[Determination of antibiotics using luminescent Escherichia coli and serum].

The methodical bases for detecting antibiotics using a bioluminescent assay and blood serum are briefed. Antibiotics inhibit the luminescence of a genetically engineered Escherichia coli strain. The degree of inhibition depended on the type of antibiotic, its concentration, and the time of cell incubation with antibiotic. The highest cell sensitivity was recorded towards the aminoglycoside antibiotics, which amounted to 85 +/- 10 ng/ml for gentamicin and streptomycin. The sensitivity of this system to a number of antibiotics essentially increased when the cells were previously activated with blood serum. The sensitivity of this method for gentamicin and streptomycin in the presence of blood serum amounted to 2.5 +/- 0.5 ng/ml; for tetracycline, 45 +/- 8 ng/ml. Use of the sera containing specific antibodies to the antibiotic detected provided a high sensitivity of the biosensor tested. Comparison of the luminescences of E. coli cells activated with normal and specific antisera upon incubation with an antibiotic allows the type of antibiotic and its quantitative content in the sample to be determined. Characteristic of the analysis of antibiotics with the help of recombinant E. coli are a high accuracy, sensitivity, specificity, simplicity, and a short time needed for measurement.

[Photoquenching of the bioluminescence of the genetically engineered Escherichia coli TG1 (pXen7) strain in the presence of photodithazine]. / Fotoindutsirovannoe podavlenie bioliuminestsentsii genno-inzhenernogo shtamma bakterii Escherichia coli TG1 (pXen7) v prisutstvii fotoditazina.

The photoquenching of the bioluminescence of the genetically engineered Escherichia coli TG1 (pXen7) strain was studied in the presence of the photosensitizer photodithazine, a glucosamine salt of chlorin e6. The photosensitized quenching of the bioluminescence was found to correlate with the colony-forming ability of the strain. The data obtained are discussed from the standpoint of using biosensor luminescent bacterial systems for the assessment of the efficiency of photosensitizers in antimicrobial photochemotherapy.

[FMN-reductase from Escherichia coli and its effect on the activity of luciferase from marine bacterium Vibrio fischeri]. / FMN-reduktaza iz Escherichia coli i ee vliianie na aktivnost' liutsiferazy iz morskikh bakterii Vibrio fischeri.

Interactions of luciferases isolated from Vibrio fischeri 6 and Escherichia coli JM109(pF3) (bearing cloned V. fischeri luxAB genes) with FMN reductase isolated from E. coli JM109 were studied. FMN reductase formed a stable complex with luciferase, suggesting similar properties of the FMN reductases in the taxonomically close families Vibrionaceae and Enterobacteriaceae.

[Use of bacterial luciferase for determining monoamine oxidase activity]. / Ispol'zovanie bakterial'noi liutsiferazy dlia opredeleniia monoaminooksidaznoi aktivnosti.

A bioluminescence assay is proposed for measuring monoamine oxidase activity in different biological specimens (platelets, mitochondria). The assay is based on the bioluminescent reaction catalysed by bacterial luciferase and coupled to monoamine oxidase. Two modifications of the bioluminescence assay were used. In the first case, the bioluminescent system was added to monoamine oxidase preincubated with the substrates, while in the second case, all the components of the coupled enzymatic systems were directly mixed in a cell. The proposed bioluminescence assay is simple, highly sensitive and rapid, and could be especially useful for biomedical examinations.

Bioluminescence decay kinetics in the reaction of bacterial luciferase with different aldehydes.

At 22 degrees C the bioluminescence decay kinetics in the in vitro reaction catalysed by Vibrio harveyi luciferase in the presence of different aldehydes--nonanal, decanal, tridecanal and tetradecanal did not follow the simple exponential pattern and could be fitted to a two-exponential process. One more principal distinction from the first-order kinetics is the dependence of the parameters on aldehyde concentration. The complex bioluminescence decay kinetics are interpreted in terms of a scheme, where bacterial luciferase is able to perform multiple turnovers using different flavin species to produce light. The initial phase of the bioluminescent reaction appears to proceed mainly with fully reduced flavin as the substrate while the final one results from the involvement of flavin semiquinone in the catalytic cycle.

[Action of phenobarbital on bacterial luciferase of Photobacterium fischeri]. / Deistvie fenobarbitala na bakterial'nuiu liutsiferazu Photobacterium fischeri.

The effect of phenobarbital, a typical substrate of monooxygenases from higher organisms, on bioluminescence of the marine bacterium Photobacterium fischeri and bacterial luciferase was studied. Phenobarbital was shown to be an effective quenching agent owing to the interaction with cytochrome P-450, a terminal luciferase component. A competitive interrelation was found between phenobarbital and an aliphatic aldehyde, the substrate of the luminescent reaction.

The inhibition of bacterial bioluminescence by xenobiotics.

