Yeast diphosphopyridine nucleotide specific isocitrate dehydrogenase. Purification and some properties

Abstract

Yeast diphosphopyridine nucleotide specific isocitrate dehydrogenase (EC 1.1.1.41) has been purified to homogeneity by the criteria of disc gel electrophoresis, sedimentation velocity, and sedimentation equilibrium. Purification of the enzyme is facilitated by preferentially eluting the enzyme from ion-exchange columns with citrate, a modifier of the enzyme. Citrate also stabilizes the enzyme during isolation and preserves the kinetic cooperativity with respect to isocitrate. The enzyme has a n apparent molecular weight of 3 X 105 as determined by sedimentation equilibrium. A subunit molecular

Subunit structure, expression, and function of NAD(H)-specific isocitrate dehydrogenase in Saccharomyces cerevisiae

“…The purified yeast enzyme retains the property of positive allosteric regulation of AMP in the presence of subsaturating concentrations of isocitrate as originally reported (1) (data not shown). The molecular weight differences for the subunits were not observed in previous studies of the yeast enzyme (1,15), although similar differences in isoelectric points for the subunits were reported by Illingworth (15). We observed the same molecular weights and isoelectric characteristics for the subunits on immunoblots of either total cellular or VOL.…”

“…Also, presumably due to a combination of low levels of glycolytic enzymes and to a dependence on oxidative metabolism, the specific activity of NAD(H)-specific isocitrate dehydrogenase is two-to threefold higher in extracts from Sg7 than in extracts from yeast strains lacking the gcrl mutation. A rapid and reproducible purification scheme was developed based on techniques described by Barnes et al (1), including phosphocellulose and DEAE-cellulose chromatography followed by Bio-Gel A-1.5m gel filtration. These steps resulted in a 200-fold purification and yielded approximately 5 mg of the enzyme per 100 g of yeast cells ( Table 1).…”

Suppression of metabolic defects of yeast isocitrate dehydrogenase and aconitase mutants by loss of citrate synthase

“…For derivatization, 10 µl samples of clarified supernatants or of standard metabolite mixtures (including TCA cycle intermediates plus aspartate and glutamate at concentrations ranging from 0-2.0 mM) were diluted with 10 µl of water containing labeled internal standards (1.0 mM [1,[5][6][7][8][9][10][11][12][13] Laboratories, respectively) and dried in a microfuge for 3 h using a Speed-Vac. The dry residues were dissolved in 50 µl of 20 mg/ml O-ethylhydroxylamine hydrochloride (Sigma-Aldrich) in pyridine, and reacted for 90 min at 30°C.…”

“…The enzyme is subject to extensive allosteric control, and the reaction is essentially irreversible under physiological conditions [1,2]. Yeast IDH is an octamer composed of four catalytic IDH2 subunits and four homologous regulatory IDH1 subunits [3,4].…”

NAD+-Linked Isocitrate Dehydrogenase: Isolation, Purification, and Characterization of the Protein from Pea Mitochondria

“…This isoform appears to be exclusively mitochondrial and has been more difficult to work with than the NADP+ enzyme. The enzyme has been purified from several eucaryotes where it appears to be an octamer of 39-kD monomers in yeast (1), 41-kD monomers in beef heart (14), and 39-and 46-kD monomers in blowfly flight muscle (22) mitochondria. The enzyme from beef heart mitochondria was also observed in higher molecular mass forms apparently made up of two and four octamers (14).…”