Purification and characterization of 3'-phosphoadenosine-5'-phosphosulfate:GalCer sulfotransferase from human renal cancer cells.
We have purified 3'-phosphoadenosine-5'-phosphosulfate:GalCer sulfotransferase [EC 2.8.2.11] from a human renal cancer cell line SMKT-R3 through a combination of affinity chromatographies using galactosylsphingosine, 3',5'-bisphosphoadenosine and heparin as ligands. The purified sulfotransferase showed a specific activity of 1.2 mumol/min/mg, which is 300 times more than the highest activity among the enzyme preparations purified so far from other sources. Homogeneity of the purified sulfotransferase was supported by the facts that the enzyme preparation showed a single protein band with an apparent molecular mass of 54 kDa on reducing SDS-PAGE and that protein bands coincided with the enzyme activity on both native PAGE and nonreducing SDS-PAGE. GalCer was the best acceptor for the purified enzyme. LacCer, GalAAG, and GalDG were also good acceptors. GlcCer, Gg3Cer, Gg4Cer, Gb4Cer, and nLc4Cer did serve as acceptors although the relative activities were low. On the other hand, the enzyme could not act on Gb3Cer, which possesses alpha-galactoside at the nonreducing terminus. Neither galactose nor lactose served as an acceptor. These observations suggest that the sulfotransferase prefers beta-glycoside, especially beta-galactoside, at the nonreducing termini of sugar chains attached to a lipid moiety.
Honke
K
K
Biochemistry Laboratory, Hokkaido University School of Medicine, Sapporo.
Yamane
M
M
Ishii
A
A
Kobayashi
T
T
Makita
A
A
D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000494:pharmacology; D006017:Glycolipids / Q000378:metabolism; D006801:Humans; D007680:Kidney Neoplasms / Q000201:enzymology*; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D006017:Glycolipids; D004815:Epidermal Growth Factor; D015238:Sulfotransferases; C020556:galactosylceramide sulfotransferase