Catalytic activity of isolated cytochromes P-450d and 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.     

Changes in the ratio of microsomal electron carriers in reconstituted microsomal membranes

The reconstitution of microsomal membrane monooxygenase system with variable contents of the hydroxylating chain enzymatic components was carried out. It was found that during self-assembly of microsomal membranes solubilized with 4% sodium cholate and gel filtration through Sephadex LH-20 in the presence of isolated microsomal enzymes, two forms of cytochrome P-450, i. e. phenobarbital- and 3-methylcholantrene-induced ones, and NADPH-cytochrome P-450 reductase, the exogenous enzymes are incorporated into the microsomal membrane matrices of control and methyl-cholantrene-treated animals. In the membranes reconstituted from the microsomes of the methylcholantrene-induced animals the catalytic activity of cytochrome P-448 in the metabolism of benz(a)pyrene at varying cytochrome P-448 and NADPH-cytochrome P-450 reductase contents were investigated.     

Induction of the phenobarbital form of cytochrome P-450 by chemically unrelated compounds

The induction of the phenobarbital form of cytochrome P-450 by xenobiotics (phenobarbital, PB, hexachlorobenzene, HCB; hexachlorocyclohexane. HCCH, and aroclor 1016, Ar) was studied. It was demonstrated that administration of these compounds to animals is accompanied by an increase in the total cytochrome P-450, NADPH-cytochrome P-450 reductase, benzphetamine-N-demethylase and aldrin-epoxidase activities. Using monospecific antibodies against the cytochrome P-450 form isolated from PB-induced microsomes (PB-cytochrome P-450), a double immunodiffusion test revealed immunological identity of cytochrome P-450 forms induced by phenobarbital and other xenobiotics. The content of this form determined by rocket immunoelectrophoresis increased markedly and made up to 20-40% of the total cytochrome P-450 content. Antibodies against PB-cytochrome P-450 inhibited by 50-70% the benzphetamine-N-demethylase and aldrin-epoxidase activities, whereas the antibodies to methylcholanthrene-induced cytochrome P-450 were fairly ineffective. It was concluded that the chemically unrelated compounds induce in liver microsomes a cytochrome P-450 form, whose immunological properties and substrate specificity are close to the PB-form of cytochrome P-450.     

An efficient method for extracting plasma ions in laser isotope separation systems

The possibility of using a Hall-current accelerator to extract ions from a partially ionized plasma produced by selective laser isotope photoionization of atomic vapor is examined. A mechanism for ion acceleration is investigated using one-dimensional time-dependent equations of two-fluid magnetohydrodynamics. The current cutoff due to the ion space charge is prevented by electron emission. It is shown that, at an accelerating voltage of 25–50 V and emission current density of several mA/cm², the ion component is accelerated throughout the entire plasma volume up to a velocity of ～10⁵ cm/s in a few microseconds. The influence of resonant charge exchange and secondary ionization by electrons on both the acceleration dynamics and selectivity degradation is taken into account. It is shown that the Hall-current extractor allows one to avoid selectivity degradation even when the plasma size exceeds the charge-exchange mean free path by one order of magnitude.     

Correlation between cytochrome P-448 content and benzopyrene hydroxylase activity during the induction of microsomal monooxygenases using methylcholanthrene-type xenobiotics

Using antibodies against electrophoretically homogeneous cytochrome P-448 from rat liver microsomes induced by 3-methylcholanthrene, the changes in the immunologic identity and contents by cytochrome P-448 induced by 3-methylcholanthrene, 3.4-benzpyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), were studied. No cytochrome P-448 was detected in the liver microsomes of control or phenobarbital-induced rats. This form of the cytochrome makes up to about 35% of the total content of the CO-binding hemoprotein during TCDD induction and up to 90% during 3-methylcholanthrene and 3,4-benzpyrene induction. On the other hand, 3-methylcholanthrene, 3,4-benzpyrene and TCDD significantly and equally activates the cytochrome P-448-dependent benzpyrene hydroxylase, since the antibodies against cytochrome P-448 inhibit benzpyrene metabolism in the microsomes by 85-90%. The possible reasons for the TCDD-induced increase in the catalytic activity of cytochrome P-448 as compared to the immunologically identical cytochrome P-448 induced by 3-methylcholanthrene and 3,4-benzpyrene, are discussed.     

