[Fractionation of a chalone-containing substance from Ehrlich's ascites tumor using high pressure chromatography]. / Fraktsionirovanie keilonsoderzhashchego preparata iz astsitnoi opukholi érlikha s pomoshch'iu khromatografii vysodogo davleniia.

The effect of various fractions of chalone--containing preparation from ascyte Ehrlich's tumour obtained by high performance liquid chromatography (HPLC) on mitotic activity and DNA synthesis in the tumour has been studied. After filtration the division of active chalone component which inhibits entering cells into M-phase and S-phase took place. The component inhibiting DNA synthesis eluated with G1-chalone.

[Oxidative modification of cytochrome P-450 during its function. I. Comparative study of the inactivation of cytochrome P-450 LM2 in various systems]. / Okislitel'naia modifikatsiia tsitokhroma P-450 pri ego rabote. I. Sravnitel'noe issledovanie inaktivatsii tsitokhroma P-450 LM2 v razlichnykh sistemakh.

The changes in the content of purified isolated cytochrome P-450 LM2 under the action of hydrogen peroxide and during its operation in a soluble reconstituted system were studied. It was found that cytochrome P-450 LM2 inactivation by hydrogen peroxide is accompanied by a decrease in the hemoprotein activity, loss of heme, oxidation of SH-groups and changes in the oligomeric state of the enzyme. There were some differences in the mechanisms of cytochrome P-450 LM2 inactivation under the action of H2O2 and during catalysis.

[Oxidative modification of cytochrome P-450 during its function. II. Study of the mechanism of cytochrome P-450 LM2 inactivation in a soluble reconstructed monooxygenase system]. / Okislitel'naia modifikatsiia tsitokhorma P-450 pri ego rabote. II. Issledovanie mekhanizma inaktivatsii tsitokhroma P-450 LM2 v rastvorimoi rekonstruirovannoi monooksigenaznoi sisteme.

Inactivation of cytochrome P-450 LM2 induced by hydrogen peroxide formed in the active site of the enzyme was studied. Catalase did not protect cytochrome P-450 LM2 from inactivation during its operation in a soluble reconstituted system. The hemoprotein inactivation in this system was found to depend on the ratio of hemo- to flavoproteins. It was demonstrated that cytochrome P-450 LM2 inactivation during catalysis is accompanied by cleavage of the hemoprotein molecule. It is probable that this fact plays a key role in regulation of enzyme decay.

[Induction of cytochrome P-450 by triterpensaponins in Vietnamese ginseng]. / Induktsiia tsitokhroma P-450 triterpensaponinami V'etnamskogo zhen'shenia.

It was found that rat liver cytochrome P-450 is induced by the Vietnamese ginseng triterpensaponines mixture (TSM) as well as by K5VN Panaxozides-11 (VP-11) purified from this mixture. Addition of TSM and VP-11 accelerates benz(alpha)pyrene and aminopyrine hydroxylation and increases the content of cytochrome P-450 isoforms with Mr of 57 kDa and 54 kDa in rat liver microsomes. Since VP-11 accounts for about 50% of TSM, the results obtained suggest that the microsomal monooxygenase system induction is caused by this triterpensaponine. Induction by TSM and VP-11 was compared to that by phenobarbital (PB) and 3-methylcholanthrene (MC). It was shown that according to their inductive action TSM and VP-11 belong neither to the PB- nor to the MC-type. Cytochrome P-450 induction may play an important role in the triterpensaponine action on the organism, because this enzyme participates in the metabolism of such endogenous compounds as prostaglandins, steroid hormones, cholesterol, etc.

[Cytochrome P-450 and oxidative modification of macromolecules]. / Tsitokhrom P-450 i okislitel'naia modifikatsiia makromolekul.

Depending on the localization and functional value, the reactions of the oxidative modification of macromolecules may be classified into two large groups: intracellular and extracellular. To exemplify the first-group reactions, cytochrome P-450 was studied, which is capable of generating different active forms of oxygen during the catalytic cycle. These forms were found to have bactericidal effects and to be able to cause DNA molecule break. In the course of the reaction, cytochrome P-450 also became inactivated under the effect of active oxygen. The involvement of hydrogen peroxide forming directly during peroxycomplex breakdown was proved. It was shown using a soluble reconstructed system that cytochrome P-450 inactivation is attended with the hemoprotein molecule decomposition, destruction and loss of heme, and a rise in the proteolytic vulnerability of the protein. The second reaction group was exemplified by human leucocyte myeloperoxidase which generated hypochlorite along with the active forms of oxygen. Myeloperoxidase showed its bactericidal effect against different strains of bacteria and fungi. The Ames test revealed mutagenic effects of the active oxygen forms generated by myeloperoxidase. In the course of the myeloperoxidase reaction, DNA degradation followed by splitting of the polynucleotide chain and specific pulling out of thymine residues were observed.

Microheterogeneity of cytochrome P450(LM2) as revealed by isoelectric focusing.

