EF-hand motifs of a , b and c isoforms of diacylglycerol kinase bind calcium with different affinities and conformational changes

The three diacylglycerol kinase isoenzymes (DGK a , DGK b and DGK c ) cloned so far contain in common a tandem repeat of EF-hand motifs. However, the Ca (cid:35) + dependences of the DGK activities are known to be variable between isoenzymes, and the Ca (cid:35) + -binding activities of these motifs have not been tested except for those present in DGK a . We therefore attempted to define the intrinsic properties of EF-hands occurring in the DGK isoenzymes. For this purpose we bacterially expressed and purified the EF-hand motifs (termed DKE forms) of the three DGKs. Equilibrium dialysis with the purified DKE forms showed that all of the expressed proteins could bind approx. 2 mol of Ca (cid:35) + per mol. However, the apparent dissociation constant ( K d )