Isolation, purification, and characterization of catalase from the methylotrophic yeast Pichia pastoris.

Catalase (CATpp) with molecular weight 223 kD was isolated from the methylotrophic yeast Pichia pastoris and purified 90-fold by ion-exchange chromatography and gel filtration. Quantitative parameters of absorption and CD spectra of CATpp solutions and of its membrane-concentrated form (CATpp-conc) were studied. Rates of H2O2 decomposition and kinetic characteristics Km and kcat of CATpp and CATpp-conc were determined in 10 mM phosphate buffer (pH 7.4) at 30 degrees C, as well as the effective constant kin of the enzyme inactivation rate during the catalysis and the constant k2 of the interaction rate of the Complex I catalases with H2O2. Thermal inactivation of CATpp in solutions at 45 degrees C was characterized by the effective rate constant kin*, and the low-frequency (27 kHz) ultrasonic inactivation of CATpp at 20 degrees C was characterized by the first-order rate constant kin(US). All spectral and kinetic characteristics of CATpp and CATpp-conc were compared with the corresponding values for catalase from bovine liver (CAT) and for catalase from the methylotrophic yeast Candida boidinii (CATcb). All three catalases were rather similar in their spectral properties but strongly varied in their kinetic parameters, and their comparison suggests that CATpp should be the best enzyme in its overall properties as it displayed the maximal efficiency in terms of kcat/Km, thermal stability comparable with the thermal stability of CAT in terms of kin*, the minimal kin, and high stability in the ultrasonic cavitation field at the US power of 60 W/cm2.

Thiamine status in liver and brain of rats genetically selected for different sensitivity to hypnotic effect of alcohol.

BACKGROUND: The mechanisms of the different sensitivity or resistance of animals and humans to alcohol are still not completely understood. For further biochemical characterization of animals genetically selected for high-alcohol sensitivity (HAS) and low-alcohol sensitivity (LAS) with the hypnotic effect of alcohol, the thiamine status and thiamine metabolizing enzymes in these animals have been studied. METHODS: We investigated thiamine diphosphate and thiamine triphosphate levels as well as the activity of thiamine-dependent enzyme, transketolase, and thiamine-metabolizing enzymes, thiamine kinase, and thiamine triphosphatase in the liver and brain of HAS, LAS, and CAS (control) rats by standard biochemical techniques. RESULTS: It was found that the activity of transketolase, and the level of the coenzyme form of thiamine, thiamine diphosphate (TDP), were significantly lower in HAS versus LAS rats. The activation of transketolase by the exogenous TDP (TDP-effect) was significantly higher in the liver and brain regions of HAS rats compared with LAS rats. The level of TDP in the liver and cerebellum of HAS rats was significantly lower compared with LAS rats. These results indicate a severe deficiency of TDP in HAS rats. HAS rats have a significantly lower activity of thiamine triphosphatase, the additional source of TDP. Accordingly, HAS rats have much higher thiamine triphosphate levels in the liver and brain, compared with LAS rats. There were no significant differences between groups with respect to the thiamine diphosphatase and thiamine kinase activity. Most of the above parameters had the intermediate values in CAS rats, compared with LAS and HAS rats. These data indicate the possible role of the thiamine phosphate esters and related enzymes in the mechanisms that bring about the differential sensitivity to the hypnotic effect of alcohol. CONCLUSIONS: HAS rats have the genetically mediated thiamine diphosphate deficiency and increased thiamine triphosphate levels, probably due to reduced activity of thiamine triphosphatase in the liver and brain, compared with LAS rats. It can be related with the higher initial sensitivity of HAS rats to hypnotic effect of ethanol.

Ligand-protein interaction in plant seed thiamine-binding proteins. Binding of various thiamine analogues to the sepharose-immobilized buckwheat-seed protein.

Soluble thiamine-binding proteins occur in microorganisms, some animal tissues, and plant seeds. Their representative, the buckwheat-seed protein, was chosen as a model for chemical studies on the mechanism of ligand-protein interaction in these systems. In this work, in order to refine a concept of the chemical topography of the thiamine-binding center, the buckwheat seed protein was immobilized in Sepharose gel and probed with a new set of thiamine-related compounds. In terms of the standard change of Gibbs free energy on the complex formation, the following energetic contributions were specifically assigned to major structural features of the thiamine molecule: (i) 35-45% to the specific electronic structure of planar, unsaturated thiazolium ring with positive charge asymmetrically delocalized, one half of that contribution being attributable to the S(1) atom, (ii) 11-18% to nitrogen atoms and their electronic coupling within the pyrimidine ring, (iii) 15% to the 4'-amino group, and (iv) less than 10% to the hydroxyethyl chain.

