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Thiamin pyrophosphate was synthesized in 71% yield, on a multi-milligram scale, using overexpressed thiazole kinase, pyrimidine kinase, thiamin phosphate synthase, and thiamin phosphate kinase. This provides a facile route to isotopically labeled thiamin pyrophosphate from its readily available pyrimidine and thiazole precursors.
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We show here that TPP --> TMP conversion can take place in rat liver mitochondria. This occurs via the novel, putative TPP pyrophosphatase localised in the mitochondrial matrix, as shown both by digitonin titration and by an HPLC enzyme assay carried out on the mitochondrial matrix fraction. Certain features of the reaction, including the substrate and pH dependence, are reported. Additional evidence is given that externally added TMP can cross the mitochondrial membrane in a manner consistent with the occurrence of a carrier-mediated process. This can occur both via the TPP translocator and via a novel translocator, inhibited by CAT but different from the ADP/ATP carrier.
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The growth of a thiamine pyrophosphate auxotroph of Escherichi coli was inhibited by either thiamine or thiamine monophosphate, and the growth of a thiamine monophosphate auxotroph was inhibited by thiamine. The thiamine pyrophosphate-dependent oxidation of pyruvate was inhibited by thiamine with whole cells of the thiamine pyrophosphate auxotroph but not with cell extracts prepared from the same organism. In addition, the thiamine pyrophosphate uptake of the thiamine pyrophosphate auxotroph was inhibited by either thiamine or thiamine monophosphate. Although the thiamine pyrophosphate uptake of a revertant, selected for prototrophy from the thiamine monophosphate auxotroph, was inhibited by thiamine to an extent comparable to that observed with the thiamine monophosphate auxotroph, its growth was no longer inhibited by thiamine. A possible mechanism for the inhibition by thiamine and thiamine monophosphate in the utilization of thiamine pyrophosphate is discussed.
Metal complexes of thiamine pyrophosphate (TPP) of the general formula [M2(TPPH)2Cl2]x4H2O (M = Zn2+, Cd2+) were isolated from methanolic solutions and characterized by elemental analysis, FT-IR, and multinuclear NMR spectroscopies. The data provide evidence for the bonding of the metals to the N(1') atom of the pyrimidine ring and to the pyrophosphate group. The stability constant measurements of TPP and 2-(alpha-hydroxyethyl)thiamine pyrophosphate (HETPP) metal complexes in aqueous solution imply the formation of dimeric complex species similar to the isolated solid products. They indicate also that HETPP forms more stable metal complexes than does TPP. To evaluate the coenzyme action of TPP and HETPP metal complexes, enzymic studies have been done using pyruvate decarboxylase apoenzyme. TPP metal complexes do not bind to the apoenzyme, unlike the Zn(II)-HETPP complex which can act as coenzyme. Considering these results, possible functional implications for thiamine involvement in catalysis are discussed.
Two types of mutants of Escherichia coli were isolated, one of which (mutant 70-23-107) responded to thiamine pyrophosphate, and the other (mutant 70-23-102) to thiamine monophosphate and thiamine pyrophosphate. They were produced by further mutation of a thiamine auxotroph of E. coli 70-23 with N-methyl-N'-nitro-N-nitrosoguanidine. The parent organism required thiamine because phosphohydroxymethylpyrimidine kinase activity was lacking in this organism, and hydroxymethylpyrimidine pyrophosphate was not permeable through the cell membrane of E. coli. Thiamine, thiamine monophosphate, and thiamine pyrophosphate were all equally active for the parent, whereas mutants 70-23-102 and 70-23-107 lost their ability to grow on thiamine. Both mutants differed only in the growth response to thiamine monophosphate: the former could grow on thiamine monophosphate, whereas the latter could not. Experimental results with the newly isolated mutants indicate that in E. coli the free form of thiamine is not involved in de novo synthesis of thiamine pyrophosphate, but thiamine monophosphate, an exclusive product formed by the reaction between hydroxymethylpyrimidine pyrophosphate and hydroxyethylthiazole monophosphate, is directly phosphorylated to form thiamine pyrophosphate. Exogenous thiamine, on the other hand, is converted to thiamine pyrophosphate via the intermediate formation of thiamine monophosphate.
