Pig Heart Isocitrate Dehydrogenase Research Articles

The mechanism of the inhibition of dehydrogenases by salicylate

Abstract Salicylate inhibits rabbit muscle lactate dehydrogenase, horse liver alcohol dehydrogenase, pig heart malate dehydrogenase and pig heart isocitrate dehydrogenase in vitro. The inhibitions are reversible, involving competition with nad, nadh2 or nadp. The results are discussed with reference to some of the in vivo actions of the drug.

Characterization of tRNA-dependent Peptide Bond Formation by MurM in the Synthesis of Streptococcus pneumoniae Peptidoglycan

MurM is an aminoacyl ligase that adds l-serine or l-alanine as the first amino acid of a dipeptide branch to the stem peptide lysine of the pneumococcal peptidoglycan. MurM activity is essential for clinical pneumococcal penicillin resistance. Analysis of peptidoglycan from the highly penicillin-resistant Streptococcus pneumoniae strain 159 revealed that in vivo and in vitro, in the presence of the appropriate acyl-tRNA, MurM(159) alanylated the peptidoglycan epsilon-amino group of the stem peptide lysine in preference to its serylation. However, in contrast, identical analyses of the penicillin-susceptible strain Pn16 revealed that MurM(Pn16) activity supported serylation more than alanylation both in vivo and in vitro. Interestingly, both MurM(Pn16) acylation activities were far lower than the alanylation activity of MurM(159). The resulting differing stem peptide structures of 159 and Pn16 were caused by the profoundly greater catalytic efficiency of MurM(159) compared with MurM(Pn16) bought about by sequence variation between these enzymes and, to a lesser extent, differences in the in vivo tRNA(Ala):tRNA(Ser) ratio in 159 and Pn16. Kinetic analysis revealed that MurM(159) acted during the lipid-linked stages of peptidoglycan synthesis, that the d-alanyl-d-alanine of the stem peptide and the lipid II N-acetylglucosaminyl group were not essential for substrate recognition, that epsilon-carboxylation of the lysine of the stem peptide was not tolerated, and that lipid II-alanine was a substrate, suggesting an evolutionary link to staphylococcal homologues of MurM such as FemA. Kinetic analysis also revealed that MurM recognized the acceptor stem and/or the TPsiC loop stem of the tRNA(Ala). It is anticipated that definition of the minimal structural features of MurM substrates will allow development of novel resistance inhibitors that will restore the efficacy of beta-lactams for treatment of pneumococcal infection.

Subunit Interactions of Yeast NAD+-specific Isocitrate Dehydrogenase

Yeast mitochondrial NAD(+)-specific isocitrate dehydrogenase is an octamer composed of four each of two nonidentical but related subunits designated IDH1 and IDH2. IDH2 was previously shown to contain the catalytic site, whereas IDH1 contributes regulatory properties including cooperativity with respect to isocitrate and allosteric activation by AMP. In this study, interactions between IDH1 and IDH2 were detected using the yeast two-hybrid system, but interactions between identical subunit polypeptides were not detected with this or other methods. A model for heterodimeric interactions between the subunits is therefore proposed for this enzyme. A corollary of this model, based on the three-dimensional structure of the homologous enzyme from Escherichia coli, is that some interactions between subunits occur at isocitrate binding sites. Based on this model, two residues (Lys-183 and Asp-217) in the regulatory IDH1 subunit were predicted to be important in the catalytic site of IDH2. We found that individually replacing these residues with alanine results in mutant enzymes that exhibit a drastic reduction in catalysis both in vitro and in vivo. Also based on this model, the two analogous residues (Lys-189 and Asp-222) of the catalytic IDH2 subunit were predicted to contribute to the regulatory site of IDH1. A K189A substitution in IDH2 was found to produce a decrease in activation of the enzyme by AMP and a loss of cooperativity with respect to isocitrate. A D222A substitution in IDH2 produces similar regulatory defects and a substantial reduction in V(max) in the absence of AMP. Collectively, these results suggest that the basic structural/functional unit of yeast isocitrate dehydrogenase is a heterodimer of IDH1 and IDH2 subunits and that each subunit contributes to the isocitrate binding site of the other.

