Glutamate dehydrogenase from Pisum sativum L.

A 2–8-fold increase in the activity of glutamate dehydrogenase (GDH), accompanied by an alteration of the GDH isoenzyme pattern, was observed in detached pea shoots floated on tap water (preincubated shoots). Sugars supressed the process, whereas NH ₊ ⁴ and various metabolites as well as inhibitors of energy metabolism and protein synthesis were ineffective. The subcellular distribution pattern revealed evidence that the GDH isoenzymes are exclusively located in the mitochondrial matrix. The alterations in GDH activity occurring in preincubated shoots are restricted to the mitochondria.An experimental device suitable for studying the GDH function in isolated intact mitochondria has been established. Using [¹⁴C] citrate as the carbon source and hydrogen donor, the mitochondria synthesized considerable amounts of glutamate upon addition of NH ₊ ⁴ . The rates of glutamate formation in dependency of increasing NH ₊ ⁴ levels follow simple Michaelis-Menten kinetics. Half-saturation concentrations of NH ₊ ⁴ of 3.6±1.2 mM; 1.9±0.06 mM and 1.6±0.1 mM were calculated for the mitochondria isolated from pea shoots, roots, and preincubated shoots, respectively. The results are discussed in relation to the possible role of GDH in NH_+/4 assimilation at elevated intracellular NH_+/4 levels.Abbreviations GDH Glutamate dehydrogenase - MDH malate dehydrogenase - GOT aspartate aminotransferase - SDH succinate dehydrogenase - HEPES 4-(2-hydroxyethyl)-1-piperazineethan-sulfonic acid - BSA bovine serum albumin - TPP thiamine pyrophosphate - DNP 2,4-dinitrophenol - CCCP carbonyl cyanide m-chlorophenylhydrazone - DCPIP 2,6-dichlorophenolindophenol Dedicated to Professor Dr. Maximilian Steiner on the occasion of his 75th birthday     

Synthesis of [N]glutamate from [N]h(4) and [N]glycine by mitochondria isolated from pea and corn shoots

Metabolically competent mitochondria were isolated from pea and corn shoots on Percoll discontinuous density gradients. Rates of synthesis of [¹⁵N]glutamate were measured by gas chromatography-mass spectrometry after the incubation of mitochondria with either 2 millimolar [¹⁵N] H₄⁺ or [¹⁵N]glycine in the presence of 1 millimolar citrate as the respiratory substrate. When [¹⁵N]H₄⁺ was provided, mitochondria isolated from light-grown pea shoots synthesized [¹⁵N]glutamate with a rate of 2.64 nanomoles per hour per milligram mitochondrial protein. Corn mitochondria produced [¹⁵N]glutamate at a rate approximately 11 times greater than the pea mitochondria. Dark treatment during growth for the last 24 hours caused a slight reduction in the rate of synthesis in both species. When [¹⁵N]glycine was used, pea mitochondria synthesized [¹⁵N]glutamate with a rate of 6.32 nanomoles per hour per milligram protein. Rapid disappearance of [¹⁵N]glycine and synthesis of [¹⁵N]serine was observed with a molar ratio of 2 glycine to 0.78 serine. The rate of glutamate synthesis was only 0.2% that of serine, due in part to the dilution of [¹⁵N]H₄⁺ by the [¹⁴N]H₄⁺ pool in the mitochondria. The majority of the [¹⁵N]H₄⁺ released from glycine appears to have been released from or remains unmetabolized in the mitochondria. Corn mitochondria showed no apparent disappearance of [¹⁵N]glycine and little synthesis of [¹⁵N]serine, indicating that our preparation originated primarily from mesophyll cells. Under our conditions of glycine/serine conversion, [¹⁵N]glutatmate was synthesized at a rate of 7% of that of [¹⁵N]serine synthesis by corn mitochondria.     

