Purification and characterization of two amine N-sulfotransferases, AST-RB1 (ST3A1) and AST-RB2 (ST2A8), from liver cytosols of male rabbits.
Two sulfotransferases (STs), designated as AST-RB1 (ST3A1) and AST-RB2 (ST2A8), with high a amine N-sulfonating activity, were purified from male rabbit liver cytosols. AST-RB1 and AST-RB2 were purified to homogeneity by the anion-exchange, affinity, and hydroxyapatite chromatography. The N-terminus of both enzymes were blocked. The subunit molecular mass of both enzymes was estimated to be 34 kDa on SDS-PAGE. AST-RB1 efficiently catalyzed N-sulfonation of alicyclic, alkyl, and arylamines such as 4-phenyl-1,2,3, 6-tetrahydropyridine, 1-[(5-chloro-2-oxo-3(2H)-benzothiazolyl)acetyl]-piperazine, desipramine, and aniline, whereas its catalytic activities toward 2-naphthol and dehydroepiandrosterone (DHEA) were very low. On the other hand, AST-RB2 efficiently catalyzed sulfonation of desipramine and DHEA, but had no activity toward 2-naphthol. Amino acid sequences of peptide fragments derived from the purified AST-RB1 showed no significant homology with previously reported STs, but those from the purified AST-RB2 shared a high similarity with those of the ST2 family. Both enzymes were expressed specifically in the liver. The present results strongly suggest that the purified AST-RB1 is a novel enzyme in terms of structure and catalytic properties showing high selectivity for amine substrates, and AST-RB2 is a quite unique from among ST2A enzymes of other species in its substrate specificity.
Shiraga
T
T
Biopharmaceutical and Pharmacokinetic Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., 1-6, Kashima 2-chome, Yodogawa-ku, Osaka, 532-8514, Japan.
Iwasaki
K
K
Hata
T
T
Yoshinari
K
K
Nagata
K
K
Yamazoe
Y
Y
Ohno
Y
Y
D000588:Amines / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002853:Chromatography, Liquid; D003429:Cross Reactions; D003600:Cytosol / Q000201:enzymology*; D003687:Dehydroepiandrosterone / Q000378:metabolism; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008969:Molecular Sequence Data; D009284:Naphthols / Q000378:metabolism; D009928:Organ Specificity; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism