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Results for “THIAMINE PYROPHOSPHATE”

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At least 433 records · Page 24Linked to original sources

PubMed1980

Regulation of the 2-oxoglutarate dehydrogenase lipoate succinyltransferase complex from cauliflower by nucleotide. Pre-steady state kinetics and physical studies.

Analysis of the binding of thiamin pyrophosphate to the 2-oxoglutarate dehydrogenaselipoate succinyltransferase multienzyme complex using pre-steady state kinetic methods revealed that the presence of 2-oxoglutarate is not necessary for binding, although it does stabilize the complex by slowing the rate of dissociation of the holoenzyme. The rate of binding of thiamin-PPi to the enzyme and the subsequent enzyme activation are not limited by a reaction at C-2 of the thiazolium ring of thiamin-PPi since no kinetic isotope effect is observed when 2-D-thiamin-PPi is substituted for the protonated cofactor. The presence of 5'-AMP, which activates the reaction producing both a V and a Km response, causes a significant increase in kon for thiamin-PPi. The AMP analog 1,N6-ethenoadenosine-5'-monophosphate (epsilon-AMP) also activates the reaction, but shows only a K effect, with no influence on V. This effector reduces Kd for the thiamin-PPi2-oxoglutarate dehydrogenase complex by increasing kon. The change in kon for thiamin-PPi in response to changes in hydrogen ion concentration shows pK values which are unaffected by the addition of AMP, in this respect resembling the steady state kinetic response of V/Km and differing from the pH profile of V. The dissociation constant of holoenzyme is relatively insensitive to pH over the range pH 6 to 9, but in the presence of AMP the Kd, which is decreased in the range from pH 7 to 8, increases sharply at higher or lower pH values.

Acyltransferases↗

PubMed1980

Furosemide-induced thiamine deficiency.

Male Wistar rats were separated into 4 groups: group 1, thiamine sufficient diet (control); group 2, thiamine sufficient diet with intraperitoneal administration of furosemide (20 mg . kg-1 of body weight); group 3, thiamine deficient diet; group 4, thiamine deficient diet within tra-peritoneal administration of furosemide. After 4 weeks, the rats were killed and the thiamine levels and activity of transketolase were assayed. Thiamine concentration and transketolase activity were significantly decreased and thiamine pyrophosphate effect was significantly increased in the blood, and various tissues in group 2 and 4 compared with group 1 and 3, respectively. The intraperitoneal administration of various concentrations of furosemide (20 mg, 10 mg, and 2 mg . kg-1 of body weight) resulted in a significant increase in urinary thiamine excretion. Thus, it is assumed that long-term administration of furosemide could induce a thiamine deficiency.

Animals↗

PubMed1978

[Isolation and some properties of transketolase from rat liver].

A new procedure transketolase isolation from rat liver using sievorptive chromatography and adsoption chromatography on hydroxylapatite is described. An enzyme preparation is obtained, which is homogenous under polyacrylamide gel electrophoresis. Molecular weight of native enzyme, estimated by sedimentation equilibrium method, is 130 000. Electrophoresis in polyacrylamide gel in the presence of sodiumdodecylsulphate has revealed a single protein fraction with molecular weight of 70 000. The extinction coefficient A1%1cm at 280 nm is found to be 13. Thiamine pyrophosphate content in native holoenzyme is 1.8--2 moles per 1 mole of protein.

Animals↗

PubMed1979

Effects of 5-fluorouracil on the thiamin status of adult female rats.

The effect of 5-fluorouracil on the thiamin status of normal female adult rats has been investigated. Pre-treatment of the animals with the cytotoxic drug daily for 3 successive days resulted in a significant decrease in hepatic concentrations of thiamin concomitant with a decrease in thiamin-dependent transketolase enzyme activity and an increase in thiamin-pyrophosphate-(TPP-)stimulating effect in whole blood when compared with those of pair-fed control animals. The TPP effect of transketolase enzyme activity was also increased by 5-fluorouracil in vitro. Furthermore, the treatment with 5-fluorouracil resulted in decreased liver and spleen concentrations without affecting the urinary excretory levels of thiamin in animals supplemented with large doses of the vitamin. Giving a dose comparable to a human therapeutic dose caused a similar increase in the TPP effect. These results indicate that treatment with 5-fluorouracil may be associated with thiamin deficiency by increasing either the utilization or the breakdown of thiamin.

Animals↗

PubMed1999

Characterization of point mutations in patients with pyruvate dehydrogenase deficiency: role of methionine-181, proline-188, and arginine-349 in the alpha subunit.

