Separation and determination of thiamin and its phosphate esters by SP-Sephadex chromatography.
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Animals↗
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Results for “THIAMINE PYROPHOSPHATE”
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Enzymatic synthesis of thiamin triphosphate.
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Animals↗
Improved purification of the pyruvate dehydrogenase complex from Ascaris suum body wall muscle and characterization of PDHa kinase activity.
An improved purification scheme for the isolation of the Ascaris suum pyruvate dehydrogenase complex directly from body wall muscle has been developed which yields a fully activated pyruvate dehydrogenase complex with substantial PDHa kinase activity. The apparent Km for coenzyme A (CoA) is much lower than previously reported and can only be accurately measured in the presence of a CoA-regenerating system. The alpha-pyruvate dehydrogenase subunit of the ascarid complex is unique and its migration on sodium dodecylsulfate polyacrylamide gels is altered after phosphorylation. PDHa kinase activity is inhibited by ADP, thiamine pyrophosphate, and physiological levels of pyruvate and propionate. In contrast, PDHa kinase activity is stimulated by elevated NADH/NAD+ and acetyl CoA/CoA ratios, although it appears that the NADH/NAD+ ratios required for half-maximal stimulation are more than an order of magnitude greater than those reported for mammalian pyruvate dehydrogenase complexes.
Acetyl Coenzyme A↗
Conformational studies of Escherichia coli pyruvate oxidase.
Pyruvate oxidase (pyruvate:oxygen oxidoreductase (phosphorylating), EC 1.2.3.3) is a peripheral membrane enzyme from Escherichia coli which utilizes the cofactors thiamin pyrophosphate (TPP) and flavin-adenine dinucleotide (FAD) to catalyze the decarboxylation of pyruvate to acetic acid and carbon dioxide. The specific activity of the oxidase is enhanced 25-fold when assayed in the presence of certain lipids and detergents. Previous studies have demonstrated that the affinity of pyruvate oxidase for phospholipids and detergents is substantially increased when the flavin is reduced. In this paper, several techniques are utilized to probe both the nature of the active site and the conformational changes in the protein which are concomitant with flavin reduction and with the binding of lipids to the enzyme. Analysis of the circular dichroism spectrum in the far ultraviolet region indicates that neither the binding of lipid activators to the oxidase nor reduction of the enzyme-bound flavin by pyruvate has a significant effect on the average secondary structure of the enzyme. High-resolution electron microscopy demonstrates that at low enzyme concentrations, i.e., assay conditions, incubation of the reduced flavoprotein in the presence of an amphiphilic activator does not alter the quaternary structure of pyruvate oxidase. The results indicate that the conformational changes in the protein due either to reduction of the flavin or to the binding of lipid activators are localized.
Circular Dichroism↗
Identification, purification and reconstitution of thiamin metabolizing enzymes in human red blood cells.
Thiamin and its mono- (TMP), di- (TDP) and triphosphate (TTP) were assayed in adult human whole blood using high-performance liquid chromatography (HPLC). TDP and TTP were detected in red blood cells (RBC), but not in plasma. After incubation with 20 microM thiamin and 5 mM glucose for 2 h, the TDP and TTP contents of RBC increased from 111 to 222 and 0.6 to 2.2 nmol/l of packed RBC, respectively, suggesting enzymatic conversion of thiamin to TDP and then to TTP. Thiamin pyrophosphokinase (TPK, EC 2.7.6.2) had not been isolated before from human materials, nor had cytosolic adenylate kinase (AK1, EC 2.7.4.3) in human RBC been demonstrated to catalyze the phosphorylation of TDP to TTP, although AK1 from pig and chicken skeletal muscle possess TTP-synthesizing activity. TPK and AK1 in a human RBC lysate were therefore purified by a series of the conventional techniques. The specific activity of the purified TPK, which was obtained as a single protein, was 720 nmol TDP formed/mg protein per h at 37 degrees C. A partially purified AK1 preparation catalyzed the formation of TTP from TDP (specific activity, 170 nmol/mg protein per h at 37 degrees C) in addition to its proper reaction to form ATP from ADP. After incubation of the purified TPK and AK1 with 20 microM thiamin in the presence of ATP, ADP and Mg2+ at 37 degrees C for 48 h, the amounts of TDP and TTP synthesized were 465 and 54.0 pmol/250 microliters reaction mixture, respectively. Neither TDP nor TTP was formed when TPK was omitted from the reaction mixture and an omission of AK1 resulted in the formation of TDP alone. These results indicate that thiamin is converted to TDP by TPK and, subsequently, to TTP by AK1 in human RBC.
