L-glutamate Dehydrogenase Research Articles

The Molecular Weight of the Polypeptide Chains of l-Glutamate Dehydrogenase

The Molecular Weight of the Polypeptide Chains of l-Glutamate Dehydrogenase

Photoinactivation of L-glutamate dehydrogenase in a spectrophotofluorimeter

The inactivatin of enzymes by ultraviolet radiation has been studied (Setlow and Doyle 1957; Mee 1964), but knowledge of which amino acid residues are affected is still incomplete. We have recently noted that beef liver L-glutamate dehydrogenase (GDH) lost enzyme activity as well as protein fluorescence under certain conditions of spectrophotofluorimetric assay. Since the fluorescence of proteins is due almost wholly to tryptophan residues (Teale 1960), these observations seem relevant to the mechanism of photoinactivation of enzymes. This communication documents these findings and also discusses the significance of photodecomposition in fluorimetric analysis.

Catalytic activity of sub-units of glutamic dehydrogenase.

L-GLUTAMIC dehydrogenase (GDH) has been shown to dissociate into sub-units under a variety of experimental conditions, such as dilution1, high or low pH2,3, and presence of DPNH4, urea5, diethylstilbœstrol6, or bicarbonate ions7. The relationship between the state of aggregation of the enzyme and its catalytic activity has been the subject of some controversy. Several investigators have attempted to relate the state of aggregation of the enzyme, as studied at high protein concentrations, to catalytic activity measured at protein concentrations several orders lower in magnitude6,8,9. Recent light scatter studies in this laboratory have shown that if the degree of splitting of the enzyme is plotted against pH and enzyme concentration, a quite irregular mathematical surface results2. Consideration of such surfaces plotted from experimental results obtained in the presence of various substrates, coenzymes, and other compounds suggested to us that it is, in general, quite invalid to relate catalytic activity measured under one set of conditions to degree of aggregation of the protein measured under different conditions. We therefore sought to measure both catalytic activity and the degree of aggregation of the protein in identical solutions so that these two properties might be related to each other in a valid manner.

FLAVOPROTEINS IN THE LEAVES OF HIGHER PLANTS CATALYZING THE OXIDATION OF L-GLUTAMATE

Two forms of enzyme capable of catalyzing the oxidation of L-glutamate (and L-aspartate) were isolated from the leaves of spinach and separated from each other by column-chromatographic purification on calcium phosphate and anion exchangers. They were distinguished as GD1 (L-glutamate dehydrogenase 1) and GD2 (L-glutamate dehydrogenase 2). The purification procedures and some fundamental properties of the partially purified enzymes were investigated. It was discovered that the enzymes did not require any cofactor, i e., neither dialysis nor precipitation with ammonium sulfate caused a fall in enzyme activities and the addition of DPN and TPN to the reaction mixture did not accelerate the reaction rate From the results of spectroscopic investigation GD1 and GD2 were shown to be flavoproteins, although their prosthetic group has not yet been identified The activity of GD1 was enhanced by the addition of FAD or FMN, while GD2 was not accelerated by these factors. The characteristics of the two enzymes including substrate specificity, MICHAELIS constant, optimum pH of the reaction and specificity for electron acceptors were compared. From the stoichiometric study of the oxidation of L-glutamate with these enzymes, it was confirmed that the reaction is represented by the following equation: L-glutamate+oxidized dye+h2oGD1orGD2→ Among various inhibitors tested, molecular oxygen which could function as electron acceptor of L-glutamate oxidation in the presence of GD1 was found to cause a strong inhibition upon the same reaction with TTC as el acceptor. The inhibition was confirmed to be due to hydrogen peroxide produced as a result of the aerobic oxidation of L-glutamate.

Lack of species specificity of antibody to an enzyme.

Antibodies to crystalline bovine hepatic L-glutamic dehydrogenase were induced in rabbits. These antibodies inhibited the bovine glutamic dehydrogenase used as antigen, and also inhibited glutamic dehydrogenases from rat, rabbit, human, pigeon, and frog livers, as well as frog renal and muscle glutamic dehydrogenase. The antibody did not inhibit yeast glutamic dehydrogenase which differs from the animal enzymes in cofactor requirement. The kinetic characteristics of the inhibition of the bovine glutamic dehydrogenase indicate mixed competitive and non-competitive inhibition, suggesting reaction with multiple antigenic sites; the data indicate competitive inhibition of the enzyme from other species, suggesting reaction with the catalytic site.

