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PubMed1980

Thiamin Phosphorylation by Thiamin Pyrophosphotransferase during Seed Germination.

Thiamin pyrophosphotransferase activity was present in seedling extracts from several monocot and dicot species of agronomic as well as noncultivated plants. Changes in thiamin pyrophosphotransferase activity and thiamin pyrophosphate content were followed for 6 days in soybean (Merr.) seedlings. Maximum enzyme activity occurred 48 to 96 hours from imbibition. Thiamin pyrophosphate content peaked sharply at 36 hours and was preceded by increased thiamin pyrophosphotransferase activity. Addition of pyrithiamin, an inhibitor of in vitro thiamin pyrophosphotransferase activity, to the imbibition medium at various times inhibited subsequent fresh weight gains of soybean seedlings. These results indicated that, although not among the earliest phosphorylation events after initiation of water imbibition by soybean seeds, a substantial increase in thiamin pyrophosphate content did precede the onset of rapid seedling growth and development. Since both enzyme activity and thiamin appear to be available in unimbibed soybean seeds, ATP or other nucleoside triphosphate concentration may represent an important factor in modulating thiamin phosphorylation during early seedling development.

Journal Article↗
PubMed1983

Transketolase, pyruvate and oxoglutarate dehydrogenase activities and [14C]thiamin turnover in tissues of mice fed thiamin-deficient diet.

A study was made of turnover of [14C]thiamin (5 or 2 micrograms/mouse) in mice fed a thiamin-deficient diet. Simultaneously the activities of the thiamin-dependent enzymes (transketolase, pyruvate and oxoglutarate dehydrogenases) were measured as an index of efficiency of fulfilling the coenzyme function of the vitamin under conditions of different thiamin status. After [14C]thiamin injections of 5 micrograms/mouse, kidney, spleen, stomach and pancreas tissue stores turned over completely on day 9, whereas by day 13 this process had not yet been finished in liver, heart and brain. On administration of 2 micrograms [14C]thiamin/mouse, turnover of the tissue stores proceeded at a slower rate. The tissue transketolase activity decreased after the 2-microgram injections as compared to that in the mice administered 5-microgram injections. With 2 micrograms of [14C]thiamin, the pyruvate dehydrogenase activity lowered gradually in all the tissues studied, whereas the oxoglutarate dehydrogenase decreased in liver and kidneys. The pattern of the depression of the thiamin-dependent enzyme activities after the 2-microgram [14C]thiamin injections suggests a regularity in the vitamin redistribution in different organs and subcellular fractions.

Animals↗
PubMed1996

Thiamine pyrophosphate-requiring enzymes are altered during pyrithiamine-induced thiamine deficiency in cultured human lymphoblasts.

The most common of the severe complications of thiamine deficiency are beriberi and Wernicke-Korsakoff syndrome. To help clarify the biochemical basis for these disorders, a cell culture system has been established in which pyrithiamine, a potent thiamine transport inhibitor, was used to mimic different degrees of thiamine deficiency within human lymphoblasts. Activities of both transketolase and alpha-ketoglutarate dehydrogenase (alpha-KGDH) decreased at the same rate and to roughly the same levels in response to thiamine deficiency within a given cell line. However, variation in sensitivity to thiamine deficiency, as judged by the relative percentage of loss of enzymatic activities, was found when different cell lines were compared. When exogenous thiamine pyrophosphate was added to the activity assays, differences between transketolase and alpha-KGDH became readily apparent. Only 25% of the lost transketolase activity was present as apo-enzyme, whereas 70% of the lost alpha-KGDH activity was present in the apo-enzyme form. For transketolase, the non-recoverable activity was due mainly to a decrease in the synthesis rate of the protein during thiamine deficiency, suggesting that thiamine has a direct effect on the expression of the transketolase gene and/or protein.

Cell Culture Techniques↗
PubMed1988

The evaluation of erythrocyte thiamin diphosphate as an indicator of thiamin status in man, and its comparison with erythrocyte transketolase activity measurements.

HPLC measurement of the major physiological form of thiamin, thiamin diphosphate, in erythrocytes (ETDP) has been assessed as an indicator of thiamin status and compared with erythrocyte transketolase activity (ETKA) before and after activation with exogenous thiamin diphosphate (TDP effect). The comparison was made by following the response of the parameters to incubation of erythrocytes with thiamin and by measurements in alcoholic, elderly and student groups. All parameters were responsive to the thiamin content of their environment, but ETDP was at least as sensitive as TDP effect and more sensitive than ETKA in reflecting thiamin uptake and utilisation by erythrocytes. There was a better correlation between ETDP and ETKA than between ETDP and TDP effect. All the groups showed a significant positive correlation between ETDP and ETKA, but only the elderly and the alcoholic groups showed a significant negative correlation between ETDP and TDP effect. The lack of correlation between ETDP and TDP effect in the student group may be due to the relative imprecision of TDP effect measurements at the lower end of the reference range. ETDP is more stable than ETKA in frozen erythrocytes, is easier to standardise and is not affected by various factors which can influence the enzyme assay. Measurement of ETDP is, therefore, recommended for first line assessment of thiamin status.

