Uptake of the lipophilic cation dibenzyldimethylammonium into Saccharomyces cerevisiae. Interaction with the thiamine transport system

The distribution ratio of the lipophilic cation dibenzyldimethylammonium between the cells of Saccharomyces cerevisiae and the medium appears to reflect changes in the membrane potential in a way that is qualitatively correct: the addition of a proton conductor or of an agent which blocks metabolism causes an apparent depolarization of the cell membrane; monovalent cations cause also a lowering of the equilibrium distribution, whereas the addition of divalent cations results in an increase of the partition ratio.However, uptake of dibenzyldimethylammonium and probably also of other liophilic cations proceeds via the thiamine transport system of the yeast. Dibenzyldimethylammonium transport is inducible, like thiamine transport. A kinetic analysis of the mutual interaction between thiamine and dibenzyldimethylammonium uptake shows that these compounds share a common transport system; moreover, dibenzyldimethylammonium uptake is inhibited completely by thiamine disulfide, a competitive inhibitor of thiamine transport and dibenzyldimethylammonium uptake in a thiamine-transport mutant is reduced considerably.It is concluded that one should be cautious when using lipophilic cations to measure the membrane potential of cells of S. cerevisiae.     

Role of thiamine thiol form in nitric oxide metabolism

In alkaline media the thiamine cyclic form is converted into a thiol form (pK(a) 9.2) with an opened thiazole ring. The thiamine thiol form releases nitric oxide from S-nitrosoglutathione (GSNO). Thiamine disulfide, mixed thiamine disulfide with glutathione, and nitric oxide are produced in the reaction. Free glutathione was recorded in small amounts. The concentration of formed nitric oxide agreed well with the concentration of degraded GSNO. The concentration of released nitric oxide was determined under anaerobic conditions spectrophotometrically by production of nitrosohemoglobin. In air, the release of nitric oxide was recorded by the production of nitrite or the oxidation of oxyhemoglobin to methemoglobin. The concentration of the thiol form in the body under physiological pH values (7.2-7.4) did not exceed 1.5-2.0%. We believe that due to the exchange reactions between the thiamine thiol form and S-nitrosocysteine protein residues, nitric oxide can be released and mixed thiamine-protein disulfides are formed. The mixed thiamine disulfides (including thiamine ester disulfides) as well as the thiamine disulfide form are quite easily reduced by low molecular weight thiols to form the thiamine cyclic form with a closed thiazole ring. A possible role of the thiamine thiol form in releasing deposited nitric oxide from low-molecular-weight S-nitrosothiols and protein S-nitrosothiols and in regulation of blood flow in the vascular bed is discussed.     

Binding and transport of thiamine by Lactobacillus casei.

The relationship between thiamine transport and a membrane-associated thiamine-binding activity has been investigated in Lactobacillus casei. Thiamine transport proceeds via a system whose general properties are typical of active uptake processes; entry of the vitamin into the cells requires energy, is temperature dependent, exhibits saturation kinetics, and is inhibited by substrate analogs. A considerable concentration gradient of unchanged thiamine can be achieved by the system, although the vitamin is slowly metabolized to thiamine pyrophosphate. Consistent with these results, L. casei also contains a high-affinity, thiamine-binding component which could be measured by incubation of intact cells with labeled substrate at 4 degrees C (conditions under which transport is negligible). Binding was insensitive to iodoacetate, occurred at a level (0.5 nmol per 10(10) cells) nearly 20-fold higher than could be accounted for by facilitated diffusion, and was found to reside in a component of the cell membrane. Participation of this binder in thiamine transport is supported by the observations that the processes of binding and transport showed similarities in their (i) regulation by the concentration of thiamine in the growth medium, (ii) binding affinities for thiamine, and (iii) susceptibility to inhibition by thiamine analogs.     

Effect of tunicamycin on thiamine transport in Saccharomyces cerevisiae

The activity of thiamine transport in Saccharomyces cerevisiae was decreased by the treatment with tunicamycin without affecting the growth of yeast cells. Although the total activity of a soluble thiamine-binding protein in yeast periplasm, which is known to be a glycoprotein, was decreased by tunicamycin treatment, the activity of thiamine uptake by yeast protoplasts was inhibited as much as by whole cells. Furthermore, tunicamycin decreased the activity of the membrane-bound thiamine-binding protein in a dose dependent way and in parallel with the thiamine transport activity. These findings suggested that the membrane-bound thiamine-binding protein is a glycoprotein which plays a functional role in thiamine transport in S. cerevisiae.     