The involvement of glutamate dehydrogenase in the adaptation of mitochondria to oxidize glutamate in sucrose starved pea embryos

Embryos of pea (Pisum sativum L. cv Sol) deprived of cotyledons were cultured for 3 days in medium with or without sucrose. Respiratory activity of embryos (intact) as well as the ability to oxidize glutamate by mitochondria isolated from embryos were studied. Respiration of intact embryos grown in sucrose supplemented medium was more intensive than in the starved ones. Transfer of the starved embryos to the sucrose-containing medium induced the increase in the intensity of O₂ consumption. Mitochondria isolated from both starved and control embryos exhibited respiratory control. Mitochondria isolated from embryos cultured in the absence of sucrose showed higher (about 60 %) ability to oxidize glutamate and α-ketoglutarate than mitochondria from embryos grown in sucrose containing medium. The absence of sucrose in the medium led to a rapid increase in the specific activity of glutamate dehydrogenase (NADH-GDH and NAD-GDH) and it was accompanied by changes in izoenzymatic pattern of enzyme. These results suggest that in the conditions of sucrose starvation glutamate dehydrogenase may be responsible for the increase of glutamate oxidation by mitochondria of pea embryos. Electrophoretic separation of glutamate dehydrogenase isolated from embryos cultured in medium without sucrose showed the presence of ca. 17 isoenzymes while in non-starved embryos only 7 isoenzymes were identified. However, the addition of sucrose to starved embryos after 24 hours of cultivation led to a decrease in glutamate dehydrogenase activity (up to 40 %) but it did not cause the changes in isoenzymatic pattern. These results suggest that in the conditions of sucrose starvation glutamate dehydrogenase maybe responsible for the increase of glutamate oxidation by mitochondria of pea embryos. The posibility of glutamate dehydrogenase regulation by sucrose is discussed.     

Steady-state kinetics of glutamate dehydrogenase from Pisum sativum L. mitochondria.

Glutamate dehydrogenase [l-glutamate:NAD⁺ oxidoreductase (deaminating) EC 1.4.1.2]has been purified 487-fold from pea stem mitochondria. The enzyme has a specific activity in the presence of 1 mm CaCl₂ of 54 Enzyme Commission (EC) units. Calcium, manganese, and zinc ions activate the reductive amination reaction. The [Ca²⁺]_0.5 for activation by calcium is 9 μm. The extent of activation by calcium changed during purification and storage. The oxidative deamination was slightly inhibited by calcium. The pH optimum for the reductive amination reaction was 8.0 and for the oxidative deamination was 9.2. At pH 8.0 and in the presence of 1 mm CaCl₂ with the ionic strength held constant the enzyme showed normal kinetics for the reductive amination reaction. Under identical conditions except for the absence of CaCl₂ the oxidative deamination reaction showed normal kinetics for glutamate. There was substrate activation at high NAD⁺ concentrations and these concentrations were avoided in the kinetic analysis. A steady-state kinetic analysis showed that a simple mechanism could not be in effect and a partially random mechanism is proposed.     

Light-dependent reduction of pyruvate by pea chloroplasts in the presence of glutamate dehydrogenase and C5-dicarboxylic acids

Illuminated pea chloroplasts supported (glutamine plus α-oxoglutarate (α-OG)) and (NH₃ plus α-OG)-dependent O₂ evolution. The properties of these reactions were consistent with light-coupled glutamate synthase and glutamine synthetase activities. In the presence of a glutamate-oxidizing system (component C) comprised of NAD-specific glutamate dehydrogenase (NAD-GDH), lactate dehydrogenase (LDH), 4 mM pyruvate and 0.2 mM NAD, illuminated chloroplasts supported O₂ evolution in the presence of glutamine. The reaction did not proceed in the absence of any one of the constituents of component C and the properties of O₂ evolution were consistent with light-coupled glutamate synthase activity. In the presence of component C, chloroplasts also catalysed O₂ evolution in the presence of catalytic concentrations of glutamate. Studies of O₂ evolution and metabolism of [¹⁴C]-glutamate in the presence of the inhibitors methionine sulphoximine (MSO) and azaserine suggest that O₂ evolution was dependent on the synthesis of glutamine from the products of glutamate oxidation. This was supported by polarographic studies using α-OG and NH₃ instead of glutamate.The results are consistent with a C₅-dicarboxylic acid shuttlemechanism for the export of reducing equivalents from illuminated chloroplasts (glutamate) and recycling of the oxidation products (α-OG and NH₃).     

