Glutamate dehydrogenase of lupin nodules: Purification and properties

Glutamate dehydrogenase, GDH (l-glutamate: NAD⁺ oxidoreductase (deaminating) EC 1.4.1.2) was purified from the plant fraction of lupin nodules and the purity of the preparation established by gel electrophoresis and electrofocusing. The purified enzyme existed as 4 charge isozymes with a MW of 270000. The subunit MW, as determined by dodecyl sulphate electrophoresis, was 45 000. On the basis of the results of the MW determinations a hexameric structure is proposed for lupin-nodule GDH. The pH optima for the enzyme were pH 8.2 for the amination reaction and pH 8.8 for the deamination reaction. GDH from lupin nodules showed a marked preference for NADH over NADPH in the amination reaction and used only NAD⁺ for the deamination reaction. Pyridoxal-5′-P and EDTA inhibited activity. The enzyme displayed Michaelis-Menten kinetics with respect to all substrates except NAD⁺. When NAD⁺ was the varied substrate, there was a deviation from Michaelis-Menten behaviour towards higher activity at high concentrations of NAD⁺.     

Regulation of glycerol metabolism in <Emphasis Type="Italic">Enterobacter aerogenes</Emphasis> NBRC12010 under electrochemical conditions

Enterobacter aerogenes NBRC12010 was able to ferment glycerol to ethanol and hydrogen gas. Fermentation of glycerol ceased in the stationary phase of growth, and it was activated by electrochemical reactions using thionine as an electron transfer mediator from bacterial cells to an electrode. Using resting cells of E. aerogenes NBRC12010 in only citrate buffer solution, the cells did not consume glycerol at all, but they could metabolize glucose. These results suggest that the regulation of glycerol metabolism occurred at enzymatic steps before glycolysis. In E. aerogenes NBRC12010, glycerol was metabolized via glycerol dehydrogenase (GDH) and then dehydroxyacetone kinase. The GDH-catalyzed reaction mainly depended on the ratio of NAD⁺/NADH. At a NAD⁺/NADH ratio of nearly 1 or less, it was substantially suppressed and glycerol metabolism stopped. When the ratio was higher than 1, GDH was activated and glycerol was metabolized. Thus, the reaction of glycerol metabolism depended on the balance of cellular NAD⁺/NADH. Exogenous NADH was oxidized to NAD⁺ by electrochemical reactions with thionine. We proposed the activation mechanism of glycerol metabolism under electrochemical conditions.     

Increased activity of glucose dehydrogenase co-immobilized with a redox mediator in a bioreactor with electrochemical NAD+ regeneration

An electrochemical bioreactor with glucose dehydrogenase immobilized on to the electrode surface produced gluconic acid from glucose with concomitant recycling of the NAD⁺ coenzyme at 0.7 V. Since the enzyme is deactivated during operation at this redox potential, co-immobilization of 3,4-dihydroxybenzaldehyde as mediator allowed the system to operate at 0.2 V and increased both the activity (2.4-times) and the stability of the immobilized enzyme (2.2-times). The different effective electrochemical surfaces resulting from the different mediator immobilization modes are important in determining these three properties.     

A “Stripping” Ligand Tactic for Use with the Kinetic Locking-on Strategy: Its Use in the Resolution and Bioaffinity Chromatographic Purification of NAD-Dependent Dehydrogenases

