Purification of yeast isocitrate dehydrogenase

The NAD-linked isocitrate dehydrogenase from baker's yeast was purified to homogeneity (as judged by gel filtration and polyacrylamide-gel electrophoresis) with an overall yield of 50% by using dilute solutions of the allosteric effector (AMP) to elute the enzyme specifically from CM-cellulose. This method preserves the allosteric properties of the crude enzyme. Although the pure enzyme shows only a single band on electrophoresis in the presence of sodium dodecyl sulphate, two types of subunit are observed in 8m-urea. The isoelectric point of the enzyme rises during purification, and this may reflect the partial loss of an additional low-molecular-weight component. Values are included for the amino acid composition and extinction coefficients of the pure enzyme.     

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

Mitochondrial NAD(H)-specific isocitrate dehydrogenase was purified from Saccharomyces cerevisiae for analyses of subunit structure and expression. Two subunits of the enzyme with different molecular weights (39,000 and 40,000) and slightly different isoelectric points were resolved by denaturing electrophoretic techniques. Sequence analysis of the purified subunits showed that the polypeptides have different amino termini. By using an antiserum to the native enzyme prepared in rabbits, subunit-specific immunoglobulin G fractions were obtained by affinity purification, indicating that the subunits are also immunochemically distinct. The levels of NAD(H)-specific isocitrate dehydrogenase activity and immunoreactivity were found to correlate closely with those of a second tricarboxylic acid cycle enzyme, malate dehydrogenase, in yeast cells grown under a variety of conditions. S. cerevisiae mutants with defects in NAD(H)-specific isocitrate dehydrogenase were identified by screening a collection of yeast mutants with acetate-negative growth phenotypes. Immunochemical assays were used to demonstrate that one mutant strain lacks the 40,000-molecular-weight subunit (IDH1) and that a second strain lacks the 39,000-molecular-weight subunit (IDH2). Mitochondria isolated from the IDH1 and IDH2 mutants exhibited a markedly reduced capacity for utilization of either isocitrate or citrate for respiratory O2 consumption. This confirms an essential role for NAD(H)-specific isocitrate dehydrogenase in oxidative functions in the tricarboxylic acid cycle.     

A Klebsiella pneumoniae plasmid which codes for isocitrate dehydrogenase

Abstract A naturally occurring Klebsiella pneumoniae plasmid of 114 MDa was able to complement isocitrate dehydrogenase deficiencies in K. pneumoniae and Salmonella typhimurium . The plasmid encoded isocitrate dehydrogenase, which differed from that of S. typhimurium in the kinetic parameters for NADP and isocitrate, in its isoelectric point, and in its response to repression by cAMP. This is the first report of a naturally occurring plasmid encoding a Krebs cycle enzyme.     

Binding of ampholine to transfer RNA.

The melting temperature of isoaccepting tRNAfMet is affected by Ampholine. The plot of Tm versus the logarithm of Ampholine concentration shows clearly an increasing effect of Ampholine when the pH changes from 7.4 to 4.2. This result is interpreted as binding of Ampholine to the nucleic acid. The effects of Ampholine have been compared with those of soidum, magnesium and tetraethylene pentamine. Ampholine carrier ampholytes at pH 4.2 bind to tRNA with the same affinity as magnesium; at higher pH values they are less active. An hypothesis for the mechanism of action of Ampholine on nucleic acids during isoelectric focusing is proposed.     

Purification of mitochondrial NADH dehydrogenase from Drosophila hydei and comparison with the 'heat-shock' polypeptides.

Mitochondrial NADH dehydrogenase (NADH:(acceptor) oxidoreductase, EC .6.99.3) from either Drosophila hydei larvae or embryos has been purified 150- and 120-fold, respectively. The purified enzyme appeared homogeneous and showed a molecular weight of 57 000. The molecular weight of the nondenatured enzyme was 79 000. On isoelectro-focussing of the preparation, two fractions were observed, a major one with an isoelectric point of 6.2 and a minor fraction with an isoelectric point of 4.9. Straight-line kinetics in Lineweaver-Burk plots were observed for the purified enzyme with a Km of 0.040 mM. The Km was not changed during the purification procedure, suggesting that the enzyme was not denatured or inactivated. The pH optimum of the purified enzyme was 5.6. The molecular weight of the purified mitochondrial NADH dehydrogenase does not correspond to that of one of the 'heat-shock' polypeptides.     

