Ceramide is a competitive inhibitor of diacylglycerol kinase in vitro and in intact human leukemia (HL-60) cells.

摘要: Prior studies demonstrated that ceramide was phosphorylated by a novel Ca(2+)-dependent kinase distinct from diacylglycerol (DG) in human myelogenous leukemia (HL-60) cells (Kolesnick, R. N., and Hemer, M. (1990) J. Biol. Chem. 265, 10900-10904). The present were initiated to determine whether mammalian DG purified homogeneity possessed phosphotransferase activity toward ceramide. A high molecular weight rat brain Mg(2+)-(but not Ca(2+)-) dependent did phosphorylate the presence of either cation. In contrast, served as competitive inhibitor with an inhibition constant (Ki) 2-6-fold greater than Km for DG. Inhibition noncompetitive respect ATP Mg2+. cell-permeable ceramide, N-octanoyl sphingosine (C8-cer), used study effects on intact HL-60 cells. C8-cer induced dose- time-dependent increases cellular levels. As little 1 microM increased basal level 103 177 pmol.10(6) cells-1, maximal 2.9-fold elevation 292 cells-1 occurred 10 C8-cer. detected after min, 7.5 sustained 30 min. accompanied reduction 32P incorporation phosphatidic acid short term-labeled [32P]orthophosphoric acid, consistent kinase. similar exogenous phospholipase C into acid. metabolized sphingomyelin, indicating generated through phosphatidylcholine:ceramide cholinephosphotransferase reaction. or treatment sufficient redistribute protein cytosol membrane. These findings provide evidence may serve