[PROPERTIES OF CHICKEN LIVER MEMBRANE-ASSOCIATED THIAMINE TRIPHOSPHATASE].

The enzymes involved in thiamine triphosphate (ThTP) metabolism in birds are not characterized so far. The aim of the present work was to study some properties of ThTPase in chicken liver. In liver homogenate, ThTPase activity has been found to display a bell-like pH-profile with a maximum of 5.5-6.0. Low activity was observed without divalent metal ions, while the addition of Mg2+ or Ca2+, each at 5 mM concentration, enhanced the rate of ThTP hydrolysis by a factor of 17-20. In the presence of 5 mM Mg2+ an apparent K(m) of the enzyme for ThTP was estimated by the method of non-linear regression as well as from the Hanes plot to be 1.7-2.2 mM. Monovalent anions such as I-, SCN-, NO3-, Br-, Cl- (at 150 mM concentration) showed inhibitory effect decreasing the rate of ThTPase reaction by 20-60%. After the homogenate was centrifuged, more than 85% of ThTPase activity was revealed in the fraction of insoluble particles indicating a membrane localization of the enzyme. The precipitate treatment with 1% sodium deoxycholate caused about 53% solubilization of the activity. During Toyopeal HW-55 chromatography, ThTPase activity was eluted simultaneously with ATPase and ITPase peaks in the void volume of the column. Thus, a non-specific high molecular mass protein complex seems to be involved in ThTP hydrolysis in the chicken liver. The chicken liver phosphatase is clearly distinguishable from all membrane-bound ThTPases reported previously.

[Identification of thiamine monophosphate hydrolyzing enzymes in chicken liver].

In animals, thiamine monophosphate (TMP) is an intermediate on the path of thiamine diphosphate, the coenzyme form of vitamin B1, degradation. The enzymes involved in TMP metabolism in animal tissues are not identified hitherto. The aim of this work was to study TMP hydrolysis in chicken liver. Two phosphatases have been found to contribute to TMP hydrolysis in liver homogenate. The first one, possessing a maximal activity at pH 6.0, is soluble, whereas the second one represents a membrane-bound enzyme with a pH optimum of 9.0. Membrane-bound TMPase activity was enhanced 1.7-fold by 5 mM Mg2+ ions and strongly inhibited by levamisole in uncompetitive manner with K1 of 53 µM, indicating the involvement of alkaline phosphatase. An apparent Km of alkaline phosphatase for TMP was calculated from the Hanes plot to be 0.6 mM. The soluble TMPase has an apparent Km of 0.7 mM; this enzyme is Mg2+ independent and insensitive to levamisole. As estimated by gel filtration on a Toyopearl HW-55 column, the soluble enzyme has a molecular mass of 17.8 kDa, TMPase activity being eluted simultaneously with peaks of flavinmononucleotide and p-nitrophenyl phosphatase activity. Thus, TMP appears to be a physiological substrate for a low-molecular weight acid phosphatase, also known as low-molecular-weight protein phosphotyrosine phosphatase.

[Purification and properties of a catalytically active fragment of soluble nucleoside triphosphatase from bovine kidney].

A catalytic fragment of soluble NTPase has been isolated from bovine kidneys.The 236-fold purification was carried out to obtain the preparation with a specific activity of 37.7 U/mg of protein. The purification scheme included the enzyme extraction followed by four column chromatography steps. The catalytic fragment was activated with divalent metal ions, had a pH optimum of 7.0, and possessed specificity for ITP, GTP, UTP and XTP. The apparent K(m) for Mg-ITP, Mg-GTP and Mg-UTP complexes were calculated from Hanes plots to be 1.70 mM, 0.93 mM and 0.48 mM, respectively. As estimated by gel filtration and SDS-PAAGE, the catalytic fragment has Mw 54.7 kDa being composed of two identical polypeptide chains. Our results suppose soluble NTPase from bovine kidney to consist of regulatory and catalytic structural units.