Purification and properties of NADP(+)-dependent isocitrate dehydrogenase from the corpus luteum

Cytoplasmic NADP(+)-dependent isocitrate dehydrogenase (isocitrate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42) was purified 290-fold from the 15,000 x g supernatant fraction of porcine corpora lutea. The major purification step was by anion-exchange chromatography with an FPLC mono P column. Enzyme lability was overcome by including Mg2+, DL-isocitrate, dithiothreitol and glycerol in the elution buffers. The molecular weight of the denatured enzyme was found to be 48,000 by SDS-polyacrylamide gel electrophoresis. The Stokes' radius was estimated to be 3.7 nm by gel filtration and the isoelectric point was 4.8 as determined by chromatofocusing. The purified enzyme had a specific activity of 57.8 units/mg and a broad optimal pH for activity from 7.5 to 9.0. The Km for the substrates DL-isocitrate and NADP+ were 13 and 12 microM, respectively. Polyclonal antibodies were raised against the purified enzyme. Protein (Western) blotting showed an immunological similarity between the cytoplasmic enzyme of the ovary, liver, adrenal gland and heart. A difference was demonstrated between the ovarian enzyme and the heart mitochondrial enzyme. The substrate turnover number and Mr of the ovarian enzyme were similar to those found for the enzyme from the liver and adrenal gland.     

Aggregation of ampholine on heparin and other acidic polysaccharides in isoelectric focusing.

The mechanism of complexation of pI range 3.5--5 Ampholine to heparin in isoelectric focusing has been explored by the dye-binding technique at different pH values in solution. There is no significant interaction between heparin and Ampholine at pH 6.7. Weak, or selective, binding occurs at pH 5.1, and very strong interaction at pH 3.5. In the latter system, the Ampholine components appear to behave as polycations due to their ordered sequence of positive charges, each two methylene groups apart, which favors a strong binding to polyanions. In addition, there appear to be variable stoichiometries for the strong binding between heparin and Ampholine, depending on their relative amounts. It is proposed that at a low ratio of heparin to Ampholine (Ampholine excess), aggregation is perpendicular to the heparin chain, with the end ammonium charge of each Ampholine molecule neutralizing one negative charge along the heparin molecule; at higher ratios (heparin excess), the bound Ampholine segment is aligned parallel to the heparin molecule, so that on the average one Ampholine component neutralizes approx. three negative charges. The banding of heparin in isoelectric focusing in the pH range 3.0--4.5 can be explained by aggregation of the various components on heparin in amounts dependent upon the net charge on the Ampholine species at the given pH, and upon the changing stoichiometries as a function of the variation in ratio of heparin to Ampholine along the pH gradient. Binding of Ampholine to polygalacturonate was also demonstrated in excess Ampholine in a pH range dependent on the degree of protonation of the carboxyl groups of this acidic polysaccharide as well as on the net positive charge of the Ampholine. The aggregation seen at pH 4.2--4.5 led to the prediction and subsequent demonstration that polygalacturonate would also exhibit binding upon isoelectric focusing. This supports the hypothesis that aggregation of Ampholine on polyanions having sufficient charge density is a general phenomenon which can lead to spurious banding of certain polymers at appropriate pH ranges in isoelectric focusing. On the basis of their behavior in isoelectric focusing at pH 3.0--4.5, strength of aggregation of the polyanions studied appears to be heparin A = heparin B greather than polyglutamate greater than carboxyl-reduced heparin B greater than polygalacturonic acid.     

Purification and Characterization of Cytosolic NADP Specific Isocitrate Dehydrogenase from Pisum sativum

Cytosolic NADP-specific isocitrate dehydrogenase was isolated from leaves of Pisum sativum. The purified enzyme was obtained by ammonium sulfate fractionation, ion exchange, affinity, and gel filtration chromatography. The purification procedure yields greater than 50% of the total enzyme activity originally present in the crude extract. The enzyme has a native molecular weight of 90 kilodaltons and is resolved into two catalytically active bands by isoelectric focusing. Purified NADP-isocitrate dehydrogenase exhibited K_m values of 23 micromolar for dl-isocitrate and 10 micromolar for NADP, and displayed optimum activity at pH 8.5 with both Mg²⁺ and Mn²⁺.     

