[Bioluminescent assay of total bacterial contamination of raw milk]. / Bioliuminestsentnyi metod opredeleniia obshcheo bakterial'noi obsemenennosti syrogo moloka.

A rapid (30-35 min) bioluminescence assay of total bacterial contamination (TBC) of raw milk was optimized. This method includes incubation of milk samples in the presence of Neonol-10 and medical purity grade pancreatin with further removal of nonbacterial ATP by filtration through a membrane filter, cell disruption by treatment with dimethyl sulfoxide, and measurement of ATP concentration in a reaction with the bioluminescent reagent Immolum. The TBC detection threshold is 0.5 x 10(5) colony-forming units (CFU) per ml milk. Coefficients of correlation between the standard plate count method and bioluminescence assay (R) and residual standard deviations (Sxy) in raw milk samples (n = 140) were 0.83 and 0.54, respectively. In sterilized milk samples artificially contaminated with pure cultures of the main representatives of milk microflora (coli-forms, Staphylococcus aureus, Streptococcus thermophilus, and Streptococcus group D), these values were 0.89-0.99 and 0.09-0.29, respectively. The specific content of ATP was found to be (0.8 +/- 0.1) x 10(-18) mol/CFU in coli-forms; (12.0 +/- 8.1) x 10(-18) mol/CFU in S. aureus; (35.2 +/- 16.9) x 10(-18) mol/CFU in S. thermophilus; and (42.5 +/- 1.3) x 10(-18) in Streptococcus group D.

[A bioluminescent method of determining antibiotic sensitivity of microbial cells in septic blood]. / Bioliuminestsentnyi metod opredeleniia antibiotikochuvstvitel'nosti mikrobnykh kletok v septicheskoi krovi.

A bioluminescence assay for rapid (5-7 h) determination of susceptibility to antibiotics was applied to samples of septic blood and optimized. The method comprises hemolysis of blood, reduction of osmolarity by adding concentrated nutritive media, and further incubation of the samples in the presence or absence of therapeutic doses of the antibiotic examined. Growth of bacteria is estimated by the level of bacterial ATP in the sample, which is determined by a bioluminescence assay. Hemolysis and further incubation of samples in nutrition media reduced the concentration of nonbacterial ATP to a level that did not interfere with the determination of bacterial ATP. There was a positive correlation between the levels of resistance to antibiotics determined by the bioluminescence assay and standard plate counts.

[Comparative evaluation of methods of intracellular ATP extraction from different types of microorganisms for bioluminescent determinationof microbial cells]. / Sravnenie metodov ekstraktsii vnutrikletochnogo ATF mikroorganizmov razlichnogo tipa dlia bioliuminestsentnogo opredeleniia kletok mikro organizma.

The efficiency of dimethyl sulfoxide (DMSO), trichloroacetic acid (TCA), and cetyltrimethylammonium bromide (CTAB) as extractants of intracellular ATP from various microorganisms was compared in bioluminescent measurements of microbial cell concentrations. Extraction with CTAB was found to provide an approximately ten times higher sensitivity of the bioluninescent assay of microbial cells than extraction with DMSO or TCA. In Gram-positive bacteria and yeasts, the ATP concentration in the extract was a linear function of the microbial suspension density only within a cell concentration range of D600 0.02-3.5 in the three types of tested extracts. In Gram-negative bacteria, a significant deviation from the linear dependence between ATP concentration and microbial suspension density was observed in CTAB extracts at D600 > 1.

[The ATP content of the neutrophils and whole blood in the inhabitants of regions in the Altai Territory subjected to nuclear testing exposure]. / Soderzhanie ATF v neitrofilakh i tsel'noi krovi u zhitelei raionov Altaiskogo kraia, podvergshikhsia vozdeistviiu iadernykh ispytanii.

