Cell-free synthesis and processing of a large precursor of glutamate dehydrogenase of rat liver

The mitochondrial matrix protein glutamate dehydrogenase of rat liver was synthesized in a cell-free reticulocyte lysate using mRNA from free or membrane-bound polysomes from rat liver. Immunoprecipitation of the (³⁵S)methionine labeled translation mixture was performed using rabbit anti-glutamate dehydrogenase serum. Analysis after electrophoresis of the immunoprecipitate by fluorography of a dried sodium dodecyl sulfate/polyacrylamide gel showed that the glutamate dehydrogenase is synthesized ‘in vitro’ as a large precursor. A mitochondrial extract from rat liver processed the precursor synthesized “in vitro” to the mature form.     

Crystallization and some properties of glutamate dehydrogenase from rat liver

1. Glutamate dehydrogenase (L-glutamate:NAD(P) oxidoreductase, EC 1.4.1.3) from rat liver has been crystallized with a method carefully avoiding all denaturating agents. A 236-fold purification was achieved at a yield of 20%. The specific activity was 185 units/mg protein. The enzyme was homogeneous by analytical zone electrophoresis and sedimentation studies. The s0(20),w value was 13.2. 2. Sedimentation studies in the analytical ultracentrifuge and the behaviour of the enzyme in the disc-electrophoresis revealed that glutamate dehydrogenase from rat liver did not undergo a reversible association-dissociation reaction as reported of glutamate dehydrogenase of nearly all other mammalians. 3. Using antibodies prepared against crystalline bovine liver glutamate dehydrogenase, no immunological differences between the rat and the bovine liver enzyme could be observed.     

Effect of aspartate on complexes between glutamate dehydrogenase and various aminotransferases.

In previous studies it was found that: (a) aspartate aminotransferase increases the aspartate dehydrogenase activity of glutamate dehydrogenase; (b) the pyridoxamine-P form of this aminotransferase can form an enzyme-enzyme complex with glutamate dehydrogenase; and (c) the pyridoxamine-P form can be dehydrogenated to the pyridoxal-P form by glutamate dehydrogenase. It was therefore concluded (Fahien, L.A., and Smith, S.E. (1974) J. Biol. Chem 249, 2696-2703) that in the aspartate dehydrogenase reaction, aspartate converts the aminotransferase into the pyridoxamine-P form which is then dehydrogenated by glutamate dehydrogenase. The present results support this mechanism and essentially exclude the possibility that aspartate actually reacts with glutamate dehydrogenase and the aminotransferase is an allosteric activator. Indeed, it was found that aspartate is actually an activator of the reaction between glutamate dehydrogenase and the pyridoxamine-P form of the aminotransferase. Aspartate also markedly activated the alanine dehydrogenase reaction catalyzed by glutamate dehydrogenase plus alanine aminotransferase and the ornithine dehydrogenase reaction catalyzed by ornithine aminotransferase plus glutamate dehydrogenase. In these latter two reactions, there is no significant conversion of aspartate to oxalecetate and other compounds tested (including oxalacetate) would not substitute for aspartate. Thus aspartate is apparently bound to glutamate dehydrogenase and this increases the reactivity of this enzyme with the pyridoxamine-P form of aminotransferases. This could be of physiological importance because aspartate enables the aspartate and ornithine dehydrogenase reactions to be catalyzed almost as rapidly by complexes between glutamate dehydrogenase and the appropriate mitochondrial aminotransferase in the absence of alpha-ketoglutarate as they are in the presence of this substrate. Furthermore, in the presence of aspartate, alpha-ketoglutarate can have little or no affect on these reactions. Consequently, in the mitochondria of some organs these reactions could be catalyzed exclusively by enzyme-enzyme complexes even in the presence of alpha-ketoglutarate. Rat liver glutamate dehydrogenase is essentially as active as thebovine liver enzyme with aminotransferases. Since the rat liver enzyme does not polymerize, this unambiguously demonstrates that monomeric forms of glutamate dehydrogenase can react with aminotransferases.     

