title: EF-hand motifs of alpha, beta and gamma isoforms of diacylglycerol kinase bind calcium with different affinities and conformational changes.
abstract: 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 Ca(2+)-binding activities of these motifs have not been tested except for those present in DGK alpha. 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 Ca2+ per mol. However, the apparent dissociation constant (Kd) for calcium binding to alpha-DKE (9.9 microM) was an order of magnitude greater than those estimated for beta-DKE (0.89 microM) and gamma-DKE (0.40 microM). Experiments with 2-p-toluidinyl-naphthalene 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 Ca(2+)-induced conformational changes.
authors.affiliation: Department of Liberal Arts and Sciences, School of Health Sciences, Sapporo Medical University, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Yamada
authors.forename: F
authors.initials: F
authors.lastname: Sakane
authors.forename: N
authors.initials: N
authors.lastname: Matsushima
authors.forename: H
authors.initials: H
authors.lastname: Kanoh
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D019852:Diacylglycerol Kinase; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D007527:Isoenzymes / Q000378:metabolism*; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D011487:Protein Conformation
chemical_list: D007527:Isoenzymes; D017853:Phosphotransferases (Alcohol Group Acceptor); D019852:Diacylglycerol Kinase; D002118:Calcium