[Purification and various properties of exonuclease from bacteriophage T4]. / Ochistka i nekotorye svoistva ékzonukleazy bakteriofaga T4.

Exonuclease A was isolated from bacteriophage T4-infected cells of E. coli. The molecular mass of the enzyme is approximately 42,000 Da, pH optimum is 7-8.5, pI is 4.05. The enzyme activity depends on Mg2+, the optimal concentration of Mg2+ being 1-5 mM. The enzyme splits one- and two-helical DNA in the direction of 3'----5' and is a deoxyribonuclease splitting 5'-deoxynucleotides. The enzyme shows a practically equal affinity for one and two-helical DNA. The Km value for one- and two-helical DNA is 10 +/- 1 and 11 +/- 1 pmole of chain DNA, respectively. The Vmax value for one- and two-helical DNA is 61 +/- 5 and 45 +/- 5 pmole of nucleotides per min. Exonuclease A may be used for preparing substrates for DNA-polymerase T4 and Klenow fragment, i.e., during labeling of DNA at 3'-ends.

[Use of Cibacron blue F 3GA-CL-sepharose 6B for the purification of T4 DNA- and RNA-ligases]. / Ispol'zovanie CL-sefarozy 6B-tsibakron golubogo F3GA dlia ochistki DNK- i RNK-ligaz T4.

The sorption capacity of the dye cibacron blue F3GA, immobilized on CL-Sepharose 6B and other support matrices, in respect to DNA- and RNA-ligases T4 was being studies. Cibacron blue F3GA immobilized on CL-Sepharose 6B binds a three-fold amount of DNA-ligase in comparison to RNA-ligase. The enzyme chromatography on cibbacron blue F3GA-CL-Sepharose 6B revealed a stronger linkage between DNA-ligase T4 and the sorbent than between RNA-ligase T4 and the sorbent. Elution was performed with potassium chloride. DNA-ligase T4 was eluted with 0.25-0.5 M KCl and RNA-ligase T4 with 0.08-0.18 M KCl. Since deoxyexonuclease contaminants possess stronger bonds with the sorbent than ligases, elution of deoxyexonucleases occurs at higher concentrations of KCl. Chromatography of enzymes on cibacron blue F3GA-CL-Sepharose 6B allows one to obtain DNA- and RNA-ligases essentially free of DNase and RNase contaminants.

[Interaction of DNA and RNA ligases of phage T4 with Cibacron Blue F 3GA]. / Vzaimodeistvie DNK- i RNK-ligaz faga T4 s tsibakronom golubym F3GA.

Using kinetic methods and differential spectrophotometry, the interaction between DNA and RNA ligases T4 and cibacron blue F3GA was studied. It was shown that the dye inhibits the first step of the enzymatic reaction, i. e. the formation of the AMP ligase complex. A 50% inhibition of the AMP-ligase complex by DNA ligase occurs at the dye concentration of 1 X 10(-5) M, that by RNA ligase--at 1 X 10(-4) M. Cibacron blue F3GA also inhibits the formation of end products of the reaction catalyzed by these enzymes. The dye is a noncompetitive inhibitor of RNA ligase with respect to [32P]oligoA20 and a competitive one with respect to ATP. Using differential spectrophotometry, it was found that the interaction occurs predominantly via electrostatic bonds between the SH-groups of the dye and the amino groups of lysine residues. DNA ligase possesses a higher affinity for the dye than RNA ligase.

[Biosynthesis of early enzymes induced by bacteriophage T4]. / Issledovanie biosinteza rannikh fermentov, indutsiruemykh bakteriofagom T4.

The biosynthesis of early enzymes of DNA-ligase, RNA-ligase, DNA-polymerase, polynucleotide kinase, exonuclease A induced by bacteriophage T4amN82 was studied. The maximal activity of DNA-ligase was observed at the 60th min after the infection, while that of the other enzymes was revealed at the 90th min and reached 4, 45, 529, 120 and 78 units per mg of protein for DNA-ligase, RNA-ligase, polynucleotide kinase, DNA-polymerase and exonuclease A, respectively. Bacteriophage T4amN82 induced the maximal biosynthesis of the tested enzymes, when E. coli B-23 cells were grown in medium I containing trypton bacto ("Merck", West Germany) and a yeast extract ("Difco", USA). Similar events were observed when E. coli B-23 cells infected with phage T4amN82 were grown in a medium (II) with casein hydrolysate and yeast extract. Ultrasonication used for the disruption of E. coli B-23 cells infected with bacteriophage T4 had no effect on the enzyme activities.

[Participation of 5'-deoxyadenosyl-B12 in regulating methylation of tRNA]. / Uchastie 5'-dezoksiadenozil-B12 v reguliatsii metilirovaniia tRNK

The effects of different forms of cobalamines on the activities of tRNA-methylases of Zajdela ascite hepatoma were studied. Of six cobamides studied 5'-deoxyadenosyl-B12 and factor B containing as a ligand HSO3 in the concentrations of 2.4-10(-5) and 4.8-10(-5) M inhibited the tRNA-methylase activity by 21% and 15% correspondingly. The inhibitory effect of 5'-deoxyadenosyl-B12 is probably dependent on the adenosyl part of the molecule. 5'deoxyadenosyl-B12 exerted a selective effect of Zajdela ascite hepatoma tRNA-methylases, inhibiting largely the activity of 5-methyl cytosine methylase during the methylation of the E. coli K12W6 tRNA and yeast tRNA1 Val.

[Estimation of kinetic constants and study of site specificity of Zajdela ascite hepatoma and rat liver tRNA-methylases]. / Opredelenie kineticheskikh konstant i izuchenie pozitsionnoi spetsifichnosti tRNK-metilaz astsitnoi gepatomy zaidelia i pecheni krys

Individual yeast tRNAVal1 was used as a substrate for estimation of kinetic constants and study of site specificity of m5C-and m1A-methylases of Zajdela ascite hepatoma and rat liver. It was demonstrated that the rate of yeast tRNAVal1 methylation by hepatoma tRNA-methylases is 4--5 times higher than that induced by liver tRNA-methylases. The rates of 1-hour methyl groups incorporation into tRNAVal1 were 3.7 and 4.7 times higher in case of m5C-and m1A-methylases and 9.4 and 4.5 times higher in case of m1G-and m7G-methylases of hepatoma than the respective rates obtained for corresponding liver methylases. The main products of methylation were m5C and m1A containing about 90% of total radioactivity incorporated into tRNA. m5C-methylases of liver and hepatoma had similar affinity for S-Ad-Met. The Km value for both enzymes was 2.66 micronmole; the Km values for m1A-methylases of liver and hepatoma with respect to S-Ad-Met were the same and equal to 0,25 micronmole. m5C and m1A methylases of liver and hepatoma had adequate affinity for yeast tRNAVal1; their site specificity was the same, since they methylated in yeast tRNAVal1 cytosine in the tetracytidylic sequence of C49--C52 and adenine in the 59th position from the 5'-end of the molecule.