Purification and characterization of rat kidney sphingosine kinase.
Sphingosine kinase catalyzes the formation of the bioactive sphingolipid metabolite sphingosine 1-phosphate, which plays important roles in numerous physiological processes, including growth, survival, and motility. We have purified rat kidney sphingosine kinase 6 x 10(5)-fold to apparent homogeneity. The purification procedure involved ammonium sulfate precipitation followed by chromatography on an anion exchange column. Partially purified sphingosine kinase was found to be stabilized by the presence of high salt, and thus, a scheme was developed to purify sphingosine kinase using sequential dye-ligand chromatography steps (since the enzyme bound to these matrices even in the presence of salt) followed by EAH-Sepharose chromatography. This 385-fold purified sphingosine kinase bound tightly to calmodulin-Sepharose and could be eluted in high yield with EGTA in the presence of 1 M NaCl. After concentration, the calmodulin eluate was further purified by successive high pressure liquid chromatography separations on hydroxylapatite, Mono Q, and Superdex 75 gel filtration columns. Purified sphingosine kinase has an apparent molecular mass of approximately 49 kDa under denaturing conditions on SDS-polyacrylamide gel, which is similar to the molecular mass determined by gel filtration, suggesting that the active form is a monomer. Sphingosine kinase shows substrate specificity for D-erythro-sphingosine and does not catalyze the phosphorylation of phosphatidylinositol, diacylglycerol, ceramide, DL-threo-dihydrosphingosine, or N,N-dimethylsphingosine. However, the latter two sphingolipids were potent competitive inhibitors. With sphingosine as substrate, the enzyme had a broad pH optimum of 6.6-7.5 and showed Michaelis-Menten kinetics, with Km values of 5 and 93 microM for sphingosine and ATP, respectively. This study provides the basis for molecular characterization of a key enzyme in sphingolipid signaling.
Olivera
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A
Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, D. C. 20007, USA.
Kohama
T
T
Tu
Z
Z
Milstien
S
S
Spiegel
S
S
D000818:Animals; D002850:Chromatography, Gel; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity; D000099157:Sphingosine Kinase
D017853:Phosphotransferases (Alcohol Group Acceptor); D000099157:Sphingosine Kinase