RESUMO
Thiamine triphosphate (ThTP) is found in small amounts in most organisms from bacteria to mammals, but little is known about its physiological role. In vertebrate tissues, ThTP may act as a phosphate donor for the phosphorylation of certain proteins; this may be part of a new signal transduction pathway. We have recently characterized a highly specific 25-kDa thiamine triphosphatase (ThTPase) that is expressed in most mammalian tissues. The role of this enzyme may be the control of intracellular concentrations of ThTP. As the latter has been considered to be a neuroactive form of thiamine, we have studied the distribution of ThTPase mRNA and protein in rodent brain using in situ hybridization and immunohistochemistry. With both methods, we found the strongest staining in hippocampal pyramidal neurons, as well as cerebellar granule cells and Purkinje cells. Some interneurons were also labeled and many ThTPase mRNA-positive and immunoreactive cells were distributed throughout cerebral cortical gray matter and the thalamus. White matter was not significantly labeled. ThTPase immunoreactivity seems to be located mainly in the cytoplasm of neuronal perikarya. Immunocytochemical data using dissociated cultured cells from hippocampal and cerebellum showed that the staining was more intense in neurons than in astrocytes. The protein was rather uniformly located in the perikarya and dendrites, suggesting that ThTP and ThTPase may play a general role in neuronal metabolism rather than a specific role in excitability. There was no apparent correlation between ThTPase expression and selective vulnerability of certain brain regions to thiamine deficiency.
Assuntos
Encéfalo/enzimologia , Neurônios/enzimologia , Tiamina Trifosfatase/metabolismo , Animais , Humanos , Imuno-Histoquímica , Hibridização In Situ , Masculino , Camundongos , RNA Mensageiro/análise , Ratos , Ratos Wistar , Proteínas Recombinantes/metabolismoRESUMO
In most organisms, the main form of thiamine is the coenzyme thiamine diphosphate. Thiamine triphosphate (ThTP) is also found in low amounts in most vertebrate tissues and can phosphorylate certain proteins. Here we show that ThTP exists not only in vertebrates but is present in bacteria, fungi, plants and invertebrates. Unexpectedly, we found that in Escherichia coli as well as in Arabidopsis thaliana, ThTP was synthesized only under particular circumstances such as hypoxia (E. coli) or withering (A. thaliana). In mammalian tissues, ThTP concentrations are regulated by a specific thiamine triphosphatase that we have recently characterized. This enzyme was found only in mammals. In other organisms, ThTP can be hydrolyzed by unspecific phosphohydrolases. The occurrence of ThTP from prokaryotes to mammals suggests that it may have a basic role in cell metabolism or cell signaling. A decreased content may contribute to the symptoms observed during thiamine deficiency.
Assuntos
Bactérias/metabolismo , Tiamina Trifosfatase/metabolismo , Tiamina Trifosfato/metabolismo , Sequência de Aminoácidos , Animais , Bactérias/enzimologia , Encéfalo/enzimologia , Bovinos , Fungos/enzimologia , Fungos/metabolismo , Humanos , Invertebrados , Masculino , Mamíferos , Dados de Sequência Molecular , Plantas/enzimologia , Plantas/metabolismo , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Suínos , Tiamina Trifosfatase/químicaRESUMO
We compared the thiamine and thiamine phosphate contents in the frontal, temporal, parietal, and occipital cortex of six patients with frontal lobe degeneration of the non-Alzheimer's type (FNAD) or frontotemporal dementia with five age-, postmortem delay-, and agonal status-matched control subjects. Our results reveal a 40-50% decrease in thiamine diphosphate (TDP) in the cortex of FNAD patients, whereas thiamine monophosphate was increased 49-119%. TDP synthesizing and hydrolyzing enzymes were unaffected. The activity of citrate synthase, a mitochondrial marker enzyme, was decreased in the frontal cortex of patients with FNAD, but no correlation with TDP content was found. These results suggest that decreased contents of TDP, which is essentially mitochondrial, is a specific feature of FNAD. As TDP is an essential cofactor for oxidative metabolism and neurotransmitter synthesis, and because low thiamine status (compared with other species) is a constant feature in humans, a nearly 50% decrease in cortical TDP content may contribute significantly to the clinical symptoms observed in FNAD. This study also provides a basis for a trial of thiamine, to improve the cognitive status of the patients.