Adenylate Kinase↗
Purification and characterization of pyruvate ferredoxin oxidoreductase from the hyperthermophilic archaeon Pyrococcus furiosus.
Pyrococcus furiosus grows optimally at 100 degrees C by carbohydrate fermentation. It is thought to contain a novel tungsten-dependent, NAD(P)-independent glycolytic pathway in which one of the oxidation steps is catalyzed by a tungsten-containing aldehyde ferredoxin oxidoreductase. The enzyme that catalyzes the terminal oxidation step, pyruvate ferredoxin oxidoreductase (POR), has now been purified. POR has a molecular mass of 100 kDa and is comprised of three subunits (45, 31 and 24 kDa). It lacks tungsten but contains thiamine pyrophosphate (TPP) and two ferredoxin-type [4Fe-4S] clusters per molecule which, by EPR spectroscopy, can be differentiated by their relaxation properties. The enzyme requires CoASH but not TPP for pyruvate oxidation activity and will not use 2-oxoglutarate, phenyl pyruvate or indole pyruvate as substrates. POR is virtually inactive at 25 degrees C and shows a temperature optimum for pyruvate oxidation above 90 degrees C. The apparent Km values for pyruvate, CoASH and P. furiosus ferredoxin at 80 degrees C are 460, 100 and 70 microM, respectively. Carbon monoxide was a potent inhibitor of pyruvate oxidation (apparent Ki = 7 microM). The half-life of activity (t50%) in air at 25 degrees C was 15 min and the t50% value at 80 degrees C (under anaerobic conditions) was 23 min. Based on molecular comparisons with PORs from mesophilic organisms, it is proposed that P. furiosus POR may represent an ancestral form of a pyruvate-oxidizing enzyme.
Amino Acids↗
Arginine-239 in the beta subunit is at or near the active site of bovine pyruvate dehydrogenase.
We have modified bovine pyruvate dehydrogenase (E1), the first catalytic component of the pyruvate dehydrogenase complex, with pyreneglyoxal. Treatment of E1 with pyreneglyoxal resulted in the loss of enzyme activity. Pyruvate plus thiamin pyrophosphate (TPP) afforded approximately 80% protection against this inactivation and protected two arginine residues per mol of E1 tetramer (alpha 2 beta 2) from modification. Circular dichroism spectral analysis indicated absence of any gross structural changes in the enzyme as a result of modification. Comparison of the peptide maps, monitored at 345 nm of unprotected and pyruvate plus TPP protected E1s after V8 digestion revealed that a peptide in the protected enzyme was labeled by pyreneglyoxal to a lesser extent than its counterpart in the unprotected enzyme. Sequence analysis of the peptide demonstrated that it corresponded precisely to amino-acid residues 235 to 246 in the human E1 beta sequence, with arginine residues at positions 239 and 242. Since Arg-239 is conserved in the beta-subunit of all presently known sequences of the pyruvate dehydrogenase complex and branched-chain alpha-keto acid dehydrogenase complex, it is strongly suggested that Arg-239 in the human E1 beta sequence is at or near the active site of bovine E1.
Amino Acid Sequence↗
Subcellular localization and compartmentation of thiamine derivatives in rat brain.
The subcellular distribution of thiamine derivatives in rat brain was studied. Thiamine diphosphate content was highest in the mitochondrial and synaptosomal fractions, and lowest in microsomal, myelin and cytosolic fractions. Only 3-5% of total thiamine diphosphate was bound to transketolase, a cytosolic enzyme. Thiamine triphosphate was barely detectable in the microsomal and cytosolic fraction, but synaptosomes were slightly enriched in this compound compared to the crude homogenate. Both myelin and mitochondrial fractions contained significant amounts of thiamine triphosphate. In order to estimate the relative turnover rates of these compounds, the animals received an intraperitoneal injection of either [14C]thiamine or [14C]sulbutiamine (isobutyrylthiamine disulfide) 1 h before decapitation. The specific radioactivities of thiamine compounds found in the brain decreased in the order: thiamine > thiamine triphosphate > thiamine monophosphate > thiamine diphosphate. Incorporation of radioactivity into thiamine triphosphate was more marked with [14C]sulbutiamine than with [14C]thiamine. The highest specific radioactivity of thiamine diphosphate was found in the cytosolic fraction of the brain, though this pool represents less than 10% of total thiamine diphosphate. Cytosolic thiamine diphosphate had a twice higher specific radioactivity when [14C]sulbutiamine was used as precursor compared with thiamine though no significant differences were found in the other cellular compartments. Our results suggest the existence of two thiamine diphosphate pools: the bound cofactor pool is essentially mitochondrial and has a low turnover; a much smaller cytosolic pool (6-7% of total TDP) of high turnover is the likely precursor of thiamine triphosphate.
