Human sphingosine kinase: purification, molecular cloning and characterization of the native and recombinant enzymes.
Sphingosine 1-phosphate (S1P) is a novel lipid messenger that has important roles in a wide variety of mammalian cellular processes including growth, differentiation and death. Basal levels of S1P in mammalian cells are generally low, but can increase rapidly and transiently when cells are exposed to mitogenic agents and other stimuli. This increase is largely due to increased activity of sphingosine kinase (SK), the enzyme that catalyses its formation. In the current study we have purified, cloned and characterized the first human SK to obtain a better understanding of its biochemical activity and possible activation mechanisms. The enzyme was purified to homogeneity from human placenta using ammonium sulphate precipitation, anion-exchange chromatography, calmodulin-affinity chromatography and gel-filtration chromatography. This resulted in a purification of over 10(6)-fold from the original placenta extract. The enzyme was cloned and expressed in active form in both HEK-293T cells and Escherichia coli, and the recombinant E. coli-derived SK purified to homogeneity. To establish whether post-translational modifications lead to activation of human SK activity we characterized both the purified placental enzyme and the purified recombinant SK produced in E. coli, where such modifications would not occur. The premise for this study was that post-translational modifications are likely to cause conformational changes in the structure of SK, which may result in detectable changes in the physico-chemical or catalytic properties of the enzyme. Thus the enzymes were characterized with respect to substrate specificity and kinetics, inhibition kinetics and various other physico-chemical properties. In all cases, both the native and recombinant SKs displayed remarkably similar properties, indicating that post-translational modifications are not required for basal activity of human SK.
Pitson
S M
SM
Hanson Centre for Cancer Research, Division of Human Immunology, Institute of Medical and Veterinary Science, Frome Road, Adelaide 5000, SA, Australia.
D'andrea
R J
RJ
Vandeleur
L
L
Moretti
P A
PA
Xia
P
P
Gamble
J R
JR
Vadas
M A
MA
Wattenberg
B W
BW
D000595:Amino Acid Sequence; D000645:Ammonium Sulfate / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002460:Cell Line; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000166:cytology; D004789:Enzyme Activation; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification*; D010920:Placenta / Q000201:enzymology; D011487:Protein Conformation; D020033:Protein Isoforms; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature; D014470:Umbilical Cord / Q000166:cytology; D000099157:Sphingosine Kinase
D000645:Ammonium Sulfate; D002147:Calmodulin; D010743:Phospholipids; D017853:Phosphotransferases (Alcohol Group Acceptor); D020033:Protein Isoforms; D011994:Recombinant Proteins; D000099157:Sphingosine Kinase