Use of a bisubstrate inhibitor to distinguish between isocitrate dehydrogenase isozymes

Bisubstrate inhibitors, obtained by covalently linking 2-oxoglutarate with NAD+ and NADP+, were synthesized and tested for their ability to inhibit NAD+- and NADP+-dependent isocitrate dehydrogenases from pig heart mitochondria. The NADP+-dependent enzyme was specifically inhibited by the NADP oxoglutarate adduct and not by the NAD adduct. The NADP adduct was competitive with both coenzyme and substrate, isocitrate. In contrast, the NAD+-dependent enzyme was inhibited by both adducts. NAD oxoglutarate is competitive with both NAD+ and isocitrate while the NADP adduct is competitive with isocitrate but not with NAD+. Nevertheless conditions could be set up so that use of these inhibitors would be feasible for a metabolic study.     

The role of various amino acid residues in the functioning of NADP-specific isocitrate dehydrogenase from bovine adrenal cortex cytoplasm

The modification of SH-groups in the native isocitrate dehydrogenase accessible to 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) is accompanied by the enzyme inactivation. Isocitrate rather than NADP and MnCl2 protects two SH-groups of the enzyme from modification by DTNB and attendant inactivation. The isocitrate dehydrogenase inactivation by DTNB obeys pseudofirst-order reaction kinetics. The number of DTNB-titrated sulphydryl groups does not change after the isocitrate dehydrogenase denaturation by sodium dodecyl sulphate. In the presence of manganese ions isocitrate and to a lesser extent NADP protect isocitrate dehydrogenase from the inactivation induced by 2,3-butanedione, a specific modifier of arginine residues. It has also been shown that the methylene blue-sensitized photoinactivation of the enzyme associated with the photooxidation of histidine residues decreases in the presence of NADP. These data provide evidence for an essential role of the SH-groups, arginine residues and, probably, histidine in the functioning of NADP-dependent isocitrate dehydrogenase from adrenal cortex.     

Kinetic properties of NADP-specific isocitrate dehydrogenase from bovine adrenals

At low concentrations of Mg2+ or Mn2+ the reaction catalyzed by isocitrate dehydrogenase from bovine adrenal cortex proceeds with a lag period which disappears as a result of the enzyme saturation with Mn2+ or Mg2+. The nu o versus D,L-isocitrate concentration curve is non-hyperbolic, which may be interpreted either by the presence of two active sites with different affinity for the substrate (K'mapp = 2.3 and 63 microM) within the enzyme molecule or by the "negative" cooperativity of these sites. The apparent Km value for NADP lies within the range of 3.6-9 microM. High concentrations of NADP inhibit isocitrate dehydrogenase (Ki = 1.3 mM). NADP.H inhibits the enzyme in a mixed manner with respect to NADP (Ki = 0.32 mM). In the presence of NADP.H the curve nu o dependence on NADP concentration shows a "negative" cooperativity between NADP binding sites. The reverse enzyme-catalyzed reaction of reductive carboxylation of 2-oxoglutarate does not exhibit any significant deviations from the Michaelis-Menten kinetics. The Km value for 2-oxoglutarate is 120 microM, while that for NADP.H is 10 microM.     

Equilibrium binding of coenzymes and substrates to nicotinamide-adenine dinucleotide phosphate-linked isocitrate dehydrogenase from bovine heart mitochondria.

1. The stoicheiometries and affinities of ligand binding to isocitrate dehydrogenase were studied at pH 7.0, mainly by measuring changes in NADPH and protein fluorescence. 2. The affinity of the enzyme for NADPH is about 100-fold greater than it is for NADP+ in various buffer/salt solutions, and the affinities for both coenzymes are decreased by Mg2+, phosphate and increase in ionic strength. 3. The maximum binding capacity of the dimeric enzyme for NADPH, from coenzyme fluorescence and protein-fluorescence measurements, and also for NADP+, by ultrafiltration, is 2 mol/mol of enzyme. Protein-fluorescence titrations of the enzyme with NADP+ are apparently inconsistent with this conclusion, indicating that the increase in protein fluorescence caused by NADP+ binding is not proportional to fractional saturation of the binding sites. 4. Changes in protein fluorescence caused by changes in ionic strength and by the binding of substrates, Mg2+ or NADP+ (but not NADPH) are relatively slow, suggesting conformation changes. 5. In the presence of Mg2+, the enzyme binds isocitrate very strongly, and 2-oxoglutarate rather weakly. 6. Evidence is presented for the formation of an abortive complex of enzyme-Mg2+-isocitrate-NADPH in which isocitrate and NADPH are bound much more weakly than in their complexes with enzyme and Mg2+ alone. 7. The results are discussed in relation to the interpretation of the kinetic properties of the enzyme and its behaviour in the mitochondrion.     