The isolation, purification, and some properties of NAD-dependent isocitrate dehydrogenase from the organic acid-producing yeast Yarrowia lipolytica

The NAD+-dependent isocitrate dehydrogenase of the organic acid-producing yeast Yarrowia lipolytica was isolated, purified, and partially characterized. The purification procedure included four steps: ammonium sulfate precipitation, acid precipitation, hydrophobic chromatography, and gel-filtration chromatography. The enzyme was purified 129-fold with a yield of 31% and had a specific activity of 22 U/mg protein. The molecular mass of the enzyme was found to be 412 kDa. The enzyme consists of eight identical subunits with a molecular mass of about 52 kDa. The Km for NAD+ is 136 microM, and that for isocitrate is 581 microM. The effect of some intermediates of the citric acid cycle and nucleotides on the enzyme activity was studied. The role of isocitrate dehydrogenase (NAD+) in the overproduction of citric and keto acids is discussed.     

NADP+-specific isocitrate dehydrogenase of Excherichia coli. III. Two-step purification employing affinity chromatography.

The NADP+-specific isocitrate dehydrogenase (threo-DS-isocitrate:NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42) of Excherichia coli has been purified to electrophoretic homogeneity by a two-step purification procedure employing affinity chromatography. The overall yield of enzyme was 30% with specific activity 125 mumol/min per ng protein. Electrophoretic homogeneity of the isocitrate dehydrogenase was deterimed in analytical polyacrylamide gels in a Tris/acetate/EDTA buffer system at pH 7.5 and in a citrate/phosphate buffer system at pH 6.0.     

The purification and physicochemical characterization of maize (Zea mays L.) isocitrate lyase.

A purification scheme is described for the glyoxylate cycle enzyme isocitrate lyase from maize scutella. Purification involves an acetone precipitation and a heat denaturation step, followed by ammonium sulfate precipitation and chromatography on DEAE-cellulose and on blue-Sepharose. The latter step results in the removal of the remaining malate dehydrogenase activity, and of a high molecular mass (62 kDa) but inactive degradation product of isocitrate lyase. Catalase can be completely removed by performing the DEAE-cellulose chromatography in the presence of Triton X-100. Pure isocitrate lyase can be stored without appreciable loss of activity at -70 degrees C in 5 mM triethanolamine buffer containing 6 mM MgCl2, 7 mM 2-mercaptoethanol, and 50% (v/v) glycerol, pH 7.6. Maize isocitrate lyase is a tetrameric protein with a subunit molecular mass of 64 kDa. Purity of the enzyme preparation was demonstrated by polyacrylamide gel electrophoresis in the presence of dodecylsulfate, in acid (pH 3.2) urea and by isoelectric focusing (pI = 5.1). Maize isocitrate lyase is devoid of covalently linked sugar residues. From circular dichroism measurements we estimate that its structure comprises 30% alpha-helical and 15% beta-pleated sheet segments. The enzyme requires Mg2+ ions for activity, and only Mn2+ apparently is able to replace this cation to a certain extent. The kinetics of the isocitrate lyase-catalyzed cleavage reaction were investigated, and the amino acid composition of the maize enzyme was determined. Finally the occurrence of an association between maize isocitrate lyase and catalase was observed. Such a multienzyme complex may be postulated to play a protective role in vivo.     

A novel biotin protein required for reductive carboxylation of 2-oxoglutarate by isocitrate dehydrogenase in Hydrogenobacter thermophilus TK-6

Isocitrate dehydrogenase was purified from Hydrogenobacter thermophilus, and the corresponding gene was cloned and sequenced. The enzyme had similar structural properties to the isocitrate dehydrogenase of Escherichia coli, but differed in its catalytic properties, such as coenzyme specificity, pH dependency and kinetic parameters. Notably, the enzyme catalysed the oxidative decarboxylation of isocitrate, but not the reductive carboxylation of 2-oxoglutarate. The carboxylation reaction required the addition of cell extract and ATP-Mg, suggesting the existence of additional carboxylation factor(s). Further analysis of the carboxylation factor(s) resulted in the purification of two polypeptides. N-terminal amino acid sequencing revealed that the two polypeptides are homologues of pyruvate carboxylase with a biotinylated subunit, but do not catalyse pyruvate carboxylation. Pyruvate carboxylase was also purified, but was not active in stimulating isocitrate dehydrogenase. Isocitrate dehydrogenase, the novel biotin protein, ATP-Mg and NADH were essential for the reductive carboxylation of 2-oxoglutarate. These observations indicate that the novel biotin protein is an ATP-dependent factor, which is involved in the reverse (carboxylating) reaction of isocitrate dehydrogenase.     