The kinetic locking-on strategy utilizes soluble analogues of the target enzymes' specific substrate to promote selective adsorption of individual NAD⁺-dependent dehydrogenases on their complementary immobilized cofactor derivative. Application of this strategy to the purification of NAD⁺-dependent dehydrogenases from crude extracts has proven that it can yield bioaffinity systems capable of producing one-chromatographic-step purifications with yields approaching 100%. However, in some cases the purified enzyme preparation was found to be contaminated with other proteins weakly bound to the immobilized cofactor derivative through binary complex formation and/or nonspecific interactions, which continuously “dribbled” off the matrix during the chromatographic procedure. The fact that this problem can be overcome by including a short pulse of 5′-AMP (stripping ligand) in the irrigant a couple of column volumes prior to the discontinuation of the specific substrate analogue (locking-on ligand) is clear from the results presented in this report. The general effectiveness of this auxiliary tactic has been assessed using model studies and through incorporation into an actual purification from a crude cellular extract. The results confirm the usefulness of the stripping-ligand tactic for the resolution and purification of NAD⁺-dependent dehydrogenases when using the locking-on strategy. These studies have been carried out using bovine liver glutamate dehydrogenase (GDH, EC 1.4.1.3), yeast alcohol dehydrogenase (YADH, EC 1.1.1.1), porcine heart mitochondrial malate dehydrogenase (mMDH, EC 1.1.1.37), and bovine heart -lactate dehydrogenase ( -LDH, EC 1.1.1.27).     

Nucleotide allosteric regulation of the glutamate dehydrogenases of Teladorsagia circumcincta and Haemonchus contortus

The expression of glutamate dehydrogenase (GDH; EC 1.4.1.3) in L3 of the nematode Haemonchus contortus was confirmed by detecting GDH mRNA, contrary to earlier reports. The enzyme was active in both L3 and adult H. contortus homogenates either with NAD⁺/H or NADP⁺/H as co-factor. Although it was a dual co-factor GDH, activity was greater with NAD⁺/H than with NADP⁺/H. The rate of the aminating reaction (glutamate formation) was approximately three times higher than for the deaminating reaction (glutamate utilisation). GDH provides a pathway for ammonia assimilation, although the affinity for ammonia was low. Allosteric regulation by GTP, ATP and ADP of L3 and adult H. contortus and Teladorsagia circumcincta (Nematoda) GDH depended on the concentration of the regulators and the direction of the reaction. The effects of each nucleotide were qualitatively similar on the mammalian and parasite GDH, although the nematode enzymes were more responsive to activation by ADP and ATP and less inhibited by GTP under optimum assay condition. GTP inhibited deamination and low concentrations of ADP and ATP stimulated weakly. In the reverse direction, GTP was strongly inhibitory and ADP and ATP activated the enzyme.     

Molecular and biochemical characterisation of a Teladorsagia circumcincta glutamate dehydrogenase

A full length cDNA encoding glutamate dehydrogenase was cloned from Teladorsagia circumcincta (TcGDH). The TcGDH cDNA (1614 bp) encoded a 538 amino acid protein. The predicted amino acid sequence showed 96% and 93% similarity with Haemonchus contortus and Caenorhabditis elegans GDH, respectively. A soluble N-terminal 6xHis-tagged GDH protein was expressed in the recombinant Escherichia coli strain BL21 (DE3) pGroESL, purified and characterised. The recombinant TcGDH had similar kinetic properties to those of the enzyme in homogenates of T. circumcincta, including greater activity in the aminating than deaminating reaction. Addition of 1 mM ADP and ATP increased activity about 3-fold in the deaminating reaction, but had no effect in the reverse direction. TcGDH was a dual co-factor enzyme that operated both with NAD⁺ and NADP⁺, GDH activity was greater in the deaminating reaction with NADP⁺ as co-factor and more with NADH in the aminating reaction.     

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.     

Effects of molecular weight of dextran and NAD+ density on coenzyme activity of high molecular weight NAD+ derivative covalently bound to dextran

NAD⁺ has been covalently attached to dextrans having different molecular weights to give various NAD⁺ densities (mol NAD⁺ per mol d-glucosyl residue). The effects of molecular weight of dextran and of NAD⁺ density on the coenzyme activity of the dextran-bound NAD⁺ derivatives were examined for the reactions catalysed by alcohol dehydrogenase (alcohol: NAD⁺ oxidoreductase, EC 1.1.1.1) and lactate dehydrogenase (l-lactate:NAD⁺ oxidoreductase, EC 1.1.1.27). The molecular weight of dextran had little effect on coenzyme activity in the range 10 000 to 500 000. At low NAD⁺ density (<0.05 mol NAD⁺/mol d-glucosyl residue), the coenzyme activities of the derivatives were relatively low, but higher densities had little effect on the activity. Dextran-bound NAD⁺ derivatives were twice as stable as free NAD⁺.     