The effect of various xenobiotic substrates of microsomal cytochrome P-450, including dimethylaniline, ethylmorphine, hexabarbital and aminopyrine, on the bioluminescence of the bacteria Vibrio fischeri and the bacterial luciferase mixed-function oxidase system is described. These compounds are effective inhibitors of the luminescence reaction. The inhibition provided evidence for the competitive nature of the interactions between xenobiotics and an aliphatic aldehyde, which is a substrate of bacterial luciferase, at the binding site for cytochrome P-450. The bioluminescence method is suitable for the analysis of metabolism and detoxication of various xenobiotics.

[Lactate dehydrogenase activity and its isoenzyme in a system coupled with bacterial luciferase]. / Issledovanie aktivnosti laktatdegidrogenazy i ee izofermentov v sopriazhennoi sisteme s bakterial'noi liutsiferazoi.

Properties of a coupled system "LDH-bacterial luciferase" were studied. The light-generating enzymatic system from luminescent bacteria Photobacterium fisheri was used as an indicator of the dehydrogenase activity. Kinetic parameters of the coupled system were studied using commercially available preparation of LDH and the enzyme from human blood plasma. The luminescent activity of bacterial preparation was shown to correlate with the activity of LDH and its isoenzymes within wide range of blood plasma concentrations.

[Inhibition of bacterial luminescence by cytochrome P-450 substrates]. / Ingibirovanie bakterial'noi liuminestsentsii substratami tsitokhroma P-450.

The mechanisms of luminescence quenching by various drugs, e.g. dimethylaniline, ethylmorphine, hexobarbital and aminopyrine, which are effective inhibitors of luminescence both in intact cells and in bacterial luciferase, were studied. It was shown that the inhibition of luminescence occurs due to competition of the bacterial luminescence system substrate--aliphatic aldehyde in cytochrome P-450. The functional similarity of the bacterial luminescence system to the microsomal hydroxylation system is postulated.

[Inhibitory analysis of the luminescent electron transport chain of Photobacterium fischeri]. / Ingibitornyi analiz liuminestsentnoí elektrontransportnoi tsepi Photobacterium fischeri.

The quenching of luminescence of bacterial luciferase from Photobacterium fischeri by non-specific electron acceptors and inhibitors of dehydrogenases was studied. The inhibition of the luminescent reaction obeys the non-competitive mechanism with NADH, FMN and aliphatic aldehyde. The inhibitors compete with cytochrome c for NADH -- cytochrome c oxido-reductase. It is concluded that lumiredoxin, a FeS-containing protein, is the most sensitive component of the luminescent electron transport chain.

[Isolation and purification of bacterial luciferase from Photobacterium fischeri for analytical purposes]. / Vydelenie i ochistka bakterial'noi liutsiferazy iz Photobacterium fischeridlia analiticheskih tselei.

A procedure resulting in a highly purified preparation of bacterial luciferase with a high specific activity towards FMNH2 and NADH was developed. Using SDS-electrophoresis in polyacrylamide gel, it was shown that the enzyme has a subunit composition and that its monomers do not contain the luciferase activity. The use of the obtained preparation for determining the content of NADH and FMN by the bioluminescent method allowed to increase its sensitivity up to 10(-18) and 10(-17) moles, respectively.

[Cytochromes of the luminescent bacterium, Photobacterium fischeri, their solubilization and relationship to luminescence]. / Tsitokhromy liuminestsentnykh bakterii Photobacterium fischeri, ikh soliubilizatsiia i sviaz' so svecheniem.

The hemoprotein composition of the luminescent bacterium Photobacterium fischeri was studied, in particular, the distribution of cytochromes among the bacterial fractions, viz. cell-free extract, supernatant, "particles", protein preparation. The hemochromogenic analysis has shown that the principal hemoproteins of Photobacterium fischeri are cytochromes, with hemes of the b and c type. The activity of luciferase is distributed with hemoproteins. The purified preparation of luciferase contains cytochrome of the b type that has been identified as mixed functions oxidase--P-450.

[Electron transport systems of Photobacterium fischeri]. / Elektrontransportnye sistemy Photobacterium fischeri.

The composition of cytochromes was studied in Photobacterium fischeri 6 at different growth phases and under various conditions of cultivation. The electron transport chains of the bacterium are characterized by the presence of cytochromes b and c types. The terminal oxidases are cytochromes o, a2+a1 and P-450. The hemoprotein P-450 functions as a mixed function oxidase. The qualitative composition of cytochromes does not depend on the growth phase of the bacterium but does on the conditions of cultivation. The bacterium synthesizes cytochromes a2+a1 in the conditions of oxygen deficiency. The synthesis of cytochrome o depends on the composition of the medium.

[Effect of linoleic acid and products of its free radical oxidation on succinate dehydrogenase]. / Vliianie linolevoi kisloty i produktov ee svobodnoradikal'no okisleniia na suktsinatdegidrogenazu.

Effects of linoleic acid and its oxidation products (with 75--80% content of hydroperoxide) on isolated succinate dehydrogenase were studied by means of spectrophotometry and ESR-spectroscopy. It was shown that unlike the unoxidized acid, low concentrations of its oxidation products increased catalytic activity, while its high concentrations of its oxidation products increased catalytic activity, while its high concentrations brought about a decrease of SH-group content and the amplitude of ESR signal of reduced nonheme iron in the enzyme.