Isocitrate dehydrogenase from Drosophila melanogaster was purified to apparent homogeneity (340-fold) using ammonium sulfate fractionation and Matrex Red A affinity chromatography. Substrate kinetics (Km) of IDH were determined to be 29.8 ± 7.9 µM for isocitrate and 1.05 ± 0.1 µM for NADP+. IDH activity has an absolute requirement for a divalent metal cofactor.
Native polyacrylamide gel electrophoresis indicated the presence of three active forms of IDH with molecular sizes of 103 kDa, 97 kDa, and 90 kDa. SDS polyacrylamide gel electrophoresis revealed two subunits of the enzyme. These subunits had molecular weights of 65 kDa and 51 kDa. A model for the genetic control of IDH activity in Drosophila melanogaster is proposed based on the data of this experiment and genetic data for two Idh loci.