Purification and Characterization of NADP+-Dependent 3α-Hydroxysteroid Dehydrogenase from Mouse Liver Cytosol

1988 ◽  
Vol 103 (6) ◽  
pp. 1027-1034 ◽  
Author(s):  
Akira Hara ◽  
Yoshio Inoue ◽  
Makoto Nakagawa ◽  
Fumiharu Naganeo ◽  
Hideo Sawada
Keyword(s):  
Mouse Liver ◽  
Hydroxysteroid Dehydrogenase ◽  
Purification And Characterization ◽  
Liver Cytosol

scholarly journals Purification and Characterization of Platelet-activating Factor Acetylhydrolase II from Bovine Liver Cytosol

1995 ◽  
Vol 270 (38) ◽  
pp. 22308-22313 ◽  
Author(s):  
Kenji Hattori ◽  
Mitsuharu Hattori ◽  
Hideki Adachi ◽  
Masafumi Tsujimoto ◽  
Hiroyuki Arai ◽  
...  
Keyword(s):  
Platelet Activating Factor ◽  
Bovine Liver ◽  
Purification And Characterization ◽  
Liver Cytosol ◽  
Platelet Activating Factor Acetylhydrolase

Purification and Characterization of a 3,17 β-Hydroxysteroid Dehydrogenase from Streptomyces hydrogenans

1979 ◽  
Vol 34 (7-8) ◽  
pp. 533-540 ◽  
Author(s):  
Helmut Duchmann ◽  
Lothar Träger
Keyword(s):  
Gel Electrophoresis ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecylsulfate ◽  
Sedimentation Coefficient ◽  
Hydroxysteroid Dehydrogenase ◽  
Ammonium Sulfate Precipitation ◽  
Purification And Characterization

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.

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