ARACHIDONOYL-DIACYLGLYCEROL KINASE FROM BOVINE TESTIS - PURIFICATION AND PROPERTIES

Previous work in our laboratory demonstrated the existence of a membra ne-bound diacylglycerol kinase highly selective for diacylglycerols co ntaining arachidonate as the sn-2 fatty acyl moiety (MacDonald, M. L., Mack K. F., Richardson, C. N., and Glomset, J. A. (1988) J. Biol. Che m. 263, 1575-1583). We now report the purification of arachidonoyl-dia cylglycerol kinase 34,400-fold to apparent homogeneity from bovine tes tis. High concentrations of both salt and detergent were required to e xtract the enzyme from membranes and stabilize its activity, suggestin g that in. vivo the enzyme is part of a complex with other membrane or cytoskeletal proteins. Arachidonoyl-diacylglycerol kinase had an appa rent M(r) of 58,000 both on SDS-polyacrylamide gels and by size exclus ion chromatography. The enzyme appeared to be an integral membrane pro tein. In a mixed micellar assay, arachidonoyl-diacylglycerol kinase fo llowed surface dilution kinetics with respect to diacylglycerol. The p urified enzyme retained the arachidonate selectivity observed previous ly in membranes. Kinetic analyses indicated a K-m for sn-1-stearoyl-2- arachidonoylglycerol of 2.4 mol %, as compared to 43 mol % for sn-1-pa lmitoyl-2-oleoylglycerol. Calcium, an activator of some other diacylgl ycerol kinases, had no apparent effect on the arachidonate-specific en zyme. Guanosine triphosphate could effectively substitute for ATP as t he phosphoryl donor and Mg2+ could be replaced by Mn2+ or Ca2+. Phosph atidylserine and, to a lesser extent, phosphatidylinositol inhibited t he purified enzyme. Phosphatidylcholine and phosphatidylethanolamine h ad only small effects.