Induction of hepatic microsomal cytochrome P450 by dexamethasone in rhesus monkey (Macaca mulatta).

Intraperitoneal administration of 150 mg dexamethasone (DEX) Kg-1 body wt for four days to rhesus monkeys resulted in statistically significant increases in the activities of hepatic tyrosine aminotransferase (3 fold), microsomal cytochrome P450 (2 fold) and erythromycin N-demethylase (4 fold), but no change in the activities of aminopyrine N-demethylase and NADPH cytochrome c reductase. Three peaks were obtained from control or DEX-treated monkey livers on fractionation of detergent solubilized microsomes by anion exchange chromatography on DE-52. Peak II obtained from DEX-treated monkey microsomes on DE-52 demonstrated the highest specific activity of cytochrome P450 (5.84 nmol mg-1 protein) as compared to other peaks from the same microsomes or any of the peaks obtained from the control microsomes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the microsomes from control and experimental animals and peak II obtained after anion exchange chromatography of DEX-treated microsomes demonstrated the intensification of two polypeptides of 52.5 and 50 kDa. The results indicate that DEX is an inducer of cytochrome P450 and dependent erythromycin N-demethylase in non-human primate, Macaca mulatta.

Aminopyrine-N-demethylase. II. Characterization of a unique monooxygenase isoform P-450ap.

A previously unidentified cytochrome P-450ap possessing the highest aminopyrine-N-demethylase activity has been isolated from liver microsomes of 4-isopropylaminoantipyrine-induced rats, using affinity chromatography in combination with ion-exchange chromatography with subsequent separation on a hydroxyapatite column. The isolated cytochrome P-450ap has the following characteristics: Mr = 49 kD, CO-peak maximum at 450.5 nm, rate of demethylation in a reconstituted system for aminopyrine of 25.5 nmoles of HCHO/min per nmole of P-450, and for benzphetamine a rate of 17.0 nmoles of HCHO/min per nmole of P-450. The hemoprotein synthesis is paralleled by the synthesis of a protein with Mr of 51 kD. Immunochemical analysis permitted the identification of the latter protein as cytochrome P-450b. It was, demonstrated that cytochrome P-450ap does not interact with the antibodies to the major phenobarbital induced form, i.e. with cytochrome P-450b.

[Multiple forms of cytochrome P-450. Characteristics of isolated aminopyrine-N-demethylase (cytochrome P-450AP)]. / Mnozhestvennye formy tsitokhroma P-450. Kharakteristiki izolirovannoi aminopirin-N-demetilazy (tsitokhroma P-450AP).

A previously unidentified cytochrome P-450AP possessing the highest aminopyrine-N-demethylase activity has been isolated from liver microsomes of 4-isopropylaminoantipyrine-induced rats, using affinity chromatography in combination with ion-exchange chromatography with subsequent separation on hydroxyl apatite. Using radioisotope techniques, it was found that 4-isopropylaminoantipyrine induces cytochrome P-450AP synthesis de novo. The isolated cytochrome P-450AP has the following characteristics: Mr = 49,000 Da. CO-peak maximum at 450.5 mm, rate of aminopyrine demethylation in a reconstituted system-20 nmol HCHO/min/nmol of cytochrome P-450, benzphetamine-15. The hemoprotein synthesis is paralleled with the synthesis of a protein with Mr of 51,000 Da. Immunochemical analysis permitted to identify the latter protein as cytochrome P-450b. It was demonstrated that cytochrome P-450AP does not interact with the antibodies to the major phenobarbital-induced form, i.e., with cytochrome P-450b.

Resolution of multiple P-450 forms: separation of aryl hydrocarbon hydroxylase and aminopyrine N-demethylase-associated P-450 isoenzymes by chromatofocusing.

Chromatofocusing between pH 7.4 and 5.0 was introduced as a final step for the resolution of multiple forms of cytochrome P-450 from control, phenobarbital and 3-methylcholanthrene-pretreated rat liver microsomal fractions. Altogether, chromatofocusing produced 21 P-450-containing pools, which differed from each other with respect to substrate specificity, spectral maximum and elution pH. Aryl hydrocarbon (benzo(a)pyrene) hydroxylase (AHH) activity was concentrated into low pI pools in all animal groups. 7-Ethoxycoumarin O-deethylase (ECOD) activity comigrated with AHH activity throughout the purification procedure. Aminopyrine N-demethylase (APND) activity was spread into several pools with forms of both low and high pI proteins.