The coenzyme thiamine pyrophosphate inhibits the self-splicing of the group I intron.

Effects of the coenzyme thiamine pyrophosphate and its analogs on the inhibition of self-splicing of primary transcripts of the phage T4 thymidylate synthase gene (td) were investigated. Of all compounds tested, the coenzyme thiamine pyrophosphate was the most potent inhibitor and the order of inhibitory efficiency for compounds tested was as follows: thiamine pyrophosphate>thiamine monophosphate>thiamine>thiochrome. Increasing guanosine concentration overcame the suppression of self-splicing by thiamine pyrophosphate close to the level of normal splicing. Kinetic analysis demonstrated that thiamine pyrophosphate acts as a competitive inhibitor for the td intron RNA with a Ki of 2.2mM. The splicing specificity inhibition by thiamine pyrophosphate is predominantly due to changes in Km.

Regulatory effect of thiamin pyrophosphate on pig heart pyruvate dehydrogenase complex.

The kinetic behavior of pig heart pyruvate dehydrogenase complex (PDC) containing bound endogenous thiamin pyrophosphate (TPP) was affected by exogenous TPP. In the absence of exogenous TPP, a lag phase of the PDC reaction was observed. TPP added to the PDC reaction medium containing Mg2+ led to a disappearance of the lag phase, inducing strong reduction of the Km value for pyruvate (from 76.7 to 19.0 microM) but a more moderate decrease of Km for CoA (from 12.2 to 4.3 microM) and Km for NAD+ (from 70.2 to 33.6 microM), with no considerable change in the maximum reaction rate. Likewise, thiamin monophosphate (TMP) decreased the Km value of PDC for pyruvate, but to a lesser extent (from 76.7 to 57.9 microM) than TPP. At the unsaturating level of pyruvate, the A50 values for TPP and TMP were 0.2 microM and 0.3 mM, respectively. This could mean that the effect of TPP on PDC was more specific. In addition, exogenous TPP changed the UV spectrum and lowered the fluorescence emission of the PDC containing bound endogenous TPP in its active sites. The data obtained suggest that TPP plays, in addition to its catalytic function, the important role of positive regulatory effector of pig heart PDC.

Thiamine pyrophosphate (TPP) negatively regulates transcription of some thi genes of Salmonella typhimurium.

In Salmonella typhimurium, thiamine is a required nutrient that is synthesized de novo. Labeling studies have demonstrated probable precursors for both the 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate moiety and the 4-methyl-5-(beta-hydroxyethyl) thiazole monophosphate moiety. The isolation of thiamine auxotrophs with mutations in at least five different genetic loci is reported. The majority (22 of 25) of the mutants required only the thiazole moiety of thiamine to satisfy their growth requirement. Most (14 of 25) of the mutants were affected in the thi cluster at min 90 on the S. typhimurium genetic map. Data provided herein indicate that this cluster encodes an operon whose transcription is regulated by thiamine and suggest that thiamine pyrophosphate, or a molecule derived form it, is the effector molecule. Mutants with altered regulation of this operon were isolated, and we propose that they are defective in thiamine phosphate kinase, the product of the thiL gene.

[Effect of thiamine and its metabolites on the activity of tissue and purified alcohol dehydrogenase]. / Vliianie tiamina i ego metabolitov na aktivnost' tkanevoi i ochishchennoi alkogol'degidrogenazy.

Thiamine is shown to have a stimulatory action on ++alcohol dehydrogenase activity in blood and small intestines of white rats. The same effect is obtained on the purified ++alcohol dehydrogenase as well. Thiochrome inhibits the enzyme preparation.

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.

Intestinal transport of thiamin and thiamin monophosphate in rat everted jejunal sacs: a comparative study using some potential inhibitors.

