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PubMed1982

Thiamine metabolism in the liver of mice with Ehrlich ascites carcinoma.

Thiamine pyrophosphate (TPP) content, activities of thiamine pyrophosphokinase (TPKase), thiamine pyrophosphatase, transketolase (TK), pyruvate (PDG) and oxoglutarate dehydrogenases (OGDG) were measured in the liver and cells of Ehrlich ascites carcinoma (EAC) on the 5th, 10th and 15th day after transplantation of the tumor to mice fed a thiamine-deficient diet. The TPP level gradually decreased in the liver of tumor-bearing mice but remained constant in tumor cells (1.06 +/- 0.02 microgram/g tissue). Deprivation of dietary thiamine lowered the liver TPP level even to a greater extent, and subsequent daily 10 micrograms thiamine/mouse injections did not restore it. The TPKase activity in the liver of mice with EAC decreased by 24% and in thiamine deficiency, by 44%. The liver PDG, OGDG and TK activities were reduced but slightly in mice with EAC, whereas thiamine deprivation resulted in a decrease of the enzyme activities: PDG by 60%, OGDG by 25% and TK by 45%. TK activity in tumor cells was 90 mumol S-7-P/g tissue/h, and the TPP effect amounted to 24%. Thiamine deprivation decreased the TK activity by 45% and raised the TPP effect up to 180%. Thiamine injections restored the TK activity in tumor cells and reduced the TPP effect.

Animals↗

PubMed1995

Overexpression and characterization of human tetrameric pyruvate dehydrogenase and its individual subunits.

Pyruvate dehydrogenase (E1), an alpha 2 beta 2 tetramer, is the first component of the pyruvate dehydrogenase complex which catalyzes a two-step oxidative decarboxylation of pyruvic acid. To overexpress human E1 and its subunits individually, cDNAs for the mature forms of human E1 alpha and E1 beta were subcloned either individually or together into a plasmid pQE-9 and expressed in Escherichia coli M15. A polyhistidine extension was added at the NH2-termini of the recombinant E1 alpha and E1 beta for the rapid purification of the proteins by Ni-nitrilotriacetic-agarose chromatography. The polyhistidine extension on either E1 alpha or E1 beta subunit did not affect the activity of the recombinant tetrameric E1. Highly purified recombinant human E1 catalyzed the partial reactions of the oxidative and nonoxidative conversion of pyruvic acid with the same efficiency as E1 purified from bovine kidney. Recombinant human E1 interacted with thiamin pyrophosphate by forming a charge transfer complex band at 330 nm that changed during the catalytic cycle. Recombinant human E1 was phosphorylated by E1-kinase (with concomitant inactivation) by incorporating nearly three phosphoryl groups per mole of E1. When expressed individually, E1 alpha and E1 beta subunits lacked any catalytic activity in the oxidative or nonoxidative reactions. Spectral studies demonstrated that there was no thiamin pyrophosphate binding to either recombinant E1 alpha or E1 beta subunit. The E1 alpha subunit retained the ability to be phosphorylated; however, the incorporation of phosphoryl groups into recombinant E1 alpha alone was only about 12% of that observed with the tetrameric E1. These findings show that both subunits are required for formation of the active center and catalysis.

Base Sequence↗

PubMed1983

The substrate-mediated inactivation of the pyruvate dehydrogenase component of the pigeon breast muscle pyruvate dehydrogenase complex.

Incubation of the pyruvate dehydrogenase component isolated from the pigeon breast muscle pyruvate dehydrogenase complex with Mg2+, thiamine pyrophosphate and low concentrations of pyruvic acid in the absence of electron acceptors results in irreversible time-dependent inactivation of the enzyme. The rate of the enzyme inactivation is markedly decreased in the presence of high concentrations of pyruvate; in this case acetoin and acetolactate are detected in the reaction mixture. The enzyme activity is stabilized when the artificial electron acceptor, 2,6-dichlorophenolindophenol, is present in the reaction mixture. The substrate-mediated inactivation of the enzyme is accompanied by incorporation of the 2-[14C]-substrate fragment and labelled thiamine pyrophosphate into the protein fraction. The enzyme reactivation by neutral hydroxylamine and the protective effect of dithiothreitol suggest that the SH-group(s) may be involved in the substrate-mediated inactivation of pyruvate dehydrogenase.

