Porcine 80-kDa diacylglycerol kinase is a calcium-binding and calcium/phospholipid-dependent enzyme and undergoes calcium-dependent translocation.
We attempted to assess the regulatory role of EF-hand motifs recently detected in the primary structure of porcine 80-kDa diacylglycerol kinase (DGK) (Sakane, F., Yamada, K., Kanoh, H., Yokoyama, C., and Tanabe, T. (1990) Nature 344, 345-348). By using 80-kDa DGK purified from porcine thymus cytosol, we found that this isozyme indeed bound 2 mol Ca2+ per mol enzyme with high affinity (apparent dissociation constant, kd = 0.3 microM). The Ca2+ binding was cooperative with a Hill coefficient of 1.4. We next studied the effect of 1 x 10(-5) M Ca2+ on the kinetic properties of DGK employing a beta-octyl glucoside mixed micellar assay system. In the absence of Ca2+, phosphatidylserine, so far used as an enzyme activator in various assay systems, was rather inhibitory, and Ca2+ alone activated enzyme to a limited extent. However, phosphatidylserine plus Ca2+ markedly activated the enzyme, giving approximately 4-fold higher Vmax and 10-fold less Km values for ATP. In contrast, the apparent Km values for diacylglycerol were not significantly affected (approximately 3 mol %). Furthermore, by immunoblotting using anti-80 kDa DGK antibodies we found that the soluble DGK in the homogenate of porcine thymocytes was translocated to membranes in a Ca2(+)-dependent manner. Indeed we noted the presence of a 33-residue amphipathic alpha-helix in the DGK sequence, which may account for the protein-lipid interaction. The results demonstrate that Ca2+ plays a key role in the regulation of DGK action by controlling enzyme interaction with membrane phospholipids.
Sakane
F
F
Department of Biochemistry, Sapporo Medical College, Japan.
Yamada
K
K
Imai
S
S
Kanoh
H
H
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D019852:Diacylglycerol Kinase; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010718:Phosphatidylserines / Q000494:pharmacology*; D010770:Phosphotransferases / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013552:Swine; D013950:Thymus Gland / Q000201:enzymology
D010718:Phosphatidylserines; D010770:Phosphotransferases; D019852:Diacylglycerol Kinase; D002118:Calcium