Glutamate dehydrogenase in developing endosperm, chloroplasts, and roots of castor bean

All the glutamate dehydrogenase activity in developing castor bean endosperm is shown to be located in the mitochondria. The enzyme can not be detected in the plastids, and this is probably not due to the inactivation of an unstable enzyme, since a stable enzyme can be isolated from castor bean leaf chloroplasts. The endosperm mitochondrial glutamate dehydrogenase consists of a series of differently charged forms which stain on polyacrylamide gel electrophoresis with both NAD⁺ and NADP⁺. The chloroplast and root enzymes differ from the endosperm enzyme on polyacrylamide gel electrophoresis. The amination reaction of all the enzymes is affected by high salt concentrations. For the endosperm enzyme, the ratio of activity with NADH to that with NADPH is 6.3 at 250 millimolar NH₄Cl and 1.5 at 12.5 millimolar NH₄Cl. K_m values for NH₄⁺ and NAD(P)H are reduced at low salt concentrations. The low K_m values for the nucleotides may favor a role for glutamate dehydrogenase in ammonia assimilation in some situations.     

Properties of glutamate dehydrogenase from Lemna minor

Sterile cultures of Lemna minor grown in the presence of either nitrate, ammonium or amino acids failed to show significant changes in glutamate dehydrogenase (GDH) levels in response to nitrogen source. Crude and partially purified GDH preparations exhibit NADH and NADPH dependent activities. The ratio of these activities remain ca 12:1 during various treatments. Mixed substrate and product inhibition studies as well as electrophoretic behaviour suggest the existence of a single enzyme which is active in the presence of both coenzymes. GDH activity was found to be localized mainly in mitochondria. Kinetic studies revealed normal Michaelis kinetics with most substrates but showed deviations with NADPH and glutamate. A Hill-coefficient of 1.9 determined with NADPH indicates positive cooperative interactions, whereas a Hill-coefficient of 0.75 found with glutamate may be interpreted in terms of negative cooperative interactions. NADH dependent activity decreases rapidly during gel filtration whereas the NAD⁺ and NADPH activities remain unchanged. GDH preparations which have been pretreated with EDTA show almost complete loss of NADH and NAD⁺ activities. NADPH activity again remains unaffected. NAD⁺ activity is fully restored by adding Ca²⁺ or Mg²⁺, whereas the NADH activity can only be recovered by Ca²⁺ but not at all by Mg²⁺. Moderate inhibition of GDH reactions observed with various adenylates are fully reversed by adding Ca²⁺, indicating that the adenylate inhibition is due solely to the chelating properties of these compounds.     

Some properties of glutamate dehydrogenase,glutamine synthetase and glutamate synthase from Corynebacterium callunae

Characteristics of the three major ammonia assimilatory enzymes, glutamate dehydrogenase (GDH), glutamine synthetase (GS) and glutamate synthase (GOGAT) in Corynebacterium callunae (NCIB 10338) were examined. The GDH of C. callunae specifically required NADPH and NADP⁺ as coenzymes in the amination and deamination reactions, respectively. This enzyme showed a marked specificity for -ketoglutarate and glutamate as substrates. The optimum pH was 7.2 for NADPH-GDH activity (amination) and 9.0 for NADP⁺-GDH activity (deamination). The results showed that NADPH-GDH and NADP⁺-GDH activities were controlled primarily by product inhibition and that the feedback effectors alanine and valine played a minor role in the control of NADPH-GDH activity. The transferase activity of GS was dependent on Mn⁺² while the biosynthetic activity of the enzyme was dependent on Mg²⁺ as essential activators. The pH optima for transferase and biosynthetic activities were 8.0 and 7.0, respectively. In the transfer reaction, the K _m values were 15.2 mM for glutamine, 1.46 mM for hydroxylamine, 3.5×10^-3 mM for ADP and 1.03 mM for arsenate. Feedback inhibition by alanine, glycine and serine was also found to play an important role in controlling GS activity. In addition, the enzyme activity was sensitive to ATP. The transferase activity of the enzyme was responsive to ionic strength as well as the specific monovalent cation present. GOGAT of C. callunae utilized either NADPH or NADH as coenzymes, although the latter was less effective. The enzyme specifically required -ketoglutarate and glutamine as substrates. In cells grown in a medium with glutamate as the nitrogen source, the optimum pH was 7.6 for NADPH-GOGAT activity and 6.8 for NADH-GOGAT activity. Findings showed that NADPH-GOGAT and NADH-GOGAT activities were controlled by product inhibition caused by NADP⁺ and NAD⁺, respectively, and that ATP also had an important role in the control of NADPH-GOGAT activity. Both activities of GOGAT were found to be inhibited by azaserine.Abbreviations GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase     