Reduced folate carrier transports thiamine monophosphate: an alternative route for thiamine delivery into mammalian cells

Although the reduced folate carrierRFC1 and the thiamine transporters THTR-1 and THTR-2 share ~40% oftheir identity in protein sequence, RFC1 does not transport thiamineand THTR-1 and THTR-2 do not transport folates. In the present study,we demonstrate that transport of thiamine monophosphate (TMP), animportant thiamine metabolite present in plasma and cerebrospinalfluid, is mediated by RFC1 in L1210 murine leukemia cells. Transport ofTMP was augmented by a factor of five in cells (R16) that overexpressRFC1 and was markedly inhibited by methotrexate, an RFC1 substrate, butnot by thiamine. At a near-physiological concentration (50 nM), TMP influx mediated by RFC1 in wild-type L1210 cells was ~50% ofthiamine influx mediated by thiamine transporter(s). Within 1 min, the majority of TMP transported into R16 cells was hydrolyzed to thiamine with a component metabolized to thiamine pyrophosphate, the active enzyme cofactor. These data suggest that RFC1 may be one of the alternative transport routes available for TMP in some tissues whenTHTR-1 is mutated in the autosomal recessive disorderthiamine-responsive megaloblastic anemia.

     

A constitutive thiamine metabolism mutation, thi80, causing reduced thiamine pyrophosphokinase activity in Saccharomyces cerevisiae.

We identified a strain carrying a recessive constitutive mutation (thi80-1) with an altered thiamine transport system, thiamine-repressible acid phosphatase, and several enzymes of thiamine synthesis from 2-methyl-4-amino-5-hydroxymethylpyrimidine and 4-methyl-5-beta-hydroxyethylthiazole. The mutant shows markedly reduced activity of thiamine pyrophosphokinase (EC 2.7.6.2) and high resistance to oxythiamine, a thiamine antagonist whose potency depends on thiamine pyrophosphokinase activity. The intracellular thiamine pyrophosphate content of the mutant cells grown with exogenous thiamine (2 x 10(-7) M) was found to be about half that of the wild-type strain under the same conditions. These results suggest that the utilization and synthesis of thiamine in Saccharomyces cerevisiae is controlled negatively by the intracellular thiamine pyrophosphate level.     

Molecular mechanisms of thiamine utilization

Thiamine is required for all tissues and is found in high concentrations in skeletal muscle, heart, liver, kidneys and brain. A state of severe depletion is seen in patients on a strict thiamine-deficient diet in 18 days, but the most common cause of thiamine deficiency in affluent countries is alcoholism. Thiamine diphosphate is the active form of thiamine, and it serves as a cofactor for several enzymes involved primarily in carbohydrate catabolism. The enzymes are important in the biosynthesis of a number of cell constituents, including neurotransmitters, and for the production of reducing equivalents used in oxidant stress defenses and in biosyntheses and for synthesis of pentoses used as nucleic acid precursors. Because of the latter fact, thiamine utilization is increased in tumor cells. Thiamine uptake by the small intestines and by cells within various organs is mediated by a saturable, high affinity transport system. Alcohol affects thiamine uptake and other aspects of thiamine utilization, and these effects may contribute to the prevalence of thiamine deficiency in alcoholics. The major manifestations of thiamine deficiency in humans involve the cardiovascular (wet beriberi) and nervous (dry beriberi, or neuropathy and/or Wernicke-Korsakoff syndrome) systems. A number of inborn errors of metabolism have been described in which clinical improvements can be documented following administration of pharmacological doses of thiamine, such as thiamine-responsive megaloblastic anemia. Substantial efforts are being made to understand the genetic and biochemical determinants of inter-individual differences in susceptibility to development of thiamine deficiency-related disorders and of the differential vulnerabilities of tissues and cell types to thiamine deficiency.     

