Immunochemical determination of human cholesterol 7 alpha-hydroxylase

To produce antibodies for determination of the protein mass of human cholesterol 7 alpha-hydroxylase, a fusion protein was prepared from an in-frame fusion gene containing the cDNA for human cholesterol 7 alpha-hydroxylase near the 3' terminus of the lacZ gene of Escherichia coli. The fusion protein was purified by (NH4)2SO4 fractionation and gel-filtration chromatography on a Sephacryl column. Rabbits were immunized with this fusion protein and antisera were obtained. IgG was prepared by submitting antisera to chromatography on protein-A--Sepharose. Antibodies directed against bacterial proteins including beta-galactosidase were removed by affinity chromatography on a column to which bacterial proteins of E. coli containing beta-galactosidase had been immobilized. Evidence that the antibodies are indeed reactive against human liver cholesterol 7 alpha-hydroxylase was obtained by immunoblot analysis with human cholesterol 7 alpha-hydroxylase expressed in COS cells from the coding region of the human cholesterol 7 alpha-hydroxylase cDNA. The antiserum inhibited the activity of cholesterol 7 alpha-hydroxylase in human liver microsomes by approximately 70%. On immunoblotting of solubilized human liver microsomes, a positive band was obtained at a position corresponding to the protein mass for human cholesterol 7 alpha-hydroxylase. When calibration was performed using the fusion protein, a linear relationship was observed between the density and the amount of protein. Proportionality was also observed between the density and the amount of protein for microsomes of COS cells transfected with the coding region of the human cholesterol 7 alpha-hydroxylase cDNA. Liver microsomes from patients treated with cholestyramine (n = 3) were shown to contain levels of cholesterol 7 alpha-hydroxylase protein approximately twofold higher than those of liver microsomes from untreated patients (n = 6; P < 0.02), whereas cholesterol 7 alpha-hydroxylase activity was approximately six-fold higher in liver microsomes from the cholestyramine-treated patients than in the corresponding preparations from the untreated patients (P < 0.02). The higher activities observed in cholestyramine-treated patients, therefore, cannot be explained only by an increased amount of protein, suggesting a posttranslational mechanism to increase the activity of human cholesterol 7 alpha-hydroxylase in addition to the transcriptional control.