Immunocytochemical Characterization of Glutamate Dehydrogenase in the Cerebellum of the Rat

The immunocytochemical distribution of glutamate dehydrogenase was studied in the cerebellum of the rat using antibodies made in rabbit and guinea pig against antigen purified from bovine liver. Antiserum was found to block partially enzymatic activity both of the purified enzyme and of extracts of the rat cerebellum. Using immunoblots of proteins of rat cerebellum, a major immunoreactive protein and several minor immunoreactive proteins were detected with antiserum. Only a single immunoreactive protein was detected using affinity-purified antibody preparations. This protein migrates with a molecular weight identical to that of the subunit of glutamate dehydrogenase. Further evidence that the antibodies were selective for glutamate dehydrogenase in rat cerebellum was obtained through peptide mapping. Purified glutamate dehydrogenase and the immunoreactive protein from rat cerebellum generated similar patterns of immunoreactive peptides. No significant cross-reaction was observed with glutamine synthetase. Immunocytochemistry was done on cryostat- and Vibratome-cut sections of the cerebellum of rats that had been perfused with cold 4% paraformaldehyde. Glial cells were found to be the most immunoreactive structures throughout the cerebellum. Most apparent was the intense labeling of Bergmann glial cell bodies and fibers. In the granule cell layer, heavy labeling of astrocytes was seen. Purkinje and granule cell bodies were only lightly immunoreactive, whereas stellate, basket, and Golgi cells were unlabeled. Labeling of presynaptic terminals was not apparent. These findings suggest that glutamate dehydrogenase, like glutamine synthetase, is enriched in glia relative to neurons.     

A histochemical study of changes in mitochondrial enzyme activities of rat liver after ischemia in vitro

Changes in the activity of three mitochondrial enzymes in rat liver after in vitro ischemia have been determined by enzyme histochemical methods. The changes were correlated with the appearance in the electron microscope of flocculent densities in the mitochondria indicative of irreversible cell injury. The flocculent densities were observed in rat liver after about 2 h of ischemia in vitro at 37 degrees C. At the same time the activity of glutamate dehydrogenase, localized in the mitochondrial matrix, started to decrease. However, the activities of succinate dehydrogenase localized in the inner membrane of mitochondria, as well as monoamine oxidase of the mitochondrial outer membrane did not change at that stage. It is concluded from the results of this study and those of others that flocculent densities are formed by denaturation of proteins of the mitochondrial matrix in which glutamate dehydrogenase takes part. It should be considered more as a sign than as the cause of cell death.     

A rapid and efficient new method of purification of glutamate dehydrogenase by affinity chromatography on GTP-sepharose

The very high affinity for GTP of glutamate dehydrogenase was used to purify this enzyme by affinity chromatography. After periodic acid oxidation, GTP was covalently bound to an activated Sepharose. When crude mitochondrial extracts were applied on a column of this GTP-Sepharose, glutamate dehydrogenase was retained with very few other proteins. Glutamate dehydrogenase from rat liver was eluted with a KCl gradient with only one contaminating protein. From a pig heart mitochondrial extract the enzyme was purified 300-fold in one step. A chromatography on hydroxyapatite was sufficient to achieve the purification. This very simple technique avoids the long and troublesome crystallization steps generally involved in glutamate dehydrogenase purification.     

Translation of glutamate dehydrogenase mRNA in a reticulocyte cell-free system

Messenger RNA template activity for glutamate dehydrogenase was detected in poly(A)-rich RNA extracted from rat liver polysomes. Enzyme synthesized in cell-free reticulocyte system was detected by measuring enzyme activity in the translation incubation mixture using dual wavelength spectrophotometric technique. The translation product was also identified by a partial purification of the labeled synthesized enzyme and by coelectrophoresis with the carrier enzyme preparation from mitochondrial matrix.     

Microquantitative analysis of the intra-acinar profiles of glutamate dehydrogenase in rat liver.

In adult male and female rat liver, the activity of NAD(+)-and NADP(+)-dependent glutamate dehydrogenase (GDH) was microquantitatively measured in tissue samples of 50-150 ng, microdissected continuously along the sinusoidal length. Total activity of GDH with NAD+ as co-factor was found to be higher by a ratio of about 1:2.3 than with NADP+. All intra-acinar enzyme profiles, irrespective of sex, showed an increasing gradient of GDH activity from the periportal beginning to the perivenous end. These findings are at variance with the immunohistochemical localization of GDH in rat liver. The microquantitative GDH profiles with higher perivenous values could indicate a more pronounced glutamine synthesis in Zone 3 of the liver acinus.     