Identification of the Subunits and Target Peptides of Pig Heart NAD-Specific Isocitrate Dehydrogenase Modified by the Affinity Label 8-(4-Bromo-2,3-dioxobutylthio)NAD

Pig heart NAD-dependent isocitrate dehydrogenase reacts with 8-(4-bromo-2,3-dioxobutylthio)-NAD (8- BDB-TNAD) with incorporation of 1.21 mol of reagent/mol of average subunit when the enzyme reaches the limit of 25% residual activity (Kumar, A., and Colman, R. F.,Arch. Biochem. Biophys.308, 357–366, 1994). Inclusion of NADPH decreases both the extent of inactivation and the reagent incorporation to 0.55 mol/mol of average subunit. We have now isolated the peptides labeled by radioactive 8-(4-bromo-2,3-dioxobutylthio)-[2-3H]NAD and have located them within the sequence of pig heart NAD-dependent isocitrate dehydrogenase. The enzyme is composed of three types of subunits, present as α2βγ. We have separated the subunits from unmodified and 8-BDBT[2-3H]NAD-modified enzymes by HPLC on a C4reverse-phase column, after pretreatment of the enzymes with sodium dodecyl sulfate or urea, and compared the subunit sequences of the porcine enzyme with those of the corresponding subunits from other mammalian NAD-dependent isocitrate dehydrogenases. The predominant radioactivity of 8-BDBT[2-3H]NAD is observed in the α and γ peaks, and the NADPH-protected enzyme exhibits marked reduction in incorporation into these peaks. However, evidence based on recombination of subunits from modified and unmodified enzymes indicates that only labeling of the α-subunit is responsible for inactivation by 8-BDB-TNAD. Cyanogen bromide was used to cleave the modified enzyme, and we purified one labeled peptide from the α-subunit (amino acids 84–177) as well as one from the γ-subunit (amino acids 67–186). In the α-subunit, decreased modification by [7-14C]phenylglyoxal of Arg88and Arg98after prior labeling of the enzyme by 8-BDB-TNAD indicates that these residues are the critical target sites of the reactive nucleotide analogue. We conclude that α subunit's Arg88and Arg98are both at or near the allosteric NADPH sites of the pig heart isocitrate dehydrogenase.

Activity associated with individual subunits of pig heart NAD-specific isocitrate dehydrogenase

NAD-specific pig heart isocitrate dehydrogenase is composed of three distinct types of subunits: α, β, and γ, which have molecular weights of about 40,000 but differ in amino acid composition and in isoelectric points. When the native enzyme is subjected to polyacrylamide gel electrophoresis under nondenaturing conditions, two major protein bands with M r values of about 360,000 (band 1) and 100,000 (band 2) and two minor bands (bands 3 and 4) with M r values of about 40,000 are consistently present. Enzymatic activity, as detected from NADH fluorescence, is distributed throughout the protein-staining region. Analytical isoelectric focusing in urea reveals that band 1 is composed of all three subunits in roughly the normal ratio of 2α:1β:1γ, and is probably an octamer, band 2 of an equal amount of α and β and is probably dimer, while bands 3 and 4 each consist of only the monomeric α subunit. The highest enzymatic specific activity is associated with a region intermediate between octamer and dimer, which includes the 160,000 tetramer. The protein pattern resulting from isoelectric focusing under nondenaturing conditions consists of protein bands comparable in pattern to those in the presence of urea along with bands of intermediate p I values, many of which are associated with enzymatic activity. Analysis of the subunit composition of these bands supports the activity of the α species in isolation and establishes the activity of the separated β component. No activity of the isolated γ subunit species has thus far been demonstrated. However, the highest apparent specific activity is observed when at least two types of subunits are present. These studies indicate that a range of oligomeric species of the enzyme are enzymatically active and that at least three of the four subunit chains comprising the minimum complete enzyme molecule (2α:1β:1γ) possess an active site.

Re-evaluation of molecular weight of pig heart NAD-specific isocitrate dehydrogenase.

NAD-specific pig heart isocitrate dehydrogenase was earlier reported, on the basis of gel filtration experiments, to have a molecular weight of approximately 340,000. In the present study, the enzyme is shown by equilibrium ultracentrifugation to have a weight average molecular weight of approximately 224,000 which can be attributed to a rapidly associating-dissociating protein system. The results of light-scattering measurements are consistent with the lower value of molecular weight. The enzyme exhibits an average frictional ratio, f/f0, of 1.39 as determined from ultracentrifuge experiments, and this deviation from typical proteins may account for the previous high molecular weight estimates. An average Stokes radius of 6.0 nm was calculated from the present gel filtration experiments. By use of this value and a sedimentation coefficient of 9.1 S, an average molecular weight of 245,000 has been calculated. Previous studies (Ramachandran, N., and Colman, R. F. (1980) J. Biol. Chem. 255, 8859-8864) have indicated that the enzyme is composed of three different subunits, present in the ratio 2:1:1, each of which has a molecular weight of about 40,000. These results, together with the present observations, lead to the conclusion that, under stabilizing conditions in solution, the NAD-dependent isocitrate dehydrogenase predominantly exhibits a minimum, molecular weight of 160,000 but behaves as a mixture of oligomeric species with an average Mr of about 224,000.

