[Catalytic activity of isolated cytochromes P-450d and P-450c]. / Kataliticheskaia aktivnost' izolirovannykh tsitokhromov P-450d i P-450c.

Cytochrome P-450d was isolated from isosafrol-induced rat liver microsomes by affinity chromatography on 1.8-diaminooctyl-Sepharose 4B and chromatography on hydroxylapatite using a linear potassium phosphate gradient (45-250 mM). The enzyme has a molecular mass of 54 kDa, CO-maximum 448 nm is characterized by a high spin state; the rate of 4-aminobiphenyl hydroxylation is 54 nmol/min/nmol of cytochrome P-450d (37 degrees C), those, of 7-ethoxyresorufin O-deethylation and benz (a) pyrene oxidation are 1 nmol/min/nmol of cytochrome P-450d (22 degrees C) and 2 nmol/min/nmol of cytochrome P-450d (37 degrees C), respectively. The properties of cytochrome P-450d were compared to those of cytochrome P-450c isolated from 3-methylcholanthrene-induced rats. The yield of these cytochromes under the conditions used (10% P-450d from isosafrol-induced microsomes and 15% P-450c from 3-methylcholanthrene-induced microsomes) was relatively high. Antibodies to cytochromes P-450d and P-450c were obtained. Using rocket immunoelectrophoresis the percentage of these hemoprotein forms in 3-methylcholanthrene-induced (P-450d-20%, P-450c-70%) and isosafrol-induced rat liver microsomes (P-450d-50%, P-450c-15%) was determined.

[Cytochrome P-448 induction in liver microsomes of mice of inbred strains after administration of xenobiotics of MC-type]. / Induktsiia tsitokhroma P-448 v mikrosomakh pecheni myshei inbrednykh linii posle vvedeniia ksenobiotikov tipa MKh-tipa.

Using immunochemical methods, the identity of cytochrome P-448 from liver microsomes of mice of "inducible" and "non-inducible" lines during induction by xenobiotics of MX-type (3-methylcholanthrene, 3,4-benzpyrene, 2,3,7,8-tetrachlorodibenzodioxin) was established. This hemoprotein form was shown to play a role in 3,4-benzpyrene metabolism. Monospecific antibodies to purified cytochromes P-448 and P-450 were obtained; the cytochrome P-448 content in microsomes was measured by rocket immunoelectrophoresis. The content of cytochrome P-448 in control and phenobarbital-induced microsomes makes up to 10-15% of the total hemoprotein content determinable from the CO-spectra. 3-Methylcholanthrene and 3,4-benzpyrene injected into "non-inducible" mice cause no increase in the content of this hemprotein form, whereas in mice induced with 2,3,7,8-tetrachlorodibenzodioxin it rises to 50%. Under these conditions, an almost 100% inhibition of 3,4-benzpyrene metabolism by antibodies to cytochrome P-448 is observed. Antibodies against cytochrome P-448 obtained from liver microsomes of 3-methylcholanthrene-induced mice cause a 90% inhibition of 3,4-benzpyrene in microsomes induced with 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzodioxin.

[Immunological study of the identity of cytochrome P-448 in the mouse and rat liver after induction with 3-methylcholanthrene]. / Immunokhimicheskoe issledovanie identichnosti tsitokhromov P-448 pecheni myshei i krys pri induktsii 3-metilkholantrenom.

As shown by means of Ouchterlony double immunodiffusion technique after induction with 3-methyl cholanthrene the antibodies towards cytochrome P-448 from rat liver tissue developed a clear-cut precipitation line with homologous antigen and interacted also with cytochrome P-448 from mice liver tissue although the precipitation was less distinct. Antibodies towards mice liver cytochrome P-448 reacted also with the rat liver cytochrome P-448 but less distinctly as compared with the mice cytochrome. At the same time, these antibodies inhibited similarly the benz(a)-pyrene-hydroxylase reaction, catalyzed by both mice and rat cytochromes P-448. Partial immunological identity appears to occur in the cytochromes studied. Role of total antigenic determinants in enzymatic reactions catalyzed by cytochrome P-448 is discussed.

[Isolation and characterization of the basic forms of cytochrome P-450 from liver microsomes of C57BL mice after phenobarbital and 3-methylcholanthrene induction]. / Vydelenie i kharakteristika osnovnykh form tsitokhroma P-450 iz mikrosom pecheni myshei linii C57BL pri induktsii fenobarbitalom i 3-metilkholantrenom.

Using hydrophobic and ion-exchange chromatography, cytochromes P-450 and P-448 from liver microsomes of C 57 BL mice induced by phenobarbital and 3-methylcholantrene were isolated. The cytochromes purified to homogeneity as evidenced from SDS polyacrylamide gel electrophoresis were characterized in terms of molecular weight and catalytic and spectral properties and by peptide mapping. Cytochrome P-450, in contrast to cytochrome P-448, was not bound to the ion-exchanger and was eluted in a void volume. Cytochrome P-450 (Mr = 51 000) elicits a low spin signal and reveals a high catalytic activity toward aminopyrine and a low catalytic activity toward benz(a)pyrene. Cytochrome P-448 (Mr = 55 000) elicits both high an low spin signals and reveals a high catalytic activity toward benz(a)pyrene and a low catalytic activity toward aminopyrine. Limited proteolysis with papain demonstrated the differences in the proteins primary structure.