Purification and characterization of cholesterol sulfotransferase from rat skin

Document Type

Article

Publication Title

Biochemistry and Cell Biology

Abstract

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 2700-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 3β-hydroxysteroids with a Δ5 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 3β-hydroxysteroid substrates ranged from 0.6 to 8 μM.

First Page

499

Last Page

506

DOI

10.1139/o01-132

Publication Date

1-1-2001

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