Glutamate dehydrogenase activity in developing soybean seed: Isolation and characterization of three forms of the enzyme

Three forms of glutamate dehydrogenase have been isolated from developing soybean seed. Their intracellular locations could not be determined directly because organelles and marker enzymes showed abnormal distribution on sucrose density gradient fractionation. By analogy with enzymes from other parts of the plant, glutamate dehydrogenase 2 was shown to be located in chloroplasts and glutamate dehydrogenase 3 in mitochondria. Glutamate dehydrogenase 1 could not be located in this way because it is found only in the seed. The three enzymes are similar in pH optima, molecular weight and substrate specificity with respect to 2-oxoglutarate and l-glutamate. The mitochondrial enzyme is specific for NAD⁺. The chloroplast enzyme shows low activity with NADP⁺ relative to NAD⁺ but uses NADPH readily in the aminating direction. Glutamate dehydrogenase 1 is active with both nucleotides and is the only form to show substantial deaminating activity with NADP⁺. Glutamate dehydrogenases 1 and 2 are activated and stabilized by glutathione and 2-mercaptoethanol whereas enzyme 3 is unaffected. No significant metabolic control of any of the enzymes could be detected. Malate, citrate, adenine nucleotides and long-chain fatty acyl CoA derivatives gave slight inhibition at high concentrations. Amino acids had no effect on activity. A possible role for the enzyme characteristic of the developing seed is discussed in relation to nutrient supply during the accumulation of reserve materials in the seed.     

Purification of NADP-dependent glutamate dehydrogenase from Pseudomonas aeruginosa and immunochemical characterization of its in vivo inactivation

The ‘high ammonia pathway’ enzyme glutamate dehydrogenase (NADP⁺) is inactivated in cells of Pseudomonas aeruginosa when the stationary phase of growth in reached. Purified glutamate dehydrogenase (NADP⁺) appeared to be a protein composed of six identical subunits with a molecular weight of 54 000. With antibodies raised against purified enzyme it was found that glutamate dehydrogenase (NADP⁺) inactivation is accompanied by a parallel decrease in immunologically reactive material. This suggests that glutamate dehydrogenase (NADP⁺) inactivation is caused or followed by rapid proteolysis.     

Assimilation of ammonia inParacoccus denitrificans

Two pathways serve for assimilation of ammonia inParacoccus denitrificans. Glutamate dehydrogenase (NADP⁺) catalyzes the assimilation at a high NH₄ ⁺ concentration. If nitrate serves as the nitrogen source, glutamate is synthesized by glutamate-ammonia ligase and glutamate synthase (NADPH). At a very low NH₄ ⁺ concentration, all three enzymes are synthesized simultaneously. No direct relationship exists between glutamate dehydrogenase (NADP⁺) and glutamate-ammonia ligase inP. denitrificans, while the glutamate synthase (NADPH) activity changes in parallel with that of the latter enzyme. Ammonia does not influence the induction or repression of glutamate dehydrogenase (NADP⁺). The inner concentration of metabolites indicates a possible repression of glutamate dehydrogenase (NADP⁺) by the high concentration of glutamine or its metabolic products as in the case when NH₄ ⁺ is formed by assimilative nitrate reduction. No direct effect of the intermediates of nitrate assimilation on the synthesis of glutamate dehydrogenase (NADP⁺) was observed.     

