Purification of cholesterol 7 alpha-hydroxylase from human and rat liver and production of inhibiting polyclonal antibodies.
Cholesterol 7 alpha-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 7 alpha-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 7 alpha-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 7 alpha-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 7 alpha-hydroxylase activity. Immunoblots of rat microsomes with the rabbit anti-rat cholesterol 7 alpha-hydroxylase antibody demonstrated that the antibody reacted quantitatively with the rat microsomal enzyme. Microsomes from cholesterol-fed rats showed increased cholesterol 7 alpha-hydroxylase mass, whereas treatment with pravastatin, an inhibitor of hydroxy-methylglutaryl-coenzyme A reductase, reduced enzyme mass. Microsomes from starved rats contained slightly less cholesterol 7 alpha-hydroxylase protein than chow-fed control rats. These results indicate a similarity in molecular mass, structure, and antigenicity between rat and human cholesterol 7 alpha-hydroxylases; demonstrate the production of inhibiting anti-cholesterol 7 alpha-hydroxylase antibodies that can be used to measure the change in cholesterol 7 alpha-hydroxylase enzyme mass under various conditions; and emphasize the unique structure of cholesterol 7 alpha-hydroxylase with respect to other cytochrome P-450-dependent hydroxylases.
Nguyen
L B
LB
Department of Medicine, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark 07103.
Shefer
S
S
Salen
G
G
Ness
G
G
Tanaka
R D
RD
Packin
V
V
Thomas
P
P
Shore
V
V
Batta
A
A
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology*; D002790:Cholesterol 7-alpha-Hydroxylase / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D002791:Cholesterol, Dietary / Q000494:pharmacology; D002792:Cholestyramine Resin / Q000494:pharmacology; D002845:Chromatography; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006538:Heptanoic Acids / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D007527:Isoenzymes / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D008970:Molecular Weight; D009281:Naphthalenes / Q000494:pharmacology; D017035:Pravastatin; D051381:Rats; D011919:Rats, Inbred Strains; D013217:Starvation / Q000201:enzymology; D013250:Steroid Hydroxylases / Q000302:isolation & purification*
D000906:Antibodies; D002791:Cholesterol, Dietary; D006538:Heptanoic Acids; D007527:Isoenzymes; D009281:Naphthalenes; D002792:Cholestyramine Resin; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D002790:Cholesterol 7-alpha-Hydroxylase; D017035:Pravastatin