EF-hand motifs of alpha, beta and gamma isoforms of diacylglycerol kinase bind calcium with different affinities and conformational changes

The three diacylglycerol kinase isoenzymes (DGK alpha, DGK beta and DGK gamma) cloned so far contain in common a tandem repeat of EF-hand motifs. However, the Ca2+ dependences of the DGK activities are known to be variable between isoenzymes, and the Ca2+-binding activities of these motifs have not been tested except for those present in DGK alpha. We therefore attempted to define the intrinsic properties bf 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 Ca2+ per mol. However, the apparent dissociation constant (K-d) for calcium binding to alpha-DKE (9.9 mu M) was an order of magnitude greater than those estimated for beta-DKE (0.89 mu M) and gamma-DKE (0.40 mu M). Experiments with 2-p-toluidinylnaphthalene 6-sulphonate, a probe for hydrophobic regions of proteins, showed that the binding of Ca2+ to beta-DKE resulted in the exposure of hydrophobic amino acids, whereas hydrophobic regions of alpha-DKE and gamma-DKE were masked by the addition of Ca2+. Taken together, these results indicate that DGK alpha, DGK beta and DGK gamma possess EF-hand structures with intrinsic properties different from each other with respect to affinities for Ca2+ and Ca2+-induced conformational changes.