The pathway of glutamate oxidation in isolated mitochondria from the avian hepatomatous growth induced by MC-29 virus

The mitochondria isolated from transplantable chicken hepatomatous growth induced by MC-29 virus were deficient in glutamate dehydrogenase. Oxypolarographic tests showed that glutamate oxidation in the tumor mitochondria was initiated via transamination, while glutamate was deaminated by glutamate dehydrogenase in liver mitochondria to supply adenosine triphosphate. Prominent glutamate oxidation and transformation-linked low glutamine synthetase activity may be favorable to the bioenergetics of this fast-growing tumor.     

Changes in the acinar distribution of some enzymes involved in carbohydrate metabolism in rat liver parenchyma after experimentally induced cholestasis

Extrahepatic cholestasis induced by ligation and transsection of the common bile duct caused a change in the parenchyma/stroma relationship in rat liver. Two weeks after ligation, the periportal zones of the parenchyma were progressively invaded by expanding bile ductules with surrounding connective tissue diverging from the portal areas. Parenchymal disarray developed and small clumps of hepatocytes or isolated hepatocytes were scattered within the expanded portal areas. These cells showed normal activity of lactate, succinate and glutamate dehydrogenase and may, therefore, be considered to be functionally active. After cholestasis the remainder of the liver parenchyma showed adaptational changes with respect to glucose homeostasis, as demonstrated by histochemical means. Glycogen stores disappeared completely whereas glycogen phosphorylase activity increased about ten fold. The increased glycogen phosphorylase activity and glycogen depletion indicate a greater glycogenolytic capacity in liver parenchyma after bile duct ligation to maintain as far as possible a normal plasma glucose concentration. The parenchymal distribution pattern of glucose-6-phosphatase activity did not change significantly after bile duct ligation. The isolated hepatocytes within the expanded portal tracts showed a high activity of this enzyme whereas the pericentral parenchyma was only moderately active. The distribution patterns of glucose-6-phosphate dehydrogenase and lactate dehydrogenase activity in the liver parenchyma were also largely unchanged after bile duct ligation, but the histochemical reaction for glucose-6-phosphate dehydrogenase activity demonstrated infiltration of the remainder of the parenchyma by non-parenchymal cells, possibly Küpffer cells and leucocytes as part of an inflammatory reaction. Under normal conditions the mitochondrial enzymes succinate and glutamate dehydrogenase show an opposite heterogenous distribution pattern in liver parenchyma. Following cholestasis both enzymes became uniformly distributed. The underlying regulatory mechanism for these different changes in distribution patterns of enzyme activities is not yet understood.     

Submitochondrial localization and function of enzymes of glutamine metabolism in avian liver

Glutamine synthetase (EC 6.3.1.2) was localized within the matrix compartment of avian liver mitochondria. The submitochondrial localization of this enzyme was determined by the digitonin-Lubrol method of Schnaitman and Greenawalt (35). The matrix fraction contained over 74% of the glutamine synthetase activity and the major proportion of the matirx marker enzymes, malate dehydrogenase (71%), NADP-dependent isocitrate dehydrogenase (83%), and glutamate dehydrogenase (57%). The highest specific activities of these enzymes were also found in the matrix compartment. Oxidation of glutamine by avian liver mitochondria was substantially less than that of glutamate. Bromofuroate, an inhibitor of glutamate dehydrogenase, blocked oxidation of glutamate and of glutamine whereas aminoxyacetate, a transaminase inhibitor, had little or no effect with either substrate. These results indicate that glutamine metabolism is probably initiated by the conversion of glutamine to glutamate rather than to an alpha-keto acid. The localization of a glutaminase activity within avian liver mitochondria plus the absence of an active mitochondrial glutamine transaminase is consistent with the differential effects of the transaminase and glutamate dehydrogenase inhibitors. The high glutamine synthetase activity (40:1) suggests that mitochondrial catabolism of glutamine is minimal, freeing most of the glutamine synthesized for purine (uric acid) biosynthesis.     

THE PURIFICATION AND PROPERTIES OF RAT BRAIN GLUTAMATE DEHYDROGENASE

—A method is described for the preparation of glutamate dehydrogenase in a highly purified form from rat brain. Only one protein band was detected when the enzyme was subjected to electrophoresis on SDS polyacrylamide gels. The rat brain enzyme was essentially identical to the rat liver enzyme with respect to electrophoresis on SDS polyacrylamide gels, immunochemical properties and most kinetic parameters. However, the brain enzyme was much less reactive with glutamate, was more sensitive to inhibition by haloperidol, and was considerably more stable than the liver enzyme.     

