Effect of l-leucine on the nitrogen metabolism of isolated rat liver mitochondria

1. l-Leucine strongly activated intramitochondrial glutamate dehydrogenase in the direction of glutamate synthesis. 2. In the deamination direction, the enzyme was not stimulated by leucine. This was probably due to a rate-limiting transport of glutamate across the mitochondrial membrane. 3. The effect of leucine on the kinetic constants of glutamate dehydrogenase in a mitochondrial sonicate was studied. 4. In isolated mitochondria, leucine did not stimulate the synthesis of citrulline with glutamate as the source of NH(3). 5. Leucine very markedly stimulated the synthesis of glutamate from added 2-oxoglutarate+NH(4)Cl. 6. Under conditions where glutamate and citrulline could be synthesized simultaneously from added NH(4)Cl, leucine greatly increased glutamate synthesis at the expense of citrulline synthesis. 7. It is suggested that the intramitochondrial leucine concentration may be a factor influencing the nitrogen metabolism of the liver cell.     

Regulation of insulin release by factors that also modify glutamate dehydrogenase

Leucine and monomethyl succinate initiate insulin release, and glutamine potentiates leucine-induced insulin release. Alanine enhances and malate inhibits leucine plus glutamine-induced insulin release. The insulinotropic effect of leucine is at least in part secondary to its ability to activate glutamate oxidation by glutamate dehydrogenase (Sener, A., Malaisse-Lagae, F., and Malaisse, W. J. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 5460-5464). The effect of these other amino acids or Krebs cycle intermediates on insulin release also correlates with their effects on glutamate dehydrogenase and their ability to regulate inhibition of this enzyme by alpha-ketoglutarate. For example, glutamine enhances insulin release and islet glutamate dehydrogenase activity only in the presence of leucine. This could be because leucine, especially in the presence of alpha-ketoglutarate, increases the Km of glutamate and converts alpha-ketoglutarate from a noncompetitive to a competitive inhibitor of glutamate. Thus, in the presence of leucine, this enzyme is more responsive to high levels of glutamate and less responsive to inhibition by alpha-ketoglutarate. Malate could decrease and alanine could increase insulin release because malate increases the generation of alpha-ketoglutarate in islet mitochondria via the combined malate dehydrogenase-aspartate aminotransferase reaction, and alanine could decrease the level of alpha-ketoglutarate via the alanine transaminase reaction. Monomethyl succinate alone is as stimulatory of insulin release as leucine alone, and glutamine enhances the action of both. Succinyl coenzyme A, leucine, and GTP are all bound in the same region on glutamate dehydrogenase, where GTP is a potent inhibitor and succinyl coenzyme A and leucine are comparable activators. Thus, the insulinotropic properties of monomethyl succinate could result from it increasing the level of succinyl coenzyme A and decreasing the level of GTP via the succinate thiokinase reaction.     

Ox liver glutamate dehydrogenase. The use of chemical modification to study the relationship between catalytic sites for different amino acid substrates and the question of kinetic non-equivalence of the subunits.

The effect of pyridoxal 5'-phosphate on the activity of ox liver glutamate dehydrogenase towards different amino acid substrates was investigated. Both alanine and glutamate activities decreased steadily in the presence of pyridoxal 5'-phosphate. The alanine/glutamate activity ratio increased as a function of inactivation by pyridoxal 5'-phosphate, indicating that glutamate activity is lost more rapidly than alanine activity. A mixture of NADH, GTP and 2-oxoglutarate completely protected the alanine and glutamate activities against inactivation by pyridoxal 5'-phosphate. The activity of glutamate dehydrogenase towards glutamate and leucine decreased steadily in a constant ratio in the presence of pyridoxal 5'-phosphate. The effect of leucine on the alanine and glutamate activities as a function of inactivation by pyridoxal 5'-phosphate was studied. The results are interpreted to suggest that the subunits of glutamate dehydrogenase hexamer are kinetically non-equivalent with regard to activity towards the two monocarboxylic amino acids as well as glutamate, and that all three substrates share the same active centre. However, leucine is also able to bind at a separate regulatory site.     

