[Hydrolysis of phosphoether bonds of heme-independent chloride peroxidase from Serratia marcescens]. / Gidroliz fosfoéfirnoi sviazi gemnezavisimoi khloridperoksidazoi iz Serratia marcesens.

Heme- and metal-independent chloroperoxidase from Serratia marcescens W 250 is shown to be capable of catalyzing the p-nitrophenyl phosphate hydrolysis. The parameters of the phosphatase reaction are determined and inhibitors and activators of the process are found. A hypothetical mechanism of the hydrolysis of phosphoesters by heme- and metal-independent haloperoxidases is suggested. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.

[Immobilization of chloroperoxidase from Serratia marcescens]. / Immobilizatsiia khlorperoksidazy iz Serratia marcescens.

A bacterial non-heme chloroperoxidase from Serratia marcescens W 250 was immobilized in calcium-alginate gel. Methods for stabilization of the immobilized enzyme were developed, and some kinetic parameters of the immobilized preparations were determined. The enzyme encapsulated into the gel granules in the presence of potassium ferricyanide followed by treatment with glutaraldehyde demonstrated the highest stability under the reaction conditions.

Regulation of activity of chloroperoxidase from Serratia marcescens.

The influence of various factors on the activity of chloroperoxidase from Serratia marcescens was investigated. The enzyme is active only in acetate-containing buffers within the pH range 4.2-5.8. F-, Cu2+, [Fe(CN)6]4+, and [Fe(CN)6]3+ inhibit the enzyme. The chloroperoxidase is thermostable and resistant to the effect of lower alcohols.

[Fermentation micromethod for the quantitative determination of thiamine diphosphate in biological fluids]. / Fermentativnyi mikrometod kolichestvennogo opredeleniia tiamindifosfata v viologicheskikh zhidkostiakh.

An enzymatic micromethod is proposed for quantification of thiamine biphosphate (TBP) at concentrations from 0.5 ng in 0.1-0.2 ml samples of blood or other biological liquids. The dynamics of TBP degradation in blood was studied depending on the time and conditions of storage. A high efficient complex of alcohol dehydrogenase and apopyruvate decarboxylase was isolated from baker's yeasts that can be successfully used for quantitative detection of TBP. The complex was stabilized for further application to biochemical kits for diagnosis of B1-deficiency.

[Purification and various properties of pantothenate kinase from the rat liver]. / Ochistka i nekotorye svoistva pantotenatkinazy iz pecheni krys.

Homogeneous (according to disc gel electrophoresis data) ATP: D-pantothenate-4'-phosphotransferase (pantothenate kinase, EC 2.7.1.33) was obtained from rat liver cytosol of heterogeneous stock rats. The enzyme was purified 199-fold with a 9.3% yield. The enzyme was relatively unstable but retained its activity in the presence of 10% glycerol containing 5.10(-4) M ATP over 10 days at 4 degrees C. The pH optimum was 6.5; the apparent Km values were equal to 1.2 X 10(-5) M and 1.4 X 10(-3) M for pantothenate and ATP, respectively, at the ATP/Mg2+ ratio of 1. Pantetheine produced a competitive inhibition of pantothenate kinase. Pantethine or pantetheine disulfide did not inhibit the enzyme.

[Physico-chemical properties of thiamine kinase from Saccharomyces carlsbergensis yeasts]. / Fiziko-khimicheskie svoistva tiaminkinazy drozhzhei Saccharomyces carlsbergensis.

The amino acid composition and intrinsic fluorescence were studied in thiamine kinase (ES 2.7.6.2) of brewer's yeast. The enzyme molecule is characterized by higher concentrations of amino acids which promote alpha-helix formation of the protein globule, the amount of residues (cysteine, proline) either binding or folding polypeptide chains being considerably high. Amino acids of middle and low hydrophobicities were the most frequent among the amino acid residues with nonpolar R-groups. The value for the protein isoelectric point was 6.21. The eigen pH value and isoionic point were in good agreement with the isoelectric point value and amounted to 6.28. The fluorescence spectrum has a maximum at 328 nm, half-width at 53 nm and a quantum yield at 0.14 nm. The tryptophane residues were located in hydrophobic surroundings, unexposed to anion quenchers and almost unexposed to cation ones. The fluorescence and phosphofluorescence parameters were sensitive to the conformational changes in the molecule. At pH of 5-9 the protein conformation remained unchanged. The temperature rise above 40 degrees C resulted in a disturbance in the nativity of the globule. The elevation of the enzyme concentration from 0.05 to 1 mg/ml increased the polarization degree from 0.115 to 0.194, the quantum yield and the spectrum position remaining unchanged. The results obtained develop knowledge of the equilibrium system of oligomerous forms of thiamine kinase with different catalytic properties.

