Properties of diacylglycerol kinase purified from bovine brain

Abstract

A nearly homogeneous but somewhat unstable diacylglycerol kinase (ca. MW 72,000 daltons) was purified from bovine brain by modification of the procedure of Kanoh et al. (Kanoh, H., Kondoh, H., and Ono, T. [1983]J. Biol. Chem. 258, 1767–1774). The purification consisted of four steps (brain cytosol isolation and successive chromatography on DEAE-cellulose, Sephadex G-25 for desalting and ATP-agarose) carried out in buffers stabilized with EDTA, ATP and dithiothreitol (DTT). Specific activities, determined within 4 hr of purification, ranged from 908–1857 nmol ATP incorporated/min/mg protein, with the variation reflecting the instability. Optimal activities required deoxycholate (0.1%), one of the phosphoglycerides [phosphatidylcholine (PC), phosphatidylethanolamine (PE) or phosphatidylserine (PS)] (0.025–0.25 mM), ATP (5 mM, apparent K_m=0.57 mM), 1,2-dioleoyl-rac-glycerol (5 mM, apparent K_m=1 mM) and Mg²⁺ (10 mM, apparent K_m=2.2 mM). Phosphatidylinositol (PI) was slightly less effective than PC, PE or PS and noninhibitory in combination with PC, PE or PS. Relative to PC phosphatidic acid (PA) (52%), sphingomyelin (48%), lyso-PC (1.5%) and lyso-PI (28.6%) were less effective activators. The sulfhydryl reagents,p-chloromercuribenzoic acid (PCMB) (1.0 mM),N-ethylmaleimide (NEM) (1.0 and 2.0 mM) and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) (1.0 mM), showed strong inhibition of activity which was prevented by 0.5 mM DTT. In contrast to other reports, this purified enzyme showed no monoacylglycerol kinase activity. Comparison of diacylglycerols of varying fatty acid composition indicated that the enzyme showed a preference for substrates with at least one unsaturated fatty acid, particularly in the 2-position. With saturated fatty acids the order of preference was C₁₀ and C₁₂>C₁₄>C₁₆>C₁₈. Such a pattern indicates that the enzyme shows little selectivity that favors the generation of particular molecular species of PA.