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Title: Molecular, cellular, and regulatory characterization of cholesterol 7α-hydroxylase
Author: Eldredge, Emelyn R.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1989
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This thesis is concerned with two aspects of bile acid synthesis: 1) the characterization of cholesterol 7α-hydroxylase, the rate-limiting enzyme in bile acid synthesis and 2) the characterization of a human model for bile acid synthesis. Using polyclonal antibody probes it has been possible to begin to detail the molecular nature of cholesterol 7α-hydroxylase. A polyclonal antibody raised against a male-specific cytochrome P450 (PB2a, P450h) from the P450 IIC gene subfamily was found to inhibit cholesterol 7α-hydroxylase activity in rat liver microsomes. This antibody was also found to inhibit 7α-hydroxylase activity in both hamster and mouse, idicating a common structural feature shared across species. Inhibition could be reversed by the addition of purified PB2a antigen to the assay. Using this antibody, a protein associated with 7α-hydroxylase activity was immunoprecipitated from liver microsomes of cholestyramine-treated female rats. The amino-terminal sequence from the immunoprecipated protein was distinct from that of PB2a and other P450s. These data indicate that cholesterol 7α-hydroxylase is a unique cytochrome P450 which shares structural homology with enzymes from the P450 IIC gene subfamily. The effect of substrate supply on bile acid synthesis by the human hepatoblastoma cell line HepG2 was also investigated. Incubation of HepG2 cells with the bile acid precursor 7α-hydroxycholesterol resulted in the production of bile acid, indicating that HepG2 cells have a viable bile acid synthetic pathway. Increasing the cholesterol content of the cells by the addition of the cholesterol precursor mevalonate did not stimulate bile acid synthesis. The addition of HDL to the cells also did not stimulate bile acid synthesis. Incubation of HepG2 cells with LDL, however, did increase the synthesis of bile acid. The addition of an ACAT inhibitor, 58-035, was shown to further enhance synthesis stimulated by LDL. These results are in contrast to those obtained from primary rat hepatocytes in which both mevalonate and HDL enhanced bile acid synthesis. The data presented here suggest that, in man, LDL-derived cholesterol may be preferentially utilized for bile acid synthesis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available