Glutamate is not a messenger in insulin secretion

Experiments do not support a recent claim that glutamate formed from the amination of citric acid cycle-derived alpha-ketoglutarate is a messenger in glucose-induced insulin secretion (Maechler, P., and Wollheim, C. (1999) Nature 402, 685-689). Glucose, leucine, succinic acid methyl ester, and alpha-ketoisocaproic acid all markedly stimulate insulin release but do not increase glutamate levels in pancreatic islets. Increasing the intracellular glutamate levels to 10-fold higher than basal levels by adding glutamine to islets does not stimulate insulin release. When leucine, in addition to glutamine, is applied to islets, insulin release is almost as high as with glucose alone. This is consistent with the known ability of leucine to allosterically activate glutamate deamination by glutamate dehydrogenase, which can supply alpha-ketoglutarate to the citric acid cycle. Experiments with mitochondria from pancreatic islets suggest that flux through the glutamate dehydrogenase reaction is quiescent during glucose-induced insulin secretion. These experiments support the traditional idea that when insulin release is associated with flux through glutamate dehydrogenase, the flux is in the direction of alpha-ketoglutarate.     

Regulation of aminotransferase-glutamate dehydrogenase interactions by carbamyl phosphate synthase-I, Mg2+ plus leucine versus citrate and malate

Citrate, malate, and high levels of ATP dissociate the mitochondrial aspartate aminotransferase-glutamate dehydrogenase complex and have an inhibitory effect on the latter enzyme. These effects are opposed by Mg2+, leucine, Mg2+ plus ATP, and carbamyl phosphate synthase-I. In addition, Mg2+ directly facilitates formation of a complex between glutamate dehydrogenase and the aminotransferase and displaces the aminotransferase from the inner mitochondrial membrane which could enable it to interact with glutamate dehydrogenase in the matrix. Zn2+ also favors an aminotransferase-glutamate dehydrogenase complex. It, however, is a potent inhibitor of and has a high affinity for glutamate dehydrogenase. Leucine, however, enhances binding of Mg2+ and decreases binding of and the effect of Zn2+ on the enzyme. Thus, since both metal ions enhance enzyme-enzyme interaction and Zn2+ is a more potent inhibitor, the addition of leucine in the presence of both metal ions results in activation of glutamate dehydrogenase without disruption of the enzyme-enzyme complex. Furthermore, the combination of leucine plus Mg2+ produces slightly more activation than leucine alone. These results indicate that leucine, carbamyl phosphate synthase-I, and its substrate and cofactor, ATP and Mg2+, operate synergistically to facilitate glutamate dehydrogenase activity and interaction between this enzyme and the aminotransferase. Alternatively, Krebs cycle intermediates, such as citrate and malate, have opposing effects.     

The effects L-leucine on the synthesis of urea, glutamate and glutamine by isolated rat liver cells.

With either alanine or a mixture of 15 different amino acids as nitrogen source, the addition of L-leucine inhibited the synthesis of urea by isolated rat liver cells. With alanine present leucine promoted the production of glutamate and glutamine. Comparison of effects of leucine on soluble glutamate dehydrogenase, mitochondria and isolated cells supports the postulate that leucine exerts its effect through activation of glutamate dehydrogenase. It is suggested that this latter enzyme may not be as important for the production of NH3 for carbamoyl phosphate synthesis as has been considered hitherto.     

A continuous 96-well plate spectrophotometric assay for branched-chain amino acid aminotransferases

A new, continuous 96-well plate spectrophotometric assay for the branched-chain amino acid aminotransferases is described. Transamination of L-leucine with alpha-ketoglutarate results in formation of alpha-ketoisocaproate, which is reductively aminated back to L-leucine by leucine dehydrogenase in the presence of ammonia and NADH. The disappearance of absorbance at 340 nm due to NADH oxidation is measured continuously. The specific activities obtained by this procedure for the highly purified human mitochondrial and cytosolic isoforms of BCAT compare favorably with those obtained by a commonly used radiochemical procedure, which measures transamination between alpha-ketoiso[1-14C]valerate and L-isoleucine. Due to the presence of glutamate dehydrogenase substrates (alpha-ketoglutarate, ammonia, and NADH) and L-leucine (an activator of glutamate dehydrogenase) in the standard assay mixture, interference with the measurement of BCAT activity in tissue homogenates by glutamate dehydrogenase is observed. However, by limiting the amount of ammonia and including the inhibitor GTP in the assay mixture, the interference from the glutamate dehydrogenase reaction is minimized. By comparing the rate of loss of absorbance at 340 nm in the modified spectrophotometric assay mixture containing leucine dehydrogenase to that obtained in the modified spectrophotometric assay mixture lacking leucine dehydrogenase, it is possible to measure BCAT activity in microliter amounts of rat tissue homogenates. The specific activities of BCAT in homogenates of selected rat tissues obtained by this method are comparable to those obtained previously by the radiochemical procedure.     

