[Interspecies polymorphism of peroxidase genes, localized on chromosome 5 of Arabidopsis thaliana].

Comparative analysis of nucleotide sequences in five peroxidase genes AtPrx52-AtPrx56 located in the left arm of chromosome 5 was performed by using six Arabidopsis thaliana ecotypes and lines (Columbia, Dijon-M, Blanes-M, Enkheim-M, Ler, K-156). Significal differences (up to 20 times) in the levels of nucleotide variation between these genes were detected: tandem duplicated genes AtPrx53 and AtPrx54 have the highest and the AtPrx56 gene has the lowest level of nucleotide diversity. The genes AtPrx53 and AtPrx54 were characterized by allelic dimorphism; the nonrandom association between nucleotide polymorphic sites within the AtPrx54 was shown. The connection between gaplotype of these genes and the mobility of anionic peroxidase izoforms was detected. Since two gaplotypes of AtPrx53 were coding proteins, which differed by two significant amino acid substitutions, we supposed that differences in mobility of anionic peroxidase izoforms caused by the diallelic polymorphism in amino acid sequence of AtPrx53 protein.

[Effect of the ABRUPTUS/PINOID gene on expression of the LEAFY gene in Arabidopsis thaliana].

The nucleotide sequence was analyzed for the temperature-sensitive allele abruptus (abr), which distorts polar auxin transport (PAT) in the floral shoot. The mutation C-->T was found in the second exon and led to an amino acid substitution (glycin-->glutamic acid) in the conserved domain of protein kinase encoded by the ABRUPTUS/PINOID (ABR/PID) gene. RT--PCR revealed a 100-fold decrease in transcription of the LEAFY (LFY) gene in the abr mutant with high expressiveness of the mutant character; transcription of the fused LFY::GUS gene was also low in the mutant. The results agree with data of the phenotypic analysis of the abr lfy double mutant and testify to an important role of auxin gradients in regulating expression of the LFY gene.

[The roles of the NANA and LEPIDA genes in regulating the stem growth in Arabidopsis thaliana]. / Rol' genov NANA i LEPIDA v reguliatsii rosta steblia Arabidopsis thaliana.

Genetic, physiological, and morphological studies of dwarf mutants of Arabidopsis thaliana (L.) Heynh. from the collection of the Department of Genetics and Breeding, Moscow State University, showed that the NA and LE genes are involved in regulating elongation of internode cells and sensitivity to various hormones. The na mutation suppressed stem growth only in the presence of the active LE gene. The absence of the LE activity (in the lele homozygote) restored stem growth of the na mutant to the level characteristic of the le-2 mutant, and a decrease in LE activity (in LEle heterozygote) almost completely suppressed the na phenotype. Phenotypic analysis of homozygous double mutants and heterozygotes obtained by crossing the na and le-2 mutants showed that the recessive le-2 allele has an epistatic effect on the semidominant na allele and that the genes possibly control consecutive steps of one biochemical pathway or one morphogenetic process. A hypothetical scheme was proposed for the interaction of the NA and LE gene products.

[PXD gene controls synthesis of the three anionic peroxidase isoforms in Arabidopsis thaliana]. / Gen PXD kontroliruet obrazovanie trekh izoform anionnykh peroksidaz Arabidopsis thaliana.

The pxd mutant of Arabidopsis thaliana had a changed spectrum of anionic peroxidases: three anionic isoforms in the pxd mutant plant had the same enzymatic activity but relatively high electrophoretic mobility as compared to the analogous isoforms in the wild type plants. These isoforms were the most active anionic peroxidases and could be found in most plant organs. Genetic analysis showed that all three isoforms were controlled by the PXD gene. The activity of one isoform was affected by indolyl-3-acetic acid and other stress factors. Expressed sequence tags (EST) analysis of all putative peroxidase genes of A. thaliana revealed the group of the most actively and nonspecifically expressed genes. The promoter sequences of these genes were screened to find the cis-elements. We propose that the PXD gene encodes one of the nonspecific anionic peroxidases or a protein involved in posttranscriptional or posttranslational modification of the peroxidases.

