Thiamine Triphosphatase in the Membranes of the Main Electric Organ of Electrophorus electricus: Substrate-Enzyme Interactions

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)     

Postnatal Development of Thiamine Metabolism in Rat Brain

The activities of thiamine diphosphatase (TDPase), thiamine triphosphatase (TTPase), and thiamine pyrophosphokinase and the contents of thiamine and its phosphate esters were determined in rat brain cortex, cerebellum, and liver from birth to adulthood. Microsomal TTPase activity in the cerebral cortex and cerebellum increased from birth to 3 weeks, whereas that in the liver did not change during postnatal development. Microsomal TDPase activity in the cerebral cortex showed a transient increase at 1-2 weeks, but that in the cerebellum did not change during development. In contrast to the activity of the brain enzyme, that of liver microsomal TDPase increased stepwise after birth. Thiamine pyrophosphokinase activity in the cerebellum increased from birth to 3 weeks and then decreased, whereas that in the cerebral cortex and liver showed less change during development. TDP and thiamine monophosphate (TMP) levels increased after birth and plateaued at 3 weeks whereas TTP and thiamine levels showed little change during development in the cerebral cortex and cerebellum. The contents of thiamine and its phosphate esters in the liver showed more complicated changes during development. It is concluded that thiamine metabolism in the brain changes during postnatal development in a different way from that in the liver and that the development of thiamine metabolism differs among brain regions.     

Purification and characterization of thiamine triphosphatase from bovine brain.

Soluble thiamine triphosphatase (EC 3.6.1.28) of bovine brain has been purified 68,000-fold to an electrophoretically homogeneous state with an overall recovery of 5.5% by hydrophobic chromatography on Toyopearl HW-60, Sephadex G-75 gel filtration, DEAE-Toyopearl 650M chromatography and Blue Sepharose CL-4B chromatography. The enzyme has an absolute specificity among thiamine and nucleoside phosphate esters for thiamine triphosphate and shows no nonspecific phosphatase activities. Thiamine triphosphatase is composed of a single polypeptide chain with molecular mass of 33,900 kDa as estimated by Sephadex G-100 gel filtration and SDS-polyacrylamide gel electrophoresis. The enzyme has a pH optimum of 8.7 and is dependent on divalent metal ions. Mg2+ has been found to be the most effective among cations tested. A study of the reaction kinetics over a wide range of thiamine triphosphate concentrations has revealed a biphasic saturation curve being described by higher-degree rational polynomials.     

Thiamine, Thiamine Phosphates, and Their Metabolizing Enzymes in Human Brain

Abstract: Total thiamine (the sum of thiamine and its phosphate esters) concentrations are two- to fourfold lower in human brain than in the brain of other mammals. There were no differences in the total thiamine content between biopsied and autopsied human brain, except that in the latter, thiamine triphosphate was undetectable. The main thiamine phosphate-metabolizing enzymes could be detected in autopsied brain, and the kinetic parameters were comparable to those reported in other species. Thiamine diphosphate levels were lowest in hippocampus (15 ± 4 pmol/mg of protein) and highest in mammillary bodies (24 ± 4 pmol/mg of protein). Maximal levels of thiamine and its phosphate ester were found to be present at birth. In parietal cortex and globus pallidus, mean levels of total thiamine in the oldest age group (77–103 years) were, respectively, 21 and 26% lower than those in the middle age group (40–55 years). Unlike cerebral cortex, the globus pallidus showed a sharp drop in thiamine diphosphate levels during infancy, with concentrations in the oldest group being only ∼50% of the levels present during the first 4 months of life. These data, consistent with previous observations conducted in blood, suggest a tendency toward decreased thiamine status in older people.     

Ectonucleotidase Activities Associated with Cholinergic Synaptosomes Isolated from Torpedo Electric Organ