Cysteine in the manganous-isocitrate binding site of pig heart TPN-specific isocitrate dehydrogenase: I. Kinetics of chemical modification and properties of thiocyano enzyme

Pig heart TPN-dependent isocitrate dehydrogenase is inactivated by reaction with 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB). The dependence of the rate constant for inactivation on the reagent concentration is nonlinear, and can be analyzed in terms of the existence of two mechanisms for reaction with the enzyme, one involving reversible binding prior to inactivation and the other a bimolecular reaction. Cyanide reacts with the inactive modified enzyme to yield thiocyano-isocitrate dehydrogenase without increasing the catalytic activity; this result suggests that inactivation by DTNB is not due to steric hindrance by the bulky thionitrobenzoate group bound to the enzyme. The inactive thiocyano enzyme binds manganous ion normally. In contrast to its effect on native enzyme, however, isocitrate does not strengthen the binding of Mn 2+ to the thiocyano enzyme; the tightened binding of manganous-isocitrate may be critical for the catalytic activity of the enzyme. Protection against inactivation by DTNB is provided by isocitrate plus the activator, manganous ion, or the competitive inhibitor, calcium ion. The concerted inhibitors oxalacetate and glyoxylate, when present together with Mn 2+ and TPN, also protect against loss of activity. A marked decrease in the inactivation rate constant to a finite limiting value is caused by saturating concentrations of TPNH and Mn 2+, indicating that these ligands do not bind directly at the sites attacked by DTNB. The number of cysteine residues which react with DTNB concomitant with inactivation depends on the ligands present in the reaction mixture. In all cases, the equivalent of one -SH reacts without affecting activity. In the presence of Mn 2+ and α-ketoglutarate, which do not appreciably affect the inactivation rate, loss of activity is proportional to reaction with two -SH groups. These results suggest that the integrity of a maximum of two cysteine residues is essential for the function of the pig heart isocitrate dehydrogenase, and that at least one cysteine residue may be located within the manganous-isocitrate binding site.

Effect of arginine modification on the catalytic activity and allosteric activation by adenosine diphosphate of the diphosphopyridine nucleotide specific isocitrate dehydrogenase of pig heart

Effect of arginine modification on the catalytic activity and allosteric activation by adenosine diphosphate of the diphosphopyridine nucleotide specific isocitrate dehydrogenase of pig heart

Evidence for the presence of two nonidentical subunits in NAD-dependent isocitrate dehydrogenase of pig heart

The NAD-dependent isocitrate dehydrogenase [threo-D(S)-isocitrate:NAD(+) oxidoreductase (decarboxylating); EC 1.1.1.41] from pig heart is a multisubunit enzyme with a molecular weight of approximately 340,000. Electrophoresis of the enzyme in 10% polyacrylamide gels containing sodium dodecyl sulfate reveals two discrete bands with molecular weights of 41,000 and 39,000. The two bands exhibit approximately equal intensity when stained with Coomassie Blue, Amido Black, and Bromophenol Blue, suggesting that these polypeptide chains are present in equimolar quantities in the native enzyme. The same two-band pattern is observed when the sulfhydryl groups of the enzyme are blocked by alkylation with iodoacetate prior to electrophoresis, indicating that sulfhydryl oxidation is not responsible for the observed heterogeneity. Each of the subunits appears as a single band when eluted from the gel and again subjected to electrophoresis under the same conditions. Isocitrate dehydrogenase contains a total of 41 lysine and arginine residues per average subunit of 40,000 daltons. The observation of approximately 80 peptides upon paper chromatography and high voltage electrophoresis of tryptic digests of the enzyme is consistent with the existence of two distinct polypeptide chains. Dansylation yields two NH(2)-terminal amino acid derivatives: dansyl-phenylalanine and dansyl-alanine. It is concluded that the NAD-specific isocitrate dehydrogenase is composed of equal numbers of two nonidentical subunits.