Animals↗

PubMed1978

Prevention of oxalate urolithiasis by some compounds.

Male Wistar rats were fed a basal diet, Purina Laboratory Chow, and an oxalate calculi-producing diet (CPD). The CPD was the basal diet containing 3 per cent glycolic acid. Sodium pyruvate, DL-alanine, alpha-keto glutaric acid, thiamine pyrophosphate, and L-glutamic acid were added to the CPD to determine their effectiveness in preventing calculi formation. The effectiveness of methyl glyoxal was determined by adding it to the drinking water. Rats fed CPD for 4 weeks developed calculi in the ureters, bladder, renal tubules, and/or renal pelvis and papilla. Rats in groups fed alanine and/or pyruvate had no calculi in their renal tubules or ureters; additionally, these rats had a significant reduction in incidence and amount of deposits in the renal pelvis and bladder. Rats in groups fed alpha-keto glutaric acid, thiamine pyrophosphate, L-glutamic acid, and methyl glyoxal developed equally or more severe oxalate urolithiasis than those on CPD alone. Results of this study show that either pyruvate or alanine at appropriate levels may be beneficial in preventing oxalate urolith formation.

Alanine↗

PubMed1971

Isolation of a Golgi apparatus-rich fraction from rat liver. IV. Thiamine pyrophosphatase.

The thiamine pyrophosphatase (the enzyme [s] catalyzing the release of inorganic phosphate with thiamine pyrophosphate as the substrate) activities of Golgi apparatus-, plasma membrane-, endoplasmic reticulum-, and mitochondria-rich fractions from rat liver were compared at pH 8. Activity was concentrated in the Golgi apparatus fractions, which, on a protein basis, had a specific activity six to eight times that of the total homogenates or purified endoplasmic reticulum fractions. However, only 1-3% of the total activity was recovered in the Golgi apparatus fractions under conditions where 30-50% of the UDPgalactose:N-acetylglucosamine-galactosyl transferase activity was recovered. Considering both recovery of galactosyl transferase and fraction purity, we estimate that approximately 10% of the total thiamine pyrophosphatase activity of the liver was localized within the Golgi apparatus, with a specific activity of about ten times that of the total homogenate. Cytochemically, reaction product was found in the cisternae of the endoplasmic reticulum as well as in the Golgi apparatus. This is in contrast to results obtained in most other tissues, where reaction product was restricted to the Golgi apparatus. Thus, enzymes of rat liver catalyzing the hydrolysis of thiamine pyrophosphate, although concentrated in the Golgi apparatus, are widely distributed among other cell components in this tissue.

Animals↗

PubMed1977

Auxotypes and penicillin susceptibilities of Neisseria gonorrhoeae isolated from patients with gonorrhea involving two or more sites.

A system of auxotyping described in 1973 is based on the differing nutritional requirement patterns of Neisseria gonorrhoeae strains. Our ongoing evaluation of the reliability of auxotyping has involved a study of the constancy of characteristics of gonococci isolated at one time from two or more sites of a given subject. The auxotypes and minimal inhibitory concentration (MIC) of penicillin G were determined for 181 isolates obtained from 84 patients with uncomplicated gonorrhea, for 16 isolates from 8 couples with uncomplicated gonorrhea, and for 21 isolates from 12 other patients, 9 with disseminated gonococcal infection and three consorts. The penicillin MIC served to distinguish between many members of auxotypes 1, 2, and 3, which are commonly involved in uncomplicated gonorrhea. Thus, for proline-requiring gonococci (auxotype 2) the MIC ranged from 0.01 to 1.2 IU of penicillin per ml. The profile of gonococcal responses to seven other antibacterial drugs provided useful additional information where the extent of phenotypic similarity was in doubt. In all but seven instances, the gonococci isolated from different sites of the same patient, or from a consort, had the same nutritional requirements and penicillin MIC. The gonococci isolated from one patient with disseminated gonococcal infection and from one of her two sexual contacts had nutritional requirements for arginine, hypoxanthine, uracil, and thiamine pyrophosphate, whereas the strain isolated from her second contact differed in having no requirement for thiamine pyrophosphate. The paired cervical and rectal isolates from one patient with uncomplicated gonorrhea differed only with respect to a requirement for hypoxanthine. Pairs of isolates from three patients differed slightly in degree of susceptibility to penicillin. In the remaining two instances, however, numerous differences between the isolates from the endocervix and the anal canal of a given patient indicated the presence of concomitant infections with different strains of N. gonorrhoeae.