Interaction of thiamine with rat brain synaptosomes

Thiamine has been shown to be bound specifically by a synaptosomal plasmatic membrane and transported inside to the nervous ending. Apparent K[symbol: see text] and Km for processes of binding and transport have been determined as equal 2.34 +/- 0.55 MKM and 3.92 +/- 1.3 MKM, respectively. The thiamine uptake by the isolated nervous endings (synaptosomes) at its physiological concentration is reduced in presence of metabolic inhibitors and partially depends on Mg2+ and Ca2+ ions, that can testify about the interrelation between endogenic thiamine phosphorilation and its transport through the membrane. Thiamine binding with synaptosomes is inhibited by ouabain and neurotoxins such as, latrotoxin and most significantly--with veratridin; tetrodotoxin fail to be efficient practically. In the conditions of synaptic membranes depolarisation their ability to bind thiamine is reduced and output of already uptaken with synaptosomes thiamine is observed.     

The folate and thiamine transport proteins of lactobacillus casei

Two separate binding proteins, one specific for folate and the other for thiamine, have been isolated from membrane fragments of Lactobacillus casei. Purification to homogeneity was achieved by fractionation of the Triton-solubilized proteins with microgranular silica (Quso G-32) and Sephadex G-150. Amino acid analyses revealed that the folate (M_r = 25,000) and thiamine (M_r = 29,000) binders have unusually low polarity constants, 0.32 and 0.26, respectively. Evidence obtained with intact cells has established a direct role for these binding proteins in transport of the corresponding vitamins: (A) In each case, the processes of binding and transport showed similarities in substrate affinities and repression by excess vitamin in the growth medium. (B) Competition studies employing amethopterin, 5-formyl tetrahydrofolate, and 5-methyl tetrahydrofolate (for folate) and thiamine monophosphate and thiamine pyrophosphate (for thiamine) have shown that the ability of these compounds to inhibit the transport of the corresponding vitamins is paralleled by their ability to inhibit binding. (C) Amethopterin-resistant mutants which are defective in folate transport have a comparable defect in ability to bind folate. (D) Amethopterin-resistant cells which (compared with the parent cell line) contain folate transport systems with altered affinities for amethopterin also contain binding proteins whose affinities for amethopterin have changed by equivalent amounts. (E) Both the transport and binding of folate by one of the mutants were stimulated (approximately 3-fold) in parallel by the addition of mercaptoethanol.     

Inactivation of the thiamine transport system in Saccharomyces cerevisiae with O-bromoacetylthiamine

We have synthesized and characterized O-bromoacetylthiamine (BrAcThiamine), a new reagent for inactivating the thiamine transport system in Saccharomyces cerevisiae. A Lineweaver-Burk plot of data from the transport kinetic measurements showed that BrAcThiamine was a competitive inhibitor of thiamine transport in S. cerevisiae with a Ki value of 0.60 microM. Incubating BrAcThiamine with yeast cells at 40 degrees C in 0.05 M potassium phosphate buffer, pH 5.0, caused concentration- and time-dependently a remarkable loss of thiamine transport activity. The inactivating reaction of yeast thiamine transport by BrAcThiamine proceeded most effectively at pH 5.0, coinciding with the optimal pH of the transport activity. Thiamine and thiamine analogs (pyrithiamine and O-acetylthiamine) protected yeast thiamine transport activity against the inactivation by BrAcThiamine. In addition, it was found that a membrane fraction prepared from yeast cells treated with BrAcThiamine had a thiamine-binding activity only 20% of that from control cells without inactivating the binding activity of the soluble fraction. These results suggest that BrAcThiamine inactivates the uptake activity by irreversible binding to the binding site of carrier protein(s) in the thiamine transport system.     

