Isolation of two distinct 3-hydroxysteroid sulfotransferases from the guinea pig adrenal. Evidence for 3 alpha-hydroxy versus 3 beta-hydroxy stereospecificity.
Two physically distinct hydroxysteroid sulfotransferases (HSTs) that demonstrate substrate specificity with respect to the orientation of the ring A 3-hydroxyl group have been isolated from the guinea pig adrenal gland. Nondenaturing liquid-phase isoelectric focusing permitted the separation of the activities, and reverse phase high performance liquid chromatography was used to purify the two proteins to homogeneity. The 3 beta-HST had an apparent molecular mass of 33 kDa and utilized pregnenolone, 17-hydroxypregnenolone, and dehydroepiandrosterone as substrates. The 3 alpha-HST was slightly smaller at 32 kDa and utilized allopregnanolone and androsterone as substrates. The proteins were further distinguished by isoelectric point, immunoreactivity, and tryptic peptide mapping. Peptides isolated from both guinea pig HSTs demonstrated significant amino acid sequence homology (approximately 65% identity) to rat liver HST; however, available sequence data from the two proteins did not yield differences that might account for their stereospecific substrate selectivity. This paper represents the first definitive report demonstrating the existence of discrete HSTs that exhibit substrate specificity based on the stereochemistry of the 3-hydroxyl group.
Driscoll
W J
WJ
Section on Adrenal Cell Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Martin
B M
BM
Chen
H C
HC
Strott
C A
CA
D000311:Adrenal Glands / Q000201:enzymology*; D000738:Androsterone / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003687:Dehydroepiandrosterone / Q000378:metabolism; D006168:Guinea Pigs; D007526:Isoelectric Point; D007700:Kinetics; D008297:Male; D010449:Peptide Mapping; D011280:Pregnanolone / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000302:isolation & purification; D013466:Sulfurtransferases / Q000302:isolation & purification / Q000378:metabolism*
D003687:Dehydroepiandrosterone; D011280:Pregnanolone; D000738:Androsterone; D013466:Sulfurtransferases; D015238:Sulfotransferases; C036994:pregnenolone sulfotransferase; C028152:alcohol sulfotransferase