Kinetic mechanisms of mitochondrial carriers catalysing exchange reactions.

The single-binding site or ping-pong mechanism is widely accepted for exchange reactions, catalysed by mitochondrial carriers. However, when the most relevant approach to discriminate between mechanisms, i.e., kinetic study is used, the ping-pong mechanism is eliminated in favour of the sequential or ternary complex mechanism implying two binding sites simultaneously accessible to both internal and external substrates. This is the case for the oxoglutarate carrier, the aspartate/glutamate carrier and there are very strong presumptive evidences for the adenylic carrier.

Purification and kinetic mechanism of 5,10-metliyienetetrahydrofolate reductase from sheep liver.

5,10-Methylenetetrahydrofolate reductase (EC 1.1.1.68) was purified from the cytosolic fraction of sheep liver by (NH4)2 SO4 fractionation, acid precipitation, DEAE-Sephacel chromatography and Blue Sepharose affinity chromatography. The homogeneity of the enzyme was established by sodium dodecyl sulphatepolyacrylamide gel electrophoresis, ultracentrifugation and Ouchterlony immunodiffusion test. The enzyme was a dimer of molecular weight 1,66,000 ± 5,000 with a subunit molecular weight of 87,000 ±5,000. The enzyme showed hyperbolic saturation pattern with 5-methyltetrahydrofolate. K0.5 values for 5-methyltetrahydrofolate menadione and NADPH were determined to be 132 μΜ, 2.45 μΜ and 16 μΜ. The parallel set of lines in the Lineweaver-Burk plot, when either NADPH or menadione was varied at different fixed concentrations of the other substrate; non-competitive inhibition, when NADPH was varied at different fixed concentrations of NADP; competitive inhibition, when menadione was varied at different fixed concentrations of NADP and the absence of inhibition by NADP at saturating concentration of menadione, clearly established that the kinetic mechanism of the reaction catalyzed by this enzyme was ping-pong.