Inhibition of certain mitochondrial oxidative enzymes by porphyrins and metalloporphyrins

Liver mitochondria of mice administered hematoporphyrin parenterally showed inhibition of succinate-cytochrome c reductase, primary succinate dehydrogenase, isocitrate dehydrogenase, l-glutamate dehydrogenase and DPNH-cytochrome c reductase. Cytochrome oxidase was not inhibited. Porphyrin compounds in amounts in excess of those in the control animals were found in these mouse liver mitochondria and similar amounts of hematoporphyrin, as well as protoporphyrin and metalloporphyrins, were incorporated into mouse liver mitochondria in vitro. isoCitrate dehydrogenase, primary succinate dehydrogenase and DPNH-cytochrome c reductase were inhibited to a degree similar to that observed with the in vivo-administration of the inhibitor. Similar inhibitions also were observed with isocitrate and primary succinate dehydrogenases solubilized from the acetone powders. Highly purified preparations of enolase, aldolase and lactate dehydrogenase were not inhibited. The enzyme inhibitions noted, as well as others which may be present, appear to explain, at least in part, the toxicity of these compounds, and offer a possible mechanism for a biochemical pathogenesis of the loss of cellular function noted in some of the porphyrias.

L-Glutamate dehydrogenase isoenzymes.

SEVERAL dehydrogenases have been resolved into isoenzymes by electrophoresis in starch gel. Attempts to fractionate L-glutamate dehydrogenase in this way, however, have so far failed. Markert and Moller1 found that this enzyme was electrophoretically immobile in starch gel, and Tsao2 obtained negative results in attempts to fractionate and render gluta-mate dehydrogenase visible.

Development of brain enzymes concerned with glutamic acid metabolism in fetal monkey and pig

Changes in the activity of glutamotransferase, l-glutamic dehydrogenase, and glutamic-oxaloacetic transaminase were encountered during the fetal development of the pig and monkey brain. There was an increase in the specific activity (per milligram of dried tissue) in all three enzymes during fetal growth, with the smallest relative increase in glutamic-oxaloacetic transaminase and the greatest activity in the glutamotransferase. The latter increase was especially marked in the last part of intrauterine life and more marked in the pig. An unexplained decrease in the activity of the fetal brain glutaminase was encountered in the final stages of gestation.

The oxidation of l-amino acids by mitochondria from rat liver

The oxidation of l-alanine by actively phosphorylating mitochondria from rat liver has been shown. l-Alanine alone was oxidised very slowly, but, in the presence of T.C.A. intermediates and l-glutamate, there was a marked stimulation of its oxidation. The system was very responsive to low concentrations of l-alanine and to most of the T.C.A. intermediates. Of the T.C.A. intermediates α-oxo-glutarate showed the greatest stimulation. l-Alanine and l-aspartate reduced the conversion of dl-[I- 14C]-glutamate to carbon-dioxide. It is concluded that the probable mechanism of oxidation is not by action of l-amino acid oxidase but via transamination to α-oxo-glutarate and subsequent oxidation of the pyruvate produced. The results give evidence for mitochondrial l-glutamic dehydrogenase and transaminase as a functional cyclic system in the deamination of amino acids and as mediators of the oxidation of their carbon skeletons.

Distribution of l-alanine dehydrogenase and l-glutamate dehydrogenase in Bacilli

Distribution of l-alanine dehydrogenase and l-glutamate dehydrogenase in Bacilli

L-GLUTAMIC ACID DEHYDROGENASE: STRUCTURAL REQUIREMENTS FOR SUBSTRATE COMPETITION: EFFECT OF THYROXINE

l-GLUTAMIC ACID DEHYDROGENASE: STRUCTURAL REQUIREMENTS FOR SUBSTRATE COMPETITION: EFFECT OF THYROXINE

The utilization of purines, pyrimidines, and inorganic nitrogenous compounds by effective and ineffective strains of Rhizobium meliloti.