The homogeneity of different preparations of cytochrome P450(LM2), the major form of cytochrome P450 found in the liver microsomes of phenobarbital-treated rabbits, was checked by SDS polyacrylamide gel electrophoresis and N-terminal amino acid sequencing. In SDS-PAGE all the preparations migrated as a single protein band of molecular mass 49 kDa. The electrophoretic mobility of this band corresponded to that of the major phenobarbital-inducible band in the microsomes of phenobarbital-treated rabbits. A single N-terminal sequence (Met-Glu-Phe) corresponding to the N-terminal sequence of the LM2 form of cytochrome P450 was revealed by N-terminal amino acid sequence analysis. Isoelectric focusing of electrophoretically homogeneous cytochrome P450(LM2) was conducted in polyacrylamide gel in an LKB Ampholine pH 5-8 gradient under denaturing conditions (9.1 M urea). Under these conditions cytochrome P450(LM2) was resolved into six subfractions in the pH range 6.5-7.75. The data obtained reflect the microheterogeneity of this form of cytochrome P450. The existence of different isoelectric points cannot have been due to different protein conformations because denaturing conditions were used, but seem to reflect different primary structures of the cytochrome subfractions.

[Induction of cytochrome P-450 in rat liver microsomes by perfluorodecalin]. / Induktsiia perftordekalinom tsitokhroma P-450 v mikrosomakh pecheni myshei.

Perfluorodecalin was incorporated into phospholipid liposomes and injected intraperitoneally in various dozes. The maximal cytochrome P-450 induction is reached 48 hours after perfluorodecalin injection. Cytochrome P-450 content increases 4 times after perfluorodecalin injection in dose of 0.6 ml/kg in homogenate, and 6 times after perfluorodecalin injection in a dose of 0.4 ml/kg in microsomes. Phenobarbital and perfluorodecalin induce several cytochrome P-450 isozymes and cause the appearance of a new isozyme with mass 56 kD absent in microsomes of intact CBA mice. Perfluorodecalin induction strongly increased the rate of NADPH-dependent aminopyrine nN-demethylation (6-7 times per mg of microsomal protein and 1.5 times per nmol cytochrome P-450). The rate of NADPH-dependent hydroxylation of aniline was not affected by perfluorodecalin induction.

[The effect of phenobarbital and amidopyrine on the rate of decomposition of isoforms of cytochrome P-450 in mouse liver microsomes]. / Vliianie fenobarbitala i amidopirina na skorost' raspada izoform tsitokhroma P-450 v mikrosomakh pecheni myshei.

Separation of microsomal proteins in gradient polyacrylamide gel gives 60 protein bands. The molecular mass range of 48-58 kDa corresponding to cytochrome isozymes contains 7 bands for intact mice and 8 bands for phenobarbital-induced mice. Phenobarbital treatment causes both the appearance of a new cytochrome P-450 isozyme with a molecular mass of 56 kDa and the increase in the content of three isozymes with molecular masses of 54, 52.5 and 50 kDa. The half-life time of cytochrome P-450 isozymes in the livers of intact and phenobarbital-induced mice differs from 15 to 42 hours. Phenobarbital induction results in the breakdown acceleration of the isozyme with a molecular mass of 52.5 kDa and the breakdown retardation of the isozyme with a molecular mass of 54 kDa. Aminopyrine injections to phenobarbital-pretreated mice result in the breakdown acceleration of the cytochrome P-450 isozyme with a molecular mass of 56 kDa.

[Rate of microsomal protein metabolism in the mouse liver]. / Issledovanie skorosti obmena belkov mikrosom pecheni myshei.

NaH14CO3, a poorly reutilized biosynthesis precursor, was used to study the rate of whole microsomal protein degradation in mouse liver. The use of the precursors, however, does not prevent the reutilization of labeled amino acids on phenobarbital administration. To avoid reutilization, a new method has been developed. It was shown that phenobarbital injections have no effect on the degradation rate of the whole microsomal protein. The effect of amidopyrine, a monooxygenase microsomal system substrate, on the rate of whole microsomal protein degradation was examined. An experimental model was developed, in which the monooxygenase microsomal system substrate does not exhibit the properties of its inducer. Amidopyrine administration to mice simultaneously with phenobarbital induction has no effect on the degradation rate of the whole microsomal protein.

[Effects of hydrogen peroxide on cytochrome P-450 inactivation]. / Issledovanie vliianiia perekisi vodoroda na inaktivatsiiu tsitokhroma P-450.

Inactivation rate of purified oligomeric cytochrome P-450 LM2 has been investigated in glucose oxidase system and under the action of exogenous hydrogen peroxide (400 microM). It has been found that hydrogen peroxide has a distinct inactivating effect on cytochrome P-450. The enzyme inactivation is accompanied by the loss of heme and the decrease in SH-group content in the protein molecule. Benzphetamine, a substrate specific for this enzyme isoform, exerts a protective effect by decreasing the rate of cytochrome P-450 inactivation and SH-group oxidation. Similar results have been obtained during the investigation of cytochrome P-450 inactivation in the monomerized system. It has been found that the inactivation process is accompanied by the formation of the enzyme aggregates. The changes in the aggregate state are due to the formation of intermolecular covalent bonds.