Inhibition of dehydrogenase enzymes by hexachlorophene

Low concentrations of the antibacterial agent hexachlorophene (HCP) inhibited a number of pyridine nucleotide-linked dehydrogenase enzymes, including bovine liver glutamate dehydrogenase (GDH), beef heart malate dehydrogenase (MDH), torula yeast glucose 6-phosphate dehydrogenase (G-6-P-D), horse liver alcohol dehydrogenase (ADH), pig heart isocitrate dehydrogenase (ICD), and beef heart lactate dehydrogenase (LDH). Initial velocity studies at appropriate enzyme concentrations gave I 50 values for the dehydrogenases which ranged between 1.6 μM for GDH and 105 μM for ICD and LDH. More detailed kinetic analyses of G-6-P-D, ICD and GDH showed that inhibition by HCP in most cases was of the noncompetitive type. Calculations made from the kinetic data gave apparent K i values with G-6-P-D, of 59 μM for NADP + and of 38 μM for glucose 6-phosphate; with ICD, of 1.0 μM for NADP + and of 25 μM for isocitrate; and, with GDH, of 2.0 μM for NADH, of 7.4 μM for α-ketoglutarate, and of 2.3 μM for ammonium acetate.

Kinetics of Binding of Cytochrome c Oxidase from Rat Liver to an Immunoadsorbent

We have also examined similar parameters for several other enzymes; the trend suggests that in these cases there is a correlation between apparent association constant and capacity. These enzymes include yeast glutathione reductase, glucose 6-phosphate dehydrogenase, glutamate dehydrogenase, alcohol dehydrogenase and malate dehydrogenase, Neurospora crassa glutamate dehydrogenase, pig heart isocitrate dehydrogenase and lactate dehydrogenase, and Lactobacillus casei dihydrofolate reductase.

The role of arginine in the triphosphopyridine nucleotide dependent isocitrate dehydrogenase of pig heart.

The role of arginine in the triphosphopyridine nucleotide dependent isocitrate dehydrogenase of pig heart.

Preparation of coenzymic activity of soluble polyethyleneimine-bound NADP+ derivatives.

Alkylation at N-1 of the NADP+ adenine ring with 3,4-epoxybutanoic acid gave 1-(2-hydroxy-3-carboxypropyl)-NADP+. Enzymic reduction of the latter, followed by alkaline Dimroth rearrangement and enzymic reoxidation, gave N6-(2-hydroxy-3-carboxypropyl)-NADP+. On the other hand, bromination at C-8 of the NADP+ adenine ring, followed by reaction with the disodium salt of 3-mercaptroproionic acid, gave 8-(2-carboxyethylthio)-NADP+. Carbodimide coupling of the three carboxylic NADP+ derivatives to polyethyleneimine afforded the corresponding macromolecular NADP+ analogues. The carboxylic and the polyethyleneimine derivatives synthesized have been shown to be co-enzymically active with yeast glucose-6-phosphate dehydrogenase, liver glutamate dehydrogenase and yeast aldehyde dehydrogenase. The degree of efficiency relative to NADP+ with the three enzymes ranged from 17% to 100% for the carboxylic derivatives and from 1% to 36% for the polyethyleneimine analogues. On comparing the efficiences with the three enzymes of the N-1 derivatives to the one of the corresponding N6 anc C-8 analogues, the order of activity was N-1 greater than N6 greater C-8, except in the case of the carboxylic compounds with glutamate dehydrogenase, where this order was inverted. None of these modified cofactors were active with pig heart isocitrate dehydrogenase.

Protein fluorescence and electronic energy transfer in the determination of molecular dimensions and rotational relaxation times of native and coenzyme-bound horse liver alcohol dehydrogenase

The intrinsic fluorescence lifetimes of horse liver alcohol dehydrogenase (EC 1.1.1.1) and pig heart isocitrate dehydrogenase (EC 1.1.1.42) have been determined to be 5.36 ns and 4.84 ns, respectively. When reduced coenzyme is bound, the fluorescence lifetime of alcohol dehydrogenase is reduced to 4.98 ns while that of isocitrate dehydrogenase remains unchanged. Oxidized coenzymes have no effect on fluorescence lifetimes of alcohol and isocitrate dehydrogenases. This virtual constancy of protein fluorescence lifetimes has allowed the conclusion to be reached that in protein-ligand complexes with equilibrium constants in the range of 10 4–10 6 M −1, the static mode of quenching is substantial. The observation of resonance energy transfer in alcohol dehydrogenase-NADH complex facilitates the determination of the distance between tryptophan and the reduced nicotinamide ring involved in the transfer as 30.6 Å, compared to the effective molecular radius of 36.2 Å for alcohol dehydrogenase. The increased rotational relaxation times of coenzyme-bound alcohol dehydrogenase relative to the unliganded form ( ρ h  72 ns) indicated in this protein structural fluctuations occurring in the time range of nanoseconds.

