Rat Liver Glutamate Dehydrogenase Research Articles

Image analysis and image processing as tools to measure initial rates of enzyme reactions in sections: distribution patterns of glutamate dehydrogenase activity in rat liver lobules.

To analyze regional differences in the activity of glutamate dehydrogenase in rat liver in situ, we developed an image recording and processing system for monitoring the formation of a colored final reaction product in time. All absorbance measurements of test and control reactions in time in consecutive sections were used to fit the data to a quadratic curve, with the derivative at t = 0 representing the initial velocity of formazan formation. The images of sections incubated for test and control reactions were topographically matched with an affine transformation using the positions of vessels as fiducials. Specific enzyme activity was calculated by subtracting the coefficients representing the initial velocity at corresponding locations in the test and control reactions and appeared to be 8 and 4 mumoles glutamate converted per min per cm3 of tissue at 20 degrees C in pericentral and periportal zones of fasted female rats, respectively. Those values are in agreement with biochemical data. The ability to construct two-dimensional images of cellular distribution patterns of enzyme activity in liver lobules is particularly useful for the study of metabolic zonation in this organ.

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.

Regulation of the expression of glutamate dehydrogenase in rat liver

Regulation of the expression of glutamate dehydrogenase in rat liver

Nucleotide sequence of rat liver glutamate dehydrogenase cDNA

Nucleotide sequence of rat liver glutamate dehydrogenase cDNA

Nucleotide sequence of rat liver glutamate dehydrogenase cDNA

Nucleotide sequence of rat liver glutamate dehydrogenase cDNA

Properties of purified liver glutamate dehydrogenase of aging rats

Glutamate dehydrogenase was purified from the liver of immature (4-weeks), young (22-weeks) and old (116-weeks) female rats to study the effect of age on this enzyme. From 30 g of liver tissue of each age group, ∼90-fold purified enzyme was obtained in crystalline form with an average yield of 4% of the original enzyme activity. This 4% enzyme was used extensively for analyzing its age-related properties. The elution profile on a Sephadex G-200 column, molecular weight (53000±3000), ratio of A 280 260 (1.8), optimum pH (7.8), optimum temperature (25° C), and K m values for different substrates/coenzymes of the purified enzymes did not show marked age-dependent variations. Similarly, remarkable differences were not observed in the effect of a variety of nucleotides or steroid hormones on the activity of the purified enzymes of the three ages. These findings may suggest that rat liver glutamate dehydrogenase is an unaltered enzyme with advancing age of the animal.

Transport of the precursor for rat liver glutamate dehydrogenase into mitochondria “in vitro”

Glutamate dehydrogenase of rat liver, a mitochondrial matrix enzyme, is synthesized in a larger precursor form in a rabbit reticulocyte cell-free system. When this precursor was incubated with isolated mitochondria in the absence of protein synthesis, it was processed to the mature form and rendered protease-resistant during incubation with mitochondria.

Isolation of a mitochondrial factor from rat liver which potentiates the inactivation of glutamate dehydrogenase by lysosomes

Rat liver glutamate dehydrogenase (L-glutamate: NAD(P) oxidoreductase, deaminating) E.C. 1.4.1.3.) is inactivated by the mitochondrial matrix in combination with lysosomal preparations. Neither lysosomal or mitochondrial matrix extracts per se inactivate the enzyme appreciably under the conditions used. Fractionation of the matrix indicates that a low molecular weight factor is responsible for the potentiation of inactivation of glutamate dehydrogenase by lysosomes. Its absorption spectrum and chromatographic behaviour suggest that this factor is NADP.

Crystallographic studies of glutamate dehydrogenase. Preliminary crystal data.

Native and pyridoxal phosphate modified rat liver glutamate dehydrogenase crystals have been obtained and used for a preliminary x-ray crystallographic analysis. The space group is P6222 (P6422) having unit cell dimensions a = b = 101 A, c = 724 A and gamma = 120 A. The unit cell contains 36 subunits (six hexameric molecules) of molecular weight 56,000 and there is one half-molecule, i.e. three subunits, in the asymmetric unit. Packing considerations suggest that the glutamate dehydrogenase molecule has the point group symmetry 32 and that each subunit can be represented as a particle with approximate dimensions of 45 x 45 x 60 A.

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.

Hormonal induction of glutamate dehydrogenase in rat liver

Glutamate dehydrogenase rapidly increases in microsomes and appears in the cytoplasm after administration of cortisone, cAMP, hydrocortisone-acetate. Prolonged administration of ACTH maintains high level of enzyme in the mitochondria and microsomes. Hydrocortisone-acetate, insulin and cortisone decrease drastically enzyme in mitochondria.

Quantitative Analysis of Mixed Association between Different Protein Molecules

The theoretical and experimental analysis of a reversible association-dissociation equilibrium between different proteins (mixed association) is described. The experiments were performed with glutamate dehydrogenases from beef and rat liver. These enzymes are different, especially with respect to their association behavior. The association constant of rat liver glutamate dehydrogenase has been determined by light-scattering measurements. Its value (1.3 x 10(-4) M(-1)) is much lower than that of the beef liver enzyme, but the difference in the free association energy is only 30%. Association between these two enzymes is observed, also employing light-scattering experiments. Theoretical curves for mixed associating systems have been calculated and by comparison with these curves the mixed association constant could be determined. Since the free association energy of the mixed association is very near to the arithmetic mean between the values for the pure enzymes, the association interactions appear to be additive. The model of an open association with a virial coefficient is also true for the rat enzyme and the mixed association. The ultracentrifuge data are also explained by the same model and yield a similar value for the mixed association constant. Differences in the enzyme kinetics are small, but a somewhat reduced lifetime of the ternary complexes with the coenzymes and with subs-rates or GTP can be concluded for the rat liver enzyme. The circular dichroism measurements indicate no significant difference in the dissociation constants of the nucleotides, but the different amplitudes of the ellipticity indicate small differences in the electrical environment of the active center.