Intact synaptosomes isolated from the electric organ of the electric ray Torpedo marmorata contain, at their surface, enzyme activities for the hydrolysis of externally applied nucleoside phosphates. The diazonium salt of sulfanilic acid, as a low-molecular-weight, slowly permeating, covalent inhibitory agent, selectively blocks these enzyme activities and leaves intracellular lactate dehydrogenase intact. The ectoenzymes comprise both a nucleoside triphosphate and diphosphate phosphohydrolase, as well as a 5'-nucleotidase. Activity of nonspecific ectophosphatases is absent. The nucleoside triphosphatase hydrolyzes almost equally well ATP, GTP, CTP, UTP, and ITP and is activated to a similar degree by Mg2+ or Ca2+. It has a high affinity for ATP (Km for ATP in the presence of Mg2+, 75 microM; in the presence of Ca2+, 66 microM). Maximal rates in the presence of Mg2+ and Ca2+ were very similar (34.8 and 32.5 nmol of Pi/min/mg of synaptosomal protein, respectively). Either Mg-ATP or Ca-ATP can act as a true substrate. ADP inhibits hydrolysis of ATP, but AMP is without effect. The nucleoside triphosphatase is not inhibited significantly by a number of inhibitors of mitochondrial Mg2+-ATPase or of Ca2+ + Mg2+-ATPases. It is, however, considerably inhibited by filipin and quercitin. The capacity of intact synaptosomes to hydrolyze also extracellular ADP, GDP, AMP, GMP, and IMP suggests that the nucleoside triphosphatase is part of an enzyme chain that causes complete hydrolysis of the respective nucleoside triphosphate to the nucleoside. We conclude that the cholinergic nerve terminals of the Torpedo electric organ can hydrolyze ATP released on coexocytosis with acetylcholine via an ectonucleoside triphosphatase activity that is different from known endogenous nerve terminal ATPases. The final product of the hydrolysis, adenosine, can then be salvaged by the nerve terminal for resynthesis of ATP. Other possible physiological functions of the ectonucleotidases are discussed.     

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.     

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.     

Human recombinant thiamine triphosphatase: purification, secondary structure and catalytic properties

Thiamine triphosphate (ThTP) is found in most living organisms and it may act as a phosphate donor for protein phosphorylation. We have recently cloned the cDNA coding for a highly specific mammalian 25 kDa thiamine triphosphatase (ThTPase; EC 3.6.1.28). As the enzyme has a high catalytic efficiency and no sequence homology with known phosphohydrolases, it was worth investigating its structure and catalytic properties. For this purpose, we expressed the untagged recombinant human ThTPase (hThTPase) in E. coli, produced the protein on a large scale and purified it to homogeneity. Its kinetic properties were similar to those of the genuine human enzyme, indicating that the recombinant hThTPase is completely functional. Mg2+ ions were required for activity and Ca2+ inhibited the enzyme by competition with Mg2+. With ATP as substrate, the catalytic efficiency was 10(-4)-fold lower than with ThTP, confirming the nearly absolute specificity of the 25 kDa ThTPase for ThTP. The activity was maximum at pH 8.5 and very low at pH 6.0. Zn2+ ions were inhibitory at micromolar concentrations at pH 8.0 but activated at pH 6.0. Kinetic analysis suggests an activator site for Mg2+ and a separate regulatory site for Zn2+. The effects of group-specific reagents such as Woodward's reagent K and diethylpyrocarbonate suggest that at least one carboxyl group in the active site is essential for catalysis, while a positively charged amino group may be involved in substrate binding. The secondary structure of the enzyme, as determined by Fourier-transform infrared spectroscopy, was predominantly beta-sheet and alpha-helix.     

Regulation of ion uptake in membrane vesicles from rat brain by thiamine compounds

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.     

Calorimetric studies of the interaction of magnesium and phosphate with Na+, K+) ATPase: evidence for a ligand-induced conformational change in the enzyme.

The phosphorylation of (Na+, K+)ATPase from the electric organ of the electric eel is dependent on Mg2+. The amount of phosphoenzyme formed was increased by K+ and decreased by Na+. Kinetic analyses indicate that a ternary complex of ATPase, Pi and Mg2+ is formed prior to phosphorylation of the protein. Calorimetric studies revealed extraordinarily large enthalpy changes associated with the binding of Mg2+ (-49 kcal/mol) and of Pi (-42 kcal/mol), indicating a thermodynamically significant conformational change in the enzyme. The dissociation constant for the binding of Mg2+ and Pi derived from calorimetric measurements is in good agreement with the value obtained from the kinetic studies. These results indicate that ion binding induces a conformational change in the enzyme which is a prerequisite for phosphorylation by Pi.     

Postnatal development of thiamine metabolism in rat skeletal muscle.

1. The activities of 2-oxoglutarate dehydrogenase, transketolase, thiamine pyrophosphokinase and thiamine triphosphatase and the concentrations of thiamine phosphates were almost the same between rat extensor digitorum longus and soleus muscles at 2 weeks of age. 2. These enzyme activities changed after 3 weeks of age in a different way depending on the muscle phenotype. 3. Thiamine diphosphate level and the activity of 2-oxoglutarate dehydrogenase increased only in soleus muscle and thiamine triphosphate level increased only in extensor digitorum longus during development.     