Control of glutamate dehydrogenase from Lemna minor by divalent metal ions

The NADH and NAD⁺ dependent reactions catalyzed by glutamate dehydrogenase (GDH) from sterile cultures of Lemna minor are completely inactivated by EDTA. The activities of both reactions can be fully restored by addition of Ca²⁺ and to a lesser extent Mn²⁺, Zn²⁺, Sr²⁺ or La³⁺, whereas Mg²⁺ reactivates only the NAD⁺ dependent reaction. Activation of the NADH reaction by Ca²⁺ has been studied by using partially purified, EDTA pretreated, and Mg²⁺ saturated GDH preparations. Saturation kinetic curves with Ca²⁺ were always sigmoidal, whereas saturation plots for the 3 substrates of the aminating reaction at various fixed Ca²⁺ concentrations showed normal Michaelis kinetics. However, a pronounced substrate inhibition at low Ca²⁺ levels was found, particularly with NH₄⁺ and NADH. Product inhibition studies revealed unchanged enzyme substrate binding characteristics for NADH and 2-oxoglutarate in the Ca²⁺ free enzyme. A drastic alteration was established for the third substrate NH₄⁺. The kinetic data suggest that Ca²⁺ governs an equilibrium between a catalytically inactive (Ca²⁺ free) and an active (Ca²⁺ saturated) enzyme form. Inactivation by removal of Ca²⁺ is related to an alteration in the binding characteristics or binding sequence of the substrate NH₄⁺.     

Interactions between carbamyl phosphate synthase-I-mitochondrial aspartate aminotransferase and palmitoyl-CoA

Carbamyl phosphate synthase-I and glutamate dehydrogenase both form a complex with mitochondrial aspartate aminotransferase. Instead of these two enzymes competing for the aminotransferase, carbamyl phosphate synthase-I enhances glutamate dehydrogenase-aminotransferase interaction. This suggests that a complex can be formed between all three enzymes. Since this complex is stable in the presence of substrates and modifiers of the three enzymes, it could conceivably convert NH₄⁺ produced from aspartate into carbamyl phosphate. Furthermore, since carbamyl phosphate synthase-I is the predominant protein in liver mitochondria, it could play a major role in placing the aminotransferase and glutamate dehydrogenase in close proximity. Malate removes glutamate dehydrogenase from the tri-enzyme complex and thus could play a role in determining whether glutamate dehydrogenase interacts with carbamyl phosphate synthase-I or is available to participate in reactions with the Krebs cycle. Palmitoyl-CoA has a high affinity for both carbamyl phosphate synthase-I and glutamate dehydrogenase. ATP and malate which, respectively, decrease and enhance binding of palmitoyl-CoA to glutamate dehydrogenase, respectively decrease and enhance the ability of this enzyme to compete with carbamyl phosphate synthase-I for palmitoyl-CoA. Since carbamyl phosphate synthase-I is present in high levels in liver mitochondria and has a high affinity for palmitoyl-CoA, it could play a major role as a reservoir for palmitoyl-CoA.     

Role of mitochondrial glutamate dehydrogenase in the reassimilation of ammonia produced by glycine serine transformation

The ability of isolated pea-shoot mitochondria conditioned to incorporate ammonia into glutamate to reassimilate endogenously produced ammonia from glycine transformation was investigated. In the presence of 1 mM to 20 mM glycine less than 15% of the ammonia liberated was found to be incorporated into glutamate. Thus, a prominent role of mitochondrial glutamate dehydrogenase in the reassimilation of intramitochondrially produced ammonia can be excluded.Abbreviation GDH Glutamate dehydrogenase (L-glutamate: NAD⁺ oxidoreductase (deaminating), EC 1.4.1.2)     

Intra-mitochondrial poly(ADP-ribosyl)ation: Potential role for alpha-ketoglutarate dehydrogenase