The bioluminescent method was used in the studies of the influence of ionizing irradiation and/or xenobiotics on the content of ATP in RBC and neutrophils of rats, and in whole blood and neutrophils of 80 examined women of Altai Region exposed to ionizing radiation during a series of nuclear tests in Semipalatinsk in 1949-1965. Deviations from the normal ATP content were measured with due to account of the natural variability of a given metabolite. For rats, deviations from the content of ATP in erythrocytes were short-term, those in neutrophils were long-term. For people, a statistically significant increase in the content of ATP in neutrophils as compared to the control was observed. A non-linear correlation between the content of ATP in neutrophils and the calculated dose of radiation was observed. An increase in the ATP content in whole blood, with regard to the control, was not statistically significant for all groups of examined persons.

[Bioluminescence and bioluminescent analysis: development of certain aspects of the problem over the last decade]. / Bioliuminestsentsiia i bioliuminestsentnyi analiz. (Razvitie nekotorykh aspektov problemy za poslednee desiatiletie).

Recent data from gene engineering, kinetic and fluorescent studies concerning the structure and functions of firefly luciferase are reviewed. Some new trends in the development of bioluminescent methods of control over bacterial contaminations, dynamics of intracellular processes and methods of bioluminescent detection in immuno- and DNA-assays are described. Possible applications of luciferase genes as markers are considered.

[Change in adenine nucleotide pool in E. coli 1257 bacterial cells under the action of a low intensity He-Ne laser]. / Izmenenie pula adeninovykh nukleotidov v kletkakh bakterii E. coli 1257 pri vozdeistvii nizkointensivnogo He-Ne lazera.

Evidence is presented of co-immobilized bioluminescent reagents which include firefly luciferase, pyruvate kinase and adenylate kinase used for an adenylate assay in bacterial cells. The changes in the ATP, ADP and AMP content induced by irradiation with a low-power He-Ne-laser depend on the laser power and irradiation dose. The data obtained suggest that laser irradiation causes the activation of adenine nucleotide synthesis de novo.

[The effect of low doses of ionizing radiation on intracellular pH, ATP content and synthetic activity in cultured Chinese hamster fibroblasts]. / Vliianie malykh doz ioniziruiushchei radiatsii na vnutrikletochnyi pH, soderzhanie ATF i sinteticheskuiu aktivnost' kul'tiviruemykh fibroblastov kitaiskogo khomiachka.

X irradiation of Chinese fibroblasts with doses of 0.05-0.15 Gy was shown to cause intracellular pH (pHi) changes: its diminishing during the first 40-60 min by 0.16-0.18 pH units, then the return to the control level 120 min after irradiation and, finally, the increase by 0.18-0.20 pH units. Simultaneously, the synthesizing activity of the cells changed in the same way. The ATP level changed in the opposite way: increased when pH fell and decreased when pH grew. It was shown that pHi changes were connected with the changes in Na+/H(+)-exchange system, and they seemed to be primary in the chain of the alterations observed.

[The effect of starvation on mast cell functional activity and on the content of microsomal cytochromes in the liver]. / Vliianie golodaniia na funktsional'nuiu aktivnost' tuchnykh kletok i soderzhanie mikrosomal'nykh tsitokhromov v pecheni.

Histamine secretion and ATP levels were measured in mast cells from the abdominal and pleural cavities of Wistar rat males (250-300 g) which had been starving for 2-4 days. Food deprivation had no effect on histamine levels and secretion in exposure to substance 48/80. ATP concentration got reduced from starvation day 2. Food deprivation for 2 days led to decline in the content of cytochromes P-450, P-450B, in the ratio P-450B/P-450L, but had no effect on rat liver cytochrome B5. 4-day starvation did not entail further changes.

[A bioluminescent method of determining the sensitivity of microflora to antibiotics]. / Bioliuminestsentnyi metod opredeleniia chuvstvitel'nosti mikroflory k antibiotikam.

A bioluminescent rapid method (a three-hour test) has been developed to assess the susceptibility of microflora to antibiotics. It is based on comparison of intracellular ATP content in a sample (pus) after 3 h incubation at 37 degrees C in liquid culture medium (dilution: sample/culture medium--1:9) on a shaker in the presence and absence (control) of a single antibiotic concentration. A criterion has been offered for quantitative estimation of antibiotic susceptibility of microorganisms. The authors have optimized the measurements of intracellular ATP by the rapid bioluminescent method using immobilized firefly luciferase-based ATP reagent. The results of the method are in good correlation with those of the standard agar diffusion technique.