Caracteres de l'inhibition de la glutamate, de l'isocitrate et de l'alcool deshydrogenases par les analogues formique, acetique et proprionique de la triiodo-3,5,3'- l-thyronine

3,5,3'-Triiodo-thyroformic (TF 3), 3,5,3'-triiodo-thyroacetic (TA 3) and 3,5,3'-triiodo-thyropropionic (TP 3) acids, which are structural analogs of 3,5,3'-triiodo-L-thyronine (LT 3), have been found to inhibit beef liver glutamate dehydrogenase (GlDH), horse liver alcohol dehydrogenase (ADH) and pig heart isocitrate dehydrogenase (ICDH), but different mechanisms are involved. TF 3, TA 3 and TP 3 inhibit GlDH non competitively with respect to NADP, but competitively with ADP-ribose, an activator of GlDH. For the inhibition of ADH, the three iodinated derivatives compete with NAD and ADP-ribose; ADP-ribose being itself competitive with NAD. So, it appears that TF 3, TA 3 and TP 3 interfere with the coenzyme binding by blocking the binding site of the ADP-ribose portion of the coenzyme. These iodinated inhibitors quench the enhanced fluorescence which originates from the enzyme-coenzyme binding. However, in the case of the GlDH, TP 3 exhibit an opposite effect. E.s.r. studies showed that ICDH inhibition by TA 3, did not result from the chelation of Mn 2+, but that TA 3 impaired binding of Mn 2+ to the enzyme and coenzyme.

Cyanate modification of essential lysyl residues of the diphosphopyridine nucleotide-specific isocitrate dehydrogenase of pig heart.

The DPN-specific isocitrate dehydrogenase of pig heart is totally and irreversibly inactivated by 0.05 M potassium cyanate at pH 7.4 A plot of the rate constant versus cyanate concentration is not linear, but rather exhibits saturation kinetics, implying that cyanate may bind to the enzyme to give an enzyme-cyanate complex (K equal 0.125 M) prior to the covalent reaction. In the presence of manganous ion the addition of isocitrate protects the enzyme against cyanate inactivation, indicating that chemical modification occurs in the active site region of the enzyme. The dependence of the decrease of the rate constant for inactivation on the isocitrate concentration yields a dissociation constant for the enzyme-manganese-isocitrate complex which agrees with the Michaelis constant. The allosteric activator ADP, which lowers the Michaelis constant for isocitrate, does not itself significantly affect the cyanate reaction; however, it strikingly enhances the protection by isocitrate. The addition of the chelator EDTA essentially prevents protection by isocitrate and manganous ion, demonstrating the importance of the metal ion in this process. The substrate alpha-ketoglutarate and the coenzymes DPN and DPNH do not significantly affect the rate of modification of the enzymes by cyanate. Incubation of isocitrate dehydrogenase with 14C-labeled potassium cyanate leads to the incorporation of approximately 1 mol of radioactive cyanate per peptide chain concomitant with inactivation. Analysis of acid hydrolysates of the radioactive enzyme reveals that lysyl residues are the sole amino acids modified. These results suggest that cyanate, or isocyanic acid, may bind to the active site of this enzyme as an analogue of carbon dioxide and carbamylate a lysyl residue at the active site.