Ammonium chloride was not utilized by three strains of Rhizobium meliloti as the sole source of nitrogen in a sucrose medium, unless either amino or certain non-nitrogenous carboxylic acids were also present. This was also essentially true for the utilization of nitrate, nitrite, purines, and pyrimidines, all of which are potentially able to form ammonia. These results may be interpreted on the assumption that washed cells of alfalfa – sweet clover rhizobia require, for growth initiation in a nitrogen-free medium, either preformed amino acids or compounds such as ammonia and certain carboxylic acids from which amino acids can be synthesized. Since α-ketoglutarate was extremely active in promoting growth in a medium containing ammonium chloride it was implied that the ammonia may be fixed by L-glutamic acid dehydrogenase activity, especially since this particular enzyme was located in these organisms. No aspartase activity could be demonstrated. The ineffective strain differed from the effective strains in that it was unable to use purines or pyrimidines as accessory nitrogen sources in amino acid media. This was a result of strain variation and it was not coupled with the state of ineffectiveness itself. A synthetic medium has been formulated for further growth studies on washed Rhizobium cells and for investigations on auxotrophic mutants of these bacteria.

Glutamic dehydrogenase

The preparation and crystallization of l-glutamic dehydrogenase from ox and calf liver is described. The equilibrium constant for the reaction glutamate + = + DPN + + H 2 O ⇌ α- ketoglutarate = + NH 4 + + DPNH − + H + was determined and found to vary somewhat with pH and buffer system. The rate of oxidation of glutamate was also found to vary with pH. Extension of specificity studies resulted in confirmation of the reported strict specificity for l-glutamic acid. No evidence for the nonenzymatic formation of iminoglutaric acid from ammonia and α-ketoglutarate was obtained.

Intracellular distribution of enzymes.

The localization of enzymes in cells may be studied by the differential centrifugation of tissue homogenates. This method has been used to study the distribution of L-malic and L-glutamic dehydrogenases, choline oxidase, adenosinetriphosphatase, and also of the nucleic acids and nitrogen between the fractions of the homogenate. L-Glutamic dehydrogenase is entirely m itochondrial, and malicdehydrogenase is shared almost equally, between the mitochondria and the supernatant although the true mitochondrial activity is not apparent unless unmasked, e. g. by water disruption. In the absence of this precaution intact mitochondria show only a small proportion of the activity of the whole homogenate. Choline oxidase is almost entirely mitochondrial, and adenosine triphosphatase has a large representation in all the particulate fractions. In our experiments we have found that the ribonucleic acid content of the mitochondria is higher than previously reported. Distribution studies of enzymes are misleading unless it can be shown that the methods employed are valid for all the fractions studied, and our evidence shows that serious errors of in terpretation may arise unless more than one method of determination is used. In particular, the physical state of the mitochondria affects their apparent enzyme content, as shown by the; investigation of malic and glutamic dehydrogenases by manometric and optical methods. This anomaly is due to an ‘accessibility barrier’ or ‘permeability barrier’ present in intact mitochondria, which hinders the entry of coenzyme I. In an integrated system composed of several enzymes, the rate of the whole reaction may be limited by the rate of any one of the intermediate steps. Thus intact mitochondria fail to develop their maximum oxygen up take with several substrates unless a continuous supply of phosphate acceptor is ensured. Otherwise the rate of transfer of phosphate limits the whole reaction, reducing the apparent activity of the mitochondrial fraction relative to that of the whole homogenate, and hence the apparent distribution of enzyme. Similarly, the activity of the choline oxidase of the mitochondrial fraction is more sensitive to pH changes than that of the whole homogenate so that at pH 6·8, 80% of the homogenate activity may be recovered in the mitochondria, whereas at pH 7·8, the recovery is only 50%. At pH 7·8 full activity of the mitochondria, and a recovery of over 80%, may be achieved by the addition of coenzyme I.

Significance of changes in renal and hepatic L-glutamic acid dehydrogenase activity in hypophysectomized or normal rats treated with growth hormone or thyroxin.

LARGELY as a result of studies by Braunstein and co-workers (1939, 1945), attention has been directed to a pathway of amino acid catabolism in which transaminases and L-glutamic acid dehydrogenase are involved. It seemed of obvious interest to determine whether or not the activity of the latter enzyme in tissues is diminished during growth induced with somatotrophin preparations, for it is conceivable that the observed anabolic effects might be due in part to interference with the oxidative deamination of glutamic acid formed from other amino acids by transamination. experimental For our studies of L-glutamic acid dehydrogenase, normal and hypophysectomized Sprague-Dawley rats of the same age were obtained simultaneously from the same source.1 Since these animals were also used for studies on tissue cathepsin, details concerning the design of experiments, growth hormone preparation, diet, handling of tissues, and some of the methods of analysis, have already been given (Mathies, Palm, and Gaebler, 1951).