NADP-dependent isocitrate dehydrogenase from bass (Dicentrarchus labrax L.) liver

The isocitrate dehydrogenase from bass liver was purified to homogeneity by gel filtration, affinity and ion exchange chromatographies. The molecular weight was estimated by gel filtration chromatography to about 120,000. Analysis of the enzyme on sodium dodecyl sulphate polyacrylamide gel electrophoresis showed it to be a dimeric protein. The enzyme showed maximum activity in the pH range between 7.0 and 8.0 while its maximum activity was at pH 7.5. DL-Isocitrate and Mn2+ stabilized the enzyme, while NADP had the opposite effect. The Km for isocitrate was 0.31 mM and the Km for NADP was 36 microM.     

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.     

Phosphorus-31 nuclear magnetic resonance studies of the binding of nucleotides to NADP+-specific isocitrate dehydrogenase

The interaction of the 2'-phosphate-containing nucleotides (NADP+, NADPH, 2'-phosphoadenosine 5'-diphosphoribose, and adenosine 2',5'-bisphosphate) with NADP+ -specific isocitrate dehydrogenase was studied by using 31P NMR spectroscopy. The separate resonances corresponding to free and bound nucleotides, characteristic for slow exchange of nuclei on the NMR time scale, were observed in the spectra of the enzyme (obtained in the presence of excess ligand) with NADP+ and NADPH in the absence and presence of Mg2+ and with 2'-phosphoadenosine 5'-diphosphoribose in the absence of metal or in the presence of the substrate magnesium isocitrate. The position of the 31P resonance of the bound 2'-phosphate group in these spectra is invariant (delta = 6) in the pH range 5-8, indicating that the pK of this group is much lower in the complexes with the enzyme than that (pK = 6.13) in the free nucleotides. The additional downfield shift of this resonance by 1.8 ppm beyond that (delta = 4.22) of the dianionic form of the 2'-phosphate in free nucleotides suggests interaction with a positively charged group(s) and/or distortion of P-O-P angles as the result of binding to the enzyme. A single resonance of 2'-phosphate was observed in the spectrum of the enzyme complex with 2'-phosphoadenosine 5'-diphosphoribose in the presence of Mg2+, with the chemical shift dependent on the nucleotide to enzyme ratio, characteristic for the fast exchange situation. Addition of metal does not perturb the environment of the 2'-phosphate in the complexes of NADP+ and NADPH with isocitrate dehydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective action of mycobacillin on the uptake of releasable cell materials by Aspergillus niger.

The inhibition of Escherichia coli isocitrate dehydrogenase by glyoxylate and oxaloacetate was examined. The shapes of the progress curves in the presence of the inhibitors depended on the order of addition of the assay components. When isocitrate dehydrogenase or NADP+ was added last, the rate slowly decreased until a new, inhibited, steady state was obtained. When isocitrate was added last, the initial rate was almost zero, but the rate increased slowly until the same steady-state value was obtained. Glyoxylate and oxaloacetate gave competitive inhibition against isocitrate and uncompetitive inhibition against NADP+. Product-inhibition studies showed that isocitrate dehydrogenase obeys a compulsory-order mechanism, with coenzyme binding first. Glyoxylate and oxaloacetate bind to and dissociate from isocitrate dehydrogenase slowly. These observations can account for the shapes of the progress curves observed in the presence of the inhibitors. Condensation of glyoxylate and oxaloacetate produced an extremely potent inhibitor of isocitrate dehydrogenase. Analysis of the reaction by h.p.l.c. showed that this correlated with the formation of oxalomalate. This compound decomposed spontaneously in assay mixtures, giving 4-hydroxy-2-oxoglutarate, which was a much less potent inhibitor of the enzyme. Oxalomalate inhibited isocitrate dehydrogenase competitively with respect to isocitrate and was a very poor substrate for the enzyme. The data suggest that the inhibition of isocitrate dehydrogenase by glyoxylate and oxaloacetate is not physiologically significant.     