NAD(P)+-dependent isocitrate dehydrogenases in mitochondria purified from Picea abies seedlings

Isocitrate dehydrogenase (IDH) activities were measured in mitochondria isolated from aerial parts of 21-day-old spruce (Picea abies L. Karst.) seedlings. Mitochondria were purified by two methods, involving continuous and discontinuous Percoll gradients. Whatever the method of purification, the mitochondrial outer membrane was about 69% intact, and the mitochondria contained very low cytosolic, chloroplastic and peroxisomal contaminations. Nevertheless, as judged by the recovery of fumarase activity, purification on a continuous 28% Percoll gradient gave the best yield in mitochondria, which exhibited a high degree of inner membrane intactness (91%). The purified mitochondria oxidized succinate and malate with good respiratory control and ADP/O ratios. The highest oxidation rate was obtained with succinate as substrate, and malate oxidation was improved (+ 60%) by addition of exogenous NAD⁺. Experiments using standard respiratory chain inhibitors indicated that, in spruce mitochondria, the alternative pathway was present. Both NAD⁺-isocitrate dehydrogenase (EC 1.1.1.41) and NADP⁺-isocitrate dehydrogenase (EC 1.1.1.42) were present in the mitochondrial matrix fraction, and NAD⁺-IDH activity was about 2-fold higher than NADP⁺-IDH activity. The NAD⁺-IDH showed sigmoidal kinetics in response to isocitrate and standard Michaelis-Menten kinetics for NAD⁺ and Mg²⁺. The NADP⁺-IDH, in contrast, displayed lower K_m values. For NAD⁺-IDH the pH optimum was at 7.4, whereas NADP⁺-IDH exhibited a broad pH optimum between 8.3 and 9. In addition, NAD⁺-IDH was more thermolabile. Adenine nucleotides and 2-oxoglutarate were found to inhibit NAD(P)⁺-IDH activities only at high concentrations.     

Regulation of ammonia assimilation in an obligate methylotroph Methylobacillus flagellatum under steady-state and transient growth conditions

The obligate methylotroph Methylobacillus flagellatum was grown in the presence of different ammonium concentrations and the regulation of the enzymes associated with ammonium assimilation was investigated in steady-state and transient growth regimes. As the medium changed from C-limitation to dual C/N- and finally to N-limitation, the culture passed through three definite growth phases. The NADP⁺-dependent glutamate dehydrogenase (GDH) was present under ammonium limitation of the culture growth (at 2 mmol l^-1 of ammonium in the growth medium) and increased in response to an increase in nitrogen availability. Glutamine synthetase (GS) and glutamate synthase (GOGAT) activities were negligible during C- and C/N-limitation. In N-limited cells the GOGAT activity increased as the dilution rate increased up to 0.35 h^-1, and then sharply dropped. In the N-sufficient cultures both NAD⁺- and NADP⁺-dependent isocitrate dehydrogenase (NAD-ICDH and NADP-ICDH) activities were up-regulated as dilution rate increased, but in the N-limited culture the NAD-ICDH activity was up-regulated whereas NADP-ICDH one was down-regulated. Pulse additions of ammonium and methanol demonstrated the coordinate regulation of the GDH and ICDHs activities. When pulses were added to the C/N-limited cultures, there was an immediate utilization of the nutrients, resulting in an increase in biomass; at the same time the GDH and ICDH activities increased and the GS and GOGAT activities decreased. When the same ammonium/methanol pulse was added into the N-limited culture, there was a 3-hours delay in the culture response, after which the substrates were utilized at rates close to the ones shown by the C/N-limited culture after the analogous pulse.     