Resistance of tropoelastin and elastin peptides to degradation by alpha 2-macroglobulin-protease complexes

When α-ketoglutarate is the substrate, malate is a considerably more effective inhibitor of glutamate dehydrogenase than glutamate, oxalacetate, aspartate, or glutarate. Malate is a considerably poorer inhibitor when glutamate is the substrate. Malate is competitive with α-ketoglutarate, uncompetitive with TPNH, and noncompetitive with glutamate. The above, plus the fact that malate is a considerably more potent inhibitor when TPNH rather than TPN is the coenzyme, indicates that malate is predominantly bound to the α-ketoglutarate site of the enzyme-TPNH complex and has a considerably lower affinity for the enzyme-TPN complex. Ligands which decrease binding of TPNH to the enzyme such as ADP and leucine markedly decrease inhibition by malate. Conversely, GTP, which increases binding of TPNH to the enzyme also enhances inhibition by malate. Malate also decreases interaction between mitochondrial aspartate aminotransferase and glutamate dehydrogenase. This effect of malate on enzyme-enzyme interaction is enhanced by DPNH and GTP which also increase inhibition of glutamate dehydrogenase by malate and is decreased by TPN, ADP, ATP, α-ketoglutarate, and leucine which decrease inhibition of glutamate dehydrogenase by malate. These results indicate that malate could decrease α-ketoglutarate utilization by inhibiting glutamate dehydrogenase and retarding transfer of α-ketoglutarate from the aminotransferase to glutamate dehydrogenase. These effects of malate would be most pronounced when the mitochondrial level of α-ketoglutarate is low and the level of malate and reduced pyridine nucleotide is high.     

Inhibition of glutamate dehydrogenase activity in rabbit renal mitochondria by phosphoenolpyruvate

The effect of phosphoenolpyruvate on glutamate dehydrogenase activity was studied in both intact and Triton X-100-treated rabbit renal mitochondria. The intramitochondrial phosphoenolpyruvate content was modulated by application of both 3-MPA, an inhibitor of phosphoenolpyruvate carboxykinase, and BTCA, which inhibits the tricarboxylate-transporting system. The data indicate that: (i) phosphoenolpyruvate is a potent inhibitor of glutamate dehydrogenase activity; and (ii) its inhibitory effect on the enzyme may be abolished by leucine and ADP, activators of glutamate dehydrogenase.     

Autophagy of mitochondria in rat liver assessed by immunogold procedures

Glutamate dehydrogenase and carbamoyl phosphate synthase-I were localized in rat liver by immunogold procedures, using monoclonal and polyclonal antibodies. As expected, there was extensive labeling in mitochondria. Label was also found in lysosomal autophagic vacuoles. When autophagy was stimulated by in vivo administration of the anti-microtubular agent vinblastine we found that: (a) carbamoyl phosphate synthase-I and glutamate dehydrogenase could be found in mitochondria within autophagic vacuoles; (b) the carbamoyl phosphate synthase-I and glutamate dehydrogenase content of the mitochondria sequestered into autophagic vacuoles is the same as that of the nearby "free" mitochondria; and (c) in the whole liver, autophagic vacuoles contain c. 1.5 times more glutamate dehydrogenase than carbamoyl phosphate synthase-I, in contrast to mitochondria which have c. three times more carbamoyl phosphate synthase-I than glutamate dehydrogenase. The latter finding could explain, at least partially, the difference in half-lives of these enzymes.     

Factors affecting protein synthesis during biotin deficiency inAspergillus nidulans

Biotin deficiency in Aspergillus nidulans resulted in a 70% increase of the protein content and increased levels of free and bound aspartate, glutamate, serine, leucine and methionine. Likewise, the activities of NADP+ glutamate dehydrogenase, NAD+ gluatmate dehydrogenase, aspartate aminotransferase and alanine aminotransferase were significantly increased. The total RNA content increased while the DNA content was unaffected. The rRNA/tRNA ratio remained higher in biotin-deficient cells. Supplementation of glutamate, aspartate, serine, leucine and methionine to the culture medium raised the rRNA/tRNA ratio, and the difference observed in the qualitative and the quantitative patterns of protein and dry cell mass between normal and biotin-deficient cultures was abolished.     