Animals↗
The compartmentation of phosphorylated thiamine derivatives in cultured neuroblastoma cells.
Thiamine transport in cultured neuroblastoma cells is mediated by a high-affinity carrier (KM = 40 nM). In contrast, the uptake of the more hydrophobic sulbutiamine (isobutyrylthiamine disulfide) is unsaturable and its initial transport rate is 20-times faster than for thiamine. In the cytoplasm, sulbutiamine is rapidly hydrolyzed and reduced to free thiamine, the overall process resulting in a rapid and concentrative thiamine accumulation. Incorporation of radioactivity from [14C]thiamine or [14C]sulbutiamine into intracellular thiamine diphosphate is slow in both cases. Despite the fact that the diphosphate is probably the direct precursor for both thiamine monophosphate and triphosphate, the specific radioactivity increased much faster for the latter two compounds than for thiamine diphosphate. This suggests the existence of two pools of thiamine diphosphate, the larger one having a very slow turnover (about 17 h); a much smaller, rapidly turning over pool would be the precursor of thiamine mono- and triphosphate. The turnover time for thiamine triphosphate could be estimated to be 1-2 h. When preloading the cells with [14C]sulbutiamine was followed by a chase with the same concentration of the unlabeled compound, the specific radioactivities of thiamine and thiamine monophosphate decreased exponentially as expected, but labeling of the diphosphate continued to increase slowly. Specific radioactivity of thiamine triphosphate increased first, but after 30 min it began to slowly decrease. These results show for the first time the existence of distinct thiamine diphosphate pools in the same homogeneous cell population. They also suggest a complex compartmentation of thiamine metabolism.
Animals↗
Enzyme electrode composed of the pyruvate oxidase from Pediococcus species coupled to an oxygen electrode for measurements of pyruvate in biological media.
Pyruvate oxidase from Pediococcus species was immobilized with gelatin and insolubilized in film form by tanning with glutaraldehyde. The film was fixed onto the tip of an oxygen electrode. The enzyme electrode was specific for pyruvate measurements. This electrode was sensitive to 0.1 mM and could be used up to a final pyruvate concentration of 2 mM. At each step of the enzymatic film preparation and assay 0.7 mM thiamine pyrophosphate, 10 microM flavin adenine dinucleotide, 5 mM Mg2+ and 10 mM phosphate buffer were necessary. A computerized probe allowed successive measurements every 3 min for more than 20 h with the same enzymatic film. The reproducibility for the same pyruvate concentration was 2% during 400 assays without special optimization. This enzyme electrode has many applications in basic (metabolism, enzymology) and applied (blood, yoghurt) research. Results obtained from assays carried out in yoghurt are presented.
Electrodes↗
The effects of thiamin and its phosphate esters on dopamine release in the rat striatum.
The effect of thiamin and its phosphate esters on dopamine (DA) release was examined in the rat striatum using an in vivo microdialysis. Intrastriatal administration of thiamin triphosphate (TTP) or thiamin diphosphate (TDP) induced DA release, but thiamin monophosphate (TMP) or thiamin did not show any change. In the absence of Ca2+ in the perfusate, TTP did not increase the DA release. omega-Conotoxin did not decrease the TTP-dependent DA release. These findings suggest that, in contrast to TMP and thiamin, TTP and TDP may play a specific role in DA release from nerve terminals.
3,4-Dihydroxyphenylacetic Acid↗
Properties of thiamin triphosphate-synthesizing activity of chicken cytosolic adenylate kinase and the effect of adenine nucleotides.
Cytosolic adenylate kinase synthesis thiamin triphosphate (TTP) from thiamin diphosphate (TDP) in vitro by a reversible reaction: TDP + ADP Mg2+ in equilibrium TTP + AMP. The backward (TTP----TDP) reaction rate was 3-times faster than the forward (TDP----TTP) reaction rate when all the substrate concentrations were 0.1 mM. This property of TTP-synthesizing activity of the enzyme did not explain the fact that the [TTP]/[TDP] molar ratio determined in chicken white skeletal muscle is 5.0 (Miyoshi, K., Egi, Y., Shioda, T. and Kawasaki, T. (1990) J. Biochem. 108, 267-270). To solve this problem, we have studied the properties of TTP-synthesizing activity of the purified recombinant chicken cytosolic adenylate kinase preparation and the effect of adenine nucleotides, especially of ATP. The backward reaction of the TTP synthesis did not proceed in the presence of 8.8 mM ATP, a physiological concentration in chicken white skeletal muscle, while the forward reaction proceeded at a reduced rate. The [TTP]/[TDP] ratio found after a long incubation period was 3.0 and 0.7, respectively, in the presence and absence of 8.8 mM ATP. These results indicate that the high [TTP]/[TDP] molar ratio found in chicken white muscle was demonstrated in vitro by the purified chicken cytosolic adenylate kinase and support in vivo TTP synthesis by this enzyme.