Some properties of the thiamine uptake system in isolated rat hepatocytes

A kinetic study of [14C]thiamine uptake over a concentration range from 0.1 microM to 4 mM was performed in isolated rat hepatocytes. The results showed that two processes contribute to the entry in rat hepatocytes: a low affinity process with a Kt of 34.1 microM and Vmax of 20.8 pmol/10(5) cells per 30 s and a high affinity process with a Kt of 1.26 microM and Vmax of 1.21 pmol/10(5) cells per 30 s. The uptake of thiamine by the high affinity process was concentrative and reduced in a betaine medium or K+ medium. Both ouabain and 2,4-dinitrophenol decreased the thiamine uptake by the high affinity process. These findings indicate that the transport of thiamine via a high affinity process is dependent on Na+ and biological energy. The uptake of thiamine was strongly inhibited by thiamine analogs such as dimethialium and chloroethylthiamine. Among quarternary ammonium compounds other than thiamine derivatives, choline and acetylcholine significantly inhibited thiamine uptake by rat liver cells, whereas betaine and carnitine did not. A kinetic study of thiamine uptake by rat hepatocytes preloaded with pyrithiamine, a potent inhibitor of thiamine pyrophosphokinase, revealed that the biphasic property of thiamine uptake disappeared and a single carrier system for thiamine with a Kt of 40.5 microM, which was similar to the Kt value of the low affinity process, was retained. These results strongly suggest that thiamine transport system in rat liver cells is closely connected with thiamine pyrophosphokinase, which accelerates the uptake rat of thiamine by pyrophosphorylation at physiological concentrations of thiamine.     

Reversibility of thiamine deficiency-induced partial necrosis and mitochondrial uncoupling by addition of thiamine to neuroblastoma cell suspensions

Culture of neuroblastoma cells in the presence of low thiamine concentration (6 nM) and of the transport inhibitor amprolium leads to the appearance of signs of necrosis: the chromatin condenses, the oxygen consumption decreases and is uncoupled, the mitochondrial cristae are disorganized, the thiamine diphosphate-dependent dehydrogenase activities are impaired. When 10 µM thiamine are added to these cells, the basal respiration increases, the coupled respiration is restored and mitochondrial morphology is recovered within 1 h. Addition of succinate, which is oxidized via a thiamine diphosphate-independent dehydrogenase, to digitonin-permeabilized cells immediately restores a coupled respiration. Our results suggest that the slowing of the citric acid cycle is the cause of the biochemical lesion induced by severe thiamine deficiency and that part of the mitochondria remain functional. (Mol Cell Biochem 174: 121–124, 1997)     

Cloning of the human thiamine transporter, a member of the folate transporter family.

We have isolated a cDNA from human placenta, which, when expressed heterologously in mammalian cells, mediates the transport of the water-soluble vitamin thiamine. The cDNA codes for a protein of 497 amino acids containing 12 putative transmembrane domains. Northern blot analysis indicates that this transporter is widely expressed in human tissues. When expressed in HeLa cells, the cDNA induces the transport of thiamine (K(t) = 2.5 +/- 0.6 microM) in a Na(+)-independent manner. The cDNA-mediated transport of thiamine is stimulated by an outwardly directed H(+) gradient. Substrate specificity assays indicate that the transporter is specific to thiamine. Even though thiamine is an organic cation, the cDNA-induced thiamine transport is not inhibited by other organic cations. Similarly, thiamine is not a substrate for the known members of mammalian organic cation transporter family. The thiamine transporter gene, located on human chromosome 1q24, consists of 6 exons and is most likely the gene defective in the metabolic disorder, thiamine-responsive megaloblastic anemia. At the level of amino acid sequence, the thiamine transporter is most closely related to the reduced-folate transporter and thus represents the second member of the folate transporter family.     

Oxidation of thiamine on reaction with nitrogen dioxide generated by ferric myoglobin and hemoglobin in the presence of nitrite and hydrogen peroxide

It is shown that nitrogen dioxide oxidizes thiamine to thiamine disulfide, thiochrome, and oxodihydrothiochrome (ODTch). The latter is formed during oxidation of thiochrome by nitrogen dioxide. Nitrogen dioxide was produced by incubation of nitrite with horse ferric myoglobin and human hemoglobin in the presence of hydrogen peroxide. After addition of tyrosine or phenol to aqueous solutions containing oxoferryl forms of the hemoproteins, thiamine, and nitrite, the yield of thiochrome greatly increased, whereas the yield of ODTch decreased. In the presence of high concentrations of tyrosine or phenol compounds ODTch was not formed at all. The neutral form of thiamine with the closed thiazole cycle and minor tricyclic form of thiamine do not enter the heme pocket of the protein and do not interact with the oxoferryl heme complex Fe(IV=O) or porphyrin radical. The tricyclic form of thiamine is oxidized to thiochrome by tyrosyl radicals located on the surface of the hemoprotein. The thiol form of thiamine is oxidized to thiamine disulfide by both hemoprotein tyrosyl radicals and oxoferryl heme complexes. Nitrite and also tyrosine, tyramine, and phenol readily penetrate into the heme pocket of the protein and reduce the oxyferryl complex to ferric cation. These reactions yield nitrogen dioxide as well as tyrosyl and phenoxyl radicals of tyrosine molecules and phenol compounds, respectively. Tyrosyl and phenoxyl radicals of low molecular weight compounds oxidize thiamine only to thiochrome and thiamine disulfide. The effect of oxoferryl forms of myoglobin and hemoglobin, nitrogen dioxide, and phenol on thiamine oxidative transformation as well as antioxidant properties of the hydrophobic thiamine metabolites thiochrome and ODTch are discussed.     