[Biosynthesis of thiamine triphosphate and identification of thiamine diphosphate-binding proteins in the rat liver hyaloplasm]. / Biosintez tiamintrifosfata i identifikatsiia tiamindifosfatsviazyvaiushchikh belkov gialoplazmy pecheni krys.

The nature of the thiamine diphosphate binding proteins from rat liver hyaloplasm was studied. When [14C]thiamine was used as a marker, a [14C]thiamine diphosphate-containing electrophoretically homogeneous protein preparation was isolated from the liver soluble fraction and classified as transketolase. No other non-enzymatic proteins which bind thiamine diphosphate and can serve as substrates in the reaction of thiamine diphosphate synthesis in the hyaloplasm were found. It was shown that the phosphate group is transferred by rat liver thiamine diphosphate kinase to the free (but not to the protein-bound) thiamine diphosphate as it was believed earlier.

[Thiamine triphosphate content of the liver of albino rats with different levels of thiamine]. / Soderzhanie tiamintrifosfata v pecheni belykh krys pri pazlichnoi obespechennosti tiaminom.

Content of thiamine triphosphate (TTP) was studied in rat liver tissue under conditions of normal state, thiamine deficiency and after loading with thiamine. The concentration of TTP in hepatocytes of control animals was found to be 3.2-3.6 micrograms/g, corresponding to 40% of the total thiamine pool in the cells. The TTP appears to serve as a reserve of vitamin B1 deposited in mitochondria of the cells in which the biosynthesis of thiamine pyrophosphate of their own did not take place. Administration of thiamine did not induce the TTP overproduction in hepatocytes; the content of TTP was maintained in the mitochondria of the hepatocytes at the constant level.

[Role of the sulfhydryl group in the thiamine pyrophosphokinase activity of the rat liver]. / Rol' sul'fgidril'nykh grupp v fermentativnoi aktivnosti tiaminpirofosfokinazy pecheni krys.

Amount and reactivity of SH-groups in thiamine pyrophosphokinase from rat liver tissue were studied using the mercaptide-forming reagents HgCl2 and p-chloromercury-benzoate (pCMB). Two types of SH-groups were found: the first--readily accessible and the second--partially concealed groups. Amount of these groups altered in presence of 8 M urea and 0.5% sodium dodecylsulfate. The enzymatic activity was inhibited by 30% after pCMB modification of the first type SH-groups and by 100% if second type SH-groups were blocked. All the substrates used, except of ATP, protected partially thiamine pyrophosphokinase against the inactivating effect of the reagent. The mercaptide-forming reagents exhibited dissimilar efficiency apparently due to different nature of the chemical bonds formed: pCMB developed the bond protein-S-HgR and HgCl2 formed--(protein-S)2Hg bond. The data obtained suggest that the SH-groups are localized close to the active site of thiamine pyrophosphokinase and that they participate in stabilization of the molecular structure of the enzyme.

[Radiometric method of determining ATP: D-pantothenate-4'-phosphotransferase activity]. / Radiometricheskii metod opredeleniia aktivnosti ATP: D-pantotenat-4'-fosfotransferazy.

A radiometric procedure is developed for estimation of pantothenate kinase (EC 2.7.1.33) activity in various preparations of rat liver tissue; sodium 14C-D-pantothenate was used as a substrate and the reaction end product 4'-phosphopantothenic acid was measured. Optimal separation of the substrate and the end product was achieved by means of chromatography on DEAE-Sephadex A-25. 4'-phosphopantothenic acid was eluted from the column by 0.4 N HCl thus avoiding the label dilution and possible quenching of scintillation.

