Partial Purification & Kinetic Studies of Nicotinamide Adenine Dinucleotide Phosphate-Specific Isocitrate Dehydrogenase of Phycomyces Blakesleeanus

Publication Year:
1974

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Repository URL:
https://digitalcommons.wku.edu/theses/2622
Author(s):
Meredith, Michael
Tags:
Biology; Life Sciences
artifact description
The nicotinamide adenine dinucleotide phosphate -specific isocitrate dehydrogenase (threo-Ds(+) isocitrate: NADP+: oxidoreductase [decarboxylating]; E.C. 1.1.1.42.) of Phycomyces blakesleeanus was partially purified. The method used to purify the enzyme was a combination of protamine sulphate precipitation, ammonium sulphate fractionation, and ion exchange and gel filtration chromatography. The NADP+ -specific IDH was purified from an initial specific activity of 0.05 units/mg of protein to a final specific activity of 1.5 units/mg protein in solution. Molecular and reaction characteristics were explored. Employing gel filtration the molecular weight of the enzyme was determined to be 88,000. The forward reaction was found to have a pH optimum of 7.5 to 8.5. The pH optimum for the reverse reaction was found to be 6.0. Kinetic studies performed showed the apparent Km's for Mn++. and threo-Ds(+) isocitrate to be 2.45 x 10-4 M, and 1.57 x 10-4 M, respectively. Apparent Km's for Mn++ and NADPH in the reverse reaction were found to be: Mn++, 9.7 x 10-5 M, and NADPH, 1.52 x 10-4 M. Alpha-ketoglutarate did not give a linear Lineweaver-Burk plot. Hill plots for the reverse reaction showed the binding orders for Mn" and NADPH to be 2 and 1, respectively.