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PubMed1989

Pyruvate:NADP+ oxidoreductase from Euglena gracilis: the kinetic properties of the enzyme.

The kinetic properties for the native forward reaction of pyruvate:NADP+ oxidoreductase from Euglena gracilis were determined. The substrate kinetics gave a pattern of a ping-pong mechanism involving a competitive substrate inhibition of CoA against pyruvate. The Km values for pyruvate, CoA, and NADP+ were estimated to be 27, 6.6, and 28 microM, respectively, and the Ki value of CoA against pyruvate was 28 microM. CO2 inhibited noncompetitively against pyruvate and NADP+, and uncompetitively against CoA. Acetyl-CoA showed a competitive inhibition with respect to pyruvate and an uncompetitive inhibition with respect to NADP+. NADPH inhibited competitively versus NADP+, noncompetitively versus CoA, and uncompetitively versus pyruvate. The kinetic behavior is consistent with a two-site ping-pong mechanism involving the substrate inhibition. From the kinetic mechanism, it is proposed that the enzyme has two catalytic sites linked by an intramolecular electron-transport chain. One of these is a thiamine pyrophosphate-containing catalytic site which reacts with pyruvate and CoA to form CO2 and acetyl-CoA, and the other site functions in the reduction of NADP+. In contrast, when methyl viologen was used as an artificial one-electron acceptor substituting for NADP+, the reaction gave a pattern characteristic of an octa uni ping-pong mechanism involving a competitive substrate inhibition of CoA against pyruvate.

Animals↗
PubMed1984

Some properties of the thiamine uptake system in isolated rat hepatocytes.

A kinetic study of [14C]thiamine uptake over a concentration range from 0.1 microM to 4 mM was performed in isolated rat hepatocytes. The results showed that two processes contribute to the entry in rat hepatocytes: a low affinity process with a Kt of 34.1 microM and Vmax of 20.8 pmol/10(5) cells per 30 s and a high affinity process with a Kt of 1.26 microM and Vmax of 1.21 pmol/10(5) cells per 30 s. The uptake of thiamine by the high affinity process was concentrative and reduced in a betaine medium or K+ medium. Both ouabain and 2,4-dinitrophenol decreased the thiamine uptake by the high affinity process. These findings indicate that the transport of thiamine via a high affinity process is dependent on Na+ and biological energy. The uptake of thiamine was strongly inhibited by thiamine analogs such as dimethialium and chloroethylthiamine. Among quarternary ammonium compounds other than thiamine derivatives, choline and acetylcholine significantly inhibited thiamine uptake by rat liver cells, whereas betaine and carnitine did not. A kinetic study of thiamine uptake by rat hepatocytes preloaded with pyrithiamine, a potent inhibitor of thiamine pyrophosphokinase, revealed that the biphasic property of thiamine uptake disappeared and a single carrier system for thiamine with a Kt of 40.5 microM, which was similar to the Kt value of the low affinity process, was retained. These results strongly suggest that thiamine transport system in rat liver cells is closely connected with thiamine pyrophosphokinase, which accelerates the uptake rat of thiamine by pyrophosphorylation at physiological concentrations of thiamine.

2,4-Dinitrophenol↗
PubMed1978

Affinity chromatography studies with the pyruvate dehydrogenase complex of wild-type Escherichia coli.

1. The lifetime of thiamine pyrophosphate-Sepharose 2B affinity matrices synthesized according to Matsuura et al. (Matsuura, A., Iwashina, A. and Nose, Y. (1973) Biochem. Biophys. Res. Commun. 51, 241-246) has been improved. The matrix interacts with bacterial pyruvate dehydrogenase complexes. 2. The synthesis of a stable thiochrome-Sepharose 2B matrix is described. 3. Both matrices bind the pyruvate dehydrogenase complex of Escherichia coli in a 50 mM phosphate buffer, pH 7.0. Elution is possibly by an increase in ionic strength but not by the cofactor or metal-cofactor complexes. 4. The presence of Mg2+, reduces the capacity of the affinity matrices but leads to higher specificity for the multienzyme complex. 5. The pyruvate dehydrogenase complex of E. coli has been successfully purified by combining a classical purification step with these affinity chromatography systems. The method is less suitable for large scale operation.

Chromatography, Affinity↗