Activation of Glutamate Dehydrogenase by Leucine and Its Nonmetabolizable Analogue in Rat Brain Synaptosomes

Leucine and beta-(+/-)-2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH) stimulated, in a dose-dependent manner, reductive amination of 2-oxoglutarate in rat brain synaptosomes treated with Triton X-100. The concentration dependence curves were sigmoid, with 10-15-fold stimulations at 15 mM leucine (or BCH); oxidative deamination of glutamate also was enhanced, albeit less. In intact synaptosomes, leucine and BCH elevated oxygen uptake and increased ammonia formation, consistent with stimulation of glutamate dehydrogenase (GDH). Enhancement of oxidative deamination was seen with endogenous as well as exogenous glutamate and with glutamate generated inside synaptosomes from added glutamine. With endogenous glutamate, the stimulation of oxidative deamination was accompanied by a decrease in aspartate formation, which suggests a concomitant reduction in flux through aspartate aminotransferase. Activation of reductive amination of 2-oxoglutarate by BCH or leucine could not be demonstrated even in synaptosomes depleted of internal glutamate. It is suggested that GDH in synaptosomes functions in the direction of glutamate oxidation, and that leucine may act as an endogenous activator of GDH in brain in vivo.     

Gene cloning and sequence determination of leucine dehydrogenase from Bacillus stearothermophilus and structural comparison with other NAD(P)+-dependent dehydrogenases

The gene for leucine dehydrogenase (EC 1.4.1.9) from Bacillus stearothermophilus was cloned and expressed in Escherichia coli. The selection for the cloned gene was based upon activity staining of the replica printed E. coli cells. A transformant showing high leucine dehydrogenase activity was found to carry an about 9 kilobase pair plasmid, which contained 4.6 kilobase pairs of B. stearothermophilus DNA. The nucleotide sequence including the 1287 base pair coding region of the leucine dehydrogenase gene was determined by the dideoxy chain termination method. The translated amino acid sequence was confirmed by automated Edman degradation of several peptide fragments produced from the purified enzyme by trypsin digestion. The polypeptide contained 429 amino acid residues corresponding to the subunit (Mr 49,000) of the hexameric enzyme. Comparison of the amino acid sequence of leucine dehydrogenase with those of other pyridine nucleotide dependent oxidoreductases registered in a protein data bank revealed significant sequence similarity, particularly between leucine and glutamate dehydrogenases, in the regions containing the coenzyme binding domain and certain specific residues with catalytic importance.     

Bifunctional isocitrate-homoisocitrate dehydrogenase: a missing link in the evolution of beta-decarboxylating dehydrogenase

Beta-decarboxylating dehydrogenases comprise 3-isopropylmalate dehydrogenase, isocitrate dehydrogenase, and homoisocitrate dehydrogenase. They share a high degree of amino acid sequence identity and occupy equivalent positions in the amino acid biosynthetic pathways for leucine, glutamate, and lysine, respectively. Therefore, not only the enzymes but also the whole pathways should have evolved from a common ancestral pathway. In Pyrococcus horikoshii, only one pathway of the three has been identified in the genomic sequence, and PH1722 is the sole beta-decarboxylating dehydrogenase gene. The organism does not require leucine, glutamate, or lysine for growth; the single pathway might play multiple (i.e., ancestral) roles in amino acid biosynthesis. The PH1722 gene was cloned and expressed in Escherichia coli and the substrate specificity of the recombinant enzyme was investigated. It exhibited activities on isocitrate and homoisocitrate at near equal efficiency, but not on 3-isopropylmalate. PH1722 is thus a novel, bifunctional beta-decarboxylating dehydrogenase, which likely plays a dual role in glutamate and lysine biosynthesis in vivo.     

Purification and properties of glutamate dehydrogenase from the mantle muscle of the squid, Loligo pealeii. Role of the enzyme in energy production from amino acids.