Enzyme Activities in Urtica dioica: Effects of Daylength and Leaf Age on Glutamate Dehydrogenase, Aspartate Aminotransferase and Alanine Aminotransferase

Enzymes, important to protein synthesis, were investigated in young and old leaves of Urtica dioica. The plants, divided into two groups, were exposed to either 18-hour or 12-hour photo-periods. One group of plants from each photoperiodic regime was subjected to an irradiance of 28 W × m^-2, and the other group of plants to 42 W × m^-2. The enzymes investigated were glutamate dehydrogenase (GDH), aspartate aminotransferase (glutamate-oxaloacetate transaminase, GOT), and alanine aminotransferase (glutamate-pyruvate transaminase, GPT), GDH and GOT were determined by means of electrophoretic separation on polyacrylamide and spectrophotometric measurements. GPT was determined only by the latter method. Plants exposed to 18-hour photoperiods showed much higher GDH activity than did those exposed to 12-hour photoperiods. The activity of GDH also increased with leaf age. Besides one uniform NAD⁺-dependent GDH, two other NAD⁺-independent enzymes, showing GDH activity, were identified on polyacryl-amide gel electrophoresis. The distribution of NADH and NAD⁺-dependent GDH activity between young and old leaves was similar under different growth conditions. The activity of GOT was insensitive to environmental changes. The results regarding GPT indicate that this enzyme responded to different photoperiods in the same way as GDH. A correlation coefficient of 0.928 was obtained for the relationship between GDH and GPT activity.     

Modification of aldehyde dehydrogenase with dicyclohexylcarbodiimide: Separation of dehydrogenase from esterase activity

Dehydrogenase activity of the cytoplasmic (E1) isozyme of human liver aldehyde dehydrogenase (EC 1.2.1.3) was almost totally abolished (3% activity remaining) by preincubation with dicyclohexylcarbodiimide (DCC), while esterase activity with p-nitrophenyl acetate as substrate remained intact. The esterase reaction of the modified enzyme exhibited a hysteretic burst prior to achieving steady-state velocity; addition of NAD⁺ abolished the burst. TheK _m for p-nitrophenyl acetate was increased, but physicochemical properties remained unchanged. The selective inactivation of dehydrogenase activity was the result of covalent bond formation. Protection by NAD⁺ and chloral, saturation kinetics, and the stoichiometry and specificity of interaction indicated that the reaction of DCC occurred at the active site of the E1 isozyme. The results suggested that some amino acid other than aspartate or glutamate, possibly a cysteine residue, located on a large tryptic peptide of the E1 enzyme, may have reacted with DCC.     

Photocatalyzed oxidation of NAD dimers

A mixture of dimers of nicotinamide adenine dinucleotide, largely 4,4?-linked, obtained by electrochemical reduction of NAD⁺, can be photooxidized back to NAD⁺ in the presence of oxygen. Oxygen is consumed during the photooxidation process with the production of hydrogen peroxide. The oxidation is almost pH independent and is stimulated by light whose wavelength exceeds 300 nm. Lactate dehydrogenase and alcohol dehydrogenase added to the solutions under irradiation increased the oxygen uptake by the NAD dimers in a concentration-dependent way. These observations suggest that light induces the homolytic cleavage of NAD dimers to NAD radicals which in turn are oxidized to NAD⁺ by oxygen.     

Kinetics of a hollow fiber dehydrogenase reactor

A hollow fiber module was used as a reactor for conversion of ethanol to acetaldehyde in the presence of horse liver alcohol dehydrogenase as catalyst. Mass transport rates for NAD⁺, the overall acetaldehyde generation rate, catalyst effectiveness factors, and the overall order of the reaction with respect to NAD⁺ concentration were measured. A coupled-substrate reactor with continuous in situ regeneration of cofactor was also examined. Two substrates of opposite redox state were added simultaneously to the feed stream. NADH and acetaldehyde concentrations were monitored in the effluent stream. The cofactor recycle number, or ratio of moles of product to moles of NADH produced, exceeded 10,000 under certain conditions. While decreasing the NAD⁺ concentration in the feed stream decreased reactor productivity somewhat, it greatly enhanced the ratio of product formed per mole of NAD⁺ fed to the reactor. It is suggested that high cofactor costs in dehydrogenase reactors may be overcome with efficient in situ regeneration and secondary recovery and recycling of cofactor from the process stream.     

