Thiamin triphosphate

In brain, as in most mammalian cells, thiamine occurs predominantly in the form of TDP, the remainder being made up of thiamine monophosphate (10%), thiamine triphosphate (5-10%) and trace amounts of free thiamine. Thiamine is transported into brain and phosphory-lated by the action of thiamine pyrophosphokinase, and inhibition of this enzyme by thiamine antagonists such as pyrithiamine results in decrease synthesis of TDP. Treatment of experimental animals with pyrithiamine results in a generalized reduction of TDP concentrations and an early selective loss in activity of a-KGDH in regions... 

Cooper, J.R. Nishino, K. Enzymatic synthesis of thiamin triphosphate. Methods EnzymoL, 122, 24-29 (1986)... 

Ruenwongsa, P. Cooper, J.R. The role of bound thiamine pyrophosphate in the synthesis of thiamine triphosphate in rat liver. Biochim. Biophys. Acta, 482, 64-70 (1977)... 

Nishino, K. Itokawa, Y. Nishino, N. Piros, K. Cooper, J.R. Enzyme system involved in the synthesis of thiamin triphosphate. I. Purification and characterization of protein-bound thiamin diphosphate ATP phosphoryltrans-ferase. J. Biol. Chem., 258, 11871-11878 (1983)... 

Thiamine triphosphate is present in low concentration in most tissues, but its role remains unknown. Organs and tissues that generate electrical impulses are particularly rich in this compound. 

Thiamine triphosphate, diphosphate, and monophosphate were separated as the thiochrome derivatives on Hamilton PRP-1 column. The mobile phase was 15 mill phosphate buffer (pH 8.5) containing 1% tetrahydrofuran. The solvent flow rate was 0.5 mL/min, and a 20 fiL sample loop was used. Detection was by fluorescence using excitation and emission wavelengths of 365 and 433 nm, respectively. 

The reaction mixture was composed of 50 p.L of membrane preparation, 10 mM Hepes-Tris buffer (pH 6.8), 1.5 mM MgQ2, 1.5 mM EGTA, and 0.1 mM thiamine triphosphate in a total volume of 100 fiL. After 15 minutes of incubation at 25°C, the reaction was stopped by addition of 500 fiL of 6% trichloroacetic acid. The supemate obtained by centrifugation was extracted with 4 volumes of diethyl ether. The thiamine derivatives were transformed into fluorescent thiochromes by the addition of 50 fiL of oxidant [4.3 mJW K3Fe(CN)6 in 15% NaOH] to 80 fiL of sample. Thiamine diphosphatase activity was minimized by using magnesium as the metal and EGTA to chelate calcium. 

As shown in Figure 6.1, thiamin consists of pyrimidine and thiazole rings, linked by a methylene bridge the alcohol group of the side chain can be esterified with one, two, or three phosphates, yielding thiamin monophosphate, thiamin diphosphate (also known as thiamin pyrophosphate, the metabolically active coenzyme), and thiamin triphosphate. The vitamin was originally named aneurine, the antineuritic vitamin, because of its function in preventing or... 

Figure 6.1. Thiamin and thiamin anaiogs, products of thiaminoiysis, and experi-mentai antimetahoiites. Reiative moiecuiar masses (Mr) thiamin, 266.4 (chioride-hydrochioride, 337.3) thiamin monophosphate, 345.3 thiamin diphosphate, 425.3 thiamin triphosphate, 505.3 thiochrome, 262.3 thiamin thioi, 282.4 (oxidizes to thiamin disuifide, 562.7) oxythiamin, 301.8 and pyrithiamin, 420.2.

