Different effects of sphingosine, R59022 and anionic amphiphiles on two diacylglycerol kinase isozymes purified from porcine thymus cytosol.
Porcine thymus cytosol contains two immunologically distinct forms of diacylglycerol kinase (DGK) [Yamada, K. and Kanoh, H. (1988) Biochem. J. 255, 601-608]. These 2 DGK species, having apparent molecular masses of 80 and 150 kDa, were purified from the thymus cytosol. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 150-kDa DGK gave 2 polypeptide bands of 50 and 75 kDa, whereas the 80-kDa DGK yielded a single protein band. The 80-kDa DGK was markedly activated by 10-20 microM sphingosine as well as by the known anionic activators such as phosphatidylserine and deoxycholate. In contrast, the 150-kDa DGK was fully active in the absence of the anionic activators and was strongly inhibited by sphingosine (IC50, 20 microM). The putative DGK inhibitor R59022 inhibited the 80-kDa DGK (IC50, 10 microM), but had little effect on the 150-kDa form. It is therefore clear that in the thymus cytosol there are at least 2 DGK isozymes operating under different control mechanisms.
Sakane
F
F
Department of Biochemistry, Sapporo Medical College, Japan.
Yamada
K
K
Kanoh
H
H
D000818:Animals; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D003840:Deoxycholic Acid / Q000494:pharmacology; D019852:Diacylglycerol Kinase; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D010718:Phosphatidylserines / Q000494:pharmacology; D010770:Phosphotransferases / Q000302:isolation & purification / Q000378:metabolism*; D011744:Pyrimidinones / Q000494:pharmacology*; D013110:Sphingosine / Q000494:pharmacology*; D013552:Swine; D013844:Thiazoles / Q000494:pharmacology*; D013950:Thymus Gland / Q000201:enzymology*
D007527:Isoenzymes; D010718:Phosphatidylserines; D011744:Pyrimidinones; D013844:Thiazoles; D003840:Deoxycholic Acid; C047505:R 59022; D010770:Phosphotransferases; D019852:Diacylglycerol Kinase; D013110:Sphingosine