Glutamate Dehydrogenases: The Why and How of Coenzyme Specificity

NAD⁺ and NADP⁺, chemically similar and with almost identical standard oxidation–reduction potentials, nevertheless have distinct roles, NAD⁺ serving catabolism and ATP generation whereas NADPH is the biosynthetic reductant. Separating these roles requires strict specificity for one or the other coenzyme for most dehydrogenases. In many organisms this holds also for glutamate dehydrogenases (GDH), NAD⁺-dependent for glutamate oxidation, NADP⁺-dependent for fixing ammonia. In higher animals, however, GDH has dual specificity. It has been suggested that GDH in mitochondria reacts only with NADP(H), the NAD⁺ reaction being an in vitro artefact. However, contrary evidence suggests mitochondrial GDH not only reacts with NAD⁺ but maintains equilibrium using the same pool as accessed by β-hydroxybutyrate dehydrogenase. Another complication is the presence of an energy-linked dehydrogenase driving NADP⁺ reduction by NADH, maintaining the coenzyme pools at different oxidation–reduction potentials. Its coexistence with GDH makes possible a futile cycle, control of which is not yet properly explained. Structural studies show NAD⁺-dependent, NADP⁺-dependent and dual-specificity GDHs are closely related and a few site-directed mutations can reverse specificity. Specificity for NAD⁺ or for NADP⁺ has probably emerged repeatedly during evolution, using different structural solutions on different occasions. In various GDHs the P7 position in the coenzyme-binding domain plays a key role. However, whereas in other dehydrogenases an acidic P7 residue usually hydrogen bonds to the 2′- and 3′-hydroxyls, dictating NAD⁺ specificity, among GDHs, depending on detailed conformation of surrounding residues, an acidic P7 may permit binding of NAD⁺ only, NADP⁺ only, or in higher animals both.     

Synthesis of a Highly Substituted N-Linked Immobilized NAD Derivative Using a Rapid Solid-Phase Modular Approach: Suitability for Use with the Kinetic Locking-on Tactic for Bioaffinity Purification of NAD-Dependent Dehydrogenases

This study is concerned with further development of the kinetic locking-on strategy for bioaffinity purification of NAD⁺-dependent dehydrogenases. Specifically, the synthesis of highly substituted N⁶-linked immobilized NAD⁺ derivatives is described using a rapid solid-phase modular approach. Other modifications of the N⁶-linked immobilized NAD⁺ derivative include substitution of the hydrophobic diaminohexane spacer arm with polar spacer arms (9 and 19.5 Å) in an attempt to minimize nonbiospecific interactions. Analysis of the N⁶-linked NAD⁺ derivatives confirm (i) retention of cofactor activity upon immobilization (up to 97%); (ii) high total substitution levels and high percentage accessibility levels when compared to S⁶-linked immobilized NAD⁺ derivatives (also synthesized with polar spacer arms); (iii) short production times when compared to the preassembly approach to synthesis. Model locking-on bioaffinity chromatographic studies were carried out with bovine heart -lactate dehydrogenase ( -LDH, EC 1.1.1.27), bakers yeast alcohol dehydrogenase (YADH, EC 1.1.1.1) and Sporosarcinia sp. -phenylalanine dehydrogenase ( -PheDH, EC 1.4.1.20), using oxalate, hydroxylamine, and -phenylalanine, respectively, as locking-on ligands. Surprisingly, two of these test NAD⁺-dependent dehydrogenases (lactate and alcohol dehydrogenase) were found to have a greater affinity for the more lowly substituted S⁶-linked immobilized cofactor derivatives than for the new N⁶-linked derivatives. In contrast, the NAD⁺-dependent phenylalanine dehydrogenase showed no affinity for the S⁶-linked immobilized NAD⁺ derivative, but was locked-on strongly to the N⁶-linked immobilized derivative. That this locking-on is biospecific is confirmed by the observation that the enzyme failed to lock-on to an analogous N⁶-linked immobilized NADP⁺ derivative in the presence of -phenylalanine. This differential locking-on of NAD⁺-dependent dehydrogenases to N⁶-linked and S⁶-linked immobilized NAD⁺ derivatives cannot be explained in terms of final accessible substitutions levels, but suggests fundamental differences in affinity of the three test enzymes for NAD⁺ immobilized via N⁶-linkage as compared to thiol-linkage.     

