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PubMed1993

The effect of thiamin on the activation of thiamin pyrophosphate-dependent 2-oxoglutarate decarboxylase in Euglena gracilis.

The effect of thiamin on thiamin pyrophosphate-dependent 2-oxoglutarate (2-OG) decarboxylase activity in Euglena gracilis was investigated. The total activity of 2-OG decarboxylase in thiamin-sufficient cells in 3 times that in thiamin-deficient cells. The addition of thiamin to thiamin-deficient cells causes the total enzyme and holoenzyme activities to increase and reach similar levels to that in thiamin-sufficient cells. Cycloheximide and chloramphenicol, inhibitors of protein synthesis, have no effect on the total enzyme activity. Immunochemical titration and determination of 2-OG decarboxylase mRNA by using an antibody directed against Euglena 2-OG decarboxylase indicate that the increase in the holoenzyme activity of 2-OG decarboxylase is due to activation of pre-existing protein and does not require synthesis of new proteins in thiamin-deficient cells. During the period of the increase in the total activity, the apoenzyme increases and reaches a temporary peak in 2 h. Immunoblot analysis demonstrates that the precursor form (a 65 kDa subunit) of 2-OG decarboxylase in thiamin-deficient cells is more abundant than that in thiamin-sufficient cells and the increase in the apoenzyme by addition of thiamin results from the conversion of the precursor form into the mature form (a 62 kDa subunit).

Animals↗
PubMed1980

A study of partial thiamin restriction in human volunteers.

A double blind study of partial thiamin restriction was undertaken in 19 volunteer male medical students for a period of 4 to 5 weeks. Each day the subjects followed a diet supplying 500 micrograms thiamin and took 1 capsule randomly assigned to contain 5 mg thiamin hydrochloride or thiamin placebo. This capsule also supplied physiological doses of other vitamins. At the end of the study all were repleted with thiamin hydrochloride. Ten thiamin-depleted subjects were correctly identified by low urinary thiamin excretion, decreasing erythrocyte transketolase activity, and elevation of the thiamin pyrophosphate effect. The remaining nine subjects were controls. The degree of thiamin depletion induced varied between individuals and had no measurable ill effect on health as assessed by subjective feelings, physical findings, psychological testing, nerve conduction studies, and work performance. Subclinical thiamin deficiency is defined from this study as the presence of a low urinary thiamin level (27 microgram thiamin/g creatinine) together with a thiamin pyrophosphate effect above 14.2% and below 35.4%. However, when urinary thiamin excretion is unknown, the data indicate that a thiamin pyrophosphate effect above 9% and below 41.6% is likely but not certain to represent subclinical thiamin deficiency. These guidelines, developed under experimental conditions, are a reference framework for assessment of thiamin status in population groups but should be applied with caution to individuals and to people with disease.

Adolescent↗
PubMed1983

Paracatalytic inactivation of pig heart pyruvate dehydrogenase complex.

The phenomenon of paracatalytic inactivation has been demonstrated and characterized with the pig heart pyruvate dehydrogenase complex. The enzyme became progressively inactive when it was preincubated in the presence of pyruvate, thiamine pyrophosphate, and extrinsic oxidative agent, 2,6-dichloroindophenol. Not only the overall reaction of enzyme complex but the individual reactions catalyzed by enzyme components, pyruvate dehydrogenase, and dihydrolipoamide acetyltrasferase, were reduced after preincubation. The observed inactivation is due to two factors: (i) covalent incorporation of the pyruvate atoms, and (ii) formation of a thiamine pyrophosphate analog. (i) A covalent incorporation of radioactivity from [2-14C]pyruvate into the enzyme complex, which was proportional to the inhibition of overall reaction was observed. Seventy-eight percent of the radioactivity incorporated into the dihydrolipoamide transacetylase but not into the lipoic acid. Thus, modification of the dihydrolipoamide acetyltransferase component can be attributed to the covalent incorporation of pyruvate atoms. (ii) During inactivation, the enzyme-bound thiamine pyrophosphate underwent a modification resulting in the formation of a thiamine pyrophosphate analog, presumably thiamine thiazolone pyrophosphate, which inhibited exclusively the pyruvate dehydrogenase component.

