Purification and characterization of a 3,17 beta-hydroxysteroid dehydrogenase from Streptomyces hydrogenans.
نویسندگان
چکیده
3,17 ß-Hydroxysteroid dehydrogenase has been enriched and purified from cytosol o f Strepto myces 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 chro matography. Polyaciylam ide gel electrophoresis in the presence o f sodium dodecylsulfate gave a single band o f mobility corresponding to molecular weight o f 70 200 ± 2 500. 3 ß-, 17 ßas well as 20p-hydroxy steroids were dehydrogenated by the enzyme in the presence o f N A D + . The dehy drogenation proceeded faster than the reduction o f the corresponding ketosteroids in the presence o f NA DH . The enzyme does not accent N A D P + or NADPH as co-substrates. The apparent K m values were calculated to be 11 jam for 5 a -dihydrotestosterone, 20^m for testosterone and 68 [jlm for epiandrosterone in the N A D + -driven reaction, 1.8x 10-4 m for N A D +and 1.9x 10-4 M for N A D H . The catalytic activity was influenced by the ratio o f N A D +/ATP. The inhibition by ATP appears to be o f a competitive type with respect to N A D + (K x 1.15 x 10“3 m). After sucrose gradient centrifugation in a preparative ultracentrifuge the enzyme sediments with 4.1 ± 0.1 S as estimated in comparison to other proteins o f known sedimentation coefficient. The isoelectric point was determined to be 3.9 with the LKB preparative isoelectric focusing col umn (pH 2 —11) and 4.1 with the analytical flat bed polyacrylamide isofocusing (pH 3 5). The number o f SH groups was determined to be 2 m ol/m ol enzyme. In the presence o f 6 M urea the fig ure inceases to 3 mol SH /m ol enzyme. In the presence o f an excess o f /»-chloromercuribenzoate the enzyme activity decreases only partially.
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ورودعنوان ژورنال:
- Zeitschrift fur Naturforschung. Section C, Biosciences
دوره 34 7-8 شماره
صفحات -
تاریخ انتشار 1979