Culture Media↗

PubMed1990

Thiamin status of a sample of homeless clinic attenders in Sydney.

Thiamin is one of the marginally adequate nutrients in the Australian diet. The incidence and prevalence of Wernicke-Korsakoff syndrome in this country may be the highest in the world. Homeless men could be at risk for low intakes of thiamin in association with irregular high alcohol intakes. A sample of 107 homeless men from two hostels and one clinic for homeless persons in inner Sydney was investigated for nutritional status; their thiamin status is reported here. By means of 24-hour recall methods, their mean dietary thiamin intake--0.76 mg per day--was less than the National Health and Medical Research Council's recommended dietary intake of 1.1 mg per day; at 0.076 mg/MJ it was even less than the minimal requirement of 0.08 mg/MJ. It was much lower than the mean intake of 1.38 mg per day that was found in the 1983 National Dietary Survey of adults and the distribution of thiamin intakes in this study was skewed positively, with the largest intake being in the range of 0-0.1 mg per day. On clinical examination we found a high prevalence of signs that were consistent with thiamin deficiency. Twenty-four per cent of the subjects showed three-or-more of the signs of the Wernicke-Korsakoff syndrome (ophthalmoplegia, nystagmus, ataxia, peripheral neuropathy and global confusion). In assaying for red-cell transketolase levels, this subgroup showed higher thiamin pyrophosphate effects than did the whole sample. Thirty-six per cent of the whole sample showed subnormal thiamin status by the thiamin pyrophosphate effect. Thus, in this sample, homeless men showed a high prevalence of dietary, biochemical and clinical features to indicate subclinical or early clinical thiamin deficiency.

Adult↗

PubMed1982

Metabolism of thiamin and its phosphoric esters in different regions of the nervous system: a new approach.

In the nervous system thiamin could have two main functions: coenzymatic, as thiamin pyrophosphate, and/or non-coenzymatic, but related to the nervous activity. In order to investigate the thiamin role we studied the turnover and metabolism of thiamin and its phosphoesters (mono-, pyro-, and tri-phosphate) in different regions (cerebellum, brain stem, cerebral cortex and sciatic nerve) of the nervous system, in a steady state condition. Practically, we determined the regional radioactivity of thiamin and its phosphoesters at different times (up to 240 hrs) from labeled thiamin administration, analyzing the obtained data by suitable compartmental models. From the results of two different types of experiment and from their mathematical elaboration, the cerebellum, with the highest thiamin compound content, appeared to be the most active region of the brain in different steps of thiamin metabolism (phosphorylation, dephosphorylation, uptake and release). The sciatic nerve, however, stood out for only thiamin and thiamin monophosphate metabolism, in spite of its very low total thiamin content.

Animals↗

PubMed1991

Characterization and molecular properties of 2-oxoglutarate decarboxylase from Euglena gracilis.