Poly(ADP-ribose) polymerase (PARP) is an intracellular enzyme involved in DNA repair and in building poly-ADP-ribose polymers on nuclear proteins using NAD⁺. While the majority of PARP resides in the nucleus, several studies indicated that PARP may also be located in the cytosol or in the mitochondrial matrix. In this study we found several poly-ADP-ribosylated proteins in isolated rat liver mitochondria following hydrogen peroxide (H₂O₂) or nitric oxide donor treatment. Protein poly-ADP-ribosylation was more intense in isolated mitochondria than in whole tissue homogenates and it was not associated with increased nuclear PARP activity. We identified five poly-ADP-ribose (PAR) positive mitochondrial bands by protein mass fingerprinting. All of the identified enzymes exhibited decreased activity or decreased levels following oxidative or nitrosative stress. One of the identified proteins is dihydrolipoamide dehydrogenase (DLDH), a component of the alpha-ketoglutarate dehydrogenase (KGDH) complex, which uses NAD⁺ as a substrate. This raised the possibility that KGDH may have a PARP-like enzymatic activity. The intrinsic PARP activity of KGDH and DLDH was confirmed using a colorimetric PARP assay kit and by the incubation of the recombinant enzymes with H₂O₂. The KGDH enzyme may, therefore, have a novel function as a PARP-like enzyme, which may play a role in regulating intramitochondrial NAD⁺ and poly(ADP-ribose) homeostasis, with possible roles in physiology and pathophysiology.     

Uptake and release for glutamine and glutamate in a crude synaptosomal fraction from rat brain

[14C]Glutamine uptake in a crude synaptosomal (P2) fraction, (representing the sum of [¹⁴C]glutamine accumulated and [¹⁴C]glutamate formed by hydrolysis), is distinct from glutamate uptake. Glutamine uptake is Na⁺-independent and unaffected by the Na⁺–K⁺-ATPase inhibitor ouabain, whereas glutamate uptake is Na⁺-dependent and inhibited by ouabain. The uptake of both glutamine and glutamate is unaffected by the gamma-glutamyltransferase inhibitor, Acivicin. This indicates that glutamine uptake is not mediated by a carrier, as distinct from that of glutamate, and also not linked to gamma-glutamyl-transferase. Na⁺ affects the distribution of glutamine-derived glutamate by increasing the synaptosomal content and reducing that of the medium. When glutamate release from synaptosomes preloaded with [¹⁴C]glutamate is measured by superfusion technique in order to prevent reuptake, Na⁺ has been found to inhibit release in a non-depolarizing medium (Ringer buffer with no Ca²⁺) of the [¹⁴C]glutamate as well as of endogenous glutamate. The specific activity of the [¹⁴C]glutamine-derived glutamate in the incubation medium is much higher than that in the synaptosomes, indicating that there exists a readily releasable pool of newly formed glutamate in addition to another pool. The latter glutamate pool is partially reduced by Na⁺.Special Issue Dedicated to Dr. Abel Lajtha.     

THE MITOCHONDRIAL REDOX STATE OF RAT BRAIN

The use of the glutamate dehydrogenase (EC 1.4.1.3) and β-hydroxybutyrate dehydrogenase (EC 1.1.1.30) reactions for the calculation of the mitochondrial redox state of brain has been examined. To prevent post-mortem anoxic metabolism, brains were frozen in less than a second by using a new technique. Levels of ketone bodies in brain were so low relative to the contamination by blood and extracellular fluid that calculation of the mitochondrial redox state using the β-hydroxybutyrate dehydrogenase reaction was not practical. The concentrations of the non-nucleotide substrates of the glutamate dehydrogenase reaction could be accurately measured in brain and themitochondrial [NAD⁺]/[NADH] ratio calculated from the ratio [α-oxoglutarate] [NH₄⁺]/[glutamate]. The calculation is valid if the ratio [α-oxoglutarate] [NH₄⁺]/[glutamate] in mitochondria is the same as that measured in whole tissue. The evidence supporting this conclusion is the near-equilibrium of the aspartate aminotransferase (EC 2.6.1.l) reaction in brain and the observation by others that the distribution of label between α-oxoglutarate and glutamate in brain, after administration of labelled precursors, conforms to expectation. The alanine aminotransferase (EC 2.6.1.2) reaction was not near equilibrium in brain, probably because of the low in vivo activity of the enzyme.     