The relation of the soluble thiamine triphosphatase activity of various rat tissues to nonspecific phosphatases

Polyacrylamide gel electrophoresis was used to investigate the relation of the soluble thiamine triphosphatase activity of various rat tissues to other phosphatases. This technique separated the thiamine triphosphatase of rat brain, heart, kidney, liver, lung, muscle and spleen from alkaline phosphatase (EC 3.1.3.1), acid phosphatase (EC 3.1.3.2) and other nonspecific phosphatase activities. In contrast, the hydrolytic activity for thiamine triphosphate in rat intestine moved identically with alkaline phosphatase in gel electrophoresis. Thiamine triphosphatase from rat liver and brain was also separated from alkaline phosphatase and acid phosphatase by gel chromatography on Sephadex G-100. This gave an apparent molecular weight of about 30,000 and a Stokes radius of 2.5 nanometers for brain and liver thiamine triphosphatase. The intestinal thiamine triphosphatase activity of the rat was eluted from the Sephadex G-100 column as two separate peaks (with apparent molecular weights of over 200,000 and 123,000) which exactly corresponded to the peaks of alkaline phosphatase. The isoelectric point (pI) of the brain thiamine triphosphatase was 4.6 (4 degrees C). The partially purified thiamine triphosphatase from brain and liver was highly specific for thiamine triphosphate. The results suggest that, apart from the intestine, the rat tissues studied contain a specific enzyme, thiamine triphosphatase (EC 3.6.1.28). The specific enzyme is responsible for most of the thiamine triphosphatase activity in these tissues. Rat intestine contains a high thiamine triphosphatase activity but all of it appears to be due to alkaline phosphatase.     

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.     

Blood–Brain Transport of Thiamine Monophosphate in the Rat: A Kinetic Study In Vivo

To calculate the kinetic parameters of thiamine monophosphate transport across the rat blood-brain barrier in vivo, different doses of a [35S]thiamine monophosphate preparation with a specific activity of 14.8 mCi.mmol-1 were injected in the femoral vein and the radioactivity was measured in arterial femoral blood and in the cerebellum, cerebral cortex, pons, and medulla 20 s after the injection. This short experimental time was used to prevent thiamine monophosphate hydrolysis. Thiamine monophosphate was transported into the nervous tissue by a saturable mechanism. The maximal transport rate (Jmax) and the half-saturation concentration (Km) equaled 27-39 pmol.g-1.min-1 and 2.6-4.8 microM, respectively. When compared with that of thiamine, thiamine monophosphate transport seemed to be characterized by a lower affinity and a lower maximal influx rate. At physiological plasma concentrations, thiamine monophosphate transport rate ranged from 2.06 to 4.90 pmol.g-1.min-1, thus representing a significant component of thiamine supply to nervous tissue.     

Thiamine, thiamine phosphates and thiamine metabolizing enzymes in synaptosomes of rat brain

Thiamine and thiamine mono-, pyro- and triphosphate were found at detectable levels in synaptosomes isolated from whole rat brain. Synaptosomes prepared from whole brain, cerebellum and medulla were also found to contain uridine and inosine mono- and diphosphatases as well as the thiamine pyrophosphate synthetizing and hydrolyzing enzymes, but no thiamine monophosphatase. By isoelectric focusing on thin layer polyacrylamide gel of Triton X-100 homogenates of synaptosomes, thiamine pyrophosphatase activity could be separated into 10 bands with different isoelectric points. The contents of thiamine compounds and enzymes in synaptosomes were generally lower than those found in neuronal cell bodies.     

Thiamine status in humans and content of phosphorylated thiamine derivatives in biopsies and cultured cells

Background

Thiamine (vitamin B1) is an essential molecule for all life forms because thiamine diphosphate (ThDP) is an indispensable cofactor for oxidative energy metabolism. The less abundant thiamine monophosphate (ThMP), thiamine triphosphate (ThTP) and adenosine thiamine triphosphate (AThTP), present in many organisms, may have still unidentified physiological functions. Diseases linked to thiamine deficiency (polyneuritis, Wernicke-Korsakoff syndrome) remain frequent among alcohol abusers and other risk populations. This is the first comprehensive study on the distribution of thiamine derivatives in human biopsies, body fluids and cell lines.

Methodology and Principal Findings

Thiamine derivatives were determined by HPLC. In human tissues, the total thiamine content is lower than in other animal species. ThDP is the major thiamine compound and tissue levels decrease at high age. In semen, ThDP content correlates with the concentration of spermatozoa but not with their motility. The proportion of ThTP is higher in humans than in rodents, probably because of a lower 25-kDa ThTPase activity. The expression and activity of this enzyme seems to correlate with the degree of cell differentiation. ThTP was present in nearly all brain and muscle samples and in ∼60% of other tissue samples, in particular fetal tissue and cultured cells. A low ([ThTP]+[ThMP])/([Thiamine]+[ThMP]) ratio was found in cardiovascular tissues of patients with cardiac insufficiency. AThTP was detected only sporadically in adult tissues but was found more consistently in fetal tissues and cell lines.