Human pyruvate dehydrogenase (E1), a heterotetramer (alpha2beta2), is the first component of the pyruvate dehydrogenase complex (PDC). E1 catalyzes the thiamin pyrophosphate (TPP)-dependent decarboxylation of pyruvate and the reductive acetylation of the dihydrolipoamide acetyltransferase component. Site-directed mutagenesis was employed to recreate three point mutations in the alpha subunit identified in E1-deficient patients, M181V, R349H, and P188L (P188A mutant E1 was used because of the very low level of expression of P188L), to investigate the functional roles of these three amino acid residues. P188A mutant E1 was much less thermostable than the wild-type E1. The kcats of M181V and P188A mutant E1s determined in the PDC reaction were 38 and 24% of that of the wild-type enzyme, respectively. The apparent Km for TPP for M181V increased significantly (approx 250-fold when determined in the PDC assay), while the apparent Km for pyruvate increased by only about 3-fold. In contrast, P188A had similar Kms for the coenzyme and the substrate as the wild-type. Km values for R349H were not determined due to the extremely low activity of this mutant (1.2% of the wild-type E1-specific activity measured in the PDC assay). Wild-type E1 displayed a lag phase in the progress curve of the PDC reaction measured in the presence of low TPP concentrations (below 1 microM) only. All mutants had a lag phase that was not eliminated even at very high TPP concentrations, suggesting modifications in the conformation of the active site. Kinetic analysis indicated thiamin 2-thiothiazolone pyrophosphate (ThTTPP) to be an intermediate analog for wild-type human E1. M181V required a higher concentration of ThTTPP for inactivation than the wild-type and P188A E1s. The results of circular dichroism spectropolarimetry in the far UV region indicated that there were no major changes in the secondary structure of M181V, P188A, and R349H E1s. These mutant enzymes exhibited negative dichroic spectra at about 330 nm only in the presence of high TPP concentrations. This study suggests that arginine-349 is critical for E1's activity, methionine-181 is involved in the binding of TPP, and proline-188 is necessary for structural integrity of E1.

2,6-Dichloroindophenol↗

PubMed1966

The control mechanism of thiamine biosynthesis a model for the study of control of converging pathways.

1. Thiamine or the pyrimidine moiety of thiamine added in excess to a growing culture of Salmonella typhimurium LT2 repressed subsequent thiamine synthesis in non-growing organisms. 2. A mutant unable to convert added pyrimidine moiety into thiamine was not repressible by the pyrimidine, showing that thiamine, not the pyrimidine, was the repressor. 3. Thiamine repression occurred at 40mmug. of thiamine/mg. dry wt. or above and de-repression occurred at 30mmug. of thiamine/mg. dry wt. or below. 4. Thiamine controlled the pyrimidine and thiazole pathways at the same concentration and to the same extent. 5. Biosynthesis of the thiazole moiety had, in contrast with biosynthesis of the pyrimidine moiety, an additional feedback inhibition control that allowed utilization of the exogenous thiazole. 6. The enzymes joining the pyrimidine and thiazole moieties were repressible by high concentrations of thiamine. 7. Thiamine was rapidly converted into thiamine pyrophosphate and this appeared to be the active repressor. 8. Theoretical aspects of control of converging pathways are discussed.

Biological Assay↗

PubMed1982

The protective effect of hymochromone against ethanol-induced thiamin deficiency.

Intragastric administration of ethanol to male rats given at 6 gm/kg body weight, daily for two weeks, produced no significant change in the body or liver weights. Hepatic total lipid increased but this was no more than a trend. Treatment with ethanol, however, resulted in a marked decrease in transketolase activity (TK) of the haemolysed whole blood, associated with a pronounced rise in the stimulating effect of thiamin pyrophosphate on this enzyme (TPP effect). These changes mediated by ethanol were counteracted by hymochromone.

Alkaline Phosphatase↗

PubMed1976

Blood values of some vitamins in long-stay psycho-geriatric patients.

A group of 18 long-stay hospital psycho-geriatric patients (aged 64-101 years) and a group of 10 healthy elderly subjects (aged 53-67 years) who were living in their own residence were studied. The group of geriatric patients were suffering from some degree of pressure sores, dementia, irritability and anorexia. Biochemical tests on vitamin adequacy with particular references to ascorbic acid, thiamine and retinal were performed in the subjects. The thiamine status was measured by determining the thiamine pyrophosphate stimulating effect of transketolase enzyme activity in whole blood. The ascorbic acid and retinal nutritures were determined by measuring their levels in plasma. From the biochemical data of the vitamins it would appear that a significant proportion of the long-stay hospital psycho-geriatric patients may be at risk of thiamine, ascorbic acid and retinol deficiencies.

Age Factors↗

PubMed1980

Characterization and properties of pig liver transketolase.