2-Oxoglutarate decarboxylase was purified to homogeneity, as judged by polyacrylamide gel electrophoresis. It had a molecular weight of 250,000 and consisted of four identical subunits of molecular weight 62,000. The enzyme was specific for 2-oxoglutarate, but not for other 2-oxo acids such as pyruvate and oxalacetate. Thiamin pyrophosphate and MgCl2 were required for maximum activity. The Km values of the enzyme for 2-oxoglutarate, thiamin pyrophosphate, and MgCl2 were 330, 56, and 93 microM, respectively. 2-Mercaptoethanol and NADP+ augmented significantly the enzyme activity. The amino acid composition and amino acid sequence of the amino-terminal region of 2-oxoglutarate decarboxylase were determined. On ouchterlony double-immunodiffusion gels, the anti-2-oxoglutarate decarboxylase antibody gave sharp precipitin lines against the mitochondrial fraction of E. gracilis and the purified 2-oxoglutarate decarboxylase, but not against pyruvate decarboxylase from Saccharomyces cerevisiae. On Immunoblots of the crude extract of Euglena, the antibody recognized two polypeptides whose molecular weights were 62,000 and 65,000, respectively. The polypeptide with the molecular weight of 62,000 was found only in mitochondrial fractions. In vitro translation of Euglena polyadenylated RNA in a cell-free rabbit reticulocyte lysate system explained the formation of a single polypeptide with a molecular weight of 65,000, suggesting that a putative precursor of 2-oxoglutarate decarboxylase which is about 3000 larger than the subunit of the mature enzyme is synthesized in Euglena cells.

Amino Acid Sequence↗

PubMed1998

Furosemide and digoxin inhibit thiamine uptake in cardiac cells.

Heart cells in culture were used to clarify whether furosemide or digoxin cause thiamine deficiency and if so, by what mechanism. The intracellular level of thiamine pyrophosphate gradually decreased, with a half-life of 16-19 days, after treatment of cardiac cells with furosemide or digoxin. When thiamine was excluded from the growth medium, thiamine pyrophosphate levels gradually decreased, with a half-life of 5-6 days. No additive effect was observed in the presence of the above drugs when thiamine was excluded from the medium. Thiamine uptake by cardiac cells grown in a thiamine-free medium for 7 days decreased significantly in the presence of furosemide or digoxin. The effect of furosemide or digoxin on thiamine uptake was found to be dose dependent. Co-administration of furosemide and digoxin to the cardiac cell cultures resulted in an additive effect on thiamine uptake. Our results demonstrate that furosemide and digoxin inhibit thiamine uptake by cardiac cells in culture and may therefore cause thiamine deficiency in patients undergoing chronic treatment with these drugs.

Animals↗

PubMed1993

Isolation and characterization of a thiamin pyrophosphokinase gene, THI80, from Saccharomyces cerevisiae.

The thi80 mutant of Saccharomyces cerevisiae (Nishimura, H., Kawasaki, Y., Nosaka, K., Kaneko, Y., and Iwashima, A. (1991) J. Bacteriol. 173, 2716-2719) shows markedly reduced activity of thiamin pyrophosphokinase (TPK; EC 2.7.6.2). We have isolated a DNA fragment carrying the THI80 gene from a yeast genomic library by its ability to complement constitutive synthesis of the thiamin-repressible acid phosphatase, encoded by the PHO3 gene, of thi80 mutant cells. On the other hand, the thi80 locus was found to be located 3.3 centimorgans proximal to the smp3 locus on the right arm of chromosome XV by genetic mapping analysis, and one more fragment bearing the THI80 gene trailing SMP3 gene was obtained by the plasmid eviction method. The nucleotide sequence of the overlapped region between the two isolated DNAs contained an open reading frame of 957 base pairs, encoding a 319-amino acid polypeptide with a calculated molecular weight of 36,616. When the intact THI80 open reading frame was expressed as a fusion protein carrying three vector-encoded amino acids at its N terminus in Escherichia coli lacking TPK, marked TPK activity was detected in the procaryotic cells, proving that the THI80 gene of S. cerevisiae encodes a structural gene of TPK. A gene disruption experiment demonstrated that the THI80 gene was essential for growth, and therefore, revealed that TPK is the only enzyme capable of synthesizing thiamin pyrophosphate in yeast. Studies of Northern blot analysis and the enzyme assay demonstrated that the THI80 gene expression is regulated mainly at the mRNA level by the intracellular thiamin pyrophosphate and requires the positive regulatory factors encoded by THI2 and THI3 genes. However, unlike thiamin-repressible acid phosphatase and the enzymes involved in thiamin synthesis of S. cerevisiae, TPK was found to be expressed constitutively at a low level and incompletely repressed by exogenous thiamin.

Amino Acid Sequence↗

PubMed2003

Structural biology of enzymes of the thiamin biosynthesis pathway.