2,6-Dichloroindophenol↗
PubMedDate not supplied

[Thiamine pyrophosphate-dependent enzymes in the regenerating liver of albino rats under different regimens of oxythiamine administration].

Activities of the thiamine pyrophosphate-dependent enzymes (pyruvate dehydrogenase, oxoglutarate dehydrogenase, transketolase) were increased in regenerating rat liver tissue as compared with their activities in the intact tissue. After administration of oxythiamine (20 mg/kg, within 10 days, subcutaneously) into the animals the enzymatic activity studied was decreased.

Animals↗
PubMed1985

Synthesis of thiamine triphosphate in rat brain in vivo.

Thiamine metabolism in vivo was studied by intracerebroventricular injection of labeled thiamine in rat brain. Labeled thiamine was found to be rapidly converted to the phosphorylated thiamine esters. The distribution of the radioactive thiamine compounds was reached to steady state at 3 hr after injection: thiamine, thiamine monophosphate, thiamine pyrophosphate, and thiamine triphosphate were 8-12%, 12-14%, 72-74%, and 2-3%, respectively, in cerebral cortex. The presence of labeled thiamine triphosphate in the brain was further confirmed by the treatment with thiamine triphosphatase which had an absolute substrate specificity for thiamine triphosphate. These results suggest that thiamine triphosphate is synthesized in vivo in rat brain.

Animals↗
PubMed1985

Determination of thiamine and its phosphate esters in human and rat blood by high-performance liquid chromatography with post-column derivatization.

A method for the determination of thiamine and thiamine phosphate esters in human and rat blood by liquid chromatography with post-column derivatization, and distribution of thiamine and its phosphate esters in human and various animals is demonstrated. Blood is deproteinized and centrifuged. Aliquots of the samples are applied to a mu Bondapak C18 column attached to a high-performance liquid chromatograph. Addition of potassium ferricyanide sodium hydroxyide solution to the column effluent with a proportioning pump converts thiamine phosphate into fluorophores, the intensities of which are measured with a spectrofluorophotometer. In human blood, thiamine pyrophosphate was present in the greatest concentration, followed by thiamine triphosphate; thiamine monophosphate and thiamine were present in small amounts. In rat blood, thiamine pyrophosphate was found in the largest amount, followed by thiamine monophosphate. Thiamine triphosphate and thiamine were present in small amounts.

Animals↗
PubMed1978

Demonstration of a lag period in the time-course of the reaction catalyzed by pyruvate dehydrogenase complex.

At low thiamine pyrophosphate concentrations the time-course of the reaction catalyzed by mammalian pyruvate dehydrogenase complex shows a lag period of some minutes when the reaction is started by either enzyme, pyruvate or thiamine pyrophosphate. However, started by CoASH or NAD+, the lag period disappears. An increase in enzyme concentration to 25 mU/ml causes a concomitant shortening of the duration of the lag period (tau), while above this value tau is independent of the enzyme concentration. An increase in thiamine pyrophosphate concentration decreases the value of tau and the lag period vanishes at infinite thiamine pyrophosphate concentration. It is suggested that both isomerization and aggregation-dissociation reactions may play an improtant role in the development of the lag period of pyruvate dehydrogenase complex.

Animals↗
PubMed1984

Heterogeneity of human erythrocyte transketolase: a preliminary report.

A modified procedure for the preparation of transketolase from erythrocytes by dialysis and column ion-exchange resin chromatography is described. Changes have been made in the resin used, the column dimensions and the elution procedure so as to separate the enzyme with improved resolution, prepare the apoenzyme free of thiamine pyrophosphate and study the kinetics of its activation or reactivation by the coenzyme. On the basis of the elution profile of the enzyme activity from the chromatographic column, two different samples of the transketolase have been isolated, which differ not only in their isoelectric properties, but also in the proportion of the transketolase present in the apoenzyme form. Not only do the apoenzymes isolated from each of the two fractions differ in the way in which they recombine with thiamine pyrophosphate but kinetic analysis of the results shows that each fraction contains at least two variants of transketolase differing in their affinity for thiamine pyrophosphate. Three, probably four, separate variants have been identified which differ in their affinities for thiamine pyrophosphate over a range greater than 10(4). It is concluded that these two fractions of the enzyme must contain different subsets of the eight isoenzymes of transketolase of differing isoelectric points and that some of these isoenzymes must differ also in their affinity for the coenzyme. The implications of these findings for the Blass and Gibson hypothesis about the pathogenesis of the Wernicke-Korsakoff syndrome are considered.