Conclusions and Significance

The high sensitivity of humans to thiamine deficiency is probably linked to low circulating thiamine concentrations and low ThDP tissue contents. ThTP levels are relatively high in many human tissues, as a result of low expression of the 25-kDa ThTPase. Another novel finding is the presence of ThTP and AThTP in poorly differentiated fast-growing cells, suggesting a hitherto unsuspected link between these compounds and cell division or differentiation.     

Membrane-associated thiamin triphosphatase. II. Activation by divalent cations.

Activation of membrane-associated thiamin triphosphatase from rat brain requires a divalent cation (Mg2+, Ca2+, or Mn2+). The optimum concentration of Mg2+ necessary for maximal enzyme activity varies with substrate concentration; conversely, the maximal rate of hydrolysis attainbale by increasing thiamin triphosphate concentration is directly proportional to [Mg2+] for all levels of Mg2+ below that of the substrate. Under appropriate conditions, the Km of the thiamin triphosphatase for Mg2+ and for thiamin triphosphate are shown to be identical. Dissociation constants (Kd) for the binding of Mg2+ to thiamin triphosphate, thiamin diphosphate, and thiamin were determined; kinetic data re-expressed in terms of [Mg2+-thiamin triphosphate] conform to simple single substrate predictions, suggesting that the true enzyme substrate may be the Mg2+-thiamin triphosphate complex. Excess free Mg2+ inhibits thiamin triphosphatase activity competitively while excess free thiamin triphosphate in concentrations up to 10 times Km has no effect on the membrane-bound enzyme.     

Human deoxyuridine triphosphate nucleotidohydrolase. Purification and characterization of the deoxyuridine triphosphate nucleotidohydrolase from acute lymphocytic leukemia.

A new fast assay procedure for increasing deoxyuridine triphosphate nucleotidohydrolase activity was developed. With this assay procedure, this enzyme derived from blast cells of patients with acute lymphocytic leukemia was purified at least 1218-fold. The molecular weight was estimated by gel filtration to be 43,000. The enzyme exhibited optimal activity over a pH range of 7 to 8 and the activation energy was estimated to be 6.5 kcal/mol at pH 7.5. While the enzyme had activity in the absence of added divalent cations, the activity could be inhibited by EDTA but not by phenanthroline. The inhibition caused by EDTA could be reversed by Mg2+ or Zn2+. The enzyme had maximal activity in the presence of Mg2+ (40 muM) and Mg2+ (4 mM) stabilized the enzyme at 37 degrees C. Cupric ion (0.5 mM) inhibited (50%) enzyme activity in the presence or absence of Mg2+. The substrate for the enzyme was dUTP and the apparent Km was 1 muM. No other deoxyribonucleoside or ribonucleoside triphosphate served as a substrate for the enzyme.     

Thiamine triphosphate, a new signal required for optimal growth of Escherichia coli during amino acid starvation

Thiamine triphosphate (ThTP) is present in low amounts in most organisms from bacteria to humans, but its biological role remains unknown. Escherichia coli grown aerobically in LB medium contain no detectable amounts of ThTP, but when they are transferred to M9 minimal medium with a substrate such as glucose or pyruvate, there is a rapid but transient accumulation of relatively high amounts of ThTP (about 20% of total thiamine). If a mixture of amino acids is present in addition to glucose, ThTP accumulation is impaired, suggesting that the latter may occur in response to amino acid starvation. To test the importance of ThTP for bacterial growth, we used an E. coli strain overexpressing a specific human recombinant thiamine triphosphatase as a glutathione S-transferase (GST) fusion protein (GST-ThTPase). Those bacteria were unable to accumulate measurable amounts of ThTP. On minimal medium supplemented with glucose, pyruvate, or acetate, they exhibited an intermediate plateau in cell growth compared with control bacteria expressing GST alone or a GST fusion protein unrelated to thiamine metabolism. These results suggest that the early accumulation of ThTP initiates a reaction cascade involved in the adaptation of bacteria to stringent conditions such as amino acid starvation. This is the first demonstration of a physiological role of this ubiquitous compound in any organism.     

Thiamine triphosphatase from Electrophorus electric organ is anion-dependent and irreversibly inhibited by 4,4'-diisothiocyanostilbene-2,2'disulfonic acid

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.