[Vitamin B1: metabolism and functions].

The review highlights metabolism and biological functions of vitamin B1 (thiamine). Thiamine transport systems, enzymes of its biosynthesis and degradation in various organisms, as well as molecular basis of thiamine-dependent hereditary patologies are considered. A special emphasis is given to discuss the role of thiamine triphosphate and adenylated thiamine triphosphate, a new thiamine derivative recently discovered in living cells.

[The rate of molecular evolution of mammalian soluble thiamine triphosphatase].

Thiamine triphosphate (ThTP) exists in various living cells--from bacteria to mammals. ThTP concentration in mammals is regulated by a specific soluble ThTPase, which has not been revealed experimentally, however, in other organisms. In NCBI and Ensembl databases we have found information about full-size or partial amino acid sequences of the enzyme from 38 mammal species. An average rate of amino acid substitutions (k(aa)) in ThTPase molecule was estimated from the data available to be 1.41 x 10(-9) per site per year. This corresponds to unit evolutionary period of about 4.4 million years. The evolutionary rate varies for different portions of the enzyme, C-terminal fragment being the most variable (k(aa) = 3.76 x 10(-9); calculated only for 230 aa species + elephant). An average replacement rate of 1.95 x 10(-9) per amino acid site per year was calculated for the central portion of the enzyme (residues 69-141), while N-terminal sequence (residues 1-68) and 142-210 fragment evolved with k(aa) of 1.03 x 10(-9) and 0.81 x 10(-9), respectively.

Purification and characteristics of functional properties of soluble nucleoside triphosphatase (apyrase) from bovine brain.

Soluble NTPase, differing in its properties from known proteins exhibiting NTPase activity, was purified from bovine brain to homogeneity. The enzyme has pH optimum at 7.5 and shows absolute dependence on bivalent cations and broad substrate specificity towards nucleoside-5 -tri- and -diphosphates, characteristics of apyrases. The NTPase follows Michaelis-Menten kinetics in the range of investigated substrate concentrations, the apparent K(m) values for UTP, ITP, GTP, CTP, CDP, and ATP being 86, 25, 41, 150, 500, and 260 microM, respectively. According to gel-filtration and SDS-PAGE data, the molecular mass of the enzyme is 60 kD. The NTPase is localized in the cytosol fraction and expressed in different bovine organs and tissues. Total NTPase activity of extracts of bovine organs and tissues decreases in the following order: liver > heart > skeletal muscle > lung > brain > spleen > kidney ~ small intestine. The enzyme activity can be regulated by acetyl-CoA, alpha-ketoglutarate, and fructose-1,6-diphosphate acting as activators in physiological concentrations, whereas propionate exhibits an inhibitory effect.

[Substrate specificity and kinetic properties of a soluble nucleoside triphosphatase from bovine kidneys].

Soluble nucleoside triphosphatase differing in its properties from all known proteins with NTPase activity was partially purified from bovine kidneys. The enzyme has pH optimum of 7.5, molecular mass of 60 kDa, as estimated by gel chromatography, and shows an absolute dependence on divalent metal ions. NTPase obeyed Michaelis-Menten kinetics in the range of substrate concentration tested from 45 to 440 microM; the apparent Km for inosine-5'-triphosphate was calculated to be 23.3 microM. The enzyme was found to possess a broad substrate specificity, being capable of hydrolyzing various nucleoside-5'-tri- as well as diphosphates.

[Nucleoside-5'-triphosphate hydrolysis in the liver and kidney of rats with chronic alloxan diabetes].