The effects of endurance training and thiamine supplementation on anti-fatigue during exercise

The purpose of this study was to find the effect of endurance training and thiamine supplementation on anti-fatigue during the exercise. Each nine students from K Women’s University went through three cross-over treatments: placebo treatment, training treatment and thiamine treatment. Training treatment was performed with bicycle ergometer exercise for four weeks (five days per week). Each exercise was performed for an hour with intensity set at 70% (50rpm) of maximal oxygen uptake. Thiamine treatment group was given 10mg of thiamine tetrahydrofurfuryl disulfide per one kilogram for four weeks. The bicycle ergometer exercise was performed at 70% of maximal oxygen uptake in exercise intensity which 60 minutes of exercise was performed at 50rpm . Lactate concentration was significantly decreased during 15 to 30 minutes of exercise for those with training treatment and 15 to 60 minutes of exercise for those with thiamine treatment compared to placebo treatment group. Ammonia concentration was significantly decreased during 15 to 60 minutes of exercise and 15 to 30 minutes of recovery for those with training and thiamine treatment compared to placebo treatment. Resting blood thiamine concentrations of placebo treatment were significantly lower than training treatment. 60 minutes after the exercise, plasma thiamine concentration was significantly increased in all treatment group. To sum up the previous, thiamine intake during exercise positively benefits carbohydrate metabolism in a way that will decrease lactate concentration, ammonia concentration, and anti- fatigue by reducing the RPE. Therefore, we can consider thiamine intake to be utilized as similar benefits as endurance training.     

New aspects in the study of the mechanism of intestinal absorption of thiamine in rats

Intestinal absorption of thiamine was studied in rats using such metabolic inhibitors as ouabain, sodium azide and theophylline. These substances changed certain biochemical parameters of the mucosa in the small intestine affecting the level of thiamine transport across the intestinal epithelium. It was found that the intestinal transport of thiamine at lower concentration was an active process depending on the activity of Na, K, Mg-ATPase in the intestinal microvilli, and on the activity of mitochondrial processes. The used metabolic inhibitors increased the intestinal diffusion of thiamine at its higher concentration suggesting that these inhibitors changed membrane permeability by affecting enterocyte homeostasis. At both studied concentrations thiamine was rapidly absorbed from the digestive tract reaching the state of saturation which suggested a carrier character of this transport.     

Photoinactivation of the thiamine transport system in saccharomyces cerevisiae with 4-azido-2-nitrobenzoylthiamine

A newly synthesized photoreactive thiamine derivative, 4-azido-2-nitrobenzoylthiamine was found to be a competitive inhibitor of the thiamine transport system in Saccharomyces cerevisiae, exhibiting an apparent $\text{K}{}_{\mathrm{i}}$ of 36 nM. When exposed to visible light, 4-azido-2-nitrobenzoylthiamine irreversibly inactivated the thiamine transport. 4-azido-2-nitrobenzoylthiamine-dependent photoinactivation of thiamine transport was partially protected by thiamine, but not by the nitrene-trapping reagent $\text{p-}\text{aminobenzoate}$. On the other hand, the irradiation of the yeast cells in the presence of 4-azido-2-nitrobenzoylthiamine did not significantly lead to inactivation of the biotin transport system. The results suggest that 4-azido-2-nitrobenzoylthiamine is a specific irreversible inhibitor of the thiamine transport system in Saccharomyces cerevisiae.     

