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.     

Effect of Pyrithiamine Treatment and Subsequent Thiamine Rehabilitation on Regional Cerebral Amino Acids and Thiamine-Dependent Enzymes

Pyrithiamine-induced thiamine-deficiency encephalopathy in the rat shows many neuropathological and biochemical similarities to Wernicke's encephalopathy in humans. Treatment of rats with pyrithiamine resulted in moderate reductions of glutamate in thalamus and pons and in generalized severe reductions of aspartate in pons (by 89%, p less than 0.01), thalamus (by 83%, p less than 0.01), cerebellum (by 53%, p less than 0.01), and cerebral cortex (by 33%, p less than 0.05). Alanine concentrations were concomitantly increased. Activities of the thiamine-dependent enzyme alpha-ketoglutarate dehydrogenase (alpha KGDH) were decreased in parallel with the aspartate decreases; pyruvate dehydrogenase complex activities were unchanged in all brain regions. Following thiamine administration to symptomatic pyrithiamine-treated rats, neurological symptoms were reversed and concentrations of glutamate, aspartate, and alanine, as well as alpha KGDH activities, were restored to normal in cerebral cortex and pons. Aspartate levels and alpha KGDH activities remained below normal values, however, in thalamus. Thus, pyrithiamine treatment leads to reductions of cerebral alpha KGDH and (1) decreased glucose (pyruvate) oxidation resulting in accumulation of alanine and (2) decreased brain content of glutamate and aspartate. Such changes may be of key significance in the pathophysiology of the reversible and irreversible signs of Wernicke's encephalopathy in humans.     

Regional Reductions of Transketolase in Thiamine-Deficient Rat Brain

Abstract: Thiamine deficiency impairs oxidative metabolism and causes metabolic encephalopathy. An early reduction in transketolase (TK) activity may be an important pathogenic event. To assess the role of TK, we have delineated the regional/cellular distribution of TK protein and mRNA in adult rat brain in pyrithiamine-induced thiamine deficiency. TK activity declined in both vulnerable and spared regions. Immunoblots showed a parallel reduction of TK protein. With a few exceptions, immunocytochemistry indicated an overall decline of TK immunoreactivity and the decrease was not specific to vulnerable areas. In contrast to the pronounced, general decline of TK protein, in situ hybridization revealed a regional decrease of 0–25% of TK mRNA in thiamine deficiency. Northern blots indicated a similar level of TK mRNA in whole brain in thiamine deficiency. These results show that the decline of TK activity results from a proportional decrease of TK protein, and the deficiency may be due to an instability of TK protein or an inhibition of TK mRNA translation. The lack of correlation of the distribution, and the absence of specific alteration, of TK in affected regions suggest that the reduced TK may not be linked directly to selective vulnerability in thiamine deficiency.     

Molecular Evolutionary Analysis of the Thiamine-Diphosphate-Dependent Enzyme,Transketolase

Members of the transketolase group of thiamine-diphosphate-dependent enzymes from 17 different organisms including mammals, yeast, bacteria, and plants have been used for phylogenetic reconstruction. Alignment of the amino acid and DNA sequences for 21 transketolase enzymes and one putative transketolase reveals a number of highly conserved regions and invariant residues that are of predicted importance for enzyme activity, based on the crystal structure of yeast transketolase. One particular sequence of 36 residues has some similarities to the nucleotide-binding motif and we designate it as the transketolase motif. We report further evidence that the recP protein from Streptococcus pneumoniae might be a transketolase and we list a number of invariant residues which might be involved in substrate binding. Phylogenies derived from the nucleotide and the amino acid sequences by various methods show a conventional clustering for mammalian, plant, and gram-negative bacterial transketolases. The branching order of the gram-positive bacteria could not be inferred reliably. The formaldehyde transketolase (sometimes known as dihydroxyacetone synthase) of the yeast Hansenula polymorpha appears to be orthologous to the mammalian enzymes but paralogous to the other yeast transketolases. The occurrence of more than one transketolase gene in some organisms is consistent with several gene duplications. The high degree of similarity in functionally important residues and the fact that the same kinetic mechanism is applicable to all characterized transketolase enzymes is consistent with the proposition that they are all derived from one common ancestral gene. Transketolase appears to be an ancient enzyme that has evolved slowly and might serve as a model for a molecular clock, at least within the mammalian clade. Received: 13 September 1995 / Accepted: 14 November 1996     

Regional alterations of thiamine phosphate esters and of thiamine diphosphate-dependent enzymes in relation to function in experimental Wernicke's encephalopathy

Thiamine phosphate esters (thiamine monophosphate-TMP; thiamine diphosphate-TDP and thiamine triphosphate-TTP) were measured as their thiochrome derivatives by High Performance Liquid Chromatography in the brains of pyrithiamine-treated rats at various stages during the development of thiamine deficiency encephalopathy. Severe encephalopathy was accompanied by significant reductions of all three thiamine phosphate esters in brain. Neurological symptoms of thiamine deficiency appeared when brain levels of TMP and TDP fell below 15% of normal values. Activities of the TDP-dependent enzyme -ketoglutarate dehydrogenase were more severely reduced in thalamus compared to cerebral cortex, a less vulnerable brain structure. On the other hand, reductions of TTP, the non-cofactor form of thiamine, occurred to a greater extent in cerebral cortex than thalamus. Early reductions of TDP-dependent enzymes and the ensuing metabolic pertubations such as lactic acidosis impaired brain energy metabolism, and NMDA-receptor mediated excitotoxicity offer rational explanations for the selective vulnerability of brain structures such as thalamus to the deleterious effects of thiamine deficiency.     

