NADP-linked isocitrate dehydrogenase from beef liver: a new method of purification and the effect of metal ion cofactor on its stability.
1. A more rapid and efficient method of purification of beef liver NADP-linked isocitrate dehydrogenase is reported. The method involves gel filtration, ion-exchange, and affinity chromatography techniques. The enzyme is obtained in pure form with a recovery of 33%. The specific activity is 43 U/mg at 25 degrees C in the presence of Mn2+ ions and 31 U/mg under previously reported conditions and assuming a specific absorption coefficient of 1.29 cm2/mg at 280 nm. Both the yield and the specific activity are higher than the previously reported values. The apparent Km values for isocitrate and NADP+ are 1.7 and 7.3 microM, respectively, in the presence of 0.2 mM MnCl2, from the initial rate data obtained with a sensitive fluorimeter. 2. The stability of the enzyme at 25 degrees C is investigated. The enzyme is rapidly inactivated when preincubated with isocitrate and NADP+, in the absence of metal ion, and it cannot use free isocitrate as substrate.
D000818:Animals; D002417:Cattle; D003067:Coenzymes; D004591:Electrophoresis, Polyacrylamide Gel; D007521:Isocitrate Dehydrogenase / Q000302:isolation & purification*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008670:Metals; D009249:NADP / Q000032:analysis*; D010084:Oxidation-Reduction
D003067:Coenzymes; D008670:Metals; D009249:NADP; D007521:Isocitrate Dehydrogenase