Thiamin pyrophosphate is an essential cofactor of carbohydrate and branched-chain amino acid metabolism. Although its mechanistic role is well studied, the biosynthesis of thiamin has only recently been understood. Thiamin biosynthesis in Escherichia coli and Bacillus subtilis show some similarities, but diverge at key steps of thiazole formation. The biosynthesis of thiamin in eukaryotes is at a very early stage of understanding. Structural and mechanistic studies on thiamin biosynthetic enzymes have played a key role in increasing our understanding of thiamin pyrophosphate biosynthesis and have revealed unexpected evolutionary ties.

Bacteria↗

PubMed1990

High affinity of acid phosphatase encoded by PHO3 gene in Saccharomyces cerevisiae for thiamin phosphates.

The enzymatic properties of acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) encoded by PHO3 gene in Saccharomyces cerevisiae, which is repressed by thiamin and has thiamin-binding activity at pH 5.0, were investigated to study physiological functions. The following results led to the conclusion that thiamin-repressible acid phosphatase physiologically catalyzes the hydrolysis of thiamin phosphates in the periplasmic space of S. cerevisiae, thus participating in utilization of the thiamin moiety of the phosphates by yeast cells: (a) thiamin-repressible acid phosphatase showed Km values of 1.6 and 1.7 microM at pH 5.0 for thiamin monophosphate and thiamin pyrophosphate, respectively. These Km values were 2-3 orders of magnitude lower than those (0.61 and 1.7 mM) for p-nitrophenyl phosphate; (b) thiamin exerted remarkable competitive inhibition in the hydrolysis of thiamin monophosphate (Ki 2.2 microM at pH 5.0), whereas the activity for p-nitrophenyl phosphate was slightly affected by thiamin; (c) the inhibitory effect of inorganic phosphate, which does not repress the thiamin-repressible enzyme, on the hydrolysis of thiamin monophosphate was much smaller than that of p-nitrophenyl phosphate. Moreover, the modification of thiamin-repressible acid phosphatase of S. cerevisiae with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide resulted in the complete loss of thiamin-binding activity and the Km value of the modified enzyme for thiamin monophosphate increased nearly to the value of the native enzyme for p-nitrophenyl phosphate. These results also indicate that the high affinity of the thiamin-repressible acid phosphatase for thiamin phosphates is due to the thiamin-binding properties of this enzyme.

Acid Phosphatase↗

PubMed2000

On the mechanism of action of thiamin enzymes in the presence of bivalent metal ions.

Results on the interactions between the bivalent metal ions Zn2+, Cd2+, Hg2+, Co2+, Ni2+ and 'active aldehyde' thiamin derivatives are reviewed. The techniques used in these studies include spectroscopic methods, i.e., IR-Raman, UV-Vis, multidimensional and multinuclear NMR in solution and in solid state, and X-ray crystal structure determinations. More recently, potentiometric studies on thiamin pyrophosphate and 2-(alpha-hydroxyethyl)thiamin in combination with NMR and EPR techniques were also undertaken. All these studies lead to useful conclusions on the mechanism of action of thiamin enzymes in the presence of bivalent metal ions.

Cations, Divalent↗

PubMed1989

Biotransformation of N-substituted aromatic compounds in mammalian spermatozoa. Nonoxidative formation of N-hydroxy-N-arylacetamides from nitroso aromatic compounds.

The metabolism of N-substituted aromatic compounds, i.e. aniline, acetanilide, N-hydroxyacetanilide, nitrosobenzene, and nitrobenzene in mammalian spermatozoa was investigated. In boar spermatozoa fortified with glucose, no acetylation, deacetylation, and monooxygenation of these compounds were found. Nitrobenzene was reduced slowly, but nitrosobenzene was a good substrate for this reductive activity. In the latter reaction, the products were N-hydroxyacetanilide, azoxybenzene, and an organic phase-nonextractable metabolite(s). Pyruvate was found to be involved in the formation of N-hydroxyacetanilide from nitrosobenzene, and the reaction occurred through a ping-pong mechanism. This enzymatic activity, located in the mid-piece fraction of spermatozoa, was enhanced by thiamine pyrophosphate and Mg2+ and inhibited by thiamine thiazolone pyrophosphate. N-Hydroxyacetanilide formed from [3(-13)C]pyruvate showed complete retention of the isotope at the methyl carbon of the molecule. 2-Nitrosofluorene and 4-nitrosobiphenyl were also transformed into the corresponding N-hydroxy-N-arylacetamides. N-Hydroxyacetanilide was also formed in rat and human spermatozoa. These facts suggest that the formation of N-hydroxy-N-arylacetamides from the nitroso aromatic compounds and pyruvate is mediated by a pyruvate dehydrogenase complex located in the mitochondria of spermatozoa. The formation of both azoxybenzene and the organic phase-nonextractable metabolite(s) was found to be a pyruvate-independent nonenzymatic reaction.