Both free thiamin and thiamin monophosphate circulate in plasma about 60% of the total is the monophosphate. Under normal conditions, most is bound to albumin when the albumin binding capacity is saturated, the excess is rapidly filtered at the glomerulus and excreted in the urine. Although a significant amount of newly absorbed thiamin is phosphorylated in the Uver, aU tissues can take up both thiamin and thiamin monophosphate, and are able to phosphorylate them to thiamin diphosphate and thiamin triphosphate. In most tissues, it is free thiamin that is the immediate precursor of thiamin diphosphate, which is formed by a pyrophosphokinase both the p-and y-phosphates of ATP are incorporated. Thiamin monophosphate arises mainly as a result of sequential hydrolysis of thiamin triphosphate and thiamin diphosphate. 

Two percent to 3% of the thiamin in nervous tissue is present as the triphosphate, which also occurs in significant amounts in skeletal muscle, especially in fast-twitch muscle fibers. In the nervous system, the triphosphate is found exclusively in the membrane fraction muscle thiamin triphosphate is mainly cytosoUc. There are two pathways for formation of thiamin triphosphate from the diphosphate ... 

Phosphorylation by ADP, catalyzed by adenylate kinase - this enzyme is especially important in the rapid synthesis and turnover of thiamin triphosphate in slow-twitch white muscle fibers. 

In both muscle and the central nervous system, there is an active thiamin triphosphatase, so that tissue concentrations of thiamin triphosphate are strictly regulated (Nishino et al., 1983 Miyoshi et al., 1990 Lakaye et al., 2002). 

Later studies established the coenzyme role of thiamin diphosphate in transketolase in the pentose phosphate pathway. More recent studies have shown that thiamin triphosphate acts to regulate a chloride channel in nerve tissue. 

Thiamin triphosphate is formed in brain and skeletal muscle by phosphorylation of thiamin diphosphate (Section 6.2), and its concentration is very precisely controlled, because there is also an active thiamin triphosphatase (Lakaye et al., 2002). In nervous tissue thiamin triphosphate is localized... 

Early studies showed that thiamin triphosphate had a role in electrical conduction in nerve cells more recent smdies have shown that it activates a chloride channel in the nerve membrane, acting as a phosphate donor (Bettendorff... 

On the basis of depletion/repletion studies, an intake ofO.2 mgper 1,000kcal is required to maintain normal urinary excretion, but an intake of 0.3 mg per 1,000 kcal is required for a normal transketolase activation coefficient. At low levels of energy intake, there will be a requirement for metabolism of endogenous substrates and to maintain nervous system thiamin triphosphate. 

Bettendorff L, Hennuy B, De Clerck A, and Wins P (1994) Chloride permeability of rat brain membrane vesicles correlates with thiamine triphosphate content. Brain Research 652, 157-60. 

Miyamoto T, Kakizawa T, and Hashizume K (1999) Inhibition of nuclear receptor signalling by poly(ADP-ribose) polymerase. Molecular and Cell Biology 19,2644-9. Miyoshi K, Egi Y, Shioda T, and Kawasaki T (1990) Evidence for in vivo synthesis of thiamin triphosphate by cytosolic adenylate kinase in chicken skeletal muscle./oMrnal... 

Nghiem HO, Bettendorff L, and Changeux JP (2000) Specific phosphorylation ofTorpedo 43K rapsyn by endogenous kinase(s) with thiamine triphosphate as the phosphate donor. FASEB Journal 14, 543-54. 

Thiamine uptaken into the cell is phosphorylated to TDP by the enzyme thiamine pyrophosphokinase. TDP is then further phosphorylated to thiamine triphosphate (TTP) or is dephosphorylated to thiamine monophosphate (TMP). 

Fig. 2 Intercellular trafficking and thiamine and thiamine esters in brain. TMP thiamine monophosphate, TDP thiamine diphosphate, TTP thiamine triphosphate, TPKinase thiamine pyrophosphokinase...

Elecfiical stimulation of a wide range of nerve preparations results in thiamine release suggesting a role for the vitamin in membrane function that is independent of its enzyme cofactor role mediated by TDP. TDP is further phosphorylated to thiamine triphosphate (Fig. 2). Although its precise role has yet to be elucidated, it has been proposed that TTP activates high-conductance chloride channels (Bettendorf,... 

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