The intracellular location of pyruvate carboxylase in rat liver.

Because of reported extramitochondrial location of pyruvate carboxylase in rat liver concluded from extraction studies and the importance of these findings for the concept of the regulation of gluconeogenesis, the intracellular location of this enzyme in rat liver was reinvestigated. The following results were obtained. Reported differences in extraction properties between pyruvate carboxylase and glutamate dehydrogenase in rat liver are due to secondary binding of the latter enzyme to nuclei, cell debris, mitochondria and microsomes. Rat‐liver pyruvate carboxylase is located exclusively in mitochondria, a location in another subcellular component is excluded. Rat liver mitochondria are heterogeneous with respect to their enzyme content. Rate‐zonal centrifugation reveals asymmetric distribution of mitochondrial matrix enzymes: Ornithine aminotransferase is shifted towards slower‐sedimenting mitochondria, pyruvate carboxylase, malate dehydrogenase and aspartate aminotransferase in faster‐sedimenting mitochondria. Glutamate dehydrogenase as well as monoamine oxidase (outer membrane) and adenylate kinase (inter‐membrane space) are located in an intermediate position. Electronmicrographs show that the mitochondria retain their intact outer membrane and most of them are in condensed conformation. The biological significance of the observed heterogeneities with respect to the regulation of gluconeogenesis is discussed.

Biosynthesis of glutamate dehydrogenase in rat liver. Demonstration of its microsomal localization and hypothetical mechanism of transfer to mitochondria

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTBiosynthesis of glutamate dehydrogenase in rat liver. Demonstration of its microsomal localization and hypothetical mechanism of transfer to mitochondriaCatherine Godinot and Henry A. LardyCite this: Biochemistry 1973, 12, 11, 2051–2060Publication Date (Print):May 1, 1973Publication History Published online1 May 2002Published inissue 1 May 1973https://doi.org/10.1021/bi00735a005RIGHTS & PERMISSIONSArticle Views44Altmetric-Citations47LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (2 MB) Get e-Alerts Get e-Alerts

Association behaviour of rat liver glutamate dehydrogenase

Most of the glutamate dehydrogenase (GluDH, EC 1.4.1.3) from liver mitochondria exhibit in solution an association-dissociation equilibrium [2,3] . Although the amino acid composition and the peptide maps of rat liver GluDH [4, 51 are quite similar to the beef liver enzyme this enzyme protein lacks the ability to undergo a concentration-dependent reversible association reaction even in the presence of nucleotides promoting the association of the beef liver enzyme [5], From the similarity of the peptide maps it can be assumed that the conformations are similar, too, and that the relatively small differences in the primary structure in the case of the rat liver enzyme have caused a loss of only some of the structural characteristics of the rat liver enzyme which, in the case of the beef liver enzyme, are responsible for the association reaction. Therefore, it can be expected that: 1) conditions which support the associatioc process and cause an association of the rat liver enzyme may exist, and 2) that even if self association does not occur it might be possible to hybridize rat and beef liver GluDH, although the remaining structural characteristics are not sufficient under the conditions investigated till now for an association process. In the present paper hybridization between oligomers (mixed association) and sedimentation experiments are reported indicating that rat liver GluDH has a tendency to associate and to hybridize.

The Purification and Physical Properties of Glutamate Dehydrogenase from Rat Liver

Abstract Glutamate dehydrogenase has been purified and crystallized from rat liver mitochondria and from whole rat liver preparations. No differences in the enzyme prepared by the two different methods have been observed and there is no evidence that more than one glutamate dehydrogenase occurs in rat liver. The pure enzyme has a molecular weight of 350,000 ± 20,000 and consists of six to eight subunits. In contrast to the bovine liver enzyme, rat liver glutamate dehydrogenase does not polymerize to higher molecular weight forms as the enzyme concentration is raised, even in the presence of nucleotides which promote the association of the bovine liver enzyme. Also in contrast to the bovine liver enzyme, the rat liver glutamate dehydrogenase is rather unstable during the assay at low enzyme concentrations. The amino acid composition and peptide maps of the rat and bovine liver enzymes are quite similar, indicating only a few amino acid replacements. The fact that the polymerization of the bovine liver enzyme may be important in the regulation of activity by purine nucleotides in vivo indicates that the rat liver enzyme must differ with respect to such control since polymerization is lacking.

The molecular weight and some kinetic properties of crystalline rat liver glutamate dehydrogenase

The molecular weight and some kinetic properties of crystalline rat liver glutamate dehydrogenase

An enzymatic model for some effects of methoxyethylmercury acetate on rat liver glutamate dehydrogenase

An enzymatic model for some effects of methoxyethylmercury acetate on rat liver glutamate dehydrogenase