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

Varalakshmi, K and Savithri, HS and Appaji Rao, N (1984) Purification and kinetic mechanism of 5,10-metliyienetetrahydrofolate reductase from sheep liver. In: Journal of Biosciences, 5 (4). pp. 287-299.

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Abstract

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 sulphate-polyacrylamide 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.K 0.5 values for 5-methyltetrahydrofolate menadione and NADPH were determined to be 132 ΜM, 2.45 ΜM and 16 ΜM. 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.

Item Type: Journal Article
Additional Information: Copyright for this article belongs to springer.
Keywords: 5,10-Methylenetetrahydrofolate reductase;purification; ping-pong mechanism
Department/Centre: Division of Biological Sciences > Biochemistry
Date Deposited: 20 Jan 2010 05:56
Last Modified: 19 Sep 2010 05:46
URI: http://eprints.iisc.ernet.in/id/eprint/23691

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