[Penetration of thiamine pyrophosphate into rat hepatocytes]. / Izuchenie pronitsaemosti tiamindifosfata v gepatotsity krys.

Thiamin pyrophosphate transport into rat hepatocytes has been studied using TPP-beta-33P and 14C-TPP-beta-33P synthesized in the thiamin pyrophosphokinase reaction. Thiamin pyrophosphate (cocarboxylase) did not penetrate across cytoplasmic membrane.

[Thiamine transport into rat erythrocytes]. / Transport tiamina v éritrotsity krys.

14C-thiamine transport across the membranes of erythrocytes obtained from blood of normal rats was studied. The active transport was observed at less than 0.5 microM thiamine concentration. An increase in the thiamine concentration resulted in a decrease in the active transport which was accompanied by increased diffusion. Thiamine analogs inhibited its transport into erythrocytes with different efficiency. The rate of thiamine phosphate esters formation in the course of incubation characterized the state of functional activity of the kinase and phosphatase systems. The data obtained suggest that transport and phosphorylation are independent processes in erythrocytes.

[Purification of thiamine binding protein from rat erythrocytes by affinity chromatography]. / Ochistka tiaminsviazyvaiushchego belka éritrotsitov krys affinnoi khromatografiei.

Protein with thiamine-binding activity (14 nmole/mg protein) was isolated from rat red cells by affinity chromatography. Adsorbents with varying degrees of hydrophoby containing thiamine as ligand were made use for isolation. A2300-fold purification with a 50% overall yield was attained. The protein preparation was found to be homogenous upon polyacrylamide gel electrophoresis. The role of pH of the medium, of ions of bivalent metals in vitamin B1 binding with the protein isolated has been shown.

[Blood transketolase activity and the TDP effect as indices of thiamine body allowance]. / Aktivnost' transketolazy krovi i TDF-éffekt kak pokazateli obespechennosti organizma tiaminom.

Protein-bound and free forms of thiamin diphosphate (TDP) (separated by dialysis and gel filtration) were found in rat erythrocytes. Content of TDP in blood did not correlate with the transketolase activity at the initial steps of B1 avitaminosis. Decrease of the DTP total amount in blood by more than 80% did not affect distinctly the transketolase activity. As shown by binding of 14C-TDP during the equilibrium dialysis and gel filtration, the apotransketolase did not occur in erythrocytes of thiamin-deficient rats. Activation of transketolase, which occurred after addition of 50 mg TDP into the whole blood lysates (TDP-effect), was characteristic for the later steps of the avitaminosis; it depended rather on leukocyte than on erythrocyte transketolase. Estimation of TDP concentration in blood was the most suitable assay for a body providing with thiamin at the early steps of the avitaminosis. The accuracy of the coenzyme estimation was decreased within 15-30 days of the avitaminosis due to its drastic lowering. In this case, determination of the transketolase activity was the most suitable criterion. The thiamin-binding protein, found in erythrocytes, appears to participate in transport of the vitamin across erythrocyte membranes.

[Nature of the bond between the coenzyme and protein in pig liver transketolase]. / O prirode sviazi mezhdu kofermentom i belkom v transketolaze iz pecheni svin'i.

It was shown that the bond between the coenzyme and protein in pig liver transketolase is not covalent as was reported previously. Boiling of the enzyme preparation for 2 min or treatment with 10% trichloracetic acid results in a complete split-off of thiamine pyrophosphate.

[Storage of 14C-thiamine in rat liver subcellular fractions]. / Deponirovanie 14C-tiamina v subkletochnykh fraksiiakh pecheni belykh krys.

Distribution of 14C thiamine and its incorporation into TPP-dependent enzymes of rat liver subcellular fractions were studied. Thiamine was stored mainly in hyaloplasm and mitochondria. In nuclear and microsomal fractions presence of the vitamin was due to contamination by hyaloplasm components during the destruction of liver cells. In the proteins of hyaloplasm the label was incorporated in transketolase only. On the basis of these findings a procedure for isolation of the enzyme was developed. The purification process of monitored by calculation of specific radioactivity instead of the specific enzymatic activity of transketolase. In the mitochondria 14C-TPP was incorporated only into alpha-keto acid dehydrogenase complexes; transketolase was not found in the mitochondria. Metabolism of subcutaneously injected 14C-thiamine was studied in liver mitochondria and hyaloplasm.