Physicochemical Properties of the Diphosphopyridine Nucleotide-specific Isocitrate Dehydrogenase of Pig Heart

The DPN-dependent isocitrate dehydrogenase has been purified from pig heart by ion exchange chromatography and gel filtration. The resultant preparation exhibits a single polypeptide band, with a molecular weight of about 40,000, on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the enzyme is in a homogeneous state. The amino acid composition of this polypeptide chain is reported. In contrast, polyacrylamide disc gel electrophoresis of the enzyme preparation in the absence of denaturing reagents reveals a multiple protein pattern, with more than one enzymatically active species. The addition to the electrophoresis buffer of the substrate, isocitrate and manganous ion, or the allosteric activator, ADP, alters the protein pattern so that there is one major protein band. The multiple protein pattern observed in polyacrylamide disc gel electrophoresis thus appears to reflect different forms of the same enzyme. Isocitrate dehydrogenase becomes inactivated upon incubation at 4°; however, this cold inactivation can be reversed by incubation at 25° with isocitrate and manganous ion. Electrophoresis of the cold-inactivated preparations yields an increase in the protein species with faster mobility than the major enzyme band. It is proposed that at least part of the heterogeneity observed on electrophoresis of isocitrate dehydrogenase is attributable to cold inactivation occurring during the isolation procedures.

A Glutamyl Residue in the Active Site of Triphosphopyridine Nucleotide-dependent Isocitrate Dehydrogenase of Pig Heart

Abstract The maximum velocity of the reaction catalyzed by the pig heart TPN-specific isocitrate dehydrogenase depends on the basic form of an enzymatic group of pK 5.7. This pK is independent of temperature from 10–30° and increases in 20% ethanol, suggesting the ionization of a carboxyl group. The enzyme is inactivated by incubation at pH 7.0 with 1 -cyclohexyl-3-(2-morpholinoethyl)-carbodiimide either alone or in the presence of glycine ethyl ester, glycinamide, or glycine. Both the dehydrogenase and decarboxylase functions of the enzyme are affected. The addition of manganous ion and isocitrate or α-ketoglutarate to the incubation mixture produces a striking decrease in the inactivation rate, implying that reaction takes place in the active site. The inactive glycinamide enzyme exhibits a decreased ability to bind manganous ion in the presence of isocitrate, which suggests that the integrity of those amino acid residues susceptible to the carbodiimide and glycinamide are important for the binding of the manganous-isocitrate complex by the enzyme. The most probable sites of reaction of the carbodiimide are tyrosine, cysteine, glutamic and aspartic acids. Inactivation is not reversed by hydroxylamine indicating that tyrosine modification is not responsible; and no significant change in the measurable sulfhydryl content is noted upon inactivation. Treatment of the enzyme with the carbodiimide in the presence of [14C]glycine ethyl ester leads to incorporation of 1 mole of radioactive compound per mole of enzyme, concomitant with inactivation. Exhaustive proteolytic digestion of the radioactive protein followed by paper chromatography and electrophoresis led to the identification of γ-glutamylglycine as the product of the modification reaction. It is concluded that a glutamyl residue is essential for the catalytic function of isocitrate dehydrogenase and may lie within the substrate binding site.

NADP+-isocitrat-dehydrogenase aus Idus idus (Pisces: Cyprinidae). II. Einflu� der temperatur auf substrat- und cosubstrataffinit�t

Maximum substrate and cosubstrate affinity, as judged by the Michaelis constant (K M ), of NADP+-dependent isocitrate dehydrogenase of pig heart (purchased from Boehringer, Mannheim, FRG) is attained at 37°C. If K M -values of substrate (Isocitrate, IC) and cosubstrate (NADP+) of NADP+-dependent isocitrate dehydrogenase (ICDH) of the white dorsal muscle of Idus idus L. is plotted against the experimental temperature (VT), W-shaped curves result. With increasing adaptation temperature (AT), there is a shift to increasing VT. It is suggested that the W-shaped curves are due either to the simultaneous presence of two multiple forms of the enzyme, or to the reversible temperature-dependent interconversion of one protein species.

Substrate Independence of Molecular Weight of Triphosphopyridine Nucleotide-specific Isocitrate Dehydrogenase

The techniques of gel filtration and light scattering were used to evaluate a report that the molecular weight of the TPN-specific isocitrate dehydrogenase of pig heart can be influenced by its substrates. The results here presented indicate that the elution position of the enzyme from a column of Sephadex G-150 is not appreciably altered by the addition of isocitrate, manganous ion, TPN, α-ketoglutarate, or TPNH. The Stokes radius is estimated as 39.3 A. Similarly the weight average molecular weight of the enzyme, as measured by light scattering, does not vary significantly from 58,000 when isocitrate, α-ketoglutarate, metal ion, and the coenzymes are added. It is concluded that the pig heart TPN-dependent isocitrate dehydrogenase exists as a single molecular weight species independent of the presence of substrates.