Purification of cholesterol 7α-hydroxylase from human and rat liver and production of inhibiting polyclonal antibodies

Lien B. Nguyen, Sarah Shefer, Gerald Salen, Gene Ness, Richard D. Tanaka, Virginia Packin, Paul Thomas, Virgie Shore, Ashok Batta

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26 Scopus citations


Cholesterol 7α-hydroxylase, the cytochrome P-450-dependent and rate-controlling enzyme of bile acid synthesis, was purified from rat and human liver microsomes. The purified fractions were assayed in a reconstituted system containing [4-14C]cholesterol, and cholesterol 7α-hydroxylase activities in these fractions increased 500-600-fold relative to whole microsomes. Polyacrylamide gel electrophoresis of rat microsomes followed by immunoblotting with polyclonal rabbit antisera raised against purified cholesterol 7α-hydroxylases revealed two peaks at molecular masses of 47,000 and 49,000 daltons for both rat and human fractions. Increasing amounts of rabbit anti-rat and anti-human antibodies progressively inhibited rat microsomal cholesterol 7α-hydroxylase activity up to 80%. In contrast, monospecific antibodies raised against other purified cytochrome P-450 enzymes (P-450f, P-450g, and P-450j) did not inhibit rat or human cholesterol 7α-hydroxylase activity. Immunoblots of rat microsomes with the rabbit anti-rat cholesterol 7α-hydroxylase antibody demonstrated that the antibody reacted quantitatively with the rat microsomal enzyme. Microsomes from cholesterol-fed rats showed increased cholesterol 7α-hydroxylase mass, whereas treatment with pravastatin, an inhibitor of hydroxymethylglutaryl-coenzyme A reductase, reduced enzyme mass. Microsomes from starved rats contained slightly less cholesterol 7α-hydroxylase protein than chow-fed control rats. These results indicate a similarity in molecular mass, structure, and antigenicity between rat and human cholesterol 7α-hydroxylases; demonstrate the production of inhibiting anti-cholesterol 7α-hydroxylase antibodies that can be used to measure the change in cholesterol 7α-hydroxylase enzyme mass under various conditions; and emphasize the unique structure of cholesterol 7α-hydroxylase with respect to other cytochrome P-450-dependent hydroxylases.

Original languageEnglish (US)
Pages (from-to)4541-4546
Number of pages6
JournalJournal of Biological Chemistry
Issue number8
StatePublished - Mar 15 1990
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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