabilize its activity, suggesting that vivo is part complex with other membrane or cytoskeletal proteins. Arachidonoyl-diacylglycerol had an M(r) 58,000 on SDS-polyacrylamide gels by size exclusion chromatography. The appeared be integral protein. In mixed micellar assay, followed surface dilution kinetics respect diacylglycerol. purified retained selectivity observed previously membranes. Kinetic analyses indicated Km sn-1-stearoyl-2-arachidonoylglycerol 2.4 mol %, compared 43 % sn-1-palmitoyl-2-oleoylglycerol. Calcium, activator some kinases, no effect arachidonate-specific enzyme. Guanosine triphosphate could effectively substitute ATP phosphoryl donor Mg2+ replaced Mn2+ Ca2+. Phosphatidylserine and, lesser extent, phosphatidylinositol inhibited Phosphatidylcholine phosphatidylethanolamine only small effects.