Assuntos
Córtex Cerebral/química , Córtex Cerebral/patologia , Lobo Frontal/patologia , Degeneração Neural/patologia , Tiamina Pirofosfato/análise , Adulto , Idade de Início , Idoso , Análise de Variância , Autopsia , Citrato (si)-Sintase/análise , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valores de Referência , Tiamina/análise , Tiamina Monofosfato/análiseRESUMO
We investigated the influence of the sleep/waking cycle, the effects of paradoxical sleep deprivation (PSD) and of the vigilance-promoting drug modafinil on the amino acid contents of rat brain cortex. No significant nycthemeral variations in amino acid levels could be detected. PSD (12-24 hours), using the water tank method, significantly increased the levels of glutamate and glutamine. The increase was still observed after the sleep rebound period. gamma-Aminobutyric acid (GABA) levels did not change significantly during the instrumental sleep deprivation but increased during the rebound period. Control experiments indicate that the increase in glutamate and glutamine levels is due to PSD rather than to the stress associated with the experimental procedure. The increase in glutamate content cannot arise only from transamination reactions, because the levels of other amino acids (such as aspartate) did not decrease. Modafinil treatment did not significantly modify the brain cortex content of any of the amino acids tested.
Assuntos
Córtex Cerebral/química , Glutamatos/análise , Glutamina/análise , Privação do Sono , Animais , Nível de Alerta/efeitos dos fármacos , Compostos Benzidrílicos/farmacologia , Estimulantes do Sistema Nervoso Central/farmacologia , Córtex Cerebral/efeitos dos fármacos , Masculino , Modafinila , Ratos , Vigília , Ácido gama-Aminobutírico/análiseRESUMO
GABAA receptors were characterized in cellular fractions isolated from adult bovine brain. The fraction enriched in cortical astrocytes is very rich in high-affinity binding sites for [3H]flunitrazepam and other "central-type" benzodiazepine ligands. The amount of specific [3H]flunitrazepam binding was more than five times higher in the glial fraction than in synaptosomal and perikaryal fractions. [3H]Flunitrazepam was displaced by low concentrations of clonazepam and other specific ligands for central GABAA receptors. Specific binding sites for GABA, flunitrazepam, barbiturates, and picrotoxin-like convulsants were characterized. Allosteric interactions between the different sites were typical of central-type GABAA receptors. The presence of alpha-subunit(s), as revealed by [3H]flunitrazepam photoaffinity labeling, was demonstrated in all brain fractions at molecular mas 51-53 kDa. Photoaffinity labeling was highest in the glial fraction. However, in primary cultured astrocytes from neonate rat cortex, no photoaffinity labeling was detected. Information obtained from astrocytes in culture should thus be taken with caution when extrapolated to differentiated astroglial cells. Our results actually show that, in mature brain, most of the fully pharmacologically active GABAA receptors are extrasynaptic and expressed in astroglia.
Assuntos
Envelhecimento/metabolismo , Astrócitos/metabolismo , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Receptores de GABA-A/metabolismo , Animais , Benzodiazepinas/metabolismo , Sítios de Ligação , Encéfalo/citologia , Bovinos , Células Cultivadas , Picrotoxina/metabolismo , Ratos , Ratos Wistar , Ácido gama-Aminobutírico/metabolismoRESUMO
Culture of neuroblastoma cells in a medium of low-thiamine concentration (6 nM) and in the presence of the transport inhibitor amprolium leads to the appearance of overt signs of necrosis; i.e., the chromatin condenses in dark patches, the oxygen consumption decreases, mitochondria are uncoupled, and their cristae are disorganized. Glutamate formed from glutamine is no longer oxidized and accumulates, suggesting that the thiamine diphosphate-dependent alpha-ketoglutarate dehydrogenase activity is impaired. When thiamine (10 microM) is added to the cells, the O2 consumption increases, respiratory control is restored, and normal cell and mitochondrial morphology is recovered within 1 h. Succinate, which is oxidized via the thiamine diphosphate-independent succinate dehydrogenase, is also able to restore a normal O2 consumption (with respiratory control) in digitonin-permeabilized thiamine-deficient cells. Our results therefore suggest that the slowing of the citric acid cycle is the main cause of the biochemical lesion induced by thiamine deficiency as observed in Wernicke's encephalopathy.
