|Budget Amount *help
¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1992 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 1991 : ¥1,400,000 (Direct Cost : ¥1,400,000)
1. Effect of sphingosine On Diacylglyceriol kinase(DGK) isozymes in intact jurkat cells.
Sphingoshine which is the activator of purified 80 kDa DGK markrdly activated the phosphorylation of both endogenous and exogenous DGs in intact Jurkat cells. We thought that the increased phosphatidic acid(PA) accumulation by sphingosine may be a direct activation of 80 kDa DGK which was enriched in Jurkat cells. But recently, it was suggested that the plural isozymes contributed the phosphorylation of intracellular DGs. So we reevaluated the isozyme composition in Jurkat cells using combination of different DGs (arachidonoyl-and didecanoyl DGs) as the substrate and two different assay methods (octylglucoside micellar and deoxycholate suspension assays). The results suggested the presence of at least four DGK isozymes, which could be distinguwished from each other with respect to intracellular localization, specificity to DG molecular species, responsiveness to sphingosine, and reactivity to anti-8
0 kDa DGK antibody(in submitted)., we are now examining the role of DGK during differenciation of HL 60.
2. Primary structure of 80 kDa DG kinase.
Primary structure of this enzyme contains the putative ATP-binding sites, two cysteine-rich zinc finger-like sequences and two E-F hand motifs. We found that the purified DGK binds 2 mol of Ca^<2+> per mol of enzyme with high affinity (J. Biol. Chem. 266, 7096). And to elucidate the reguratory function of E-F hand motifs, we constructed the expressed several truncation and deletion mutants of the enzyme in E.Coil or COS-7 cells(Biochem. Biophys. Res. Commun. 181, 1015). Moreover, we investigated the binding for DG, phospholipids, phorbol ester, or DNA using baculovirus expression system(in preparation).
3. Primary structure of other DGK isozymes.
We could not get the cDNA clone coding for 150 kDa DGK. And now oligonucleotides with high homology between 80 kDa and 90 kDa (nerve cell specific) DGK were prepared and used in the polymerase chain reaction. Amplified DNA fragments was subsequently used to clone the DGK isozyme cDNAs. Less