[DNA-methylase activity in human cardiac muscle. Association of DNA methylase activity with actin protein fractions]. / DNK-metilaznaia aktivnost' v serdechnoi myshtse cheloveka. Assotsiatsiia DNK-metilaznoi aktivnosti s fraktsiei aktinovykh belkov.

The column isoelectrofocusing activity of the nuclear extracts of the human cardiac muscle has revealed at pH 3.5-8.2 5 peaks of DNA-methylase. When one of these peaks (II) was analyzed by the two-dimensional gel electrophoresis 6 proteins (10, 25, 35, 43, 67 and 120 kDa) were separated. 43 kDa protein had electrophoretic properties similar to actins and was able to methylate cytosine in the DNA molecules. The comparative computer analysis of the primary structure of human actins and several bacterial DNA-methylases has shown the homology of the extensive fragments of these molecules.

[Human thyroid gland DNA methylase in nodular and diffuse goiter]. / DNK-metilazy shchitovidnoi zhelezy cheloveka pri uzlovom i diffuznom zobe.

The heterogeneity and some properties of DNA-methylases isolated from nonmalignant human thyroid formations--nodular and diffuse goiters--have been studied. Isoelectrofocusing of methylase preparations produced 6-7 distinct activity peaks distinguished by pI, activity towards Ca2+ and Mg2+, sensitivity towards dithiothreitol and capacity to methylase cytosine into mono-, di- and tripyrimidine blocks in vitro. The degree of DNA methylation in vivo depended on the origin of of the goiter. Analysis of several methylase fractions by two-dimensional electrophoresis performed according to O'Farrell revealed the presence of major polypeptides having similar molecular masses (approximately 36 kDa). Besides the major component, all the preparations under study contained 6-10 characteristic minor components. The data obtained are suggestive of a structural and functional heterogeneity of human methylases.

[DNA-methylating system of chicken liver nuclei under normal conditions and in viral transformation]. / DNK-metiliruiushchaia sistema iader pecheni tsypliat v norme i pri virusnoi transformatsii.

The DNA methylating system of cellular nuclei from intact or virus transformed chicken liver was studied. The presence of multiple forms of methylases different in hydrophobic properties and isoelectric focusing points has been proved. The isoelectrofocusing made it possible to differentiate between the enzymes methylating preferably nonmethylated in vitro or methylated in vivo DNA. The DNA-methylases pool contains both types of methylases (de novo and supporting ones) in intact cells and at neoplastic transformation, however, the specificity of methylation and level of several enzymes in transformed cells is changed in the direction of broad specificity and lower activity. The general level of methylase activity at viral transformation is by 18-20% lower, while the content of 5-methylcytosine in hepatoma cells DNA is twofold lower as compared with the content in the DNA of intact cells.

[The effect of thyroid hormones on DNA methylation in rat liver in vivo and in vitro]. / Vliianie tireoidnykh gormonov na metilirovanie DNK pecheni krys in vivo i in vitro.

Methylation of rat liver DNA was studied in vivo and in vitro in presence of various content of thyroid hormones. Both administration of triiodothyronine into intact rats and thyroidectomy led to considerable alterations in activity of endogenous DNA-methylases and in content of m5C in DNA although distinct correlation between these two factors was not detected. Alterations in the acceptor activity of endogenous DNA towards bacterial DNA-methylases of thy Mbu type demonstrated the processes occurring in vivo. The methylase probe 5'...GGA...3' proved to be the universal means for testing of all the types of thyroid status involving either free DNA or whole nuclei.

[Multiple forms of DNA methylases in hepatic cell nuclei of animals in the normal state and in pathology]. / Mnozhestvennye formy DNK-metilaziz iader pechenizhivotnykh v norme i pri patologii.

Using hydrophobic chromatography multiple nature of DNA-methylating enzymes have been revealed. It was established that the most active enzymatic fraction of rats' liver and that of chicken are completely identical.

[Use of bacterial DNA-methylases for structuro-functional analysis of the eukaryotic genome]. / Ispol'zovanie bakterial'nykh DNK-metilaz dlia strukturno- funktsional'nogo issledovaniia genoma éukariot.

