[Thymidine and thymidylate kinases from the scallop Mizuhopecten yessoensis gonads].

Thymidine and thymidylate kinases were isolated from the gonads of scallop Mizuhopecten yessoensis. The enzymes were purified 537- and 100-fold, respectively, and were free of phosphatase and ATPase impurities. Ions of bivalent metals and ATP were necessary for both the nucleoside and nucleotide kinase activities; the pH optimum fall into the range of 7.5-8.5. KCl and NaCl at a concentration of up to 100 mM had no inhibiting effect on the activities of these scallop enzymes. Thymidine kinase catalyzed thymidine, and, at a lower rate, deoxycytidine phosphorylations did not utilize ribo- and deoxyribonucleosides, as well as pyrimidine ribonucleosides, as a phosphate acceptor. Thymidylate kinase phosphorylated TMP and dCMP with an efficiency of about 30%. In addition to ATP, these enzymes can also utilize with different efficiencies dATP, dGTP, GTP, UTP, and CTP as a donor of phosphate groups. Thymidine kinase activity was inhibited by TMP, TTP, and dCTP.

[Influence of thermolabile lethal toxin of Yersinia pseudotuberculosis on development of sea urchin embryos and biosynthesis of DNA and RNA in embryonic cells].

Influence of thermolabile lethal toxin of Y. pseudotuberculosis on the development of embryos of sea urchin (Strongylocentrotus intermedius) and on biosynthesis of nucleic acids in embryonic cells was studied. Thermolabile lethal toxin affected metabolic processes of cells by inhibiting DNA and RNA synthesis. It had damaging action on developing embryos of sea urchin causing morphological changes and, as a consequence, death of embryos.

[Yersinia pseudotuberculosis nucleoside-kinase].

Enzyme capable of catalyzing the phosphorylation of thymidine and uridine was isolated from Y. pseudotuberculosis cells by fractionation with the use of ammonium sulfate, ion exchange and affinity chromatography. The degree of purification of thymidine- and uridine-kinase was approximately 350 times, and at all stages of isolation the activity of both nucleoside-kinases was detected in the same peaks. The purified enzyme was capable of the phosphorylation of thymidine and uridine at temperatures of 8-10 degrees C to 50 degrees C and exhibited the maximum enzymatic activity at pH 8-8.5 and 45 degrees C in the presence of 0.5-1.0 mM MgCl2 and 2 mM ATP. The enzyme was found to have no strict substrate specificity and transferred the phosphate group from ATP to radiolabeled thymidine, uridine and desoxycytidine with different effectiveness, but did not use thymidine-monophosphate as phosphate acceptor.

[The action of Yersinia pseudotuberculosis heat stable lethal toxin on the biosynthesis of protein in vitro]. / Deistvie termostabil'nogo letal'nogo toksina Yersinia pseudotuberculosis na biosintez belka in vitro.

The results of the in vitro action of Y. pseudotuberculosis heat stable lethal toxin on the biosynthesis of protein are presented. The toxin was shown to inhibit the inclusion of exogenous amino acid into newly synthesized peptides. The degree of the inhibition of the biosynthesis of protein in the in vitro system depends on the amount of the toxin added to the incubation mixture. The use of the conjugated transcription and translation system confirms our earlier data on the influence of the lethal toxin on the biosynthesis of protein in eukaryotic cells in vivo.

[DNA, RNA, and protein biosynthesis in cells of Yersinia pseudotuberculosis at various cultivation temperatures]. / Biosintez DNK, RNK i belka v kletkakh Yersinia pseudotuberculosis pri raznykh temperaturakh kul'tivirovaniia.

Y. pseudotuberculosis cells cultivated at temperatures of 37 degrees C and 8 degrees C were found to be capable of incorporating exogenic precursors into DNA, RNA and protein. The linear growth of thymidine incorporation occurred during 8 hours of cultivation at 37 degrees C, then the amount of the incorporated label decreased. At 8 degrees C the level of thymidine incorporation into DNA gradually increased for 80 hours and longer, but not reaching the level of incorporation observed at 37 degrees C. The incorporation of uridine into RNA of Y. pseudotuberculosis cells reached its maximum after 4 hours of cultivation at 37 degrees C, at a lower temperature of cultivation the incorporation of uridine into bacterial cells was almost linear, though slower, and lasted for 20 hours. The content of radioactive alanine in Y. pseudotuberculosis protein increased during 16 hours of cultivation at a high temperature, while at 8 degrees C the growth of the incorporation level lasted for at least 40 hours. For all precursors under study the incorporation rate into the cell biopolymers at the initial stages of cultivation was higher at 37 degrees C, than at a lower temperature.

[Thymidine kinase from sea urchin oocytes]. / Timidinkinaza iz iaitsekletok morskog ezha.

Thymidine kinase was isolated and purified 1600-fold from sea urchin (Strongylocentrotus intermedius) oocytes. The molecular mass of the enzyme is 66 kDa, pI is 5.2. The enzyme activity needs Mg2+, ATP and the corresponding phosphate acceptor. The pH optimum of the enzyme activity is at 9.0-9.5. The isolated enzyme does not exhibit any strict substrate specificity and can phosphorylate thymidine, deoxycytidine and some other pyrimidine nucleosides and their derivatives, the phosphorylation rate being maximal for thymidine, ATP or dATP. The TMP formed via the enzymatic reaction does not influence the thymidine kinase activity.

