Purification and characterization of cholesterol sulfotransferase from rat skin.
Previous work has demonstrated that the activity of the enzyme cholesterol sulfotransferase is rapidly and dramatically increased upon squamous differentiation of a variety of epithelial cells in culture, including epidermal keratinocytes. As a step toward understanding the molecular mechanisms underlying this differentiation-related change, we now report the partial purification and characterization of this enzyme activity from rat skin. Supernatant solutions from rat skin homogenates were subjected to a series of column chromatography steps including anion exchange, gel filtration, chromatofocusing and hydrophobic interaction chromatography. The purification procedure resulted in cholesterol sulfotransferase activity purified 2,700-fold with a 11% recovery. The most purified preparation yielded a major Coomassie blue-stained band on denaturing polyacrylamide gel electrophoresis of an apparent molecular weight (MW) of 40,000 Da. Photoaffinity labeling with the donor substrate, 3'-phosphoadenosine-5'-phospho-[35S]-sulfate resulted in a single radiolabeled protein band on denaturing polyacrylamide gel electrophoresis, again of apparent MW 40,000 Da, strongly suggesting that the major Coomassie blue-stained band in the most purified preparation is the cholesterol sulfotransferase protein. Among 3beta-hydroxysteroids with a A5 double bond that were tested, each served as a substrate, while androgens, estrogens, corticosteroids, p-nitrophenol and DOPA did not serve as substrates. Apparent Michaelis constants for the 3beta-hydroxysteroid substrates ranged from 0.6 to 8 microM.
Rearick
J I
JI
Department of Biochemistry, Kirksville College of Osteopathic Medicine, MO 63501, USA. JRearick@kcom.edu
Calhoun
E S
ES
D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D015335:Molecular Probes; D051381:Rats; D012867:Skin / Q000201:enzymology*; D015238:Sulfotransferases / Q000302:isolation & purification* / Q000378:metabolism
D015335:Molecular Probes; D015238:Sulfotransferases; C043456:cholesterol sulfotransferase