Analysis of two diacylglycerol kinase activities in mixed micelles

This article presents and summarizes studies on the enzymology of two DGKs, the Dictyostelium enzyme termed DGKA and the related human enzyme called DGK-θ, in commonly used mixed micellar assay systems. DGKA catalyzes the ATP-dependent phosphorylation of both medium, DiC8 (1,2-dioctanoyl-sn-glycerol) and DiC6 (1,2-dihexanoyl-sn-glycerol), and long-chain DAGs with pH optima of 7.4 and 7.0, respectively. The presence of PS increases the activity of both DGK-θ and DGKA although, interestingly, PS broadens the pH profile of DGK-θ. Importantly, in addition to PS, PA also increases DGKA activity. Kinetically, DGKA phosphorylates the medium-chain DAGs in a Michaelis-Menten manner. Interestingly, the kinetics of DGKA using physiologically relevant long-chain DAGs is dependent on bulk and surface substrate concentration, and the detergent used. At relatively low bulk concentrations, DGKA displayed Michaelis-Menten kinetics with respect to the bulk substrate concentration (1,2-dioleoyl-sn-glycerol) in octylglucoside mixed micelles when the surface substrate concentration was at or below 3.5 mol%. At higher surface concentrations and the relatively low bulk substrate concentrations, however, there was a sigmoidal relationship between the initial velocity and bulk substrate concentration. DGK-θ also showed sigmoidal kinetics when the bulk substrate concentration was low. Interestingly, DGKA displayed sigmoidal kinetics with respect to the relatively low bulk substrate concentrations at all surface concentrations in Triton X-100 mixed micelles. At higher bulk concentrations, DGKA displayed hyperbolic kinetics. It is important to reiterate that these assays will not yield "true" primary kinetic parameters but do allow us to compare our results with previously published data. Future studies are focused on liposome based assays to obtain more realistic kinetic parameters to assist us in structure function studies of these enzymes.