[Cytosolic thiamine triphosphatase from bovine brain. 2. Interaction of thiamine triphosphate ester with the enzyme]. / Tsitozol'naia tiamintrifosfataza iz mozga byka. 2. Vzaimodeistvie trifosfornogo efira tiamina s fermentom.

The analysis of the steady-state kinetics of the thiamine triphosphate ester hydrolysis reaction catalyzed by homogeneous thiamine triphosphatase (EC 3.6.1.28; thiamine triphosphate phosphohydrolase) from bovine brain enables us to suggest, that the ThTP binding to the catalytic site of the ThTPase active centre takes place by the phosphate radical. The correct orientation of the substrate molecule occurs by means of the contact of the thiamine component. The crucial role in this process belong to the amino group of the pyrimidine ring and hydrophobic forces. The quaternary nitrogen of thiazole is important for the hydrolytic splitting of the substrate. The hydrolysis of thiamine triphosphate ester occurs through the formation of the ternary enzyme-substrate complex, with the Mg2+ and Mg.ThTP adding being random.

Thiamine triphosphatase activity in bovine kidney.

Properties of soluble thiamine triphosphatase (ThTPase), adenosine triphosphatase, nucleoside triphosphatase and alkaline phosphatase activities in bovine kidney were compared. ThTPase and the other phosphatases differed clearly in their pH-dependences, K(m) and molecular masses. Apparent K(m) and pH optimum for ThTPase were determined to be 45.5 microM and 8.9, respectively. Molecular mass of the enzyme was 29.1 kDa as estimated by Sephadex G-100 gel filtration. The results obtained show bovine kidney to contain a specific soluble ThTPase, this enzyme being the only one hydrolyzing low concentrations of ThTP.

Comparative kinetic characterization of catalases from Candida boidinii yeast and bovine liver.

Catalase with molecular weight 230 +/- kD was isolated and purified from methylotrophic yeasts Candida boidinii by ion-exchange chromatography. The kinetic characteristics of yeast and bovine liver catalases were compared in the reaction of H2O2 decomposition using a wide range of H2O2 concentrations (up to 0.12 M) and PH (2-10). First order rates constants (k, sec-1) were determined for both enzymes from semi-logarithmic anamorphoses of kinetic curves of H2O2 utilization. Anamorphoses of complete kinetic curves as a function of 1/ln([H2O2]0/[H2O2]t) versus 1/t were used for calculation of the effective rate constants of catalase inactivation during the reaction (k(in), sec-1) and the rate constants of interaction of catalase complex I with the second molecule of H2O2 (k2, M-1.sec-1). The effects of initial catalase concentrations, H2O2, and pH on k, k2, and k(in) were similar for both enzymes. Catalytic constant, k2, and the efficacy expressed as a ratio kcat/Km were 1.87-, 1.45-, and 1.3-fold, respectively, higher for bovine catalase than that of yeast catalase. Operational stability of yeast catalase is 3.5-fold higher than the stability of bovine catalase and much higher during cyclic decomposition of 50 mM H2O2. Enhanced operational stability and inexpensive source of its preparation open prospects for practical applications of yeast catalase for co-immobilization with superoxide dismutase on non-toxic carriers.

[Cytosolic thiamine triphosphatase from bovine brain. 1. Regulation of enzyme activity]. / Tsitozol'naia tiamintrifosfataza mozga byka. 1. Reguliatsiia fermentativnoi aktivnosti.

The steady-state kinetics of the ThTP hydrolysis by thiamine triphosphatase (EC 3.6.1.28) from bovine brain testified to the presence of two kinetically significant conformational states of the protein, their equilibrium being determined by the substrate concentration. The ThTPase isomeric forms had different activities, affinities for ThTP and activation energies. The form with high affinity for the substrate was characterized by the Km and Vmax values of 43 microM and 9.9 mumol.s-1.mg-1 whereas for the form with lower affinity these values were equal to 298 microM and 19.3 mumol.s-1.mg-1, respectively. The activation energies of the ThTP hydrolysis reactions were 85.3 and 47.1 kJ.mol-1. Several mechanisms of the enzyme activity regulation in the cell are suggested. One of the mechanisms is related to the allosteric ThTP effect inducing reversible transition of the protein to a more active conformational state, while the others include the inhibition activity by ATP and the activation of ThTP-ase by Mg2+ free ions.