Effect of pH on the inhibition of the nicotinamide–adenine dinucleotide-specific isocitrate dehydrogenase from baker''s yeast by anions

1. The sensitivity of the NAD(+)-specific isocitrate dehydrogenase from baker's yeast towards inhibition by anions decreases with decrease in pH. The patterns of the pH-dependence of the enzymic activity can be explained by this effect. 2. In the presence of a high isocitrate concentration, citrate, unlike AMP, has no antagonizing effect on the inhibition of the enzyme by anions. In the presence of AMP, citrate inhibits the enzyme at high isocitrate concentration and activates at low isocitrate concentration. 3. The effects on the enzymic activity of the previous incubation of the enzyme were studied in relation to the substrate concentration, the chloride concentration and the presence of citrate and AMP.     

Shifted Cytosolic NADP+-Dependent Isocitrate Dehydrogenase on 2-D Gel in the Brain of Genetically Epileptic El Mice

We observed a spot on two-dimensional (2-D) gel in the epileptic mutant strain El mice with a similar molecular weight but with a different isoelectric point of approximately 0.2, compared with its mother strain ddY mice. The collected protein from the El mice was identified as cytosolic NADP⁺-dependent isocitrate dehydrogenase by internal amino acid sequencing. The enzyme is known to be maximally active during the development of the brain and to play an important role in NADPH production for fatty acids and cholesterol synthesis. In addition, alterations in cholesterol synthesis early in the development of the mammalian brain have been reported to lead to chronic epilepsy. The results in the present study therefore suggest that cytosolic NADP⁺-dependent isocitrate dehydrogenase might be involved in the epileptogenesis of the El mouse.     

Purification and properties of NADP-isocitrate dehydrogenase from the unicellular cyanobacterium Synechocystis sp. PCC 6803.

NADP-dependent isocitrate dehydrogenase activity has been screened in several cyanobacteria grown on different nitrogen sources; in all the strains tested isocitrate dehydrogenase activity levels were similar in cells grown either on ammonium or nitrate. The enzyme from the unicellular cyanobacterium Synechocystis sp. PCC 6803 has been purified to electrophoretic homogeneity by a procedure that includes Reactive-Red-120-agarose affinity chromatography and phenyl-Sepharose chromatography as main steps. The enzyme was purified about 600-fold, with a yield of 38% and a specific activity of 15.7 U/mg protein. The native enzyme (108 kDa) is composed of two identical subunits with an apparent molecular mass of 57 kDa. Synechocystis isocitrate dehydrogenase was absolutely specific for NADP as electron acceptor. Apparent Km values were 125, 59 and 12 microM for Mg2+, D,L-isocitrate and NADP, respectively, using Mg2+ as divalent cation and 4, 5.7 and 6 microM for Mn2+, D,L-isocitrate and NADP, respectively, using Mn2+ as a cofactor. The enzyme was inhibited non-competitively by ADP (Ki, 6.4 mM) and 2-oxoglutarate, (Ki, 6 mM) with respect to isocitrate and in a competitive manner by NADPH (Ki, 0.6 mM). The circular-dichroism spectrum showed a protein with a secondary structure consisting of about 30% alpha-helix and 36% beta-pleated sheet. The enzyme is an acidic protein with an isoelectric point of 4.4 and analysis of the NH2-terminal sequence revealed 45% identity with the same region of Escherichia coli isocitrate dehydrogenase. The aforementioned data indicate that NADP isocitrate dehydrogenase from Synechocystis resembles isocitrate dehydrogenase from prokaryotes and shows similar molecular and structural properties to the well-known E. coli enzyme.     

Kinetic studies of a NADP+-specific isocitrate dehydrogenase from Salmonella typhimurium. Purification and reaction mechanism

The kinetics of a Mn²⁺-requiring, NADP⁺-specific isocitrate dehydrogenase from Salmonella typhimurium have been examined by the measurement of initial velocity rates in the presence and absence of the reaction products. The binding of each of the cosubstrates, isocitrate, and NADP⁺, is not independent of the other, and the isocitrate-Mn²⁺ complex is the kinetically important substrate species. All of the reaction products, α-ketoglutarate, CO₂, and NADPH are competitive with both cosubstrates and the mechanism appears to be of the rapid equilibrium random type. The enzyme has been purified to homogeneity and has an isoelectric point at pH 4.0–4.2, and an apparent molecular weight of 102,000.     