Bacterial DNA-methylases with known recognition sites (RS) were used as probes for structural-and-functional analysis of eukaryotic genome. Adenine and cytosine DNA-methylases recognizing 4 to 6-member unique and degenerative nucleotide sequences having a symmetrical and asymmetrical structure were used for probing. The use of a set of methylases enabled the selection of a probe that was the most sensitive for the given pathology. Thus, severe hypothyrosis was found to be associated with changes in the acceptor capacity of liver DNA in the heterologous++ methylation reaction as could be evidenced from testing by two probes, CCCC and GAATGC. In the cells of chicken liver hepatoma MC29, the acceptor capacity of DNA during GGA methylation appeared to be altered in the greatest degree. DNA-methylases with degenerative SR are weakly specific probes for the study of structural changes (methylation) of the animal genome.

Factors of activation and stabilization of DNA-methylases from Shigella sonnei 47 and Mycobacterium smegmatis butyricum cells.

A comparative study of factors of activation and stabilization of individual DNA-methylases from two bacterial strains--Shigella sonnei 47 and Mycobacterium smegmatis butyricum--isolated by isoelectrofocusing in a pH gradient has been carried out. Storage of enzymes at +4 degrees C (pH 7.5) is accompanied by periodic changes in the methylating activity. No such changes are observed when the enzymes are stored at pI of the protein. In this case the methylases with alkaline or close to neutral values of pI remain stable over a period of at least two weeks, whereas acidic proteins are irreversibly inactivated by the end of a two-week period. A stabilizing effect of BSA on DNA-methylases of Sso 47 and Mbu strains has been demonstrated. A direct correlation between the stabilizing effect of BSA and the degree of enzyme purity has been established. Ca2+ appear to be a universal activator of methylases of the above strains; these cations produce a potent, although a short-term effect and can be used in the production of enzyme preparations with a high specific activity in DNA recombinant technology. Protease inhibitors do not exert any appreciable effect on the methylase activity upon storage. Storage at -20 degrees C and at neutral pH leads to complete inactivation of all DNA-methylases within 24 hours. In this case glycerol is fairly ineffective as a stabilizing agent.

[Effect of various conditions on the stability of DNA-methylases from Mycobacterium smegmatis butyricum cells]. / Vliianie razlichnykh uslovii na stabil'nost' DNK-metilaz iz kletok Mycobacterium smegmatis butyricum.

DNA-methylases from M. sm. butyricum, produced by means of isoelectrofocusing, were characterized by slow spontaneous variations in the activity with amplitude of 75-80% under conditions of storage in glycerol at -10 degrees. Effect of various conditions on stabilization of activity of adenine methylases MMbu4,2 and MMbu7,2 was studied. Alterations in the enzymatic activity were not found in presence of substances applied usually to stabilize the enzymes, blood serum albumin and cations of two valent metals as the values of the alterations occurred in the ranges of the enzyme activity variations.

Sequence specificity of isolated DNA-adenine methylases from Mycobacterium smegmatis (butyricum) and Shigella sonnei 47 cells.

A set of four individual DNA-adenine methylases differing in pI (isoelectric point) values (MMbu4.2, MMbu6.4, MMbu7.3, and MMbu8.7), and a sole methylating enzyme with the same base specificity (MSso9.5) are present in M. smegmatis (butyricum) and Sh. sonnei 47 cells, respectively. The sequence specificity of each of those was studied 'in vitro' by a combined approach that comprised isostich (purine tract) analysis and identification of the immediate neighbourhood of the methylated base within the sequence methylated. The MSso9.5 recognition site has been established as the hexanucleotide 'palindromic' 5'-G-A-A-T-T-C-3' sequence which is structurally similar to the analogous MEco RI recognition site. However, in contrast to MEco RI, MSso9.5 methylates the 5'-end adenine residue in the sequence and thus it appears to be an isometimer of MEco RI. By means of the same approach, the partial nucleotide sequences methylated by each of the four individual M. butyricum enzymes were determined. MMbu7.3 and MMbu8.7 exhibit the identical sequence specificity upon methylation of the degenerative trinucleotide 5'-Py-A-Py-3' sequence and thus these enzymes are assumed to represent the different molecular forms of the methylase. MMbu4.2 methylates the 5'-G-G-A-3' sequence and thus it is of a great value as the tool for negating effects of the RBam HI and RAva II-type restriction. MMbu6.4 is of a particular interest on account of its unique DNA methylation pattern which is distinguished in the pronounced clustering of purine bases in the 5'-Pu-Pu-Pu-Pu-Pu-3' sequence methylated.

[Isolation and fractionation of DNA methylases from Mycobacterium butyricum]. / Vydelenie i fraktsionirovanie DNK-metilaz Mycobacterium butyricum.