Activity and some properties of a soluble enzyme hydrolyzing nucleoside-5'-triphosphates were studied in the liver and kidney of normal and diabetic rats. The enzyme activity was shown to be reduced by 34% (p < 0.01) in the liver extracts of diabetic animals, while no difference was observed in the kidney. When ITP was used as substrate, the apparent Michaelis constant of the enzyme was significantly lower in the liver of controls as compared to experimental rats (32.3 +/- 1.3 microM and 54.3 +/- 1.0 microM, respectively, p < 0.01). The KM values of the enzyme in the kidney were not distinguishable in both groups. NTPase exhibits maximal activity at pH 7.0 and has a broad substrate specificity with respect to different nucleoside-5'-tri- and diphosphates. Molecular mass of the enzyme was estimated by gel filtration to be 63.7 +/- 0.9 kD.

Neuronal localization of the 25-kDa specific thiamine triphosphatase in rodent brain.

Thiamine triphosphate (ThTP) is found in small amounts in most organisms from bacteria to mammals, but little is known about its physiological role. In vertebrate tissues, ThTP may act as a phosphate donor for the phosphorylation of certain proteins; this may be part of a new signal transduction pathway. We have recently characterized a highly specific 25-kDa thiamine triphosphatase (ThTPase) that is expressed in most mammalian tissues. The role of this enzyme may be the control of intracellular concentrations of ThTP. As the latter has been considered to be a neuroactive form of thiamine, we have studied the distribution of ThTPase mRNA and protein in rodent brain using in situ hybridization and immunohistochemistry. With both methods, we found the strongest staining in hippocampal pyramidal neurons, as well as cerebellar granule cells and Purkinje cells. Some interneurons were also labeled and many ThTPase mRNA-positive and immunoreactive cells were distributed throughout cerebral cortical gray matter and the thalamus. White matter was not significantly labeled. ThTPase immunoreactivity seems to be located mainly in the cytoplasm of neuronal perikarya. Immunocytochemical data using dissociated cultured cells from hippocampal and cerebellum showed that the staining was more intense in neurons than in astrocytes. The protein was rather uniformly located in the perikarya and dendrites, suggesting that ThTP and ThTPase may play a general role in neuronal metabolism rather than a specific role in excitability. There was no apparent correlation between ThTPase expression and selective vulnerability of certain brain regions to thiamine deficiency.

[Thiamine triphosphatase activity in mammalian mitochondria]. / Tiamintrifosfataznaia aktivnost' v mitokhondriiakh mlekopitaiushchikh.

Mitochondrial preparations isolated from bovine kidney and brain as well as the liver and the brain of rat show thiamine triphosphatase (ThTPase) activity. The activity was determined from the particles by freezing-thawing suggesting that a soluble enzyme is involved. The liberation patterns of ThTPase and marker enzyme activities from mitochondria under osmotic shock or treatment with increasing Triton X-100 concentrations indicate the presence of ThTPase both in the matrix and intermembrane space. It was found, basing on gel filtration behavior, that the mitochondrial ThTPase has the same molecular mass as specific cytosolic ThTPase (EC 3.6.1.28). The enzymes, however, were clearly distinguishable in Km values, the mitochondrial one showing a higher apparent affinity for substrate. These results imply the existence of ThTPase multiple forms in mammalian cells.

Thiamine triphosphate and thiamine triphosphatase activities: from bacteria to mammals.

In most organisms, the main form of thiamine is the coenzyme thiamine diphosphate. Thiamine triphosphate (ThTP) is also found in low amounts in most vertebrate tissues and can phosphorylate certain proteins. Here we show that ThTP exists not only in vertebrates but is present in bacteria, fungi, plants and invertebrates. Unexpectedly, we found that in Escherichia coli as well as in Arabidopsis thaliana, ThTP was synthesized only under particular circumstances such as hypoxia (E. coli) or withering (A. thaliana). In mammalian tissues, ThTP concentrations are regulated by a specific thiamine triphosphatase that we have recently characterized. This enzyme was found only in mammals. In other organisms, ThTP can be hydrolyzed by unspecific phosphohydrolases. The occurrence of ThTP from prokaryotes to mammals suggests that it may have a basic role in cell metabolism or cell signaling. A decreased content may contribute to the symptoms observed during thiamine deficiency.

[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.

[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.

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.