Correction with formate of metabolic disorders in alcoholic intoxication

Formate was studied for its effect on the content of acetaldehyde, activity of the total aldehyde dehydrogenase, content of substrates of glycolysis and tricarbonic-cycle and pool of free amino acids of rat tissues during alcohol intoxication. The introduction of formate during the acute alcohol intoxication lowers the acetaldehyde content in the blood; the ethanol load being prolonged--it increases the activity of aldehyde dehydrogenase and normalizes the content of pyruvate, glutamate and malate in the liver and glutamate and oxaloacetate in the brain, that evidences for the correction of metabolic disturbances in the organism.     

The effect of 4-hydroxy-2, 3-trans-pent-en-1-al and maleylaldehyde on some mitochondrial functions

Low concentrations of HPE and MLA inhibited state 3 respiration of rat liver mitochondria in the presence of different NAD⁺-dependent substrates. MLA appeared to be more active than HPE. High aldehyde concentrations inhibited the state 3 respiration with succinate. The restraint of succinate oxidation by HPE and MLA and of glutamate plus malate oxidation by MLA correlated with the inhibition of succinate and glutamate dehydrogenase activites, respectively. HPE inhibited glutamate dehydrogenase at concentrations higher than those affecting glutamate oxidation. Malate dehydrogenase activity was slightly sensitive to HPE and MLA. Both aldehydes inhibited NADH oxidation by freeze-thawed mitochondria. These results suggest the existence of a site particularly sensitive to aldehydes in the electron transport chain between the specific NAD⁺-linked dehydrogenases and ubiquinone.     

NAD kinase interaction with the activator--glutamate dehydrogenase

An electrophoretically homogeneous preparation of the NAD kinase activating factor was isolated from rabbit liver and its physico-chemical properties were investigated. The similarity of molecular weights of the activator subunit and hexamer, pI values, the number of SH-groups to the corresponding parameters for glutamate dehydrogenase and the glutamate dehydrogenase activity demonstrated by this factor allowed for the identification of the NAD kinase activating factor as glutamate dehydrogenase. Using three independent methods, the formation of the NAD kinase--glutamate dehydrogenase complex was shown. Both the oligomeric and monomeric (subunit) forms of NAD kinase were found to be able to form complexes with glutamate dehydrogenase.     

Postnatal development of peroxisomal and mitochondrial enzymes in rat liver.

Subcellular organellles from livers of rats three days prenatal to 50 weeks postnatal were separated on sucrose gradients. The peroxisomes had a constant density of 1.243 g/ml throughout the life of the animal. The density of the mitochondria changed from about 1.236 g/ml at birth to a constant value of 1.200 g/ml after two weeks. The peroxisomal and mitochondrial fatty acid beta-oxidation and the peroxisomal and supernatant activities of catalase and glycerol-3-phosphate dehydrogenase were measured at each age, as well as the peroxisomal core enzyme, urate oxidase, and the mitochondrial matrix enzyme, glutamate dehydrogenase. All of these activities were very low or undetectable before birth. Mitochondrial glutamate dehydrogenase and peroxisomal urate oxidase reached maximal activities per g of liver at two and five weeks of age, respectively. Fatty acid beta-oxidation in both peroxisomes and mitochondria and peroxisomal glycerol-3-phosphate dehydrogenase exhibited maximum activities per g of liver between one and two weeks of age before weaning and then decreased to steady state levels in the adult. Peroxisomal beta-oxidation accounted for at least 10% of the total beta-oxidation activity in the young rat liver, but became 30% of the total in the liver of the adult female and 20% in the adult male due to a decrease in mitochondrial beta-oxidation after two weeks of age. The greatest change in beta-oxidation was in the mitochondrial fraction rather than in the peroxisomes. At two weeks of age, four times as much beta-oxidation activity was in the mitochondria as in the peroxisomal fraction. Peroxisomal glycerol-3-phosphate dehydrogenase activity accounted for 5% to 7% of the total activity in animals younger than one week, but only 1% to 2% in animals older than one week. Up to three weeks of age, 85% to 90% of the liver catalase was recovered in the peroxisomes. The activity of peroxisomal catalase per g of rat liver remained constant after three weeks of age, but the total activity of catalase further increased 2.5- to 3-fold, and all of the increased activity was in the supernatant fraction.     