Comparison of peptide maps and functional properties of monooxygenases induced by 3-methylcholanthrene and beta-naphthoflavone

A comparative study revealed a similarity of catalytic, spectral, electrophoretic and immunochemical properties of microsomal cytochromes P-448 (Mr = 56,000) synthesized de novo in 3-methylcholanthrene- and beta-naphthoflavone-treated rats. The identity of peptide maps of microsomal and isolated cytochromes P-448 confirms the validity of the limited proteolysis method for identification of the homogeneity of microsomal hemoproteins and for a comparison of their structures. The data obtained provide a way for evaluation of similarity and differences in the structure and enzymatic activity of various monooxygenase forms without their preliminary isolation from the microsomal membrane.     

Catalytic activity and association of pancreatic lipase

The authors summarize their work concerning the mechanism of pancreatic lipase activation. The activation of lipase by submicellar SDS concentrations was found to imitate closely enough its activation by an interface. Lipase activation was shown to be caused by changes in the rate constants for substrate chemical transformation and to involve conformational changes of the enzyme and its association. The complex of a conformationally modified lipase with the detergent, which acts as a 'structure-forming' agent, is associated with native lipase molecules setting up their active site. The mechanism of lipase activation at an interface both in vitro and in vivo is discussed.     

Identification of molecular forms of cytochrome P-450 isolated from liver microsomes of rats induced with phenobarbital and 3-methylcholanthrene

Eight electrophoretically homogeneous forms of cytochrome P-450 were isolated from liver microsomes of phenobarbital (PB)- and 3-methylcholanthrene (MC)-induced male Wistar rats, using chromatography on 1.8-diaminooctyl-Sepharose, SEAE-Sephacel and hydroxylapatite. These cytochrome forms were compared to those described in literature in terms of their ability to metabolize androstenedione (AD), benzphetamine (BP) and 7-ethoxyresorufin (7-ER). Cytochrome P-450b capable of catalyzing with a high specificity the 16-hydroxylation of AD and N-demethylation of BP, and cytochrome P-450e immunologically related to P-450b but incapable of catalyzing these reactions were isolated from PB-microsomes. Besides, a male-specific cytochrome P-450h catalyzing the 16 alpha-hydroxylation of AD was isolated from PB-microsomes. Cytochrome P-450c possessing a high 7-ER-O-deethylase activity, and a high spin cytochrome P-450d as well as cytochrome P-450a specifically catalyzing the 7 alpha-oxidation of AD were isolated from MC-microsomes. Two forms of cytochrome P-450 isolated from PB-microsomes possessed no such activities. Data from immunochemical analysis suggest that one of these forms can be identified as cytochrome P-450k. It is concluded that the specificity of metabolism and the molecular activity of Wistar rat liver cytochrome P-450 forms are comparable with the corresponding parameters of hemoproteins isolated from other rat species. At the same time, data from metabolic analysis are suggestive of differences in the levels of certain cytochrome P-450 forms, in particular P-450a.     

The effect of pyridine and pyridine-N-oxide on the monooxygenase system of rat liver microsomes]

Effects of pyridine and pyridine-N-oxide on the monooxygenase system of rat liver microsomes have been studied. Pyridine (200 mg/kg) increased total cytochrome P-450 content and activated metabolism of some specific substrates 24 hours after injection. There was an increase in the degree of p-nitrophenol and chlorzoxazone hydroxylation due to increasing ethanol-induced cytochrome P-450IIE1 content. Pyridine was also able to induce cytochrome P-450IIB1 in rat microsomes; this reaction was accompanied by acceleration of 7-pentoxyresorufin 0-dealkylation. Cytochrome P-450IA1 appearance in liver microsomes was associated with increasing content of cytochrome P-450IA2. Dealkylation rates for specific substrates (7-ethoxyresorufin and 7-methoxyresorufin) were also increased. Similar to pyridine, pyridine-7-oxide induced cytochromes P-450IIE1, P-450IIB1/B2, and P-450IA1/A2, resulting in activation of specific substrate metabolism. Hence, pyridine and its derivative pyridine-N-oxide can be regarded as effective inducers of cytochrome P-450.