[On vitamin B1 metabolism in avitaminosis and its correction with thiamine and taurine]. / K voprosu metabolizma vitamina B1 v usloviiakh avitaminoza i ego korrektsii tiaminom i taurinom.

The levels of phosphate esters and the activities of thiamine biotransformation enzymes in the blood and tissues of albino rats were studied during oxythiamine-induced B1 deficiency and after metabolic correction with thiamine and taurine. Among thiamine phosphates, the most informative indicators of thiamine deficiency were shown to be triphosphate esters and free thiamine diphosphate. The biosynthetic enzymes thiamine kinase and thiamine diphosphate kinase played a decisive role in maintaining the initial rate and in recovering the physiologically active forms of vitamin B1. The activation of hydrolytic enzymes of thiamine phosphate esters occurred by producing abundant free thiamine diphosphate and thiamine triphosphate. Within the first hours, taurine favoured the acceleration of phosphoester biosynthesis and, accumulating in the tissues, inhibited vitamin phosphorylation reactions.

[Spectroscopic study of the structure and intramolecular mobility of yeast pyruvate decarboxylase]. / Spektroskopicheskie issledovaniia struktury i vnutrimolekuliarnoi podvizhnosti drozhzhevoi piruvatdekarboksilazy.

Steady-state and time-resolved fluorimetry were used to study the properties of holo- and apopyruvate decarboxylase (EC 4.1.1.1, PDC) from Brewer's yeast after interaction with substrate (pyruvate), cofactor (thiamine diphosphate, ThDP) and Mg2+ ions. The analysis of the enzyme's intrinsic fluorescence as well as of its complex with the probe 2-(p-toluidinylnaphthalene)-6-sulphonate (TNS) revealed that ThDP was found at the polar region of the PDC active sites, inducing a decrease in the mobility of the protein's nearest surroundings. The fluorescent probe had three different sites of binding to the protein apoform, two of which being located at the catalytic site and having different rotation freedom. The study of the PDC complex with thiochrome pyrophosphate, a ThDP structural analogue, pointed to the occurrence of a non-polar region of the enzyme active site for pyruvate absorption besides the polar region. The binding of pyruvate to the protein does not depend upon the cofactor's binding. On the basis of the fluorescent studies a model of the ThDP and pyruvate arrangement at the PDC active site is suggested.

[Metabolism of vitamin B1 and its di- and triphosphate esters in experimental allergic encephalomyelitis]. / Metabolizm vitamina B1 i ego di- i trifosfornogo efirov pri éksperimental'nom allergicheskom éntsefalomielite.

Biological significance of thiamin in development of experimental allergic encephalomyelitis has been elucidated. It has been shown that at the late preclinical stage of the disease thiamine metabolism is predominantly directed towards the maintaining of cellular metabolic homeostasis, whereas at the stage of clinical symptoms the anabolic process gives way to catabolic decomposition. Among tested thiamine phosphates the triphosphate ester is the most informative parameter in demyelinizing processes. Thiamine injections to immunized animals accelerate the vitamin phosphorylation depleting the reducing and energy potentials of the cell. Such thiamine antagonist as oxythiamine inhibits phosphatase reactions.

[Possible ways of regulating detoxifying processes in the alcohol dehydrogenase reaction with pantothenic acid derivatives]. / Vozmozhnye puti reguliatsii proizvodnymi pantotenovoi kisloty dezintoksikatsionnykh protsessov v alkogol'degidrogenaznoi reaktsii.

Oxidation of derivatives and precursors of pantothenic acid was studied in alcohol dehydrogenase reactions. Despite the presence of free hydroxymethyl groups in a number of pantothenic acid derivatives only panthenol with Km = 8 x 10(-3) M was shown to serve as a substrate for alcohol dehydrogenase from horse liver tissue (EC 1.1.1.1) Pantethine, sodium phosphopantothenate, CoA and acetyl-CoA decreased the rate of ethanol oxidation, where pantethine and sodium phosphopantothenate were competitive inhibitors, while CoA and acetyl-CoA inhibited the enzyme noncompetitively Ki = 1.2 x 10(-2) M, 2.1 x 10(-2) M, 4.4 x 10(-4) M and 5.1 x 10(-4) M, respectively. Metabolic precursors, which were different from pantothenic acid in their structure, were not involved in the alcohol dehydrogenase reaction. Possible regulation of alcohol intoxication using derivatives and precursors of vitamin B3 is discussed.

Purification and characterization of thiamine triphosphatase from bovine brain.