[Inhibition of various stages of DNA replication in sea urchin embryos]. / Ingibirovanie otdel'nykh stadii protsessa replikatsii DNK émbrionov morskikh ezhei.

The embryos of the sea urchin Strongylocentrotus intermedius possess the ability to incorporate into their DNAs 2'-deoxynucleosides together with all their bases, i.e., adenine, guanine, cytosine and thymine. This incorporation is inhibited by 3'-amino-2',3'-dideoxynucleosides with the same bases. 5'-Amino-5'-deoxynucleosides and 5'-amino-2',5'-dideoxynucleosides moderately inhibit the incorporation of [3H]2'-deoxynucleosides into the DNAs by competing with the latter, presumably at the phosphorylation stage. The most potent inhibiting effect is exerted by 2'-amino-2'-deoxynucleosides and 2'-asido-2'-deoxynucleosides; the mechanism of this inhibition is still obscure, however.

[Isolation and various properties of beta DNA polymerase from the embryos of the sea urchin Strongylocentrotus intermedius]. / Vydelenie i nekotorye svoistva DNK-polimerazy beta iz émbrionov morskogo ezha Strongylocentrotus intermedius.

DNA-polymerase beta was isolated from embryonic cells of the sea urchin S. intermedius and purified 1040-fold. The molecular weight of the enzyme is 40 000, sedimentation coefficient 3.2S, pI 8.5. The SH-reagent--N-ethylmaleimide--has no appreciable influence on the enzyme activity. The enzyme is thermolabile and needs four deoxyribonucleoside triphosphates, bivalent metal ions (Mg2+ or Mn2+) and primer template for its activity. The maximal activity is observed when a synthetic polymer--poly(dA).oligo(dT) is used. DNA-polymerase performs DNA synthesis via a distributive mechanism. In terms of physico-chemical properties, the enzyme can be related to DNA-polymerases beta.

[Inhibition of DNA biosynthesis in sea urchin embryos by 2',3'-dideoxy-3'-aminonucleosides]. / Ingibirovanie biosinteza DNK v émbrionakh morskikh ezhei s pomoshch'iu 2',3'-didezoksi-3'-aminonukleozidov.

It was shown that 3'-deoxy-3'-aminothymidine and 2',3'-dideoxy-3'-aminoadenosine are effective inhibitors of DNA synthesis of sea urchin (Paracentrotus lividus) embryos during the early steps of development. The most probable mechanism of inhibition includes the blocking of the template-directed polycondensation, in which the inhibitors act as analogs of the substrates--thymidine and 2'-deoxyadenosine 5'-triphosphates. An efficient degradation of the newly synthesized DNA was observed during the inhibition.

[DNA-polymerase from sea urchin (Strongylocentrotus intermedius. Embryos]. / Dnk-polimeraza iz embrionov morskogo ezha Strongylocentrotus intermedius. Ochistka i nekotorye svoistva.

Using DEAE-cellulose chromatography, three peaks of the DNA-polymerase activity were found in a homogenate of the sea urchin Strongylocentrotus intermedius embryos at stage 32 of the blastomer. The isolation and purification of DNA-polymerase making up the bulk of he DNA-synthesizing activity of the sea urchin embryo cells included fractionation by ammonium sulfate, chromatography on DEAE-cellulose, hydroxyapatite and DNA-cellulose, resulting in a 2750-fold purification of the enzyme. The enzyme activity requires the presence of two-chain DNA activated by pancreatic DNAse, four dNTP and Mg2+. The enzyme is inhibited by a high ionic strength (150 mM KCl or NaCL) and the sulfhydryl reagent--N-ethylmaleimide; the pH optimum is 8.0. The molecular weight of the enzyme as determined by gel-filtration is about 150 000. It is assumed that the enzyme under study can be related to DNA-polymerases of the alpha-type.

[Isolation and some properties of ATP-dependent DNAse from sea urchin (Strongylocentrotus intermedius) embryo]. / Vydelenie i nekotorye svoistva ATP-zavisimoi DNKazy iz embrionov morskogo ezha (Strongylocentrotus intermedius).

An ATP-dependent DNAse was isolated from the cells of the sea urchin Strongylocentrotus intermedius embryos by chromatography on DEAE-cellulose and gel chromatography on Sepharose 4B. The enzyme was found homogeneous during polyacrylamide gel electrophoresis. The molecular weight of the enzyme was determined by gel filtration through Sepharose 6B and was equal to approximately 450,000. The sedimentation coefficient as determined by ultracentrifugation was equal to approximately 15S. The pH optimum during native DNA hydrolysis lies within the pH range of 6,5--9,0 and that during hydrolysis of denaturated DNA--within the pH range of 9,0--9,5. The enzyme was activated by ATP and dATP at the optimal concentration of 10(-4) M. Other nucleoside triphosphates did not substitute for ATP in this reaction. The hydrolysis of denaturated DNA occurred via the exonuclease way with a formation of short (di-, tri,- tetra- and penta-) oligonucleotides. The enzyme hydrolyzed native DNA according to the endonuclease type with predominant formation of high molecular weight polynucleotides.