Assuntos
Mitocôndrias/fisiologia , Neuroblastoma/patologia , Neuroblastoma/fisiopatologia , Deficiência de Tiamina/patologia , Deficiência de Tiamina/fisiopatologia , Tiamina/farmacologia , Amprólio/farmacologia , Microscopia Eletrônica , Mitocôndrias/ultraestrutura , Necrose , Células Tumorais CultivadasRESUMO
When neuroblastoma cells were transferred to a medium of low (6 nM) thiamine concentration, a 16-fold decrease in total intracellular thiamine content occurred within 8 days. Respiration and ATP levels were only slightly affected, but addition of a thiamine transport inhibitor (amprolium) decreased ATP content and increased lactate production. Oxygen consumption became low and insensitive to oligomycin and uncouplers. At least 25% of mitochondria were swollen and electron translucent. Cell mortality increased to 75% within 5 days. [3H]PK 11195, a specific ligand of peripheral benzodiazepine receptors (located in the outer mitochondrial membrane) binds to the cells with high affinity (KD = 1.4 +/- 0.2 nM). Thiamine deficiency leads to an increase in both Bmax and KD. Changes in binding parameters for peripheral benzodiazepine receptors may be related to structural or permeability changes in mitochondrial outer membranes. In addition to the high-affinity (nanomolar range) binding site for peripheral benzodiazepine ligands, there is a low-affinity (micromolar range) saturable binding for PK 11195. At micromolar concentrations, peripheral benzodiazepines inhibit thiamine uptake by the cells. Altogether, our results suggest that impairment of oxidative metabolism, followed by mitochondrial swelling and disorganization of cristae, is the main cause of cell mortality in severely thiamine-deficient neuroblastoma cells.
Assuntos
Mitocôndrias/fisiologia , Neuroblastoma/metabolismo , Receptores de GABA-A/metabolismo , Tiamina/administração & dosagem , Trifosfato de Adenosina/metabolismo , Amprólio/farmacologia , Animais , Morte Celular , Meios de Cultura , Isoquinolinas/metabolismo , Lactatos/biossíntese , Ácido Láctico , Camundongos , Microscopia Eletrônica , Dilatação Mitocondrial , Neuroblastoma/ultraestrutura , Consumo de Oxigênio , Tiamina/metabolismo , Células Tumorais CultivadasRESUMO
At pH 7.4, 36Cl- uptake by neuroblastoma cells was Na(+)-independent, saturable and blocked by submicromolar concentrations of DIDS. This suggests that at this pH, Cl- transport is mediated by an exchanger analogous to erythroid band 3. At pH 6.2, 36Cl- uptake was markedly activated by external carboxylate anions such as acetate. Acetate-stimulated 36Cl- uptake was blocked by DIDS (IC50 = 0.15 microM). Saturation by external 36Cl- was observed with K0.5 = 8 mM. K0.5 was not modified by acetate. As 36Cl efflux is also activated by acetate, we suggest the presence, in neuroblastoma cells, of an anion exchanger activated by carboxylic anions. This exchanger is active when the extracellular pH is 6.0-6.5.