Adult↗
PubMed1996

Mechanism of ligand-protein interaction in plant seed thiamine-binding proteins. Preliminary chemical identification of amino acid residues essential for thiamine binding to the buckwheat-seed protein.

Thiamine-binding protein, isolated from buckwheat seeds, was chemically modified in an attempt to identify amino acid residues involved in protein-thiamine interaction. No evidence was found in support of specific roles of arginine residues, sulfhydryl groups, amino groups and tyrosine residues. Under carefully controlled reaction conditions (Tris pH 5-6), the modification with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide caused a complete loss of thiamine-binding capacity. Thus, the carboxyl groups seemed to be essential for binding, possibly for ionic interaction with protein-bound thiamine cation. A selective modification of histidine residues using diethylpyrocarbonate correlated with a loss of thiamine-binding capacity; the modification and the loss of binding capacity could be reversed with hydroxylamine; some ligand-protection against modification was observed. From Tsou analysis of diethylpyrocarbonate modification and resulting loss of thiamine-binding it was suggested that 1-2 of 20 histidine residues of the protein were essential for thiamine binding. The essential histidine(s) might be present in the binding site and possibly were involved in hydrogen bonding(s) with protein-bound thiamine molecule.

Binding Sites↗
PubMed2000

Fast, isotope-free methods for the assay of thiamine-binding proteins and for the determination of their affinities to thiamine-related compounds.

A fast, isotope-free method for the determination of parameters for the interactions of proteins with thiamine and related compounds was developed. The free and bound forms of a ligand (thiamine or a fluorogenic analogue) were separated by ultrafiltration using commercially available centrifugal protein microconcentrators (Nanosep, Pall Filtron). The free thiamine concentration in the filtrate was analysed by (i) a pre-column derivatisation of thiamine to thiochrome with the use of alkaline potassium hexacyanoferrate(III) followed by reverse-phase HPLC (isocratic, analytical ODS column, 10 mM potassium phosphate, pH 7.8, 5% tetrahydrofuran) with fluorometric detection (excitation at 365 nm, emission at 430 nm), or (ii) an ion-pair reverse-phase HPLC (isocratic, ODS column, 0.08% trifluoroacetic acid-0.08% sodium octanesulfonate-25% tetrahydrofuran) with post-column derivatisation and fluorometric detection. The 'saturation-binding' version (single ligand added in increasing doses to the protein samples) of this method allowed the determination of low micromolar concentrations of thiamine-binding proteins and of the dissociation constants of their complexes with thiamine or fluorogenic thiamine analogues in the range of 0.3-10 microM. Using the other, 'competitive displacement' version (constant amount of thiamine plus increasing doses of a competing ligand), dissociation constants at least one order of magnitude higher could successfully be determined.

Biochemistry↗
PubMedDate not supplied

Male infertility and thiamine-dependent erythroid hypoplasia in mice lacking thiamine transporter Slc19a2.

Thiamine-responsive megaloblastic anemia with diabetes and deafness (TRMA) is an autosomal recessive disease caused by mutations in the high-affinity thiamine transporter gene SLC19A2. To study the role of thiamine transport in the pathophysiology of TRMA syndrome and of each of the component disorders, we created a targeted disruption of the Slc19a2 gene in mice. Slc19a2 -/- mice are viable and females are fertile. Male -/- mice on a pure 129/Sv background are infertile with small testes (testis/body weight=0.13 +/- 0.04 knockout vs. 0.35 +/- 0.05 wild type, P<0.000005). The lack of developing germ cells beyond primary spermatocytes suggests an arrest in spermatogenesis prior to meiosis II. Nuclear chromatin changes indicative of apoptosis are present. No mature sperm are found in the tubules or epididymis. This phenotype suggests a previously unknown role for thiamine transport in spermatogenesis and male fertility. Slc19a2 -/- mice on a pure 129/Sv background develop reticulocytopenia after two weeks on thiamine-depleted chow with a virtual absence of reticulocytes in the peripheral blood (0.12% knockout vs. 2.58% wild type, P=0.0079). Few erythroid precursors are found in the bone marrow. Contrary to human TRMA syndrome, we see no evidence of megaloblastosis or ringed sideroblasts in the bone marrow of Slc19a2 -/- mice in thiamine-replete or thiamine-deficient dietary states. Phenotypic differences between TRMA patients and Slc19a2 -/- mice might be explained by dissimilar tissue expression patterns of the transporter, as well as by differing metabolic needs and possible different species-specific contributions of the related thiamine transporter Slc19a3.

Anemia, Megaloblastic↗
PubMed1985

Comparison of the effects of some thiamine analogues upon thiamine transport across the blood-brain barrier of the rat.

