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Characterization of NADP + ‐isocitrate dehydrogenase from the host plant cytosol of lucerne ( Medicago sativa ) root nodules
Author(s) -
Henson Cynthia A.,
Duke Stanley H.,
Collins Michael
Publication year - 1986
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1986.tb05052.x
Subject(s) - isocitrate dehydrogenase , cytosol , idh1 , divalent , enzyme , biochemistry , dehydrogenase , glutamine , biology , chemistry , amino acid , organic chemistry , mutation , gene
NADP + ‐isocitrate dehydrogenase (EC 1.1.1.42) was purified more than 1500‐fold from the host‐plant cytosol of Medicago sativa L. cv. Saranac root nodules by ion exchange and affinity chromatography. The forward reaction was characterized. The enzyme exhibited an absolute requirement for a divalent cation (preferably Mn 2+ ), had a broad activity optimum from pH 7.5 to 9.0, and was most stable at pH 7.5. The apparent Arrhenius energy of activation was 70.7 kJ mol −1 (20 to 30°C) indicating that the reaction rate of the enzyme was relatively sensitive to temperature. The K m for isocitrate was 20 μ M and for NADP + 10.7 μ M . Initial velocity and end product inhibition studies of the forward reaction indicate a random bi ter mechanism. End product studies indicated that NADPH was a competitive inhibitor and α‐ketoglutarate was a non‐competitive inhibitor with respect to isocitrate and NADP + . Citrate was a competitive inhibitor with respect to isocitrate. Glutamine was identified as a positive effector when assays were conducted at non‐saturating isocitrate concentrations. The potential significance of glutamine regulation of α‐ketoglutarate production in a dinitrogen‐fixing tissue is discussed.