Alcohol Amnestic Disorder↗
PubMed1997

Characterization of thiL, encoding thiamin-monophosphate kinase, in Salmonella typhimurium.

Thiamin pyrophosphate is an essential cofactor that is synthesized de novo by Salmonella typhimurium. In bacteria, the end product of the de novo biosynthetic pathway is thiamin monophosphate, which is then phosphorylated by thiamin-monophosphate kinase (EC 2.7.4.16) to form thiamin pyrophosphate. We have isolated and characterized the thiL gene of S. typhimurium and showed that thiL is a 978-base pair open reading frame encoding a 35-kDa protein with thiamin-monophosphate kinase activity. thiL was located in the 10-centisome region of the S. typhimurium chromosome. We demonstrated that altered thiamin-monophosphate kinase activity resulted in decreased repression of transcription of thiamin pyrophosphate-regulated thiamin biosynthetic genes. In contrast to other thi loci, thiL is not transcriptionally regulated by thiamin pyrophosphate. This result is consistent with a dual role for ThiL in de novo biosynthesis and in salvage of exogenous thiamin.

Alleles↗
PubMed1988

Distribution of thiamine, thiamine phosphates, and thiamine metabolizing enzymes in neuronal and glial cell enriched fractions of rat brain.

The distribution of thiamine, thiamine phosphoesters, and the thiamine pyrophosphate synthetizing [thiamine-pyrophosphokinase (TPKase)] as well as hydrolyzing [thiamine pyrophosphatase (TPPase) and thiamine monophosphatase (TMPase)] enzymes was determined in neuronal and glial enriched fractions prepared from rat brain. Nucleoside diphosphatases [inosine diphosphatase (IDPase) and uridine diphosphatase (UDPase)] and nucleoside monophosphatases [uridine monophosphatase (UMPase) and inosine monophosphatase (IMPase)] were also determined. Thiamine and thiamine mono- and pyrophosphate were present in neuronal enriched fractions at concentrations 2.8, 3.6, and 4.6 times higher than in glial fractions. TMPase was found only in glial enriched fractions, whereas the levels of TPKase, UMPase, IMPase, IDPase, UDPase, and TPPase were 2.0-, 2.2-, 1.3-, 2.8-, 3.7-, and 20.8-fold higher in neuronal than in glial fractions.

Animals↗
PubMed1982

thiK and thiL loci of Escherichia coli.

Mutants of Escherichia coli K-12 auxotrophic for thiamine phosphates were produced in stepwise fashion from the polyauxotrophic F- strain JC1552, via intermediate production of thiamine auxotrophs that had lost the enzymatic activity of either phosphomethylpyrimidine kinase or thiamine phosphate pyrophosphorylase. They include two types: one responds to thiamine monophosphate or thiamine pyrophosphate, and the other responds to thiamine pyrophosphate only; the former lacks thiamine kinase activity, and the latter lacks thiamine monophosphate kinase activity, in addition to the enzymatic defects caused by the first mutations. We found two genes, for which we propose the designations thiK and thiL, which govern the activities of thiamine kinase and thiamine monophosphate kinase, respectively. By conjugation and P1 transduction, the thiK locus was mapped at about 25 min, between pyrC and purB and close to fabD. The relative order of thiK with respect to nearby genes was tentatively established as pyrC-ptsG-fabD-thiK-purB. In the case of thiL, the locus was situated at about 9 min, between tsx and acrA and probably 0.2 min clockwise from the former.

Chromosome Mapping↗
PubMed1993

[The effect of phosphorylation on catalytic function of muscle pyruvate dehydrogenase complex].