[Bioluminescent methods of analysis in microbiology]. / Bioliuminestsentnye metody analiza v mikrobiologii.

The prospects for application of bioluminescent ATP-metry in microbiology are considered. A bioluminescent assay is proposed to analyse biomass by measuring the content of intracellular ATP by means of immobilized firefly luciferase after ATP extraction with dimethylsulfoxide. The assay can be used for plotting the growth curves of microorganisms and for determining the sensitivity of microorganisms to antibiotics. The detection limit of the assay is 700 cells per ml of the measured solution.

[Bioluminescent analysis in medicine and biotechnology]. / Bioliuminestsentnyi analiz v meditsine i biotekhnologii.

The possible applications of the immobilized bioluminescent systems of bacteria and fireflies in microassay are shown. The immobilization resulted in 10-100-fold stabilization of luciferases permitting their multiple use in flow column reactors. Reagents containing luciferases coimmobilized with other enzymes were used for determination of the picomolar concentrations of ATP, AMP, ADP, NADH2 and NAD+. ATP-metry was used for monitoring the growth of the microorganisms, determination of the biocide effect and estimation of the activity of ATP-dependent enzymes such as creatine kinase in medical diagnosis.

[Immobilized luciferase of Luciola mingrelica fireflies. The kinetic properties and thermostability of luciferase immobilized on cellulose films]. / Immobilizovannaia liutsiferaza svetliakov Luciola mingrelica. Kineticheskie svoistva i termostabil'nost' liutsiferazy, immobilizovannoi na tselliuloznykh plenkakh.

Luciferase of fireflies Luciola mingrelica was immobilized on cellulose films activated by cyanuric chloride or sodium periodate. Kinetic properties and the contribution of diffusional obstacles to the kinetics of the immobilized enzyme were examined. External and internal diffusion were found to influence the kinetic parameters. The stability of the enzyme was investigated at 25 degrees C and pH 7.8. Thermoactivation of the immobilized enzyme was shown to proceed in two stages: fast and slow. Dithiotreitol and cystein stabilized the enzyme at the fast stage while salt supplements at both stages. The fast thermoinactivation stage was apparently associated with the oxidation of luciferase SH-groups. It is demonstrated that the immobilized enzyme of Luciola mingrelica can be employed to measure ATP traces with the detection limit 0.1 mM. The enzyme immobilized on cellulose films can be used repeatedly.

[Subunit interactions in luciferase from the firefly Luciola mingrelica. Their role in the manifestation of enzyme activity and during thermoinactivation]. / Sub"edinichnye vzaimodeistviia v liutsiferaze svetliakov Luciola mingrelica. Ikh rol' v proiavlenii aktivnosti fermenta i protsesse termoinaktivatsii.

It was shown that the dimers of the firefly luciferase possess the catalytic activity, whereas the monomers do not. The dissociation constant (Kd) for active dimers was determined at pH 7.0--8.4 within the temperature range of 15--35 degrees and at MgSO4 and Na2SO4 concentrations varying from 37 to 370 mM and 49 to 490 mM, respectively. Under variable conditions the Kd value changed only insignificantly and made up to 13 nm. The substitution of Na2SO4 for MgSO4 decreased Kd 2.5 times. The effective rate constant for the enzyme inactivation (kin) was increased more than 5-fold, when the luciferase concentration was decreased from 200 down to 3.5 nM in the presence of 37 mM MgSO4. When the concentration of the latter was increased up to 185 mM, the value of kin ceased to depend on the enzyme concentration. The decrease of kin was also observed at an increase in Na2SO4. An inactivation pattern for the enzyme in solution was determined both for the monomer and for the dimer of the enzyme. The equations allowing to calculate the inactivation constant for the monomer (Ki) and dimer (k2) at different pH values, temperatures and salt concentrations were obtained. The enzyme was found to be stabilized by salts more than 10-fold, the stabilizing effect being far more pronounced for the enzyme monomer than for the dimer. The dependence of the effective kin value on pH and temperature was primarily influenced by the dependence of the inactivation rate constant for the dimer.