Assuntos
Antiporters/metabolismo , Cloretos/metabolismo , Neuroblastoma/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Animais , Antiporters/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Concentração de Íons de Hidrogênio , Cinética , Camundongos , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Incubation of rat brain homogenates with thiamine or thiamine diphosphate (TDP) leads to a synthesis of thiamine triphosphate (TTP). In membrane vesicles subsequently prepared from the homogenates, increased TTP content correlates with increased 36Cl- uptake. A hyperbolic relationship was obtained with a K0.5 of 0.27 nmol TTP/mg protein. In crude mitochondrial fractions from the brains of animals previously treated with thiamine or sulbutiamine, a positive correlation between 36Cl- uptake and TTP content was found. These results, together with other results previously obtained with the patch-clamp technique, suggest that TTP is an activator of chloride channels having a large unit conductance.
Assuntos
Química Encefálica/fisiologia , Canais de Cloreto/metabolismo , Tiamina Trifosfato/metabolismo , Animais , Permeabilidade da Membrana Celular/fisiologia , Cloro , Cromatografia Líquida de Alta Pressão , Técnicas In Vitro , Fosforilação , Psicotrópicos/farmacologia , Radioisótopos , Ratos , Tiamina/análogos & derivados , Tiamina/farmacologiaRESUMO
Nerve cells are particularly sensitive to thiamine deficiency. We studied thiamine transport in mouse neuroblastoma (Neuro 2a) cells. At low external concentration, [14C]thiamine was taken up through a saturable high affinity mechanism (Km = 35 nM). This was blocked by low concentrations of the Na+ channel activators veratridine (IC50 = 7 +/- 4 microM) and batrachotoxin (IC50 = 0.9 microM). These effects were not antagonized by tetrodotoxin and were also observed in cell lines devoid of Na+ channels, suggesting that these channels are not involved in the mechanism of inhibition. At high extracellular concentrations, thiamine uptake proceeds essentially via a low affinity carrier (Km = 0.8 mM), insensitive to veratridine but blocked by divalent cations. In both cases, the uptake was independent on external sodium, partially inhibited (10-35%) by depolarization and sensitive to metabolic inhibitors. A linear relationship between the rate of thiamine transport and intracellular ATP concentration was found. When cells grown in a medium of low thiamine concentration (6 nM) were exposed to 100 nM extracellular thiamine, a 3-fold increase in intracellular thiamine diphosphate was observed after 2 h while the concomitant increase in intracellular free thiamine was barely significant. These data suggest a secondary active transport of thiamine, the main driving force being thiamine phosphorylation rather than the sodium gradient.
Assuntos
Trifosfato de Adenosina/metabolismo , Batraquiotoxinas/farmacologia , Potenciais da Membrana/fisiologia , Neuroblastoma/metabolismo , Canais de Sódio/fisiologia , Tiamina/metabolismo , Veratridina/farmacologia , Aconitina/farmacologia , Amilorida/farmacologia , Animais , Ligação Competitiva , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Cinética , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Canais de Sódio/efeitos dos fármacos , Tetrodotoxina/farmacologia , Tiamina/análogos & derivados , Tiamina/farmacologia , Células Tumorais CultivadasRESUMO
The subcellular distribution of thiamine derivatives in rat brain was studied. Thiamine diphosphate content was highest in the mitochondrial and synaptosomal fractions, and lowest in microsomal, myelin and cytosolic fractions. Only 3-5% of total thiamine diphosphate was bound to transketolase, a cytosolic enzyme. Thiamine triphosphate was barely detectable in the microsomal and cytosolic fraction, but synaptosomes were slightly enriched in this compound compared to the crude homogenate. Both myelin and mitochondrial fractions contained significant amounts of thiamine triphosphate. In order to estimate the relative turnover rates of these compounds, the animals received an intraperitoneal injection of either [14C]thiamine or [14C]sulbutiamine (isobutyrylthiamine disulfide) 1 h before decapitation. The specific radioactivities of thiamine compounds found in the brain decreased in the order: thiamine > thiamine triphosphate > thiamine monophosphate > thiamine diphosphate. Incorporation of radioactivity into thiamine triphosphate was more marked with [14C]sulbutiamine than with [14C]thiamine. The highest specific radioactivity of thiamine diphosphate was found in the cytosolic fraction of the brain, though this pool represents less than 10% of total thiamine diphosphate. Cytosolic thiamine diphosphate had a twice higher specific radioactivity when [14C]sulbutiamine was used as precursor compared with thiamine though no significant differences were found in the other cellular compartments. Our results suggest the existence of two thiamine diphosphate pools: the bound cofactor pool is essentially mitochondrial and has a low turnover; a much smaller cytosolic pool (6-7% of total TDP) of high turnover is the likely precursor of thiamine triphosphate.