[Free and bound thiamine pyrophosphate level in rat liver mitochondria in various saturation of the body with thiamine]. / Soderzhanie svobodnogo i sviazannogo tiamindifosfata v mitokhondriiakh pecheni krys pri razlichnoi obespechennosti organizma tiaminom.

Content of tree and bound thiamine pyrophosphate (TPP) was measured by means of gel filtration and equilibrium dialysis procedures in mitochondria of normal rats, the rats under conditions of alimentary B1 avitaminosis and loading with thiamine. The content of protein-bound TPP was stable and equal to 20% of its total level in the mitochondria of control rats and in the rats loaded with thiamine. The content of free form of TPP was decreased in B1 avitaminosis; a severe form of the avitaminosis was accompanied by a decrease in content of the bound form. Concentration of the free form of TPP did not increase if the organism of the rats was loaded with thiamine. These data suggest existence of specific systems controlling the vitamin uptake in mitochondria. The active transport of thiamine across the mitochondrial membranes is considered as the most important process. Administration of the high doses of the vitamin led only to an increase in the non-coenzymatic form of the vitamin, indicating the absence of thiamine pyrophosphokinase in rat liver mitochondria. A possible mechanism of TPP transport in mitochondria is discussed.

[Isolation and radiometric determination of rat liver ATP: thiamine diphosphate phosphotransferase activity]. / Vydelenie i radiometricheskii metod opredeleniia aktivnosti ATP: tiamindifosfatfosfotransferazy iz pechpeni krys.

A radioassay was developed for estimation of the thiamin diphosphate kinase activity (EC 2.7.4.15); 14C-TDP was used as a substrate. After termination of the reaction (formation of TTP), the products obtained were separated using ion exchange chromatography on SP-Sephadex C-25. The method developed was very sensitive and enabled to estimate the enzymatic activity in tissue homogenates. TDP-kinase was isolated from rat liver with a 70-fold purification. Dependence of the reaction rate on pH value, concentrations of the enzyme and thiamin were studied.

[Molecular-kinetic parameters of thiamine enzymes and the mechanism of antivitamin action of hydroxythiamine in animal organisms]. / Molekuliarno-kineticheskie parametry Tiaminovykh fermentov i mekhanizm antivitaminnogo deistviai oksitiamina v organizme.

The molecula-kinetic parameters (Km, Ki) of three thiamine enzymes, e. g. thiamine pyrophosphokinase (EC 2.7.6.2), pyruvate dehydrogenase (EC 1.2.4.1) and transketolase (EC 2.2.1.1) with respect to the effects of the thiamine antimetabolite hydroxythiamine in the whole animal organism have been compared. It has been shown that only the first two enzymes, which interact competitively with the vitamin, antivitamin or their pyrophosphate ethers, obey the kinetic parameters obtained for the purified enzymes in vitro. The anticoenzymic effect of hydroxythiamine pyrophosphate with respect to transketolase is not observed in vivo at maximal concentration of the anticoenzyme in tissues due to the absence of competitive interactions with thiamine pyrophosphate. The incorporation of the true and false coenzymes into transketolase occurs only during de novo transketolase synthesis (the apoform is absent in tissues, with the exception of erythrocytes) and proceeds slowly with a half-life time equal to 24--30 hrs. After a single injection of hydroxythiamine at a large dose (70--400 mg/kg) the maximal inhibition of the transketolase activity in tissues (liver, heart, kidney, muscle, spleen, lungs adrenal grands) manifests itself by the 48th--72nd hour, when the concentration of free hydroxythiamine and its pyrophosphate is minimal and the whole anticoenzyme is tightly bound to the protein, forming the false holoenzyme. The use of hydroxythiamine for inhibition of pyruvate dehydrogenase or transketolase in animal organism is discussed.