Purification and properties of a soluble nicotinamide adenine dinucleotide-linked isocitrate dehydrogenase from Crithidia fasciculata.
A soluble NAD+-linked isocitrate dehydrogenase has been isolated from Crithidia fasciculata. The enzyme was purified 128-fold, almost to homogeneity, and was highly specific for NAD+ as the coenzyme. There is also a cytoplasmic NADP+-linked and a mitochondrial isocitrate dehydrogenase in the organism. Studies of the physical and kinetic properties of the soluble NAD+-isocitrate dehydrogenase from this organism showed that it resembled microbial NADP+-isocitrate dehydrogenases in general, all of which are cytoplasmic enzymes. The enzyme appeared not to be related to other NAD+-isocitrate dehydrogenases, which are found in the mitochondria of eukaryotic cells. The molecular weight of the soluble NAD+-isocitrate dehydrogenase was 105,000 which is within the range of the values for microbial NADP+-isocitrate dehydrogenases. Similar to the NADP+-isocitrate dehydrogenase in this organism, the enzyme was inhibited in a concerted manner by glyoxalate plus oxalacetate. Kinetic analysis revealed that Mn2+ was involved in the binding of isocitrate to the enzyme. Inhibition of the NAD+-linked isocitrate dehydrogenase by p-chloromercuribenzoate could be prevented by prior incubation of the enzyme with both Mn2+ and isocitrate; however, neither ion alone conferred protection. Free isocitrate, free Mn2+, and the Mn2+-isocitrate complex could all bind to the enzyme. Four different mechanisms with respect to the binding of isocitrate to the enzyme were tested. Of these, the formation of the active enzyme-Mn2+-isocitrate complex from (a) the random binding of Mn2+, isocitrate, and the Mn2+-isocitrate complex, or (b) the binding of Mn2+-isocitrate with free Mn2+ and isocitrate acting as dead-end competitors were both in agreement with these data.
D000818:Animals; D001665:Binding Sites; D002729:Chloromercuribenzoates / Q000494:pharmacology; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004589:Electrophoresis, Disc; D056890:Eukaryota / Q000201:enzymology*; D007521:Isocitrate Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D008433:Mathematics; D008970:Molecular Weight; D009243:NAD / Q000494:pharmacology*; D011485:Protein Binding; D013997:Time Factors
D002729:Chloromercuribenzoates; D009243:NAD; D008345:Manganese; D007521:Isocitrate Dehydrogenase