Assuntos
Córtex Cerebral/metabolismo , Frações Subcelulares/metabolismo , Tiamina/análogos & derivados , Animais , Radioisótopos de Carbono , Compartimento Celular , Feminino , Ratos , Ratos Wistar , Tiamina/farmacocinética , Tiamina Pirofosfato/análise , Tiamina Trifosfato/análiseRESUMO
Several membrane fractions were prepared from rat brain by differential and sucrose density gradient centrifugation. Most fractions took up 36Cl- rapidly at a rate linear with time during the first 30-60 s, then the rate progressively slowed down. The lowest rate of uptake was found in the mitochondrial fraction. Oxythiamin partially inhibited 36Cl- uptake in all fractions. In P2 (crude synaptosomal fraction), oxythiamin decreased the initial rate of uptake by 32%, the apparent Ki being 1.5 mM. Thiamin and amprolium were less effective as inhibitors. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (0.1-1 mM) inhibited 36Cl- uptake by 40-50%. In the presence of this compound at a concentration > or = 5 x 10(-4) M, oxythiamin became ineffective. 36Cl- uptake was increased by GABA (0.1 mM) and this effect was antagonized by picrotoxin as expected, but not by oxythiamin. The rate of 36Cl- uptake did not appreciably depend on the external chloride concentration and was unaffected by bumetanide or by replacement of external Na+ by choline. Taken together, these data suggest that the oxythiamin-sensitive 36Cl- influx is essentially diffusional and is not related to the GABA receptor or the Na:K:2Cl co-transport. Partial replacement of external Na+ by K+ or treatment with 0.1 mM veratridine (which should both result in membrane depolarization) increased 36Cl- uptake by 50 and 30% respectively; the inhibitory effect of oxythiamin was enhanced to the same proportion.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Encéfalo/metabolismo , Cloretos/metabolismo , Canais Iônicos/fisiologia , Proteínas de Membrana/fisiologia , Tiamina/análogos & derivados , Tiamina/farmacologia , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/análogos & derivados , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/farmacologia , Adenosina/farmacologia , Amprólio/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Canais de Cloreto , Colina/farmacologia , Feminino , Canais Iônicos/efeitos dos fármacos , Cinética , Proteínas de Membrana/efeitos dos fármacos , Especificidade de Órgãos , Oxitiamina/farmacologia , Cloreto de Potássio/farmacologia , Ratos , Ratos Wistar , Frações Subcelulares/metabolismo , Veratridina/farmacologiaRESUMO
Our results show that a net synthesis of thiamine triphosphate (TTP) can be demonstrated in vitro using rat brain extracts. The total homogenate was preincubated with thiamine or its diphosphate derivative (TDP), centrifuged, and washed twice. With TDP (1 mM) as substrate, a 10-fold increase in TTP content was observed in this fraction (nuclear fraction, membrane vesicles). A smaller, but significant, increase was observed in the P2 fraction (mitochondrial/synaptosomal fraction). In view of the low TTP content of our fractions, it was carefully assessed that authentic TTP was being formed. Incorporation of radioactivity from [beta-32P]TDP and [gamma-32P]ATP in TTP suggests that these two compounds are its precursors. Furthermore, TTP synthesis was inhibited by ADP and relatively low concentrations of Zn2+. These results suggest that TTP synthesis is catalyzed by an ATP:TDP transphosphorylase rather than by the cytoplasmic adenylate kinase that may be present in the vesicles. After osmotic lysis of the vesicles at alkaline pH, TTP was recovered in protein-bound form. Concomitantly, a soluble thiamine triphosphatase, with alkaline pH optimum, was also released from the vesicles. No net synthesis could be obtained in the cytosolic fraction or in detergent-solubilized systems. Like TTP synthesis, chloride permeability of the vesicles was increased when the homogenate had been incubated with thiamine and particularly with TDP. Our results suggest a regulatory role of TTP on chloride permeability, but the target remains to be characterized.