Adenine Nucleotides↗
Purification and characterization of purple acid phosphatase from rat bone.
An acid phosphatase, which was immunochemically identical to splenic purple acid phosphatase, was purified to homogeneity from rat bone. The enzyme was a two iron-containing monomeric glycoprotein with a mol. wt of 36,000. The enzyme hydrolyzed aryl phosphates, nucleoside di- and triphosphates, thiamine pyrophosphate, phosphoenolpyruvic acid and acidic phosphoproteins. The enzyme was inhibited by ammonium molybdate, NaF and CuSO4 but not by tartrate and SH-reagents.
Acid Phosphatase↗
Difference in thiamine metabolism between extensor digitorum longus and soleus muscles.
1. Thiamine diphosphate level was higher in soleus muscle than in extensor digitorum longus muscle in various animals, whereas thiamine triphosphate level was less in the former muscle than in the latter except for mouse. 2. 2-Oxoglutarate dehydrogenase, transketolase and thiamine pyrophosphokinase activities were higher in soleus muscle than in extensor digitorum longus in rat and guinea pig. 3. The differences between rat two muscle phenotypes in thiamine diphosphate, but not thiamine triphosphate, level and the thiamine-related enzyme activities disappeared after denervation. 4. Tenotomy had little effect on thiamine phosphate levels and the thiamine-related enzyme activities in rat skeletal muscles.
Animals↗
Tissue difference in cellular localization of thiamine phosphate esters.
1. Thiamine phosphate levels were determined in the soluble and particulate fractions of various rat tissues. 2. There was marked tissue difference in the cellular localization of thiamine phosphates. 3. Brain thiamine triphosphate was localized only in the particulate fraction, whereas skeletal muscle thiamine triphosphate was in the soluble fraction as a protein-unbound form.
Animals↗
The route of lysine breakdown in Candida tropicalis.
Candida tropicalis was found to contain high levels of the following enzymes after growth in defined medium on L-lysine as sole nitrogen source: L-lysine N6-acetyltransferase, N6-acetyl-lysine aminotransferase, and aminotransferase activity for 5-aminovalerate and 4-aminobutyrate. Extracts were also capable of converting 5-acetamidovalerate (and 4-acetamidobutyrate) to acetate. N6-Acetyllysine however, only gave rise to acetate in the presence of 2-oxoglutarate, NAD+ and thiamine pyrophosphate. These activities were undetectable or present in much lower concentrations in cells that had been grown on ammonium sulphate as sole nitrogen source. It is concluded that L-lysine is degraded in this organism via N6-acetyllysine, 5-acetamidovalerate and 5-aminovalerate, both nitrogen atoms being removed by transamination.
Acetates↗
Cloning and sequencing of the gene encoding benzaldehyde lyase from Pseudomonas fluorescens biovar I.
The gene (bzl) encoding benzaldehyde lyase (BL) from Pseudomonas fluorescens biovar I has been cloned and characterized. The nucleotide sequence contains an open reading frame encoding a protein of 563 amino acids. The deduced BL protein shares little homology with proteins contained in the NBRF-PIR data bank. However, the higher homologies (up to about 28%) were obtained with enzymes that also utilize thiamine pyrophosphate as a cofactor.
Aldehyde-Lyases↗
Concomitant determination of thiamin and its phosphate esters in human blood and serum by high-performance liquid chromatography.
A high-performance liquid chromatographic method for the simultaneous determination of thiamin and thiamin phosphate esters in human blood or serum has been developed. The eluent consists of acetonitrile and phosphate buffer, in the ratios 90:10 (v/v) for the elution of thiamine and 60:40 (v/v) for the phosphate esters. The four compounds are eluted within 15 min. The detection limit is 13-16 fmol. Between-assay variation is 5-11%. Samples of whole blood and serum from 30 healthy adults were analysed. The following reference values were obtained for 15 females 15 males (nM, mean +/- S.D.). In serum: thiamin, 10.9 +/- 2.9/16.9 +/- 3.3; thiamin monophosphate, 8.3 +/- 1.5/3.7 +/- 1.5. In whole blood: thiamin, 29.6 +/- 10.0/33.4 +/- 10.4; thiamin monophosphate, 9.7 +/- 2.3/10.9 +/- 5.1; thiamin diphosphate, 121 +/- 29.6/165 +/- 40.4.
Chromatography, High Pressure Liquid↗