Inhibition of sphingosine kinase in vitro and in platelets. Implications for signal transduction pathways.
Sphingosine kinase was partially purified and characterized from rat brain microsomes. A new assay, utilizing octyl-beta-D-glucopyranoside and sphingosine mixed micelles, was developed to quantitate formation of the sphingosine-1-phosphate product. The assay was proportional with respect to time and protein, displayed Michaelis-Menten kinetics, and was subject to surface dilution in regard to the sphingosine substrate. Investigations into substrate specificity showed that the enzyme is specific for the erythro-enantiomers of sphingosine and dihydrosphingosine. Neither of the threo-enantiomers were phosphorylated in this system, but both were found to be potent competitive inhibitors of sphingosine kinase activity. Human platelet sphingosine kinase activity displayed substrate and inhibitor specificities similar to the rat brain enzyme. A mixture of DL-threo-dihydrosphingosine competitively inhibited sphingosine kinase activity in a dose dependent manner in isolated platelets. DL-Threo-dihydrosphingosine caused a prolongation of the inhibition of thrombin-induced protein kinase C-dependent 40 (47)-kDa protein phosphorylation in platelets. D-, L-, or DL-Threo-dihydrosphingosine may be useful as a tool to investigate D-Erythrosphingosine metabolism and the function of sphingosine-1-phosphate in signal transduction processes.
Buehrer
B M
BM
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
Bell
R M
RM
D000818:Animals; D001792:Blood Platelets / Q000201:enzymology*; D001921:Brain / Q000201:enzymology*; D006801:Humans; D007536:Isomerism; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D051381:Rats; D013110:Sphingosine / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors; D000099157:Sphingosine Kinase
D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); D013110:Sphingosine; D000099157:Sphingosine Kinase