Assuntos
Encéfalo/metabolismo , Cloretos/metabolismo , Tiamina Trifosfato/metabolismo , Animais , Transporte Biológico , Membrana Celular/metabolismo , Cromatografia Líquida de Alta Pressão , Cinética , Permeabilidade , Ratos , Ratos Wistar , Tiamina Trifosfatase/metabolismo , Tiamina/isolamento & purificação , Tiamina/metabolismo , Tiamina Monofosfato/isolamento & purificação , Tiamina Monofosfato/metabolismo , Tiamina Pirofosfato/isolamento & purificação , Tiamina Pirofosfato/metabolismoRESUMO
A sensitive method, based on fluorescence detection, for the determination of thiamin derivatives after precolumn derivatization is described. The separation is achieved on a PRP-1 column using ion-pair reversed-phase HPLC. This method is especially well adapted to the detection of thiamin triphosphate in complex mixtures such as tissue extracts. The detection limit for TTP is 50 fmol. The contents of thiamin derivatives were determined in primary cultures of rat cerebellar granule neurons and cerebral astrocytes. The amount of TTP is about five times higher in neurons than in astrocytes. Thus in rat brain TTP seems to be essentially associated with neurons and the intracellular concentration is estimated to be about 0.2 microM. Our results suggest the existence, in nerve cells, of specific regulatory mechanisms not related to the blood-brain barrier and responsible for the maintenance of thiamin homeostasis in brain.
Assuntos
Química Encefálica , Cromatografia Líquida de Alta Pressão/métodos , Neurônios/química , Espectrometria de Fluorescência/métodos , Tiamina Trifosfato/análise , Tiamina/análise , Animais , Células Cultivadas/química , Cerebelo/química , Proteína Glial Fibrilar Ácida/imunologia , Imuno-Histoquímica , Oxirredução , Ratos , Tiamina/química , Tiamina Monofosfato/análise , Tiamina Monofosfato/química , Tiamina Pirofosfato/análise , Tiamina Pirofosfato/química , Tiamina Trifosfato/químicaRESUMO
The membrane-associated, anion-regulated thiamine triphosphatase from Electrophorus electricus electric organ can be solubilized by various neutral detergents. Polyoxyethylene ethers are the most effective. Anionic detergents readily inactivate the enzyme. A 6.4-fold increase in specific activity is obtained by successive treatment of crude membranes with octanoyl-N-methylglucamide, which solubilized other proteins, and Lubrol-PX with releases 60% of the thiamine triphosphatase (TTPase) activity. Solubilization by Lubrol-PX rapidly modifies kinetic parameters. The Km, Vmax and pH optimum are decreased. However, the solubilized TTPase may be kept at 0 degrees C for many hours without further change in specific activity. At 35 degrees C, the half-life is still 83 min at pH 5.0, but denaturation becomes rapid at pH greater than or equal to 7. Solubilization modifies anion effects on TTPase activity. The activating effect of nitrate is nearly lost, while inhibition by sulfate is no longer time-dependent.