1. The activity of glutamate dehydrogenase was measured in the tissues of the squid, Loligo pealeii. The enzyme occurs in high activity in digestive pouch, systemic heart, and all muscle tissues. 2. Glutamate dehydrogenase from mantle muscle is located intra-mitochondrially, has a molecular weight of 310,000, and is electrophoretically similar to the enzyme from all other squid tissues. 3. The enzyme from mantle muscle was purified 40-fold by elution from DEAE-cellulose and used for kinetic studies. The enzyme is NAD+-specific, activated by ADP, AMP, and leucine, and inhibited by GTP, GDP, ATP, and reaction products (in particular NADH). 4. Squid glutamate dehydrogenase shows an almost absolute dependence on ADP. The purified enzyme is activated over 100-fold by saturating concentrations of ADP (Ka = 0,75 7M); The pH optima are also altered significantly by ADP. 5. The enzyme appears to be kinetically adapted to favour glutamate oxidation in comparison to glutamate dehydrogenase from other resources. The evidence indicates that the primary role of glutamate dehydrogenase in squid mantle muscle is in regulating the catabolism of amino acids for energy production.     

Enzyme activities in the plasma of rainbow trout, Salmo gairdneri Richardson; the effects of nutritional status and salinity

The levels of lactate dehydrogenase, hydroxy butyric dehydrogenase, glutamic oxalacetic transaminase, glutamic pyruvic transaminase, glutamate dehydrogenase, alkaline phosphatase and leucine amino peptidase were determined in the plasma of rainbow trout. The protein concentration and the amount of alkaline phosphatase were reduced in starving trout. Fed trout showed reduced lactate dehydrogenase activity. There was a significant correlation between the condition factor, most of the enzyme activities and the protein concentration. At 10 parts per thousand salinity the activities of alkaline phosphatase and leucine amino peptidase were significantly elevated, while lactate dehydrogenase activity was significantly decreased.     

Enzyme activities in the serum of rainbow trout,Salmo gairdneri Richardson; the effects of water temperature*

Activities of lactate dehydrogenase, hydroxy butyric dehydrogenase, glutamic oxalacetic transaminase, glutamic pyruvic transaminase, glutamate dehydrogenase, creatinine kinase, alkaline phosphatase, and leucine amino peptidase were determined in the sera of rainbow trout. The animals had previously been adapted to temperatures of 3.5, 6, 8, 10, 12.5, 15, 17, 19, 21.5 and 23° C. Most of the enzyme activity increased with the rise in temperature. The activity of alkaline phosphatase decreased in the range 6–19° C, while the changes in the glutamate dehydrogenase activity took a complex course. The results are compared with the findings of other authors.     

Activity of several erythrocyte enzymes in chick embryos and chicks after gamma-irradiation

After irradiation of chick embryos and chicks (1,000 rad), the activity of some erythrocyte enzymes undergoes significant changes. During the 1st day after irradiation of chick embryos, the activity of lactate dehydrogenase leucine aminopeptidase and glutamate pyruvate transaminase decreases. At the 3rd day, the decrease in the activity of glucose-6-phosphate dehydrogenase and acid phosphatase is also observed. In irradiated chicks, the activity of lactate dehydrogenase, leucine aminopeptidase and aldolase decreases within the 1st and the 3rd days, the decrease being most significant for the former two enzymes. At later period (10 and 15 days after irradiation), most significant decrease was found in the activity of glucose-6-phosphate dehydrogenase. The activity of the same enzymes in the blood plasma of irradiated embryos and chicks increases, the increase being most evident for glucose-6-phosphate dehydrogenase.     

A combination of intracellular leucine with either glutamate or aspartate inhibits autophagic proteolysis in isolated rat hepatocytes

It has been shown previously that the inhibition of autophagic proteolysis in liver by a physiological mixture of amino acids can be mimicked completely by addition of leucine in combination with alanine [Leverve, X. M., Caro, L. H. P., Plomp, P. J. A. M. and Meijer, A. J. (1987) FEBS Lett. 219, 455-458]. We have now further defined conditions which lead to this inhibition. Isolated rat hepatocytes were incubated in the perifusion system in which the cells can be maintained at a steady state in the presence of low amino acid concentrations. Combinations of leucine (0.5 mM) with either alanine, glutamine, asparagine or proline (2 mM) inhibited proteolysis by 40-50%. Under these conditions, both in the absence and presence of the transaminase inhibitor, aminooxyacetate, a correlation was found between the extent of inhibition of proteolysis and the sum of the total intracellular amounts of aspartate and glutamate. Inhibition of proteolysis by leucine and leucine analogues did not correlate with their ability to activate glutamate dehydrogenase.