The flux of thiamine from the blood into the brain has been measured by a specially devised procedure in which a steady raised level of the vitamin, with or without radioactive labelling, was achieved rapidly and maintained steadily in the circulating blood plasma. This was done by a single rapid I.V. injection followed by a continuous injection given at a rate adjusted according to a pre-determined programme, so as to replace the injected material at the rate at which it had been found to leave the circulation in preliminary experiments. A series of four chemical analogues of thiamine were given to see how each affected the flux of thiamine into the brain and the findings are compared with those for a fifth analogue studied in previous work. Pyrithiamine, thiamine disulphide and acetylthiamine, like amprolium, inhibited thiamine transport across the blood-brain barrier. Kinetic analysis shows that they compete mainly for the saturable component of thiamine flux across the blood-brain barrier, with only a slight inhibition of the non-saturable component, most clearly seen with pyrithiamine. Oxythiamine, despite its close chemical similarity to thiamine did not have any significant effect upon the flux of the vitamin into the brain. These findings help to explain the efficacy of pyrithiamine administration, especially in conjunction with a thiamine-deficient diet, in rapidly producing central neurological signs of deficiency.

Amprolium↗
PubMed2004

Thiamine transporter gene expression and exogenous thiamine modulate the expression of genes involved in drug and prostaglandin metabolism in breast cancer cells.

In previous studies, we have shown that RNA levels of the thiamine transporter THTR2 were down-regulated in breast cancer tumors in comparison with normal tissues and that THTR2-mediated increases in thiamine uptake activity contributed to increased apoptosis after exposure to ionizing radiation. To further understand the biological effects of the alteration of THTR2 expression, we conducted a DNA microarray study of gene expression in THTR2-transfected breast cancer cells and found that, in addition to increased expression of THTR2 attributable to the transgene, three other genes were up-regulated >2.5-fold in the transfected cells: cytochrome P450 isoform CYP4B1, 15-hydroxyprostaglandin dehydrogenase (15-PGDH), and transcription factor CRIP1. In addition, two genes were confirmed to be down-regulated in THTR2-transfected cells: trefoil factor 1 (TFF1) and Rho-GDP dissociation inhibitor (RGDI). Up-regulation of 15-PGDH and CYP4B1 expression was observed in other breast cancer cell lines transfected with THTR2, and down-regulation was observed after suppression of THTR2 with siRNA vectors. To determine the role of exogenous thiamine in the expression of these genes, we analyzed THTR2-transfected breast cancer cells grown in thiamine-depleted medium by quantitative reverse transcription-PCR and showed that three of these five genes showed evidence of regulation by exogenous thiamine in a manner concordant with the effects of THTR2 overexpression. One of the genes up-regulated by THTR2 transfection was down-regulated by thiamine depletion (CYP4B1), and two genes with decreased expression in THTR2-transfected breast cancer cells were up-regulated by thiamine depletion (TFF1 and RGDI). In summary, these studies show unexpected relationships between thiamine metabolism and genes that may be involved in the oncogenesis of breast and lung cancer.

Blotting, Western↗
PubMed2006

Thiamine pyrophosphate: an essential cofactor for the alpha-oxidation in mammals--implications for thiamine deficiencies?

The identification of 2-hydroxyphytanoyl-CoA lyase (2-HPCL), a thiamine pyrophosphate (TPP)-dependent peroxisomal enzyme involved in the alpha-oxidation of phytanic acid and of 2-hydroxy straight chain fatty acids, pointed towards a role of TPP in these processes. Until then, TPP had not been implicated in mammalian peroxisomal metabolism. The effect of thiamine deficiency on 2-HPCL and alpha-oxidation has not been studied, nor have possible adverse effects of deficient alpha-oxidation been considered in the pathogenesis of diseases associated with thiamine shortage, such as thiamine-responsive megaloblastic anemia (TRMA). Experiments with cultured cells and animal models showed that alpha-oxidation is controlled by the thiamine status of the cell/tissue/organism, and suggested that some pathological consequences of thiamine starvation could be related to impaired alpha-oxidation. Whereas accumulation of phytanic acid and/or 2-hydroxyfatty acids or their alpha-oxidation intermediates in TRMA patients given a normal supply of thiamine is unlikely, this may not be true when malnourished.

Anemia, Megaloblastic↗
PubMed1995

The thiamine-dependent hysteretic behavior of human transketolase: implications for thiamine deficiency.

We have investigated the hysteretic properties of human transketolase with emphasis on its dependency on thiamine pyrophosphate concentration. As demonstrated previously, the reaction progress curves revealed a slow transition from an initial low velocity to a faster final steady-state velocity, characterized by the rate constant tau-1. The rate of the transition was dependent on the concentration of the thiamine pyrophosphate cofactor, with progressively longer transition times found as the concentration of thiamine pyrophosphate was decreased. At physiological thiamine pyrophosphate concentrations, the inverse rate constant was in the range of 10 to 20 min for fibroblast-derived transketolase and increased dramatically with only small decreases from these levels of thiamine pyrophosphate. Variation in the lag was found when transketolase from different individuals was examined. Moreover, at low levels of thiamine, the rate of the transition was different between fibroblast- and lymphoblast-derived transketolase. The substantial lag in formation of active holoenzyme and the findings of interindividual variation and cell type variation in the lag period suggest mechanisms for the loss of transketolase activity during thiamine deficiency and may explain, at least in part, the differential sensitivity to deficiency demonstrated by tissues and individuals.

Cells, Cultured↗