Molecular cloning, expression, and enzymatic characterization of rabbit hydroxysteroid sulfotransferase AST-RB2.
Cytosolic sulfotransferases, which consist of at least three gene families, play a major role in activation and detoxification of both endogenous and exogenous chemicals. We recently purified a rabbit sulfotransferase, AST-RB2, showing high activities to both hydroxysteroids and amines. To characterize this enzyme, a rabbit cDNA library was screened using anti-AST-RB2 antibodies. The isolated cDNA was judged to encode AST-RB2 (ST2A8) based on the amino acid sequences of peptide fragments obtained from purified AST-RB2. The cDNA showed high similarity to other mammalian hydroxysteroid sulfotransferases (ST2) at the amino acid level (58-68%), but low similarity to aryl sulfotransferases (ST1) (less than 37%). The protein expressed in Escherichia coli catalyzed sulfation of typical ST2 substrates. Therefore, ST2A8 was judged to belong to the ST2 family from both its primary structure and substrate specificity. The ST2A8 protein expressed in E. coli clearly differed from rat ST2A1 and ST2A2 on its localization (cytosol/insoluble fraction ratio). ST2A8 had no activity to lithocholate, but showed the highest catalysis on dehydroepiandrosterone and testosterone among the four forms (ST2A1, ST2A2, ST2A3, and ST2A8), indicating a clear difference between ST2A forms in substrate specificity to endogenous chemicals.
Yoshinari
K
K
Division of Drug Metabolism and Molecular Toxicology, Faculty of Pharmaceutical Sciences, Tohoku University, Aramaki-Aoba, Aoba-ku, Sendai 980-8578.
Nagata
K
K
Shiraga
T
T
Iwasaki
K
K
Hata
T
T
Ogino
M
M
Ueda
R
R
Fujita
K
K
Shimada
M
M
Yamazoe
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D008297:Male; D008969:Molecular Sequence Data; D011817:Rabbits; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D015238:Sulfotransferases; C112968:hydroxysteroid sulfotransferase AST-RB2