Characterization of NADP-Isocitrate Dehydrogenase from Nodules of Pigeonpea (Cajanus cajan)

NADP-isocitrate dehydrogenase from nodules of pigeonpea (Cajanus cajan L. cv UPAS-120) was partially purified to about 57 folds and its properties were studied. The enzyme showed an absolute requirement for a divalent cation which was fulfilled either by Mn⁺² or Mg⁺² and to a smaller extent by Co⁺². The enzyme exhibited a sigmoidal response to increasing concentrations of Mn²⁺ (S_0.5=0.3mM). The apparent Km values for isocitrate, NADP and Mg²⁺ were 21, 23 and 280 μM, respectively. It had an optimum pH of 8.0–8.2. The enzyme activity was not affected by various organic acids, amino acids and amides. NADH inhibited the activity non-competitively with respect to NADP. An apparent inhibition by ATP and ADP was due to chelation of divalent cation. NADPH acted competitively against NADP and non-competitively against isocitrate. Glutamate caused uncompetitive inhibition with respect to NADP and competitive against isocitrate. Kinetic studies suggested the reaction mechanism to be probably random sequential. Possible regulation of the enzyme activity in the nodules via cellular redox state and the levels of reaction products is discussed.     

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.     

Role of NADPH in the regulation of NADP-specific isocitrate dehydrogenase from pig heart.

The present results show that the NADP specific isocitrate dehydrogenase from pig heart exhibits a time lag before the reaction rate approaches a constant value at low metal ion concentrations. Addition of NADPH or EDTA to the assay mixture abolished the lag, and will under certain conditions activate the enzyme.The lag time increased with increasing concentrations of isocitrate and decreased with increasing enzyme concentration. The NADP and metal ion concentration affected the lag in a complex manner. At low NADP and isocitrate concentration, the lag was reduced 50% by an NADPH concentration of less than 2 μm. Stopped flow experiments showed that premixing of NADP or NADPH with the enzyme abolished the effect of NADPH on the lag time. NADPH activated the enzyme at high NADP concentrations. This activating effect could be accounted for by removal of substrate inhibition by NADP.Evidence was obtained to show that the effect of NADPH on the activity was caused by binding of the reduced coenzyme to a site separate from the normal coenzyme binding site. Binding of metal ions by the reduced coenzyme is probably of importance as EDTA affects the lag time and activity in a manner similar to NADPH. The NADPH effect seems to be a general property of NADP-linked isocitrate dehydrogenases.     

Coenzyme binding by triphosphopyridine nucleotide dependent isocitrate dehydrogenase from beef liver. Equilibrium and kinetics studies.

The binding of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide phosphate (NADP) dependent isocitrate dehydrogenase from beef liver cytoplasm was studied by several equilibrium techniques (ultracentrifugation, molecular sieving, ultrafiltration, fluorescence). Two binding sites (per dimeric enzyme molecule) were found with slightly different dissociation constants (0.5 and 0.12 muM) and fluorescence yields (7.7 and 6.3). A ternary complex was formed between enzyme, isocitrate, and NADPH, in which NADPH dissociation constant was 5 muM. On the contrary, no binding of NADPH to the enzyme took place in the presence of magnesium isocitrate. Dialysis experiments showed the existence of 1 NADP binding site/dimer, with a dissociation constant of 26 muM. When NADPH was present with the enzyme in the proportion of 1 molecule/dimer, the dissociation constant of NADP was decreased fourfold, reaching a value quantitatively comparable to the Michaelis constant. The kinetics of coenzyme binding was followed using the stopped-flow technique with fluorescence detection. NADPH binding to the enzyme occurred through one fast reaction (k1 = 20 muM-1 s-1). Dissociation of NADPH took place upon NADP binding; however, equilibrium as well as kinetic data were incompatible with a simple competition scheme. Dissociation of NADPH from the enzyme upon magnesium isocitrate binding was preceded by the formation of a transitory ternary complex in which the fluorescence of NADPH was only about 30% of that in the enzyme-NADPH complex. Then interaction between the conenzymes and the involvement of ternary complexes in the catalytic mechanism are discussed in relation with what is known about the regulatory role of the coenzyme (Carlier, M. F., and Pantaloni, D. (1976), Biochemistry, 15, 1761-1766).     

