Purification and characterization of taurodeoxycholate 7 alpha-monooxygenase in rat liver.
Taurodeoxycholate 7 alpha-monooxygenase was purified from rat liver microsomes of phenobarbital-treated rats. The purification was carried out by solubilization of microsomes by cholate, fractionation with polyethylene glycol, affinity chromatography, and hydroxylapatite chromatography. The purified preparation gave a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained 13.5 nmol of cytochrome P-450/mg of protein, which corresponded to 6.8-fold purification from microsomes on the basis of specific heme content. The specific activity of the enzyme expressed by the enzyme activity per mg of enzyme protein was increased 72-fold from microsomes. The molecular weight of the enzyme was estimated to be 54,000 from calibrated polyacrylamide gel electrophoresis. The enzyme activity-pH curve gave a broad peak between pH 7.0 and 7.5. The Michaelis constant for taurodeoxycholate of the enzyme was 33 microM. Absorption spectra of the oxidized form of the enzyme showed a Soret band at 518 nm, typical of the low spin state of cytochrome P-450 and alpha and beta band at 571 and 534 nm, respectively. The CO-difference spectrum of the reduced enzyme showed a band at 450 nm characteristic of cytochrome P-450. Taurodeoxycholate 7 alpha-monooxygenase was reconstituted from the purified enzyme, NADPH-cytochrome P-450 reductase, dilauroylglyceryl-3-phosphorylcholine, and NADPH. The purified enzyme was free from steroid 12 alpha-hydroxylase, 25-hydroxylase, and 26-hydroxylase activities. The enzyme activity was seriously inhibited by nonionic detergents, but ot by aminoglutethimide or by metapyrone.
D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification* / Q000378:metabolism; D004355:Drug Stability; D004789:Enzyme Activation; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D051381:Rats; D013657:Taurodeoxycholic Acid / Q000302:isolation & purification / Q000378:metabolism
D013657:Taurodeoxycholic Acid; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C022717:taurodeoxycholate 7 alpha-hydroxylase