Microsomal 17beta-hydroxysteroid dehydrogenase of guinea pig liver: detergent solubilization and a comparison of kinetic and stability properties of bound and solubilized forms.

Abstract

Abstract Microsomal 17β-hydroxy steroid dehydrogenase of guinea pig liver, solubilized in Triton X-100, was obtained in phospholipid-free form and physical parameters were estimated by agarose gel chromtography and density gradient centrifugation. The values for the sedimentation constant, s20,w = 5.2 s, partial specific volume, v = 0.78 ml/g , and Stokes radius, a = 66 A, indicated a molecular weight of 176,000 for the solubilized enzyme and were consistent with the presence of a significant amount of bound detergent. Triton X-100 was an inhibitor competitive with testosterone with a K1 of 0.11%. With solubilization the apparent km for testosterone was increased from 2.2 μM to 7.3 μM and the apparent KM for NAD+ reduced from 164 to 100 μM. The susceptibility to urea or trypsin inactivation was increased after solubilization. Enzymatic activity was stable for at least 48 h in 1% (v/v) Triton X-100 but was lost within 24 h in deoxycholate. The results show that phospholipids are not absolutely required for activity but the changes in kinetic and stability properties with solubilization are consistent with a role for membrane components or bound detergent in affecting the structure and catalytic properties of the enzyme.