Biomedical subjects
Publications and source records attributed to L A Luchkina.
Universal primer ability of generating conservative and variable UP-PCR (universally primed polymerase chain reaction) species-specific patterns was analysed on bacteria to serve as an example. Also, two important properties of the UP-PCR patterns (species/primer DNA hybridization specificity) are characterized.
Cationic proteins--cytochrome c and pancreatic RNAase--possess the apurinic-apyrimidinic DNA-endonuclease activity. The affinity of these proteins for DNA-apurinic sites does not differ from that of specific apurinic DNA-endonucleases described in literature. The main features of the apurinic activity of cationic proteins are as follows: low specific activity, high temperature optimum of the reaction, absence of primer-stimulated activity. The feasibility of participation of cationic proteins and some other nucleophilic compounds in single-stranded breaks production in apurinic DNA is discussed.
An activity which released free uracil from dUMP-containing DNA was purified approximately 1,700-fold from extracts of Thermothrix thiopara, the first such activity to be isolated from extremely thermophilic bacteria. The enzyme appeared homogeneous, according to the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It had a native molecular weight of 26,000 and existed as a monomer protein in water solution. The enzyme had an optimal activity at 70 degrees C, between pH 7.5 and 9.0, and in the presence of 0.2% Triton X-100. It had no cofactor requirement and was not inhibited by EDTA, but it was sensitive to N-ethylmaleimide. The purified enzyme did not contain any nuclease that acted on native or depurinated DNA. The Arrhenius activation energy was 76 kJ/mol between 30 and 50 degrees C and 11 kJ/mol between 50 and 70 degrees C. The rate of heat inactivation of the enzyme followed first-order kinetics with a half-life of 2 min at 70 degrees C. Ammonium sulfate and bovine serum albumin protected the enzyme from heat inactivation. One T. thiopara cell contains enough activity to release about 2 X 10(8) uracil residues from DNA during one generation time at 70 degrees C.
An endonuclease specific for apurinic, apyrimidinic (AP) sites in DNA was purified nearly to homogeneity from the extremely thermophilic bacterium Thermothrix thiopara. The enzyme has a molecular weight of approximately 26,000. It cleaves neither native nor UV- or gamma-irradiated DNAs and has no contaminating exonuclease or uracil-DNA glycosylase activities. The enzyme has no cofactor requirement and is not inhibited by EDTA or N'-ethylmaleimide. It shows maximal activity at 70 degrees C and a pH between 7.5 and 9.0. The Arrhenius activation energy of the reaction is 17 kJ/mol, and the apparent Km for AP sites is 38 nM. The rate of heat inactivation of the enzyme followed first-order kinetics, with a half-life of 10 min at 70 degrees C but about 150 min in the presence of 0.5 M ammonium sulfate or 0.5 mg of bovine serum albumin per ml at the same temperature. One cell of T. thiopara contains sufficient AP endonuclease activity for hydrolysis of about 10(6) phosphodiester bonds per h at 70 degrees C. An extract of these bacteria does not contain detectable Mg-dependent AP endonuclease activity, and the above-mentioned enzyme appears to be the main AP endonuclease of T. thiopara.
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A method for producing spheroplasts from yeast cells has been developed. The method involves: 1) prefreezing and thawing of cells in the minimal nutrient medium; 2) treatment with the mixture of 86 mM 2-mercaptoethanol, 5% sodium dodecylsulphate and 6% Triton X-100; 3) enzymic digestion by the digestive juice of grape helix. The formation of spheroplasts was controlled by microscopy and measurement of the optic density of the spheroplast suspension in the incubation medium and in 1% sodium dodecylsulphate. The method has been successfully tested on some bakery yeast: Saccharomyces cerevisiae, Saccharomyces paradoxus, Schizosaccharomyces pombe, Saccharomyces globosus, Saccharomyces carlsbergensis.
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