Properties of total preparation of methylases from M. butyricum strain were studied using various methods of column chromatography. Distinct heterogeneity of the methylase preparation was demonstrated by gel filtration, anion exchange chromatography on diethyl aminoethyl cellulose and affinity chromatography on Sepharose blue. Several methylases, dissimilar both in physico-chemical properties and in their specificity to nitrogenous bases, were found in M. butyricum cells. In the strain studied methylases of adenine and cytosine were found, which were responsible for biosynthesis of 6-methyl adenine and 5-methyl cytosine in the acceptor DNA at the ratio 9 : 1.

Isoelectric focusing of bacterial DNA methylases.

The multiplicity of bacterial DNA methylases has been shown for new microorganisms, Mycobacteria and Shigella, by a double-step procedure including column chromatography followed by isoelectric focusing of the total methylase fraction. The profiles of the DNA methylating activity of Sh. sonnei 47 and M. butyricum strains were studied. Sh. sonnei 47 cells were found to contain five different proteins responsible for DNA methylation and having pI 4.2, 5.3, 6.2, 8.4 and 9.2. Four M. butyricum methylases were represented by proteins with pI 4.2, 6.0, 8.0 and 9.0.

[Relations between the condition of growth and activity of DNA-methylases of Mycobacteria butyricum]. / Aktivnost' DNK-metilaz Mycobacteria butyricum v zavisimosti ot uslovii vyrashchivaniia.

Conditions for cultivation of Mycobacteria butiricum were studied. Optimal conditions were selected to maintain maximal rate of DNA-metylase activity keeping of unspecific nuclease activity in cells at a low level. Analyses of plots of the time- dependent DNA methylation by the enzymes, isolated from bacterial preparations cultivated under various conditions (solid and liquid synthetic and organic media with earation and without of it), demonstrated that aeration and organic medium were the deciding conditions responsible for high level of the methylase activity. The shape of plot of DNA methylation by the enzymes from M. butiricum was similar irrespective of the conditions of cultivation--maximal rate of methylation occurred within 2-3 hrs of incubation and was maintained within 22 hrs. The stable content of methylated DNA-acceptor, within long-term periods of incubation, correlated with the low level of nucleases since viscosity of DNA from Cd phage was unaltered after incubation with the enzyme preparation from M. butiricum. The M. butiricum strain appears to be valuable for identification of restrictases.

[Enzymes of citrate and isocitrate conversion in the heart and skeletal muscle mitochondria of embryos and adult rabbits]. / Fermenty prevrashcheniia tsitrata i izotsitrata v mitokhondriiakh serdechnoi i skeletnoi myshtsy émbrionov i vzroslykh krolikov.

Activities of citrate synthase, aconitase, NAD- and NADP-dependent isocitrate dehydrogenases were studied in mitochondria of heart and skeletal muscles of embryos and adult rabbits. Activity of these enzymes was some times lower in embryonal skeletal muscles as compared with the muscles of adult animals. Differences in activities of citrate synthase, aconitase and NADP-dependent isocitrate dehydrogenase were unsignificant in heart muscles of embryos and adult animals. Activity of NAD-dependent isocitrate dehydrogenase was distinctly higher in embryonal heart than in adult rabbits. The kinetic parameters enabled to conclude that in vitro regulation of NAD-dependent oxidation of isocitrate by substrate and activator ADP, characteristic for the enzyme from tissues of adult animals, was also found in embryos.

[The correlation between activity of enzymes participating in the formation and utilization of acetyl CoA in rabbit heart mitochondria in myocarditis and normal state]. / Sopostavlenie aktivnostei fermentov, uchastvuiushchikh v obrazovanii i ispol'zovanii atsetil-KoA v mitokhondriiakh serdatsa krolika v norme i pri miokardite

Activities in rabbit heart mitochondria of acetoacetyl-CoA-thyolase, pyruvate dehydrogenase, acetyl CoA-synthetase, citrate synthase and acetyl carnitine transferase were compared. These enzymes participate in formation and utilization of acetyl-CoA. The acetoacetyl-CoA-thyolase and acetyl CoA-synthetase were shown to possess the more distinct capacity in vitro to form acetyl CoA. The co-enzyme was most efficiently utilized under these experimental conditions by the citrate synthase. The enzymes studied were localized within the mitochondria fraction in both the subfractions of soluble and membrane-bound proteins. In myocarditis a distinct decrease in activities of the acetoacetyl-CoA-thyolase and citrate synthase was observed.