It has been shown that phosphorylation of the pyruvate dehydrogenase complex from pigeon breast muscle by endogenous ATP-dependent protein kinase suppresses the substrate conversion in the pyruvate: acceptor oxidoreductase reactions and nonoxidative reactions monitored by pyruvate decline in the absence of CoA and NAD. To identify the catalytic step blocked by phosphorylation, CD spectroscopy was used which revealed the appearance and decay of the charge transfer complex between component E1 and thiamine pyrophosphate during the enzymatic reaction. Phosphorylation of the pyruvate dehydrogenase complex while lowering the affinity for thiamine pyrophosphate does not preclude the formation of holo-E1 but inhibits its interaction with pyruvate. Phosphorylated pyruvate dehydrogenase, like the dephosphorylated enzyme, reacts with 2-hydroxyethyl thiamine pyrophosphate in half of the active sites. In the presence of deacylating agents (CoA or dithiothreitol) all the sites are reactive. A conclusion is drawn that the alternating functioning of the active centers is preserved in reductive acetylation of the acceptor substrates by phospho-E1.

Animals↗
PubMed1977

Acyl group and electron pair relay system: a network of interacting lipoyl moieties in the pyruvate and alpha-ketoglutarate dehydrogenase complexes from Escherichia coli.

The dihydrolipoyl transacetylase component of the Escherichia coli pyruvate dehydrogenase complex [pyruvate:lipoate oxidoreductase (decarboxylating and acceptor-acetylating), EC 1.2.4.1] bears two sites on each of its 24 polypeptide chains that undergo reductive acetylation by [2-(14)C]pyruvate and thiamin pyrophosphate, acetylation by [1-(14)C]acetyl-CoA in the presence of DPNH, and reaction with N-ethyl[2,3-(14)C]maleimide in the presence of pyruvate and thiamin pyrophosphate. The data strongly imply that these sites are covalently bound lipoyl moieties. The results of similar experiments with the E. coli alpha-ketoglutarate dehydrogenase complex [2-oxoglutarate:lipoate oxidoreductase (decarboxylating and acceptor-succinylating), EC 1.2.4.2] indicate that its dihydrolipoyl transsuccinylase component bears only one lipoyl moiety on each of its 24 chains. Charging of the 48 acetyl acceptor sites on the transacetylase or the 24 succinyl acceptor sites on the transsuccinylase by pyruvate or alpha-ketoglutarate, respectively, and thiamin pyrophosphate was observed in the presence of only a few functionally active pyruvate dehydrogenase or alpha-ketoglutarate dehydrogenase chains. Extensive crosslinking of the transacetylase chains was observed when the pyruvate dehydrogenase complex was treated with pyruvate and thiamin pyrophosphate or with DPNH in the presence of N,N'-o- or N,N'-p-phenylenedimaleimide, respectively. When the alpha-ketoglutarate dehydrogenase complex was treated with DPNH in the presence of N,N'-p-phenylenedimaleimide, only transsuccinylase monomers and crosslinked transsuccinylase dimers were detected. It appears that the 48 lipoyl moieties in the transacetylase and the 24 lipoyl moieties in the transsuccinylase comprise an interacting network that functions as an acyl group and electron pair relay system through thiol-disulfide and acyl-transfer reactions among all of the lipoyl moieties.

Acetyl Coenzyme A↗
PubMed1977

[Study of the kinetic mechanism of the pyruvate-2,6-dichlorophenolindophenol reductase activity of muscle pyruvate dehydrogenase].

The mechanism of pyruvate-2,6-dichlorophenol-indophenol (2,6-CPI) reductase reaction catalyzed by the pyruvate dehydrogenase complex from pigeon breast muscle and by its pyruvate dehydrogenase component was studied. The K'm values for 2,6-DCPI in both cases were found equal to 1.3--1.4-10(-5) M. The double reverse values plots obtained at a fixed concentration of the first substrate and a variable concentration of the second one were linear and had a constant K'm/V'max ratio. The substitution of thiamine pyrophosphate and pyruvate by the substrate decarboxylation product, i.e. 2-oxyethyl thiamine pyrophosphate under similar conditions resulted in kinetic plots, typical for the "ping-pong" mechanism of enzymatic reactions. A mechanism of the pyruvate 2,6-DCPI reductase reaction, providing for the interaction of 2-oxyethyl thiamine pyrophosphate after its binding to the apoenzyme with a certain protein group of the pyruvate dehydrogenase active centre, was postulated. The reaction was shown to result in the production of acetyl-substituted reduced form of the enzyme. Regeneration of free enzyme required the presence of 2,6-DCPI as oxidizing agent.

