Physico-Chemical Characterization of the NADPH Dependent Soluble 3α-Hydroxysteroid Oxidoreductase in the Rat Epididymis

The present paper describes some important physico-chemical characteristics of the NADPH dependent, 3α-hydroxysteroid oxidoreductase (3α-oxidoreductase) in the rat epididymis. This enzyme activity is stable in the presence of reducing agents (1–10 mM 2-mercaptoethanol), EDTA (1 mM), and glycerol (10% v/v). The pH optium is in the physiological range (pH 6.0–8.0).
Chromatography of epididymal cytosol fractions on calibrated G-200 columns indicated a molecular weight of 34 000, and sucrose gradient centrifugation a sedimentation coefficient of 3.3 S20,w. The enzyme has a Stokes radius of approximately 25 Å. Calculation of molecular weight using both the sedimentation rate and the Stokes radius indicated a molecular weight of 34 400 and a frictional ratio (f/f₀) of 1.17. The enzyme migrated with an R_x of 0.37 (relative to bromophenol blue) in 6.5% polyacrylamide gels and the iso-electric point (pI) determined by iso-electric focussing in 3.5 % polyacrylamide gels was 5.9. Further indication that the enzyme is negatively charged at neutral pH was obtained by DEAE-cellulose ion exchange chromatography, from which the enzyme activity was eluted between 0.05 and 0.08M KCl.
The epididymal 3α-oxidoreductase is temperature sensitive, showing a greatly reduced activity after preincubation at 50°C for 30 min. Preincubation at 60°C caused a complete loss of enzyme activity. Interestingly, preincubation at 25°C for 30 min seemed to cause a significant increase in enzyme activity. Whether this is due to "temperature activation", "cold inactivation" or to a temperature dependent disappearance of inhibitors is not known.