Subcellular distribution and properties of 17 beta-hydroxysteroid dehydrogenase in pregnant rabbit uterus.
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Biomedical subjects
Publications and source records attributed to H Hesslinger.
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Utilizing male rat liver cells we describe a method for isolating and fractionating chromosomal proteins. About 99% of chromosomal proteins was dissociated using a three step dissociation procedure. DNA was removed by sedimentation and the histone fractions were separated from the non-histone chromosomal proteins by Bio Rex 70 chromatography. The non-histone chromosomal proteins were fractionated by micro-gradient electrophoresis on SDS-polyacrylamide gels, which proved to be superior to the electrophoretic procedures currently in use. The histones were further separated on polyacrylamide-SDS slab gels using a micro-two-dimensional electrophoretic system. The high resolution of these fractionation procedures greatly enhances the possibility of observing small changes in proteins which may play a role in gene regulation.
17 beta-Hydroxysteroid dehydrogenase activity towards estradiol-17 beta has been demonstrated in the 105,000 x g supernatant of rabbit uterus. Hydroxylapatite chromatography of the enzyme activity isolated by ammonium sulfate precipitation, gel filtration and DEAE-cellulose chromatography yielded a single 17 beta-hydroxysteroid dehydrogenase activity. Further purification of the enzyme preparation by isoelectric focusing resulted in multiple peaks of activity. The molecular weight of the enzyme, caculated from mobility data on Sephadex gel, is approximately 64,000. Some properties of partially purified 17 beta-hydroxysteroid dehydrogenase activity have been studied. Estradiol-17 beta reacts at a faster rate than testosterone. The Km for estradiol is 4.16 x 10(-5) mol/l for the NAD-linked enzyme activity and 4.37 x 10(-5) mol/l when NADP as cofactor was used. The ratio of the maximal velocity for NADP to that for NAD was 1.42. The pH-optimum for estradiol appears between 9.5 and 10.5 and for estrone between 5.5 and 6.5. The enzyme appears to be of the sulfhydryl type.