The availability of carnitine acetyltransferase in mitochondria from guinea-pig liver and other tissues

The carnitine acetyltransferase and glutamate dehydrogenase activities of guinea-pig liver and other tissues were estimated. Both enzymes are wholly mitochondrial, and can only be fully observed after disruption of the mitochondrion. Triton X-100 (0.1%) or freeze-drying revealed more activity than other methods tried. In mitochondria prepared and suspended in 0.25m-sucrose and in cell cytoplasm only small fractions of the total enzymic activity could be observed in guinea-pig liver: on average 7.5% of carnitine acetyltransferase and 5.5% of glutamate dehydrogenase. It is concluded that, in liver or mammary gland of goat, guinea pig or rat, little or no carnitine acetyltransferase is available in vivo to acetyl-CoA outside the mitochondrion.     

Glutamate dehydrogenase and glutamine synthetase activity in some organs of ruminants and monogastric animals.

A comparative study of glutamate dehydrogenase (GLDH 1.4.1.2) and glutamine synthetase (GS 6.3.1.2.) activity in liver, kidney and spleen homogenates from cattle, sheep, pigs and chickens showed that chicken liver contained on an average 3.5%, pig liver 8.3% and bovine liver 45.6% of the glutamate dehydrogenase activity present in sheep liver. Relatively low trace activity was found in the spleen and kidneys, except for the renal cortex of cattle (32% of activity in the liver). GS activity was the highest in chicken liver; in pigs it amounted to 33.40%, in cattle to 24.2% and in sheep to 19.7% of this activity. No marked interspecies differences were found in the values in the kidneys and spleen. It can be concluded from the results that the relatively high GLDH activity in the liver of ruminants compared with pigs and chicken is associated with the greater ability of ruminants to utilize ammonia. The higher GS activity and lower GLDH activity in chicken liver can be attributed to higher uric acid synthesis from ammonia via glutamine and purine bases and the lower ability of birds to utilize ammonia for protein synthesis. The presence of alanine dehydrogenase was not demonstrated in chicken liver, where the maximum oxidation of NADH after the addition to pyruvate and ammonia substrate was found.     

A new biosynthetic route of porphyrin precursors in common between animals and plants

An additional enzyme, 4-oxo-5-hydroxyvalerate (OHV) dehydrogenase was identified and characterized. This enzyme catalyzes the conversion of OHV to 4,5-dioxovalerate, a direct precursor of 5-aminolevulinate. The enzyme was partially purified from rat liver supernatant as two isoenzyme (ca. 40,000 and 70,000 dalton). 5-Aminolevulinate was formed from OHV via 4,5-dioxovalerate by this dehydrogenase and alanine-4,5-dioxovalerate aminotransferase (EC 2.6.1.43). This dehydrogenase required NADP of NAD as a hydrogen acceptor. The enzyme was heat sensitive and catalyzed the reaction reversibly. The dehydrogenase was present in the high speed supernatants of liver and kidney of rat, rabbit and human, and that of spinach leaf.     

The formation of ornithine from proline in animal tissues

1. Homogenates of liver or kidney from rat, mouse, dog and guinea pig formed ornithine from proline but not from glutamate. Rat kidney was most active in this reaction and was used for further studies. 2. The overall reaction was found to be catalysed by proline oxidase to yield glutamic gamma-semialdehyde, followed by transamination of this product with glutamate as catalysed by ornithine-keto acid aminotransferase. 3. The unfavourable equilibrium of the ornithine-keto acid aminotransferase reaction was overcome chiefly by glutamate dehydrogenase in the tissue, which removed the alpha-oxoglutarate produced, by reduction with endogenous ammonia and NADH. 4. Aspartate aminotransferase in these preparations also aided in the removal of alpha-oxoglutarate. In this case the overall reaction was driven also by the rapid decarboxylation of oxaloacetate. 5. No evidence could be found for a pathway of ornithine synthesis involving acylated intermediates as has been observed in some micro-organisms. 6. The rate of ornithine synthesis in homogenates of several rat tissues paralleled the activity of ornithine-keto acid aminotransferase in these tissues, indicating that this enzyme was rate-determining for the synthesis. 7. The possible influence of these reactions on urea synthesis is discussed.