Rat everted jejunal sacs were incubated for 15 and 30 min at 37 degrees C in oxygenated Krebs-Henseleit buffer, pH 7.4, containing 0.2 microM [3H]-thiamin (3H-T) or [3H]-thiamin monophosphate (3H-TMP) with and without 10 mM 1-phenylalanine (PAL) or 2.5 mM levamisole (LEV). The concentrations of 3H-T and its phosphoesters in sac wall and serosal fluid were determined by a radiometric method after electrophoretic separation. In separate experiments, thiamin pyrophosphokinase (TPKase) and thiamin pyrophosphatase (TPPase) activities were determined in mucosal scrapings, with and without PAL or LEV, by using a radiometric and a colorimetric method, respectively. 3H-TMP was transported partly unchanged by an active mechanism similarly to 3H-T, but less efficiently. During transport, 3H-TMP was also enzymatically transformed to thiamin (T) and thiamin pyrophosphate, which accumulated in the tissue. In the serosal fluid, the concentration of 3H-TMP exceeded that of 3H-T. Presence of PAL or LEV with 3H-T or 3H-TMP in the incubation medium reduced the serosal transport and the tissue content of T compounds. LEV caused a dose-dependent inhibition of TPKase without affecting TPPase, whereas PAL inhibited both activities to about the same extent. These results indicate that the transport of TMP involves a number of different processes similar to those responsible for T transport. The effects of PAL and LEV underline the importance of phosphorylation-dephosphorylation coupling.

Thiamine pyrophosphate (cocarboxylase) as a growth factor for Haemophilus somnus.

The effect of a commercially available, chemically defined enrichment (Iso-VitaleX; BBL Microbiology Systems, Cockeysville, Md.) on the growth of 10 strains of Haemophilus somnus was studied. A 6- to 10-fold increase in growth, as measured turbidimetrically, was observed when Iso VitaleX was added to a basal medium of brain heart infusion broth to a final concentration of 1% (vol/vol). Thiamine pyrophosphate (cocarboxylase), a constituent component of Iso VitaleX, was found to be the only growth-promoting factor, and it could be used as a substitute for Iso VitaleX. An equimolar concentration (2.2 microM) of thiamine monophosphate promoted growth equal to that of thiamine pyrophosphate. Thiamine was nonstimulatory for all 10 strains tested. When alkaline thermal-treated brain heart infusion broth was used as the basal medium, 7 of the 10 strains had an absolute requirement for thiamine monophosphate or thiamine pyrophosphate. The three remaining strains showed minimal growth when thiamine was added to this basal medium; however, excellent growth was observed when thiamine monophosphate or thiamine pyrophosphate was utilized. Factor X (hemin) was found to further enhance the growth when concentrations of 5 to 10 micrograms/ml were coupled with thiamine pyrophosphate. No increase in growth was observed when factor V (nicotinamide adenine dinucleotide) was coupled with thiamine pyrophosphate. This is the first report of a growth factor requirement for H. somnus.

Alterations in uptake and metabolism of aspartate and glutamate in brain of thiamine deficient animals.

The high affinity uptake systems of aspartate, glutamate, glycine and taurine were studied in synaptosomal preparations isolated from the cerebellum, medulla-pons and the telencephalon of rats made thiamine deficient (TD) by diet or pyrithiamine (PT). There was a significant enhancement in the uptake of asparate/glutamate (probably transported by the same carrier) by the synaptosomal preparations of the cerebellum only, in both groups of thiamine-deficient animals as compared to controls. This was due to an increase in the number of uptake sites and not to an alteration of the binding affinity. Aspartate levels decreased significantly in all three brain areas of PT-treated animals and this change was greatest in the medulla-pons and the cerebellum and least in the cortex. Glutamate and serine levels were significantly decreased only in the medulla-pons whilst the concentration of glutamine was significantly increased in the three brain regions studied. The changes in both uptake and levels of amino acids in TD rats were reversed by thiamine therapy. Though the uptake studies do not discriminate between an altered aspartergic or glutamergic system, the changes in the levels of aspartate in the cerebellum suggest that the aspartergic system is involved. Since earlier studies showed a selective impairment in the high affinity uptake of serotonin by cerebellar synaptosomes, thiamine deficiency could impair cerebellar function by inducing an imbalance in its neurotransmitter systems.

Metabolism and biological activity of a thiamine new salt. I. Urinary excretion and tissue content of vitamin B1 in rats treated with thiamine chloride ester monophosphate of Bis-glucosamine.

Research has been carried out on the metabolism of thiamine chloride ester monophosphate of bis-D-glucosamine (TCMPG) evaluating the urinary excretion, the blood and the hepatic content of thiamine in the rat after intraperitoneal injection of this compound. The results obtained have shown a greater retention of vitamin B1 when it is administered as glucosamine salt. In fact, the amount of thiamine excreted by the rats which have received the TCMPG is lower while the blood and liver levels of vitamin are higher, compared with the control rats treated with thiamine monophosphate only, or with thiamine and glucosamine. These results permit the conclusion that glucosamine increases the utilization of the vitamin and its penetration into the cells.