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Thiamine pyrophosphate-ATP phosphoryltransferase, the enzyme that catalyzes the synthesis of thiamine triphosphate, has been found in the supernatant fraction of rat liver. The substrate for the enzyme is endogenous, bound thiamine pyrophosphate, since the addition of exogenous thiamine pyrophosphate had no effect. Thus, when a rat liver supernatant was incubated with gamma-labelled [32P]ATP, thiamine [32P]triphosphate was formed whereas the incubation of thiamine [32P]pyrophosphate with ATP did not produce thiamine [32P]triphosphate. The endogenous thiamine pyrophosphate was found to be bound to a high molecular weight protein which comes out in the void volume of Sephadex G-75, and is not dialyzable. The activity that catalyzes the formation of thiamine triphosphate has an optimum pH between 6 and 6.5, a linear time course of thiamine triphosphate synthesis up to 30 min, and is not affected by Ca2+, cyclic GMP and sulfhydryl reagents.
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Thiamin pyrophosphate transport into rat hepatocytes has been studied using TPP-beta-33P and 14C-TPP-beta-33P synthesized in the thiamin pyrophosphokinase reaction. Thiamin pyrophosphate (cocarboxylase) did not penetrate across cytoplasmic membrane.
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Thiamin pyrophosphate was added to human plasma and serum. After various incubations, aliquots were assayed by the thiochrome method. This revealed that fresh human plasma dephosphorylated thiamin pyrophosphate by a reaction that was time and temperature dependent and inhibited by the presence of ethylenediaminetetraacetic acid (EDTA). Fresh plasma did not dephosphorylate pyridoxal-5-phosphate nor the protein bound thiamin pyrophosphate of red cells.
Thiamin status in patients with an alcohol problem was studied before and after intramuscular thiamin hydrochloride. Results for erythrocyte transketolase activity and plasma thiamin pyrophosphate are compared. Plasma thiamin pyrophosphate values for healthy human subjects are reported for the first time. Advantages of plasma thiamin pyrophosphate in the assessment of thiamin status of patients are discussed.
The fact that thiamine pyrophosphate is synthesized in cytosol necessitates its uptake into mitochondria. The ability of mitochondria to take up externally added thiamine pyrophosphate was investigated by measuring the intramitochondrial thiamine pyrophosphate content using an enzymatic method. Thiamine pyrophosphate uptake by isolated rat liver mitochondria was found to occur in a time- and temperature-dependent manner. Uptake shows saturation characteristics with Km and Vmax values equal to about 20 microM and 700 pmol/min x mg protein, respectively, and is inhibited by certain nonpenetrating compounds. The inhibition of thiamine uptake by thiamine pyrophosphate and the efflux of endogenous thiamine pyrophosphate, caused by externally added thiamine, suggest the existence of a thiamine pyrophosphate/thiamine antiporter which could play an active role in the turnover of intramitochondrial thiamine pyrophosphate linked enzymes.
The thiamin pyrophosphate effect has been used as a reliable index to evaluate the nutritional status of thiamin. But there has not been any report concerning whether or not the thiamin pyrophosphate effect really reflects the saturation status of transketolase with thiamin pyrophosphate. In this report we studied the relationship between the thiamin pyrophosphate effect and the saturation status of transketolase. First, we determined the thiamin pyrophosphate concentrations, transketolase activities, thiamin pyrophosphate effects, and transketolase concentrations in human hemolysates from 16 apparently healthy subjects. The molar ratio of thiamin pyrophosphate to transketolase was in inverse proportion to the thiamin pyrophosphate effect. Second, we prepared apotransketolase preparations and reconstituted it with various concentrations of thiamin pyrophosphate. The thiamin pyrophosphate effects in these preparations were in good correspondence with the ratios of apotransketolase. These results indicate that the thiamin pyrophosphate effect really reflects the saturation status of transketolase with coenzyme.