Some properties of homogeneous transketolase (sedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphate glycolaldehydetransferase, EC 2.2.1.1) from pig liver were studied. It was shown that the pH optimum of the transketolase reaction lies within the range of 7.8-8.2; the isoelectric point is at pH 7.6-7.8. The molecular weight of transketolase is 138 000 +/- 3000 as determined by the sedimentation equilibrium method. The enzyme molecular is a tetramer of the alpha 2 beta 2 type. The molecular weights of the alpha- and beta-subunits determined by polyacrylamide gel in the presence of sodium dodecyl sulphate are 52 000-56 000 and 27 000-29 000, respectively. Transketolase contains about 2 mol of thiamine pyrophosphate per mol of protein and does not require metal ions for its catalytic activity.

Animals↗

PubMed1996

Recurrent aphthous stomatitis and thiamine deficiency.

Recurrent aphthous stomatitis is a disease of unknown cause. To examine whether thiamine (vitamin B1) deficiency is associated with recurrent aphthous stomatitis, we studied vitamin B1 levels in 70 patients with recurrent aphthous stomatitis and in 50 members of a control group. The vitamin B1 level was determined as thiamine pyrophosphate effect on transketolase activity in red blood cell lysates. Low levels of vitamin B1 were detected in 49 patients but in only two members of the control group (p < 0.0001). These low levels were not associated with patient age, sex, or underlying disease causing recurrent aphthous stomatitis. Our finding suggests an association between thiamine deficiency and recurrent aphthous stomatitis.

Adolescent↗

PubMed1974

Thiamine deficiency and oxalosis.

Type I hyperoxaluria results from reduced activity of alpha-ketoglutarate: glyoxylate carboligase, which is necessary for the synergistic decarboxylation of glyoxylate and alpha-ketoglutarate to alpha-hydroxy-beta-keto-adipate. Since thiamine pyrophosphate is a cofactor in the reaction, thiamine deficiency might be expected to result in tissue oxalosis. However, there was no significant increase in the incidence of renal oxalosis in 15 patients with Wernicke's encephalopathy at necropsy compared with controls. It is possible that hyperoxaluria was present in these thiamine-deficient patients but at a urine concentration below that necessary for calcium oxalate deposition. It is also possible that the severity of the thiamine deficit required for hyperoxaluria exceeds that for the neuronal and cardiac manifestations.

Acute Kidney Injury↗

PubMed2003

Thiamine deficiency in congestive heart failure patients receiving long term furosemide therapy.

OBJECTIVE: To assess the presence of thiamine deficiency in congestive heart failure patients receiving furosemide therapy. DESIGN: Prospective, biochemical analysis of thiamine status was performed in outpatients and inpatients of the University of Ottawa Heart Institute. SUBJECTS: Thirty-two patients with congestive heart failure who received at least 40 mg/day of furosemide were included. Patients were then separated into two groups depending on whether the dose of furosemide was greater than or equal to 80 mg/day. METHODS: The primary measure was actual thiamine status as assessed by the erythrocyte transketolase enzyme activity and the degree of thiamine pyrophosphate effect. RESULTS: Biochemical evidence of severe thiamine deficiency was found in 98% (24 of 25) patients receiving at least 80 mg/day of furosemide and in 57% (four of seven) of patients taking 40 mg furosemide daily, odds ratio (OR) 19.0 (1.13<OR<601.29). Thiamine status was not associated with any other clinical variables. CONCLUSIONS: These findings suggest that thiamine deficiency occurs in a substantial proportion of congestive heart failure patients being treated with furosemide.

Aged↗

PubMed1980

Thiamin status during pregnancy.

Thiamin status of 20 nonpregnant women and 60 pregnant women was assessed. Of the 60 pregnant women, only 49 and 25 were examined for the biochemical thiamin status in the 3rd trimester and postpartum period respectively. Thirty per cent of nonpregnant women and 28 to 39 per cent of pregnant mothers (in either the 2nd trimester, 3rd trimester, or postpartum phase of pregnancy) had a deficient thiamin status with the thiamin pyrophosphate effect (TPP effect) greater than 20 per cent. None of the pregnant subject were deficient in all the three periods. Follow up of the pregnant subjects in the 2nd trimester, 3rd trimester and postpartum period revealed that the deficiency was not necessarily established in the early pregnancy nor was aggravated with the progress of pregnancy. Thiamine intake was over 2/3 of the recommended in all the pregnant subjects and revealed no significant correlation with the biochemical indices. A history of oral contraceptive usage before conception had no significant effect on the incidence of deficiency in the 2nd or 3rd trimester. The occurrence of deficient thiamin status demonstrated an increase with number of previous pregnancies. No correlation was noted between the vitamin B1 status and anthropometric measurements of neonates.

