IMMUNOCHEMICAL DETERMINATION OF HUMAN CHOLESTEROL 7-ALPHA-HYDROXYLASE

To produce antibodies for determination of the protein mass of human c holesterol 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)(2)SO4 fractionation and gel -filtration chromatography on a Sephacryl column. Rabbits were immuniz edwith this fusion protein and antisera were obtained. IgG was prepare d by submitting antisera to chromatography on protein-A-Sepharose. Ant bodies directed against bacterial proteins including beta-galactosidas e were removed by affinity chromatography on a column to which bacteri al proteins of E. coli containing beta-galactosidase had been immobili zed. Evidence that the antibodies are indeed reactive against human li ver cholesterol 7 alpha-hydroxylase was obtained by immunoblot analysi s with human cholesterol 7 alpha-hydroxylase expressed in COS cells fr om the coding region of the human cholesterol 7 alpha-hydroxylase cDNA . The antiserum inhibited the activity of cholesterol 7 alpha-hydroxyl ase 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 pro tein, a linear relationship was observed between the density and the a mount of protein. Proportionality was also observed between the densit y and the amount of protein for microsomes of COS cells transfected wi th the coding region of the human cholesterol 7 alpha-hydroxylase cDNA . Liver microsomes from patients treated with cholestyramine (n = 3) w ere shown to contain levels of cholesterol 7 alpha-hydroxylase protein approximately twofold higher than those of liver microsomes from untr eated patients (n = 6; P < 0.02), whereas cholesterol 7 alpha-hydroxyl ase activity was approximately sixfold higher in liver microsomes from the cholestyramine-treated patients than in the corresponding prepara tions from the untreated patients (P < 0.02, The higher activities obs erved in cholestyramine-treated patients, therefore, cannot be explain ed only by an increased amount of protein, suggesting a posttranslatio nal mechanism to increase the activity of human cholesterol 7 alpha-hy droxylase in addition to the transcriptional control.