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.     

Thiamine and Cholinergic Transmission in the Electric Organ of Torpedo

The electric organ of Torpedo marmorata was found to contain as much as 120 +/- 24 nmol of thiamine per g of fresh tissue. The vitamin was distributed as nonesterified thiamine (32%), thiamine monophosphate (22%), thiamine diphosphate (8%), and an important proportion of thiamine triphosphate (38%). A high level of thiamine triphosphate was found in synaptosomes isolated from the electric organ. In contrast, the synaptic vesicles did not show any enrichment in thiamine, whereas they contained a marked peak of acetylcholine (ACh) and ATP. Thus thiamine seems to be very abundant in cholinergic nerve terminals; its localization is apparently extravesicular, either in the axoplasm or in association with plasma membrane. When calcium was reduced and magnesium increased in the external medium, the efficiency of transmission was diminished, owing to inhibition of ACh release; in a parallel manner the degree of thiamine phosphorylation was found to increase--this condition is known to modify the repartition of ACh between vesicular and extravesicular compartments. Electrical stimulation, which causes periodic variations of the level of ACh and ATP, also caused significant changes in thiamine esters. In addition, related changes of the vitamin and the transmitter were observed under other conditions, suggesting a functional link between the metabolism of thiamine and that of ACh in cholinergic nerve terminals.     

Stimulation of thiamine diphosphate activity by ascorbic acid in rat brain microsomes

The effect of ascorbic acid on microsomal thiamine diphosphate activity in rat brain was examined. Ascorbic acid at 0.02–0.1 mM increased the thiamine diphosphate activity by 20–600% and produced a significant amount of lipid peroxide, which was measured with thiobarbiturate under the same conditions as the enzyme. A lag period of about 10 min was observed in the process of stimulation of enzyme activity by ascorbic acid. The stimulation of enzyme activity and the lipid peroxidation induced by ascorbic acid were blocked by metal-binding compounds (EDTA, α,α′-dipyridyl, o-phenanthroline) and an antioxidant (N,N′-diphenyl p-phenylenediamine). GSH significantly enhanced the stimulation of enzyme activity and formation of lipid peroxide by 0.02–0.05 mM ascorbic acid. The effect of GSH was due in part to maintenance of the concentration of ascorbic acid in the medium, since GSH could convert dehydroascorbic acid, an oxidized form of ascorbic acid, to ascorbic acid.     

Cation Transport Specificity at the Blood–Brain Barrier

The molecular identification, expression and cloning of membrane-bound organic cation transporters are being completed in isolated in vitro membranes. In vivo studies, where cation specificity overlaps, need to complement this work. Method: Cross-inhibition of [³H]choline and [³H]thiamine brain uptake by in situ rat brain perfusion. Results: [³H]Choline brain uptake was not inhibited by thiamine at physiologic concentrations (100 nM). However, choline ranging from 100 nM to 250 M inhibited [³H]thiamine brain uptake, though not below levels observed at thiamine concentrations of 100 nM. Conclusion: (1) The molecular family of the blood–brain barrier (BBB) choline transporter may be elucidated in vitro by its interaction with physiologic thiamine levels, and (2) two cationic transporters at the BBB may be responsible for thiamine brain uptake.     

The role of cysteine 160 in thiamine diphosphate binding of the Calvin-Benson-Bassham cycle transketolase of Rhodobacter sphaeroides

The transketolase gene (cbbT) that encodes the Calvin-Benson-Bassham pathway transketolase (CbbT) of Rhodobacter sphaeroides was overexpressed in Escherichia coli and the recombinant protein purified to homogeneity. Like other transketolases, R. sphaeroides CbbT was found to be inactivated in the presence of oxygen. At its optimal pH of 7.8, CbbT displays a specific activity of 37 U/mg, a KR5P of 949 microM, a KXu5P of 11 microM, and a KThDP of 1.8 microM. Cysteine 160, equivalent to Cys159 of the yeast enzyme, is found within the active site and is loosely conserved amongst several sources of transketolase. To investigate the role of cysteine 160 found in the active site of R. sphaeroides CbbT, this residue was targeted for mutagenesis. Cys160 was changed to alanine, serine, aspartate, and glutamate. To compare the effect of these mutations on ThDP binding, spectral techniques were employed in addition to analysis by enzymatic activity. Fluorescence quenching was used to measure both equilibrium binding constants as well as first order rates of binding. The results of these studies indicated that Cys160 played an important and substantial role in cofactor binding, revealing the importance of this loosely conserved residue. In addition, the Cys160 mutants did not appear to alter oxygen-mediated inactivation.