[Immobilized luciferase from the fireflies Luciola mingrelica. Changes in the pH-dependence of the enzyme catalytic activity and stability during its immobilization on various polysaccharide carriers]. / Immobilizovannaia liutsiferaza svetliakov iz Luciola mingrelica. Izmenenie pH-zavisimosti kataliticheskoi aktivnosti i stabil'nosti fermenta pri immobilizatsii na razlichnykh polisakharidnykh nositeliah.

The luciferase from the fireflies luciola mingrelica was immobilized on different CNBr-activated polysaccharide carriers, e. g. Sepharose, Ultrodex, cellophane, Ultrogel. The catalytic activity and the inactivation rate constants of the soluble and immobilized enzyme at 25 degrees (pH 6.0--9.0) were determined. During immobilization on Sepharose the pH profile of activity shifts towards lower pH values, while upon immobilization on Ultrogel and Ultrodex it is considerably broadened. Immobilization on Ultrogel and Ultrodex results in a 3--100 -fold stabilization of the enzyme (pH less than 7.5). whereas the stability of luciferase immobilized on Sepharose and cellophane within the same pH range is 10--1000 times higher than that of the soluble enzyme. It was shown that inactivation of luciferase immobilized on Sepharose and cellophane is limited by oxidation of the SH-groups of the enzyme and is inhibited by dithiothreitol. The inactivation of luciferase immobilized on Ultrogel and Ultrodex and that of soluble enzyme is not limited by oxidation of the SH-groups.

[Kinetics and mechanism of inactivation and reactivation of immobilized luciferase from fireflies Luciola mingrelica and the role of sulfhydryl in these processes]. / Kinetika i mekhanizm inaktivatsii i reaktivatsii immobilizovannoi liutsiferazy svetliakov Luciola mingrelica i rol' sul'fgidril'nykh grupp v etikh protsessakh.

The role of SH-groups of the enzyme in the catalytic and inactivation processes of soluble and immobilized on Sepharose 4B luciferase from the fireflies Luciola mingrelica was studied. A significant role of SH-groups of the soluble and immobilized enzyme was established by titrating the enzyme with 5,5'-dithiobis-2-nitrobenzoic acid. It was shown that the rate of inactivation of immobilized luciferase is limited by oxidation of the SH-groups of the enzyme; therefore dithiothreitol effectively protects the immobilized enzyme against inactivation. The kinetics of inactivation and reactivation of immobilized luciferase in the presence of dithiothreitol were studied. Dithiothreitol had no protective effect on soluble luciferase, which is indicative of another limiting step of inactivation of soluble luciferase. A mechanism of inactivation of Luciola mingrelica luciferase is proposed. The kinetic pattern of these processes is postulated and the rate constants of individual steps of inactivation of luciferase immobilized on Sepharose 4B were determined.

[Effect of chemical modification on thermal stability of horseradish peroxidase]. / Vliianie khimicheskoi modifikatsii na termicheskuiu stabil'nost' peroksidazy iz khrena.

Soluble preparations of horse radish peroxidase are obtained by means of its amino groups modification with glutaric aldehyde, maleic anhydride and inert proteins including albumin. The enzyme activity is found to decrease under the modification with glutaric aldehyde and to be unchanged at all other cases. Thermal stability of the enzyme preparations obtained is studied within the temperature range from 56 to 80 degrees C. Thermostability of glutaric aldehyde-modified peroxidase is approximately 2.5-fold decreased at 56 degrees C. Thermostability of other preparations exceeds the stability of native peroxidase in 25--90 times at 56 degrees C. Thermodynamic parameters of activation for the process of irreversible thermoinactivation of native and modified enzyme are calculated. A strong compensation effect between activation enthalpy and entropy values is observed, which were changed in 1.5--2 times, while the free activation energy is changed by 2--3 kcal/mol only. Possible mechanism of the change of the enzyme thermal stability under its chemical modification is discussed.