Adolescent↗

PubMed1998

Chemical modification of lysine and arginine residues of bovine heart 2-oxoglutarate dehydrogenase: effect on the enzyme activity and regulation.

Chemical modification of arginine and lysine residues of bovine heart 2-oxoglutarate dehydrogenase with phenylglyoxal and pyridoxal 5'-phosphate inactivated the enzyme, indicating the importance of these residues for the catalysis. Inactivation caused by pyridoxal 5'-phosphate was prevented in the presence of thiamine pyrophosphate and Mg2+ allowing the assumption that lysine residues participate in binding of the cofactor.

Adenosine Diphosphate↗

PubMed1979

[Small angle X-ray scattering studies on the structure and symmetry of yeast pyruvate decarboxylase in solution].

The holo-enzyme of the pyruvate decarboxylase EC 4.1.1.1 isolated from brewer's yeast was studied by small-angle X-ray scattering. The point-group symmetry 222 deduced from electron microscopic investigations was confirmed for molecules in solution. The best approximation of the overall shape of the enzyme was reached by an homogeneous triaxial ellipsoid, with the half-axes of a = 2.47 nm, b = 5.08 nm and c = 8.0 nm. The ellipsoid is halved vertical to the a-axis, and both halves are turned from each other by about 30 degrees. As an explanation of the existence of four binding sites for Mg2+ and thiamine pyrophosphate on the dimeric molecule the hypothesis of a gene duplication is discussed.

Binding Sites↗

PubMed2002

Metabolic flux in both the purine mononucleotide and histidine biosynthetic pathways can influence synthesis of the hydroxymethyl pyrimidine moiety of thiamine in Salmonella enterica.

Together, the biosyntheses of histidine, purines, and thiamine pyrophosphate (TPP) contain examples of convergent, divergent, and regulatory pathway integration. Mutations in two purine biosynthetic genes (purI and purH) affect TPP biosynthesis due to flux through the purine and histidine pathways. The molecular genetic characterization of purI mutants and their respective pseudorevertants resulted in the conclusion that <1% of the wild-type activity of the PurI enzyme was sufficient for thiamine but not for purine synthesis. The respective pseudorevertants were found to be informational suppressors. In addition, it was shown that accumulation of the purine intermediate aminoimidazole carboxamide ribotide inhibits thiamine synthesis, specifically affecting the conversion of aminoimidazole ribotide to hydroxymethyl pyrimidine.

Aminoimidazole Carboxamide↗

PubMed1997

Isolation and characterization of a thiamin transport gene, THI10, from Saccharomyces cerevisiae.

We isolated a thiamin transporter gene, THI10, from a genomic library of Saccharomyces cerevisiae by the complementation of a yeast mutant defective in thiamin transport activity. The THI10 gene contained an open reading frame of 1,794 base pairs encoding a 598-amino acid polypeptide with a calculated molecular weight of 66, 903. The nucleotide sequence of THI10 is completely identical to that of an anonymous DNA (open reading frame L8083.2) mapped to chromosome XII; two other genes (open reading frames YOR071c and YOR192c) in chromosome XV are extremely similar to THI10. Moreover, the THI10 gene product showed significant sequence homology with yeast allantoin and uracil transporters. Hydropathy profile suggested that THI10 product is highly hydrophobic and contains many transmembrane regions. Gene disruption of the THI10 locus completely abolished the thiamin transport activity and thiamin binding activity in yeast plasma membrane fraction. Both the transport and thiamin binding activities were restored in the disrupted cells when the THI10 open reading frame was expressed by yeast GAL1 promoter, suggesting that the THI10 gene encodes for the thiamin transport carrier protein. Northern blot analysis demonstrated that THI10 gene expression is regulated at the mRNA level by intracellular thiamin pyrophosphate and that it requires a positive regulatory factor encoded by THI3 gene.

Amino Acid Sequence↗

PubMed1997

Identification and characterization of an operon in Salmonella typhimurium involved in thiamine biosynthesis.

Thiamine pyrophosphate (TPP) is synthesized de novo in Salmonella typhimurium and is a required cofactor for many enzymes in the cell. Five kinase activities have been implicated in TPP synthesis, which involves joining a 4-methyl-5-(beta-hydroxyethyl)thiazole (THZ) moiety and a 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) moiety. We report here identification of a 2-gene operon involved in thiamine biosynthesis and present evidence that the genes in this operon, thiMD, encode two previously identified kinases, THZ kinase and HMP phosphate (HMP-P) kinase, respectively. We further show that this operon belongs to the growing class of genes involved in TPP synthesis that are transcriptionally regulated by TPP. Our data are consistent with ThiM being a salvage enzyme and ThiD being a biosynthetic enzyme involved in TPP synthesis, as previously suggested.

Amino Acid Sequence↗