2,6-Dichloroindophenol↗
PubMed1989

Comparison of methods for thiamin and riboflavin nutriture in man.

Thiamin and riboflavin nutriture were assessed in 127 apparently healthy individuals aged between 20 and 82 yr, living in small villages near the town of Gubbio. The methods of erythrocyte transketolase activity and HPLC determination of thiamin pyrophosphate (TPP) for thiamin and erythrocyte glutathione reductase and HPLC determination of flavin adenine dinucleotide (FAD) for riboflavin were used. A significant correlation between enzymatic and HPLC methods was found. This was not the case for the correlation between status test and vitamin intake, particularly for thiamin. The vitamer determination in blood by HPLC can be considered a reliable, fast and reasonably accurate method for long-term thiamin and riboflavin nutritional status assessment.

Adult↗
PubMed1993

Structural genes for thiamine biosynthetic enzymes (thiCEFGH) in Escherichia coli K-12.

Escherichia coli K-12 synthesizes thiamine pyrophosphate (vitamin B1) de novo. Two precursors [4-methyl-5-(beta-hydroxyethyl)thiazole monophosphate and 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate] are coupled to form thiamine monophosphate, which is then phosphorylated to make thiamine pyrophosphate. Previous studies have identified two classes of thi mutations, clustered at 90 min on the genetic map, which result in requirements for the thiazole or the hydroxymethylpryimidine. We report here our initial molecular genetic analysis of the thi cluster. We cloned the thi cluster genes and examined their organization, structure, and function by a combination of phenotypic testing, complementation analysis, polypeptide expression, and DNA sequencing. We found five tightly linked genes, designated thiCEFGH. The thiC gene product is required for the synthesis of the hydroxymethylpyrimidine. The thiE, thiF, thiG, and thiH gene products are required for synthesis of the thiazole. These mutants did not respond to 1-deoxy-D-threo-2-pentulose, indicating that they are blocked in the conversion of this precursor compound to the thiazole itself.

Amino Acid Sequence↗
PubMed1995

Roles of amino acid residues surrounding phosphorylation site 1 of branched-chain alpha-ketoacid dehydrogenase (BCKDH) in catalysis and phosphorylation site recognition by BCKDH kinase.

Branched-chain alpha-ketoacid dehydrogenase is regulated by reversible phosphorylation of serine 293 (site 1) on the E1 alpha subunit. Alanine-scanning mutagenesis was used to examine the roles of residues surrounding serine 293 in catalysis by the dehydrogenase and in substrate recognition by branched-chain alpha-ketoacid dehydrogenase kinase. Alanine substitution of serine 293 resulted in a 10-fold increased Km for alpha-ketoisovalerate, a less increased (2.8-fold) Km for alpha-ketoisocaproate, but no change in Vmax or the Km for thiamine pyrophosphate. Alanine substitutions of arginine 288, histidine 292, and aspartate 296, residues highly conserved among alpha-ketoacid dehydrogenases, resulted in inactive enzymes. Each of the inactive E1 mutants bound to the E2 core subunit with equal affinity as wild-type E1, and each produced circular dichroism spectra identical to that of wild-type E1. Two mutations, H292A and S293E, abolished the ability of E1 apoenzyme to reconstitute with thiamine pyrophosphate. Each alanine-substituted E1 was phosphorylated at site 1 by branched-chain alpha-ketoacid dehydrogenase kinase with similar rates, with the exception of the R288A mutant, which displayed no detectable phosphorylation. Thiamine pyrophosphate inhibited the phosphorylation of all mutant enzymes with the exception of H292A, the mutant E1 that did not bind thiamine pyrophosphate.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