Purification and characterization of NADP+-linked isocitrate dehydrogenase from an alkalophilic Bacillus

We have succeeded in purifying to homogeneity a very labile NADP+-linked isocitrate dehydrogenase (isocitrate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42) from a strain of alkalophilic Bacillus, by a simple method, with an overall yield over 76% of the original activity. The molecular weight on Sephadex G-200 was around 90,000; and that by electrophoresis on SDS-polyacrylamide gels was about 44,000. The sedimentation coefficient (s020,w) and isoelectric point of the enzyme were determined to be 3.22 S and pH 4.7, respectively. The enzyme required Mn2+ for the reaction and for stability. The optimum pH for the reaction was in the range 7.8-8.4 at 30 degrees C; the optimum temperature at pH 8.0 was 75 degrees C; the activation energy of the reaction was 6.2 kcal/mol. The Km values for threo-Ds-isocitrate, DL-isocitrate, and NADP+ were 5.4 microM, 9.9 microM, and 7.3 microM, respectively. This enzyme was inhibited by NADPH, glyceraldehyde 3-phosphate, 3-phosphoglycerate, phosphoenol pyruvate, cis-aconitate, alpha-ketoglutarate, and oxaloacetate. In addition, it was subject to a concerted inhibition by a combination of glyoxylate and oxaloacetate, and also to a cumulative inhibition by nucleoside triphosphates.     

Comparison of malate dehydrogenase isoenzymes in someAllium species by isoelectric focusing

Malate dehydrogenase isoenzymes were studied in tenAllium species and in six cultivars ofA. cepa by isoelectric focusing in polyacrylamide gel with Ampholine pH 3.5–10.0. Using this method better resolution was obtained than by polyacrylamide gel electrophoresis. The number of MDH isoenzymes obtained by isoelectric focusing is from five to ten in the range of pH 3.65 to 6.75. MDH isoenzymes can be used for characterization on the level of species and cultivars (inA. cepa), but its use on the level of sections and subgenera is questionable.     

Proteins of bacterial membranes. Purification of NADH-dehydrogenase by electrofocusing]

A highly purified preparation of NADH dehydrogenase was isolated from bacteria M. lysodeikticus membranes. The purification procedure involved extraction of the enzyme complex from isolated membranes by EDTA, solubilization of the complex by non-ionogenic detergent (1% Triton X-100), chromatography on DEAE-cellulose and electrofocussing in the pH gradient 4-6. The isoelectric point of the preparation is at 4.5; its main component is a protein with m.w. of about 76.000.     

Isolation of L-dynurenine 3-hydroxylase from the mitochondrial outer membrane of rat liver.

Outer membrane preparations of rat liver mitochondria were isolated, after the mitochondria had been prepared by mild digitonin treatment under isotonic conditions. L-Kynurenine 3-hydroxylase [EC 1.14.13.9] was solubilized on a large scale from outer membrane by mixing with 1% digitonin or 1% Triton X-100, followed by fractionation into a minor fraction I and a major fraction II by DEAE-cellulose column chromatography. The distribution of total L-Dynurenine 3-hydroxylase was roughly 20 and 80% in fraction I and II, respectively. Fraction I consisted of crude enzyme loosely bound to anion exchanger. In the present investigation, fraction I was not used because of its low activity and rapid inactivation. In contrast, fraction II consisted of crude enzyme with high activity, excluded from DEAE-cellulose column chromatography in the presence of 1 M KC1. In addition, fraction II was purified by Sephadex G-200 gel filtration and DEAE-Sephadex A-50 column chromatography with linear gradient elution, adding 1 M KC1 and 1% Triton X-100 to 0.05 M Tris-acetate buffer, pH 8.1. After isoelectric focusing, the purified enzyme preparation was proved to be homogeneous, since the L-kynurenine 3-hydroxylase fraction gave a single band on disc gel electrophoresis. The molecular weight of this enzyme was estimated to be approximately 200,000 or more by SDS-polyacrylamide gel electrophoresis and from the elution pattern on Sephadex G-200 gel filtration. A 16-Fold increase of the enzyme activity was obtained compared with that of the mitochondrial outer membrane. The isoelectric point of the enzyme was determined to be pH 5.4 by Ampholine isoelectric focusing.