Assuntos
Órgão Elétrico/enzimologia , Electrophorus/metabolismo , Tiamina Trifosfatase/isolamento & purificação , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/análogos & derivados , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/farmacologia , Animais , Cátions Bivalentes/farmacologia , Detergentes/química , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Proteínas de Membrana/metabolismo , Nitratos/farmacologia , Desnaturação Proteica , Solubilidade , Sulfatos/farmacologia , Tiamina Trifosfatase/química , Tiamina Trifosfatase/metabolismoRESUMO
We examined the effects of thiamine derivatives on ion uptake in rat brain membrane vesicles. Thiamine triphosphate (1 mM) and pyrithiamine (0.1 mM) increase chloride uptake. Preincubation of crude homogenate with thiamine or pyrithiamine increases chloride uptake while oxythiamine has the reverse effect. Thiamine and oxythiamine also affect 22Na+ and 86Rb+ uptake in the same way as for 36Cl- but to a lesser extent. Thiamine-dependent 36Cl- uptake is activated by sodium bicarbonate (10 mM) and partially inhibited by bumetanide (0.1 mM) and 2,4-dinitrophenol (0.1 mM). Preincubation with thiamine increases the thiamine triphosphate content of the vesicles. The hypothesis that TTP is the activator of a particular chloride uptake mechanism is discussed.
Assuntos
Encéfalo/metabolismo , Cloretos/metabolismo , Rubídio/metabolismo , Sódio/metabolismo , Tiamina/análogos & derivados , Tiamina/farmacologia , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Feminino , Cinética , Oxitiamina/farmacologia , Piritiamina/farmacologia , Ratos , Ratos Endogâmicos , Tiamina Monofosfato/farmacologia , Tiamina Pirofosfato/farmacologia , Tiamina Trifosfato/farmacologia , Ácido gama-Aminobutírico/farmacologiaRESUMO
The main electric organ of Electrophorus electricus is particularly rich in thiamine triphosphate (TTP). Membrane fractions prepared from this tissue contain a thiamine triphosphatase that is strongly activated by anions and irreversibly inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an anion transport inhibitor. Kinetic parameters of the enzyme are markedly affected by the conditions of enzyme preparation: In crude membranes, the apparent Km is 1.8 mM and the pH optimum is 6.8, but trypsin treatment of these membranes or their purification on a sucrose gradient decreases both the apparent Km (to 0.2 mM) and the pH optimum (to 5.0). Anions such as NO3- (250 mM) have the opposite effect, i.e., even in purified membranes, the pH optimum is now 7.8 and the Km is 1.1 mM; at pH 7.8, NO3- increases the Vmax 24-fold. TTP protects against inhibition by DIDS, and the KD for TTP could be estimated to be 0.25 mM, a value close to the apparent Km measured in the same purified membrane preparation. Thiamine pyrophosphate (0.1 mM) did not protect against DIDS inhibition. At lower (10(-5)-10(-6) M) substrate concentrations, Lineweaver-Burk plots of thiamine triphosphatase activity markedly deviate from linearity, with the curve being concave downward. This suggests either anticooperative binding or the existence of binding sites with different affinities for TTP. The latter possibility is supported by binding data obtained using [gamma-32P]TTP. Our data suggest the existence of a high-affinity binding site (KD of approximately 0.5 microM) for the Mg-TTP complex.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Órgão Elétrico/enzimologia , Electrophorus/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Tiamina Trifosfatase/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/análogos & derivados , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/farmacologia , Animais , Ânions , Fracionamento Celular , Membrana Celular/enzimologia , Órgão Elétrico/ultraestrutura , Concentração de Íons de Hidrogênio , Cinética , Tiamina Trifosfatase/antagonistas & inibidores , Tiamina Trifosfato/metabolismo , Tiamina Trifosfato/farmacologia , TripsinaRESUMO
Thiamine triphosphatase (TTPase) from membranes isolated from the main electric organ of E. electricus is activated about 8 fold by NO3-, I- and SCN- while SO42- is inhibitory. Activating anions shift the pH optimum of the enzyme from 5.0 to 8.0. The enzyme is irreversibly inactivated by low concentrations of 4,4'-diisothiocyano-2,2' disulfonic acid (DIDS), an inhibitor of anion transport. Anions protect from DIDS inactivation. These and other results suggest that the membrane-bound TTPase activity is tightly controlled, possibly through mechanisms involving anion transport.