Purification and characterization of cytosolic diacylglycerol kinases of human platelets.
Three isozymes of diacylglycerol kinase (DGK), DGK-I, DGK-II, and DGK-III, were purified from the cytosol of human platelets by successive chromatography on DEAE-cellulose, Ultrogel AcA34, heparin-Sepharose, ATP-agarose, Mono Q, phenyl-Superose, HCA-hydroxyapatite, Wakopak G40, and TSK-3000SW columns. Two DGK species (DGK-I and DGK-III) were purified to apparent homogeneity, and upon SDS-polyacrylamide gel electrophoresis, they showed a single band of apparent molecular mass of 152 kDa (DGK-I) or 58 kDa (DGK-III). The peptide mapping analysis showed that DGK-I and DGK-III are structurally different. DGK-II was only partially purified, and its apparent Mr was estimated to be 75,000 by gel filtration. The specific enzyme activities of the three isozymes were increased 1,480-fold (DGK-I), 690-fold (DGK-II) and 2,100-fold (DGK-III) over original platelet cytosol. The activities of DGK-II and DGK-III were markedly enhanced by the presence of deoxycholate or phosphatidylserine, whereas DGK-I activity was not much affected by the anionic compounds. All of the three activities were strongly suppressed by phosphatidylcholine. Triton X-100 and octyl glucoside were strongly inhibitory to all of the enzymes, although to different extents. The DGK inhibitor, R59022, inhibited DGK-II and to a lesser extent DGK-III, but little affected DGK-I activity. DGK-I was much more heat-stable than DGK-II and DGK-III. The Km values for ATP were 150 microM for DGK-I, 245 microM for DGK-II, and 450 microM for DGK-III. The apparent Km values for suspended diolein were not much different among the DGKs and were in the range of 50-80 microM. These observations indicate that human platelet cytosol contains DGK isozymes with different enzymological properties. Furthermore, the three DGKs isolated from human platelets were found not to cross-react with the antibody raised against porcine brain 80-kDa DGK, thus indicating that human platelets contain novel species of DGK.
Yada
Y
Y
Department of Biochemistry, Gifu University School of Medicine, Japan.
Ozeki
T
T
Kanoh
H
H
Nozawa
Y
Y
D001792:Blood Platelets / Q000201:enzymology*; D002845:Chromatography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D019852:Diacylglycerol Kinase; D017886:Durapatite; D006801:Humans; D006882:Hydroxyapatites; D007527:Isoenzymes / Q000097:blood* / Q000302:isolation & purification; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D010770:Phosphotransferases / Q000097:blood* / Q000302:isolation & purification
D006882:Hydroxyapatites; D007527:Isoenzymes; D017886:Durapatite; D010770:Phosphotransferases; D019852:Diacylglycerol Kinase; D008274:Magnesium