Eubacterial isocitrate dehydrogenase with dual specificity for NAD and NADP from Rhodomicrobium vannielii

Abstract Cell-free extracts of the photosynthetic eubacterium Rhodomicrobium vannielii contained both NADP and NAD-linked isocitrate dehydrogenase activities. Apparent K _m values of 12 μM for NADP, 0.75 mM for NAD, 9.3 μM for isocitrate (NADP utilising) and 8.2 μM for isocitrate (NAD utilising) were determined in such extracts. Four lines of evidence indicated that one enzyme was responsible for the two activities; (i) non-additivity of reaction rates in the presence of both NADP and NAD (ii) the presence of one band which stained for activity with both cofactors on non-denaturing polyacrylamide gels (iii) identical heat inactivation kinetics for both activities (iv) co-elution of both activities after ion-exchange and hydrophobic interaction chromatography. This is the first report of a eubacterial isocitrate dehydrogenase with dual cofactor specificity.     

Characterization of an active site peptide modified by the substrate analogue 3-bromo-2-ketoglutarate on a single chain of dimeric NADP+-dependent isocitrate dehydrogenase

The substrate analogue 3-bromo-2-ketoglutarate reacts with pig heart NADP+-dependent isocitrate dehydrogenase to yield partially inactive enzyme. Following 65% inactivation, no further inactivation was observed. Concomitant with this inactivation, incorporation of 1 mol of reagent/mol of enzyme dimer was measured. The dependence of the inactivation rate on bromoketoglutarate concentration is consistent with reversible binding of reagent (KI = 360 microM) prior to irreversible reaction. Manganous isocitrate reduces the rate of inactivation by 80% but does not provide complete protection even at saturating concentrations. Complete protection is obtained with NADP+ or the NADP+-alpha-ketoglutarate adduct. By modification with [14C]bromoketoglutarate or by NaB3H4 reduction of modified enzyme, a single major radiolabeled tryptic peptide was obtained by high performance liquid chromatography with the sequence: Asp-Leu-Ala-Gly-X-Ile-His-Gly-Leu-Ser-Asn-Val-Lys. Evidence in the following paper (Bailey, J.M., Colman, R.F. (1987) J. Biol. Chem. 262, 12620-12626) indicates that X is glutamic acid. Enzyme modified at the coenzyme site by 2-(bromo-2,3-dioxobutylthio)-1,N(6)-ethenoadenosine 2',5'-biphosphate in the presence of manganous isocitrate is not further inactivated by bromoketoglutarate. Bromoketoglutarate-modified enzyme exhibits a stoichiometry of binding isocitrate and NADPH equal to 1 mol/mol of enzyme dimer, half that of native enzyme. These results indicate that bromoketoglutarate modifies a residue in the nicotinamide region of the coenzyme site proximal to the substrate site and that reaction at one catalytic site of the enzyme dimer decreases the activity of the other site.     

A comparison of the phosphorylated and unphosphorylated forms of isocitrate dehydrogenase from Escherichia coli ML308

NADP+ can protect active isocitrate dehydrogenase against attack by several proteases. Inactive phosphorylated isocitrate dehydrogenase is much less susceptible to proteolysis than the active enzyme, and it is not protected by NADP+. The results suggest that binding of NADP+ to, or phosphorylation of, active isocitrate dehydrogenase induces similar conformational states. Fluorescence titration experiments show that NADPH can bind to active but not to inactive isocitrate dehydrogenase. It is suggested that the phosphorylation of isocitrate dehydrogenase may occur close to its coenzyme binding site.     

The role of malic enzyme in the malate dependent biosynthesis of progesterone in the mitochondrial fraction of human term placenta

Mitochondria isolated from human term placenta were able to form citrate from malate as the only added substrate. While mitochondria were incubated in the presence of Mn2+ the citrate formation was stimulated significantly both by NAD+ and NADP+ and was inhibited by hydroxymalonate, arsenite, butylmalonate and rotenone. It is concluded that NAD(P)-linked malic enzyme is involved in the conversion of malate to citrate in these mitochondria. It has also been shown that the conversion of cholesterol to progesterone by human term placental mitochondria incubated in the presence of malate was stimulated by NAD+ and NADP+ and inhibited by arsenite and fluorocitrate. This suggests that the stimulation by malate of progesterone biosynthesis depends not only on the generation of NADPH by NAD(P)-linked malic enzyme, but also on NADPH formed during further metabolism of pyruvate to isocitrate which is in turn efficiently oxidized by NADP+-linked isocitrate dehydrogenase.     