[The relation between oxidative processes and the glycogen content in the heart and liver of rabbits with chronic ischemic heart disease]. / Sviaz' mezhdu okislitel'nymi protsessami i soderzhaniem glikogena v serdtse i pecheni krolikov pri khronicheskoi ishemicheskoi bolezni serdtsa.

While laboratory experimental model of coronary heart disease (according to Frol'kis et al.) is developed, activity of succinate dehydrogenase, alpha-ketoglutarate dehydrogenase, Na+, Ka(+)- and Mg2+ ATPase decreases, but activity of lactate dehydrogenase and concentrations of lactic and pyruvic acids in the heart tissue increase. At the same time concentration of glycogene increases more than twice. As far as we can see there is an evidence of a decrease of glycogene utilization due to change in levels of regulatory processes. Despite a decrease of ATP synthesis by the inhibition of tricarboxylic acid cycle the ATP:ADP relation reduces to ATP, as emphatic inhibition of ATPase in the heart tissues takes place in development of the model of the coronary heart disease. The relation between ATP and ADP is considered as a regulator of glycogene utilization. In the liver tissue activity of succinate dehydrogenase, alpha-ketoglutarate dehydrogenase, Na+, K(+)- and Mg2+ ATPase falls, while concentrations of lactic acid grow. No accumulation of glycogen is observed. It is obvious that there are controversial metabolic processes. Experimental data are discussed.

[Glycogen phosphorylase from human leukocytes. Isolation and kinetic properties]. / Glikogenfosforilaza iz leikotsitov cheloveka. Vydelenie i kineticheskie svoistva.

Homogeneous glycogen phosphorylase from human leukocytes has been obtained. A one-step bioluminescent procedure for the enzyme activity assay has been developed. This method is based on a continuous recording of the product of the glycogen phosphorylase-catalyzed reaction using a coimmobilized multienzyme system (phosphoglucomutase, glucose-6-phosphate dehydrogenase, NADH:FMN oxidoreductase and bacterial luciferase). The method sensitivity is 10 times as high compared to earlier described methods. The Km values for glycogen (0.2 mg/ml) and phosphate (3.9 mM) at pH 7.9 were determined. AMP was shown to be the enzyme effector.

Bioluminescent microassay of various metabolites using bacterial luciferase co-immobilized with multienzyme systems.

Co-immobilization methods have been developed for a bienzymatic system of luminescent Beneckea harveyi bacteria with formate dehydrogenase, glucose-6-phosphate dehydrogenase, and phosphoglucomutase. Bioluminescent assays have been devised for NADH, NAD, FMN, glucose 6-phosphate, and glucose 1-phosphate using the co-immobilized enzyme preparation. The lowest detection limits were in the picomole range with the bacterial extract and in the femtomole range with the partially purified enzymes, bacterial luciferase, and NADH:FMN oxidoreductase.

Immobilized bacterial luciferase and its applications.

This review discusses the properties of the bioluminescent bacterial system as well as the methods for immobilization of bacterial luciferases and for their co-immobilization with other enzymes. The analytical systems using immobilized bacterial luciferases and their applications in analytical biochemistry and biotechnology have been described.

[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.

[Bioluminescent method of determining picomolar amounts of nicotinamide-adenine dinucleotide using an immobilized extract of the luminescent bacterium Beneckea harveyi]. / Bioliuminestsentyi metod opredeleniia nikotinamidadenindinukleotida s pomoshch'iu svetiashchikhsia bakterii Beneckea harveyi.

