坂根 郁夫

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.

All mammalian diacylglycerol kinase (DGK) isoenzymes so far cloned consist of four conserved regions, namely C1, C2 (tandem EF-hand structures), C3 (tandem cysteine-rich zinc finger sequences) and the C-terminal C4 domains. To determine the catalytic domain we expressed in COS-7 cells various truncation mutants of pig DGK alpha and assessed their enzyme activities. We found that the C4 domain lacking the whole N-terminal region including the zinc fingers possessed DGK activity that was dependent on the concentrations of diacylglycerol and ATP very similarly, as did the wild-type DGK alpha. Furthermore the DGK activity of the wild-type DGK and that expressed by the C4 domain were similarly activated by anionic amphiphiles such as phosphatidylserine, phosphatidylinositol and deoxycholate. It was also shown that a DGK mutant consisting of the zinc fingers and the C4 domain has enzymological properties very similar to those expressed by the C4 domain alone. We also confirmed that the intact DCKs alpha, beta and gamma expressed in COS-7 cells displayed no detectable phorbol ester binding. These results show that the C4 domain of DGK is the catalytic region that is responsible for the enzyme activities sensitive to different activators. We cannot exclude the possibility that the N-terminal portion including the zinc fingers can still interact with diacylglycerol and activators without affecting the enzyme activity measured in vitro. However, it is quite likely that the DGK zinc fingers do not serve as diacylglycerol-binding sites, in contrast with those present in other proteins such as protein kinases C and n-chimaerin. Site-directed mutagenesis of all six putative ATP binding sites (Lys(248), Lys(383), Lys(395), Lys(483), Lys(492), and Lys(554)) did not significantly affect the enzyme activity. We therefore suggest that DGK does not contain a typical P-loop of ATP binding sites.

Diacylglycerol kinase (DGK) may regulate glycerolipid biosynthesis as well as cellular signal transduction by phosphorylating diacylglycerol back to phosphatidic acid. The cDNA cloning of DGK has revealed the presence of a novel enzyme family having unique structures. Already 4 species (designated α-δ) of animal DGK family members have been cloned and much more to be sequenced in the near future. Further work based on the knowledge obtained by the cDNA cloning would reveal the biological implication of the multiple DGK subspecies.

Cytochrome-b and flavin adenine dinucleotide (FAD) were measured in neutrophils from a male patient and his family with the X-linked form of Chronic Granulomatous Disease (CGD). The membrane fraction of the patient's neutrophils contained 93pmol/mg protein of FAD (control 102±20, mean±sd), and 32pmol/mg protein of cytochrome-b (control 88±20, mean±sd). In the spectrophotometric study, the maximal absorbance peak of cytochrome-b was at 421 nm, as that from control was at 427 nm. Cytochrome-b in neutrophils from his mother and a sister, who were thought to be carriers, had their maximal absorbance peak at 425 nm. These findings suggest that the spectral abnormality of cytochrome-b contribute to the pathogenesis of CGD in this case. © 1985, The Japan Society for Clinical Immunology. All rights reserved.