Evaluation of blood-brain barrier thiamine efflux using the in situ rat brain perfusion method

Thiamine is an essential, positively charged (under physiologic conditions), water-soluble vitamin requiring transport into brain. Brain thiamine deficiency has been linked to neurodegenerative disease by subsequent impairment of thiamine-dependent enzymes used in brain glucose/energy metabolism. In this report, we evaluate brain uptake and efflux of [3H]thiamine using the in situ rat brain perfusion technique. To confirm brain distribution was not related to blood-brain barrier endothelial cell uptake, we compared parenchymal and cell distribution of [3H]thiamine using capillary depletion. Our work supports previous literature findings suggesting blood-brain barrier thiamine uptake is via a carrier-mediated transport mechanism, yet extends the literature by redefining the kinetics with more sensitive methodology. Significantly, [3H]thiamine brain accumulation was influenced by a considerable efflux rate. Evaluation of the efflux mechanism demonstrated increased stimulation by the presence of increased vascular thiamine. The influx transport mechanism and efflux rate were each comparable throughout brain regions despite documented differences in glucose and thiamine metabolism. The observation that [3H]thiamine blood-brain barrier influx and efflux is regionally homogenous may have significant relevance to neurodegenerative disease linked to thiamine deficiency.     

Thiamine deficiency in rats affects thiamine metabolism possibly through the formation of oxidized thiamine pyrophosphate

BackgroundThiamine deficiency (TD) has a number of features in common with the neurodegenerative diseases development and close relationship between TD and oxidative stress (OS) has been repeatedly reported in the literature. The aim of this study is to understand how alimentary TD, accompanied by OS, affects the expression and level of two thiamine metabolism proteins in rat brain, namely, thiamine transporter 1 (THTR1) and thiamine pyrophosphokinase (TPK1), and what factors are responsible for the observed changes.MethodsThe effects of OS caused by TD on the THTR1and TPK1 expression in rat cortex, cerebellum and hippocampus were examined. The levels of active and oxidized forms of ThDP (enzymatically measured) in the blood and brain, ROS and SH-groups in the brain were also analyzed.ResultsTD increased the expression of THTR1 and protein level in all studied regions. In contrast, expression of TPK1 was depressed. TD-induced OS led to the accumulation of ThDP oxidized inactive form (ThDP_ox) in the blood and brain. In vitro reduction of ThDP_ox by dithiothreitol regenerates active ThDP suggesting that ThDP_ox is in disulfide form. A single high-dose thiamine administration to TD animals had no effect on THTR1 expression, partly raised TPK1 mRNA and protein levels, but is unable to normalize TPK1 enzyme activity. Brain and blood ThDP levels were increased in these conditions, but ThDP_ox was not decreased.General significanceIt is likely, that the accumulation of ThDP_ox in tissue could be seen as a potential marker of neurocellular dysfunction and thiamine metabolic state.     

A positive regulatory gene, THI3, is required for thiamine metabolism in Saccharomyces cerevisiae.

We have isolated a thiamine auxotrophic mutant carrying a recessive mutation which lacks the positive regulatory gene, THI3, which differs in the regulation of thiamine transport from the THI2 (PHO6) gene described previously (Y. Kawasaki, K. Nosaka, Y. Kaneko, H. Nishimura, and A. Iwashima, J. Bacteriol. 172:6145-6147, 1990) for expression of thiamine metabolism in Saccharomyces cerevisiae. The mutant (thi3) had a markedly reduced thiamine transport system as well as reduced activity of thiamine-repressible acid phosphatase and of several enzymes for thiamine synthesis from 2-methyl-4-amino-5-hydroxymethylpyrimidine and 4-methyl-5-beta-hydroxyethylthiazole. These results suggest that thiamine metabolism in S. cerevisiae is subject to two positive regulatory genes, THI2 (PHO6) and THI3. We have also isolated a hybrid plasmid, pTTR1, containing a 6.2-kb DNA fragment from an S. cerevisiae genomic library which complements thiamine auxotrophy in the thi3 mutant. This gene was localized on a 3.0-kb ClaI-BglII fragment in the subclone pTTR5. Complementation of the activities for thiamine metabolism in the thi3 mutant transformed by some plasmids with the THI3 gene was also examined.