[Mechanism of intestinal vitamin-D-dependent Ca absorption. Correlation between active Ca transport and release of inorganic phosphate on the basal side of intestinal epithelia]. / Izuchenie mekhanizma vitamin-D-zavisimogo vsasyvaniia Ca v kishechnika. Vzaimosviaz, aktivnogo transporta Ca s osvobozhdeniem neorganicheskogo fosfata na bazal'noi storone kishechnogo épiteliia.

Incubation of isolated sections of rat small intestine in air-saturated medium, deprived of inorganic phosphate (Pi) was accompanied by liberation of endogenous Pi from both sides of the intestinal wall as well as by development of the anion concentration gradient, which was directed from apical to basal side of the wall. After addition into medium of 0.25 mM CaCl2 triggering the Ca2+ absorption in the isolated intestinal section by an active transport mechanism, distinct output of Pi was observed at the basal side of the intestinal epithelium against the concentration gradient of the anion. Under anaerobic conditions block of Ca2+ active transport by means of inhibitors of energy supply (1 . 10(-5) M DNP, 4 . 10(-6) M p-chloromercuribenzoate) inhibited simultaneously the Pi output; the same phenomenon was observed in aged rats and in rats maintained on D-avitaminous diet, thus demonstrating the intimate relation of the processes studied. The data obtained are consistent with the idea on existence of a phosphorylated carrier of Ca2+, dephosphorylation of which on basal-lateral membrane of enterocyte is accompanied by simultaneous liberation of Ca2+ and Pi from the mucosal cells providing the energy-dependent transition of the ions against the concentration gradient.

[Functional activity of mitochondria and intensity of nucleic acid and protein synthesis in rat liver and small intestine mucosa with different supplies of vitamin D]. / Funktsional'naia aktivnost' mitokhondrii i intensivnost' sinteza nukleinovykh kislot i belka v slizistoi obolochke tonkikh kishok i pecheni krys pri razlichnoi obespechennosti vitaminom D.

Decrease in total content of mitochondrial protein in mucosa of small intestine, an alteration of distribution of the protein between the fractions of mitochondria, distinct decrease in the respiratory activity of mitochondria and in the activity of succinate- and NADH-dehydrogenases were observed in rats deficient in vitamin D. Deficiency in vitamin D was accompanied by decreased incorporation of labelled precursors into total, nuclear and mitochondrial DNA and RNA by 20-50% and into mitochondrial proteins--by 50% in mucosa of small intestine; these patterns were unaltered in liver tissue. Administration of ergocalciferol (at a dose 1000 IU) into rats normalized the impairments studied, whose alteration correlated with the increase of calcium concentration in blood serum.

[Biochemical heterogeneity of mitochondria from mucosa of rat small intestine]. / Biokhimicheskaia geterogennost' mitokhondrii slizistoi obolochki tonkogo kishechnika krys.

Mitochondria from mucosa of rat small intestine were separated (0-11% gradient of ficoll) into fractions, differing by the content of protein, DNA and cytochrome a+a3 as well as by the respiratory activity and the activity of succinate- and NADH-dehydrogenases. Content of DNA and cytochrome a+c was shown to be increased in "heavy" mitochondrial fractions. Mitochondria od "midle" fractions possessed the highest functional activity. The data obtained suggest that the mitochondrial preparations are not contaminated by other cytoplasmic structures; they were also not impaired during isolation and fractionation. The activity of RNA and DNA synthesis in vivo and distribution of labelled mitochondrial RNA and DNA in the fractions were studied. The possible relation of mitochondrial heterogeneity to, their biogenesis is discussed.

[Biochemical heterogeneity of mitochondria]. / K voprosu o biokhimicheskoi geterogennosti mitokhondrii.

Rat liver mitochondria were fractionated on the basis of their sedimentation coefficients in the gradient of ficoll. The fractions ("heavy", "middle" and "light" mitochondria) were heterogeneous with regard to the content of protein, DNA, cytochrome a + a3 and respiratory activity. Heterogeneity of mitochondria did not result from the damage or microsomal and lysosomal contamination. The biosynthesis of DNA, RNA and proteins in the different fractions of mitochondria was studied. In vivo incorporation of radioactive precursor into RNA was highest in the fractions of "middle" mitochondria, whereas in vitro the "heavy" mitochondria showed maximum activity in the synthesis of RNA. In vitro DNA synthes was maximum in the fractions of "heavy" mitochondria, protein synthesis in "heavy" and "light" mitochondria. Activity of the synthesis of RNA, DNA and proteins in vitro depends on the content of DNA and cytochrome a + a3 in the different fractions of mitochondria. It is supposed that heterogeneity of mitochondria may be connected with their biogenesis.