An Improved Method for the Determination of Cytoplasmic 3α- and 17β- and Microsomal 3α-Hydroxysteroid Dehydrogenase Activities in Rat Liver

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Abstract
A modified method was described for the radiometric determination of hydroxysteroid dehydrogenase activities in rat liver. The principal advantages of this method are the improved precision and a radical reduction in the time involved in performing the assay. The procedure comprises incubation of 14C-labeled substrate with coenzyme and cell fraction under optimized conditions; termination of the reaction by addition of organic solvent containing a defined amount of 3H-labeled reaction product; removal of precipitated protein and coenzyme by centrifugation; paper chromatographic isolation of the product; direct quantitation of 14C activity in the product zone of the paper chromatogram. The assay systems were applied to elucidate and quantitate sex and strain differences in the activities of the above enzymes in Chbb/THOM and Sprague-Dawley rats.