Purification and characterization of glutamate dehydrogenase as another isoprotein binding to the membrane of rough endoplasmic reticulum

Glutamate dehydrogenase (GDH) was purified from rough endoplasmic reticulum (RER) in rat liver using anion‐exchange and affinity chromatography. As GDH has been known as an enzyme that exists mainly in the matrix of mitochondria, the properties of purified GDH were compared with those of mitochondrial GDH. The GDH activity in 0.1% Triton X‐100‐treated RER subcellular fraction was nearly the same as intact RER, whereas that of the mitochondrial fraction increased by 50% after the detergent treatment. In kinetic values, in addition, mitochondrial GDH had a higher K_m value for NADP⁺ than NAD⁺, whereas the K_m value for NAD⁺ was higher than that for NADP⁺ in the case of GDH of RER, which showed a difference in specificity to cofactors. Moreover, when two GDH isoproteins were incubated at 42°C or treated with trypsin, GDH from RER was more stable against heat inactivation and less susceptible to proteolysis than mitochondrial GDH in both cases. In addition, GDH of RER had at least five amino acids different from mitochondrial GDH when sequences of N‐terminal and several internal peptide fragments were analyzed. These results showed that GDH of RER is another isoprotein of GDH, of whose properties are different from those of mitochondrial GDH. J. Cell. Biochem. 76:244–253, 1999. © 1999 Wiley‐Liss, Inc.     

Preparation of 12-ketochenodeoxycholic acid from cholic acid using coimmobilized 12α-hydroxysteroid dehydrogenase and glutamate dehydrogenase with NADP cycling at high efficiency

12-Ketochenodeoxycholic acid, an essential intermediate in the synthesis of chenodeoxycholic acid, has been enzymatically prepared from cholic acid. The specific oxidation of the 12α-hydroxyl group of cholic acid with NADP⁺ was catalysed by 12α-hydroxysteroid dehydrogenase (12α-hydroxysteroid: NAD⁺ oxidoreductase, EC 1.1.1.176), and the regeneration of NADP⁺ was obtained through the glutamate dehydrogenase (l-glutamate:NADP⁺ oxidoreductase, EC 1.4.1.4) catalysed reduction of α-ketoglutarate. The two enzymes were immobilized onto Sepharose CL-4B activated with tresyl chloride. The coimmobilized enzymes showed a cycling efficiency for the coenzyme similar to that of the free enzymes. High concentrations of cholic acid (up to 4%, w/v) were completely and specifically transformed into the 12-keto derivative using amounts of cofactor about 1600 times lower on a molar basis. The immobilized enzymes maintained 70% of the initial activity after 2 months of continuous use.     

NADP+-specific 2-oxoglutarate dehydrogenase in denitrifying Pseudomonas species

Several denitrifying Pseudomonas strains contained an NADP⁺-specific 2-oxoglutarate dehydrogenase, in contrast to an NAD⁺-specific pyruvate dehydrogenase, if the cells were grown anaerobically with aromatic compounds. With non-aromatic substrates or after aerobic growth the coenzyme specificity of 2-oxoglutarate dehydrogenase changed to NAD⁺-specificity. The reaction stoichiometry and the apparent K _m-values of the enriched enzymes were determined: pyruvate 0.5 mM, coenzyme A 0.05 mM, NAD⁺ 0.25 mM; 2-oxoglutarate 0.6 mM, coenzyme A 0.05 mM, NADP⁺ 0.03 mM. Isocitrate dehydrogenase was NADP⁺-specific. The findings suggest that these strains contained at least two lipoamide dehydrogenases, one NAD⁺-specific, the other NADP⁺-specific.     

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)