Soluble thiamine triphosphatase (EC 3.6.1.28) of bovine brain has been purified 68,000-fold to an electrophoretically homogeneous state with an overall recovery of 5.5% by hydrophobic chromatography on Toyopearl HW-60, Sephadex G-75 gel filtration, DEAE-Toyopearl 650M chromatography and Blue Sepharose CL-4B chromatography. The enzyme has an absolute specificity among thiamine and nucleoside phosphate esters for thiamine triphosphate and shows no nonspecific phosphatase activities. Thiamine triphosphatase is composed of a single polypeptide chain with molecular mass of 33,900 kDa as estimated by Sephadex G-100 gel filtration and SDS-polyacrylamide gel electrophoresis. The enzyme has a pH optimum of 8.7 and is dependent on divalent metal ions. Mg2+ has been found to be the most effective among cations tested. A study of the reaction kinetics over a wide range of thiamine triphosphate concentrations has revealed a biphasic saturation curve being described by higher-degree rational polynomials.

[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.

[Metabolism of thiamine and its di- and triphosphorus esters in the blood of patients with neurological symptoms of osteochondrosis]. / Metabolizm tiamina, ego di- i tri-fosfornogo éfirov v krovi bol'nykh s nevrologicheskimi proiavleniiami osteokhondroza.

Concentrations of phosphate esters in blood of patients with vertebral osteochondrosis and activities of enzymes involved in thiamine metabolism have been investigated. The pathological processes were characterized by a decrease in content of thiamine triphosphate while concentration of coenzyme, thiamine diphosphate, was relatively constant. The enzymes involved in the initial and final steps of biosynthesis and degradation of thiamine phosphorylated derivatives play the main role in maintaining vitamin B1 metabolic homeostasis.

[Features of thiamine diphosphate-dependent enzyme inhibition by oxodihydrothiochrome and its phosphorylated derivatives]. / Osobennosti ingibirovaniia tiamindifosfatzavisimykh fermentov oksodigidrotiokhromom i ego fosforilirovannymi proizvodnymi.

Anticoenzyme action of new derivatives of thiamine: oxodihydrothiochrome and its mono- and diphosphoric esters has been studied in the experiments on mice. It is shown that the given compounds exert an inhibiting action on transketolase and pyruvate dehydrogenase and do not change activity of 2-oxoglutarate dehydrogenase in the animal organism. Antivitamin effect of the studied inhibitors is observed with the lower doses and in the earlier terms as compared with the other known inhibitors of thiamine-diphosphate-dependent enzymes. The preparations inhibit activity of the yeast pyruvate-decarboxylase by the mixed (with respect to thiamine-diphosphate) type (Ki for oxodihydrothiochrome and its mono- and diphosphoric esters: 2.3 x 10(-3), 7.2 x 10(-4), 5.6 x 10(-5) M, respectively). Possible mechanisms of the action of the mentioned compounds as thiamine antimetabolites are discussed.

[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.

Studies on thiamine diphosphate kinase (EC 2.7.4.15) from brewer's yeast: purification and some properties.

Radiometric and fluorescent methods for detection of thiamine triphosphate have been used to show the presence of thiamine diphosphate kinase activity in brewer's yeast and to determine optimal conditions for its manifestation. A method of enzyme purification has been developed which involves glycerol-EDTA solution extraction, heat treatment, 2-fold ammonium sulphate fractionation, Sephadex G-200 gel filtration and ion-exchange chromatography on Sephadexes CP-C-50 and QAE-A-25. The protein has been purified 2000-fold in a 19% yield. The isoelectric and isoionic points, the amino acid composition and the molecular weight have been determined. Hydrophobic amino acids and those responsible for alpha-helix formation of the protein globule are predominant. The isoelectric point, as calculated by the amino acid composition and found by the maximum of the changes in fluorescence, is 5.8. The isoionic point value is identical with the isoelectric point. Upon gel filtration thiamine diphosphate kinase is eluted as two protein peaks with molecular weights of 162,000 +/- 8,000 and 81,000 +/- 4,000. After treatment with urea or sodium dodecyl sulphate, the protein dissociates into subunits with molecular weights of 12,500 and 14,000. The purified enzyme has some properties typical of a dissociating enzyme system.

[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.

Identification of thiamine diphosphate-binding proteins from rat liver supernatant.

Transketolase (EC 2.2.1.1) was shown to be the sole enzyme protein bound to thiamine diphosphate (ThDP) as coenzyme in the soluble fraction of rat liver. No new ThDP-binding proteins with the molar ratio of ThDP to protein of 1.0 were detected in the rat liver supernatant under the purification and assay conditions employed.

[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.