Expression and distribution of cholesterol 7 alpha-hydroxylase in rat liver.
The hydroxylation of cholesterol by cholesterol 7 alpha-hydroxylase (CYP7) to 7 alpha-hydroxycholesterol is the rate-limiting step in the production of bile acids. An anti-peptide antibody targeted to the C-terminus of CYP7 was produced by immunising rabbits with the synthetic peptide Tyr-Lys-Leu-Lys-His. The antibody bound to a single band of 54 kDa from rat hepatic microsomal fractions. The intensity of the band was subject to a diurnal variation and showed a significant increase (P < 0.01) in apoprotein at night. Treatment of rats with cholestyramine increased CYP7 apoprotein in the morning (P < 0.005) and at night (P < 0.005), but diurnal variation was maintained. CYP7 catalytic activity, measured using a specific gas chromatography/mass spectrometry assay, showed similar changes in the pattern of diurnal variation and induction. The distribution of CYP7 in rat liver tissue sections was investigated by immunocytochemistry. In sections from rats treated with cholestyramine, there was an even distribution of immunoreactivity, except in the proximal perivenous hepatocytes where immunoreactivity was slightly more intense. A similar distribution was found in sections from untreated rat liver, except immunoreactivity was overall slightly less intense. This study shows that the C-terminus of CYP7 is a useful epitope for the targeting of anti-peptide antibodies.
Brassil
P J
PJ
Department of Clinical Pharmacology, Royal Postgraduate Medical School, London, U.K.
Edwards
R J
RJ
Davies
D S
DS
D000085:Acetates; D019342:Acetic Acid; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D002725:Chloroform; D002790:Cholesterol 7-alpha-Hydroxylase / Q000032:analysis* / Q000276:immunology; D002792:Cholestyramine Resin / Q000008:administration & dosage; D000939:Epitopes / Q000276:immunology; D005260:Female; D005404:Fixatives; D015870:Gene Expression; D007150:Immunohistochemistry; D008099:Liver / Q000201:enzymology*; D000432:Methanol; D008969:Molecular Sequence Data; D051381:Rats; D017208:Rats, Wistar
D000085:Acetates; D000906:Antibodies; D000939:Epitopes; D005404:Fixatives; C046544:methacarn; D002792:Cholestyramine Resin; D002725:Chloroform; D002790:Cholesterol 7-alpha-Hydroxylase; D019342:Acetic Acid; D000432:Methanol