The luminous bacteria Beneckea Harveyi were immobilized on BrCN-sepharose and cellulose films activated with cyanuric chloride. Preparations with high luciferase and FMN-reductase activities were obtained, which showed no background luminescence without NADH being added. The storage conditions for the preparations obtained were optimized, and their kinetic parameters and thermostability were studied. Standard curves for NADH determining within the concentration range 1 nM-1 microM were plotted with the detection level of 1 picomol NADH. The preparations are very promising for bioluminescent assay due to their high activity, simple production, high stability during storage and a possibility for the repeated use.

[Cooxidation of potassium ferrocyanide and o-dianisidine by hydrogen peroxide catalyzed by horseradish peroxidase. Substrate-substrate activation]. / Sovmestnoe okislenie ferrotsianina kaliia i o-dianizidina perekis'iu vodoroda, kataliziruemoe peroksidazoi iz khrena. Substrat-substratnaia aktivatsiia.

The kinetics of cooxidation of potassium ferrocyanide and o-dianisidine by hydrogen peroxide catalyzed by horseradish peroxidase were studied. The peroxidation of potassium ferrocyanide is activated by o-dianisidine. A scheme illustrating the direct involvement of the enzyme in substrate-substrate activation is proposed. A method for determination of the rate constants of the first electron transfer from o-dianisidine to peroxidase (i. e. reduction of peroxidase E1 to E2) and of the constants for o-dianisidine binding by E1 was developed.

[Kinetics and mechanism of nucleophilic effect on the oxidation of o-dianisidine catalyzed by horseradish peroxidase]. / Kinetika i mekhanizm deistviia nukleofilov na reaktsiiu okisleniia o-dianizidina, kataliziruemuiu peroksidazoi nz khrena.

The effects of benzimidazole and 4-nitroimidazole on the reaction of o-dianisidine peroxidase oxidation within the pH range of 3.7--9.0 were studied. Both substituted imidazoles activate the reaction at less than 0.6. In the presence of 4-nitroimidazole the activation is non-competitive, whereas in the presence of benzimidazole it is of a mixed type, which is close to the non-competitive one. The kinetic parameters (kAcat, alpha, KA) for the reaction activated by both imidazoles were determined. It was assumed that the activators interact with the protein group (pK approximately to 6.5), which limits the enzyme activity. This results in the increase of pKapp of the protein group in question, resulting in the appearance of the maximal peroxidase activity in the alkaline region of pH. It was shown that the intermolecular interactions involved in the peroxidase-induced oxidative catalysis are largely due to electrostatic rather than to hydrophobic factors.

[Effect of N-alkylimidazoles on oxidation of o-dianizidine catalyzed by horseradish peroxidase]. / Vliianie N-alkilimidazolov na okislenie o-dianizidina, kataliziruemoe peroksidazoi iz khrena.

Effect of a number of N-alkylimidazoles (from N-methyl to N-octylimidazole) on peroxidase oxidation of o-dianizidine at pH 8.0 is studied. Alkylimidazoles studied are found to activate the reaction under given conditions, the activation type being non-competitive KA and (alpha) values are similar for all the activators, which suggests a similar mechanism of their action. Similar KA values suppose an insignificant role of hydrophobic interactions in the binding of N-alkylimidazoles with the enzyme.

[Kinetic study of o-dianisidine oxidation by hydrogen peroxide in the presence of horseradish peroxidase]. / Kineticheskoe izuchenie reaktsii okisleniia o-dianizidine perekis'iu vodoroda v prisutstvii peroksidazy iz khrena.

A kinetic study of o-dianisidine oxidation by hydrogen peroxide in the presence of horseradish peroxidase within the pH range of 3.7-9.0 has been carried out. It was shown that the reaction of o-dianisidine peroxidase oxidation obeys the Michaelis--Menten kinetics; the kcat and Km values within the pH range used were determined. The optimum of peroxidase catalytic activity during o-dianisidine oxidation was observed at pH 5.0-6.0. The kinetic pattern of the reaction is discussed. It was demonstrated that deprotonation of the group at pK 6.5 decreases the kcat value 60 times. At pH greater than 8.0 an additional ionogenic group controls the enzyme activity.