Purification and characterization of NADP+-specific glutamate dehydrogenase fromEscherichia coli

The NADP⁺-specific glutamate dehydrogenase fromEscherichia coli has been purified to electrophoretic homogeneity. The enzyme was purified 40-fold and has a specific activity of 23. Glutamate dehydrogenase fromE. coli is a hexameric enzyme with a native molecular weight of 275 KDa composed of monomers each with a molecular weight of 44.5 KDa. In nondenaturing isoelectric focusing gels, the purified enzyme is resolved into six catalytically active species, each with a molecular weight of 275 KDa and with isoelectric points ranging between pH 5.3 and 5.7. The K_m values for substrates and coenzymes have been determined, and the effect of several divalent ions on catalytic activity has been investigated.     

Subcellular Location of NADP-Isocitrate Dehydrogenase in Pisum sativum Leaves

The subcellular location of NADP⁺-isocitrate dehydrogenase was investigated by preparing protoplasts from leaves of pea seedlings. Washed protoplasts were gently lysed and the whole lysate separated on sucrose gradients by a rate-zonal centrifugation. Organelles were located by marker enzymes and chlorophyll analysis. Most of the NADP⁺-isocitrate dehydrogenase was in the soluble fraction. About 10% of the NADP⁺-isocitrate dehydrogenase was present in the chloroplasts as a partially latent enzyme. Less than 1% of the activity was found associated with the peroxisome fraction. NADP⁺-isocitrate dehydrogenase was partially characterized from highly purified chloroplasts isolated from shoot homogenates. The enzyme exhibited apparent K_m values of 11 micromolar (NADP⁺), 35 micromolar (isocitrate), 78 micromolar (Mn²⁺), 0.3 millimolar (Mg²⁺) and showed optimum activity at pH 8 to 8.5 with Mn²⁺ and 8.8 to 9.2 with Mg²⁺. The NADP⁺-isocitrate dehydrogenase activity previously claimed in the peroxisomes by other workers is probably due to isolation procedures and/or nonspecific association. The NADP⁺-isocitrate dehydrogenase activity in the chloroplasts might help supply α-ketoglutarate for glutamate synthase action.     

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

The NAD⁺-dependent isocitrate dehydrogenase from etiolated pea (Pisum sativum L.) mitochondria was purified more than 200-fold by dye-ligand binding on Matrix Gel Blue A and gel filtration on Superose 6. The enzyme was stabilized during purification by the inclusion of 20% glycerol. In crude matrix extracts, the enzyme activity eluted from Superose 6 with apparent molecular masses of 1400 ± 200, 690 ± 90, and 300 ± 50 kD. During subsequent purification steps the larger molecular mass species disappeared and an additional peak at 94 ± 16 kD was evident. The monomer for the enzyme was tentatively identified at 47 kD by sodium dodecyl-polyacrylamide gel electrophoresis. The NADP⁺-specific isocitrate dehydrogenase activity from mitochondria eluted from Superose 6 at 80 ± 10 kD. About half of the NAD⁺ and NADP⁺-specific enzymes remained bound to the mitochondrial membranes and was not removed by washing. The NAD⁺-dependent isocitrate dehydrogenase showed sigmodial kinetics in response to isocitrate (S_0.5 = 0.3 mm). When the enzyme was aged at 4°C or frozen, the isocitrate response showed less allosterism, but this was partially reversed by the addition of citrate to the reaction medium. The NAD⁺ isocitrate dehydrogenase showed standard Michaelis-Menten kinetics toward NAD⁺ (K_m = 0.2 mm). NADH was a competitive inhibitor (K_i = 0.2 mm) and, unexpectedly, NADPH was a noncompetitive inhibitor (K_i = 0.3 mm). The regulation by NADPH may provide a mechanism for coordination of pyridine nucleotide pools in the mitochondria.