NADP-specific isocitrate dehydrogenase of Mycobacterium phlei ATCC 354: purification and characterization

NADP-dependent isocitrate dehydrogenase (EC 1.1.1.42) from Mycobacterium phlei ATCC 354 was purified to homogeneity by ammonium sulphate fractionation, followed by DEAE cellulose and Sephadex G-200 chromatography. The pH optimum of the enzyme was 8.5. The Km values for isocitrate and NADP were 74 and 53 microM, respectively. Mn2+ was essential for enzyme activity. The enzyme lost all activity on incubation at 70 degrees C for 15 min; isocitrate and NADP protected against this thermal inactivation. p-Chloromercuribenzoate inhibited the enzyme; pre-incubation of enzyme with isocitrate + Mn2+ prevented this inhibition. The purified enzyme showed concerted inhibition by glyoxylate + oxaloacetate and was inhibited by oxalomalate.     

Activation of NADP-specific isocitrate dehydrogenase by chelating agents.

The effects of different metal chelating agents on the activity of the NADP-linked isocitrate dehydrogenase from pig heart have been studied. Addition of ethylene glycolbis(β-aminoethyleter) N,N′-tetraacetic acid, N-hydroxyethylenediamine triacetic acid, and ethylenediamine tetraacetic acid (EDTA) under certain conditions could enhance the activity by a factor of nearly 3. Moreover, the time lag occurring before the reaction rate approached a constant value at suboptimal metal-ion concentrations was abolished by the metal chelating agents. S^0.5 for isocitrate increased slightly in the presence of the metal-chelating agents. The substrate inhibition occurring at high NADP concentrations was abolished by the activator. The pH optimum was the same in the absence and presence of EDTA. The extent of activation increased on a relative basis with increasing pH. Studies of the sedimentation behavior of the enzyme under different conditions suggested that the effect of the metal-chelating agents could not be accounted for by aggregation or depolymerization of the enzyme. NADPH affects the enzyme activity in a similar way, although less efficiently than the metal chelating agents. The results indicate that most organic metal complexes can activate the enzyme. It has previously been suggested that isocitrate complexed with a metal ion is the real substrate for the enzyme. If this holds true, the activation found with other organic metal complexes can be accounted for by a reduction in the apparent K_m for the isocitrate metal complex and by an increase in the maximum rate of the reaction by removal of the substrate inhibition at high NADP concentrations.     

Isocitrate Dehydrogenases and NADP-Alcohol Dehydrogenase of Euglena gracilis var. bacillaris*

SYNOPSIS. Nicotinamide adenine dinucleotide phosphate (NADP) and nicotinamide adenine dinucleotide (NAD) linked isocitrate dehydrogenase and NADP linked alcohol dehydrogenase have been detected in Euglena gracilis var. bacillaris. The NADP isocitrate dehydrogenase showed half-maximal activity at a concentration of 3 × 10^?5 M DL-isocitrate, but did not follow simple Michaelis-Menten kinetics with respect to substrate concentration. The optimal NADP concentration was about 0.06 mM, and activity fell off sharply on either side of this optimum. Fresh preparations of the enzyme migrated as single bands in disc electrophoresis, but two enzymatically active bands were present after frozen storage. The NAD isocitrate dehydrogenase followed Michaelis-Menten kinetics with respect to substrate. In crude extracts, no requirement for adenosine monophosphate, adenosine diphosphate, or sulfhydryl compounds could be found. NADP alcohol dehydrogenase activity could be found with either ethanol or propanol as substrate. Low concentrations of coenzyme A were moderately inhibitory. In tris(hydroxymethyl) aminomethane buffer (tris buffer), Euglena extracts reduced NAD slowly in the absence of exogenous substrate. In the absence of tris, no such reduction occurred. A similar phenomenon was observed with NADP.     

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.