Purification and characterization of a 3,17 beta-hydroxysteroid dehydrogenase from Streptomyces hydrogenans
| Autor: | Lothar Träger, Helmut Duchmann |
|---|---|
| Rok vydání: | 1979 |
| Předmět: |
Gel electrophoresis
Chromatography 3-Hydroxysteroid Dehydrogenases biology Chemistry Isoelectric focusing Dehydrogenase Dihydrotestosterone General Biochemistry Genetics and Molecular Biology Enzyme assay Streptomyces Substrate Specificity Molecular Weight Kinetics Isoelectric point Sephadex biology.protein Polyacrylamide gel electrophoresis Chloromercuribenzoates Ammonium sulfate precipitation |
| Zdroj: | Zeitschrift fur Naturforschung. Section C, Biosciences. 34(7-8) |
| ISSN: | 0341-0382 |
| Popis: | 3,17 β-Hydroxysteroid dehydrogenase has been enriched and purified from cytosol of Streptomyces hydrogenans. After ammonium sulfate precipitation and filtration on Sephadex G-100 the enzyme was finally purified by preparative gel electrophoresis and DEAE-Sephadex A-50 chromatography. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate gave a single band of mobility corresponding to molecular weight of 70 200 ± 2 500. 3 β-. 17 β- as well as 20 β-hydroxy steroids were dehydrogenated by the enzyme in the presence of NAD+. The dehydrogenation proceeded faster than the reduction of the corresponding ketosteroids in the presence of NADH. The enzyme does not accent NADP+ or NADPH as co-substrates. The apparent Km values were calculated to be 11 μᴍ for 5 α-dihydrotestosterone, 20 μᴍ for testosterone ana 68 μᴍ for epiandrosterone in the NAD+-driven reaction, 1.8 x 10-4 m for NADH+ and 1.9 x 10-4 ᴍ for NADH. The catalytic activity was influenced by the ratio of NAD+/ATP. The inhibition by ATP appears to be of a competitive type with respect to NAD+ (Ki 1.15 x 10-3 ᴍ). After sucrose gradient centrifugation in a preparative ultracentrifuge the enzyme sediments with 4.1 ± 0.1 S as estimated in comparison to other proteins of known sedimentation coefficient. The isoelectric point was determined to be 3.9 with the LKB preparative isoelectric focusing column (pH 2-11) and 4.1 with the analytical flat bed polyacrylamide isofocusing (pH 3 - 5). The number of SH groups was determined to be 2 mol/mol enzyme. In the presence of 6 M urea the figure inceases to 3 mol SH/mol enzyme. In the presence of an excess of p-chloromercuribenzoate the enzyme activity decreases only partially. |
| Databáze: | OpenAIRE |
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