Amines↗

PubMed1989

Leaky pantothenate and thiamin mutations of Salmonella typhimurium conferring suphometuron methyl sensitivity.

The herbicide suphometuron methyl inhibits the utilization of pyruvate and 2-ketobutyrate by the branched-chain amino acid biosynthetic enzyme acetolactate synthase. Eighteen insertions of the transposon Tn10 into the genome of Salmonella typhimurium LT2 caused hypersensitivity to this herbicide. Five of these insertions conferred a partial auxotrophic requirement. Concurrent herbicide sensitivity and heat-labile pantothenate auxotrophy was due to panD::Tn10 mutations, while coincident sulphometuron methyl sensitivity and thiamin auxotrophy was attributable to thiA::Tn10 mutations. The phenotypes of these mutations suggested that coenzyme A and thiamin pyrophosphate availability modulated the cells' response to sulphometuron methyl. A model suggesting a key role for 2-ketobutyrate accumulation in herbicide action is supported by the function of thiamin pyrophosphate in 2-ketoacid metabolism and the known role of a 2-ketoacid in coenzyme A synthesis.

Acetolactate Synthase↗

PubMed1976

The thiamin status of early cancer patients with particular reference to those with breast and bronchial carcinomas.

The thiamin status of early bronchial and breast carcinomas has been assessed using "thiamin pyrophosphate" (TPP) stimulating effect of transketolase enzyme activity. The TPP stimulating effect was found to be greater in a high proportion of the cancer patients than in normal subjects, suggesting that these patients may be at risk of thiamin deficiency. The urinary excretory level of thiamin was high in the cancer patients; it is, therefore, unlikely that the biochemical deficiency of the vitamin was due to lack of the vitamin intake. It is, however, possible that the cancer patients were unable to convert thiamin to thiamin pyrophosphate, the active form of the vitamin.

Adult↗

PubMed1987

Thiamine triphosphate and membrane-associated thiamine phosphatases in the electric organ of Electrophorus electricus.

The main electric organ of Electrophorus electricus is particularly rich in thiamine triphosphate, which represents 87% of the total thiamine content in this tissue. The thiamine pyrophosphate concentration, however, is very low in the eel electric organ and skeletal muscle as compared with other eel or rat tissues. Furthermore, electroplax membranes contain a whole set of enzymes responsible for the dephosphorylation of thiamine tri-, pyro- and monophosphate. Thiamine triphosphatase has a pH optimum of 6.8 and is dependent on Mg2+. The real substrate of the enzyme is probably a 1:1 complex of Mg2+ and thiamine triphosphate. Thiamine pyrophosphatase is activated by Ca2+. The apparent Km for thiamine triphosphate and Vmax are found to be, respectively, 1.76 mM and 5.95 nmol/mg of protein/min. Thiamine triphosphatase activity is inhibited at physiological K+ concentrations (up to 90 mM) and increasing Na+ concentrations (50% inhibition at 300 mM). ZnCl2 (10 mM) inhibits 90% of the enzyme activity. ATP and ITP are also strongly inhibitory. No significant effect of neurotoxins is seen. Membrane-associated thiamine triphosphatase is affected differently by proteolytic enzymes and is partially inactivated by pretreatment with phospholipase C and neuraminidase. The physiological significance of thiamine triphosphatase is discussed in relation to a specific role of thiamine in the nervous system.

Animals↗