DOCSLIB.ORG

Serum Glutamate Dehydrogenase Is Not a Reliable Marker of Liver Cell Necrosis in Alcoholics

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Serum Glutamate Dehydrogenase Is Not a Reliable Marker of Liver Cell Necrosis in Alcoholics J Clin Pathol: first published as 10.1136/jcp.35.2.207 on 1 February 1982. Downloaded from J Clin Pathol 1982;35;207-210 Serum glutamate dehydrogenase is not a reliable marker of liver cell necrosis in alcoholics WJ JENKINS, SB ROSALKI, Y FOO, PJ SCHEUER, E NEMESANSZKY,* S SHERLOCK From the Departments of Medicine, Chemical Pathology and Histopathology, Royal Free Hospital and School of Medicine, Pond Street, London NW3 2QG SUMMARY Serum glutamate dehydrogenase (EC 1.4.1.3.) activity was measured in 73 hospital patients who had a history of chronic alcohol abuse and who all had a liver biopsy performed. High levels of serum GDH activity occurred in those patients with recent excess alcohol con- sumption independently of the underlying liver histology, and did not discriminate between those patients with and those without alcoholic hepatitis. Since alcoholic hepatitis is thought to be a precursor of GDH reliably reflects liver cell necrosis, and that of cirrhosis, a convenient and reliable marker of liver a serum value two and a half times the normal value cell necrosis would be extremely useful in following is indicative of alcoholic hepatitis.8 For these reasons the progress of liver disease in alcoholic patients. it was suggested that the serum GDH activity should Repeated liver biopsiesare impractical for monitoring be estimated routinely in alcoholics. However, the copyright. purposes, and raised activities of the so-called possibility that recent alcohol consumption might hepatic enzymes commonly measured in serum are increase serum activities of GDH independent of found in all histological stages of alcoholic liver histological change was not investigated. GDH disease from steatosis to cirrhosis.' Conversely in activity is considerably raised in liver biopsies from alcoholic hepatitis the serum transaminase activities recently drinking patients with alcoholic steatosis,9 may be only moderately raised or even normal.2 and high activities of the enzyme in serum may at Furthermore, the transaminases are not specific to least partly reflect this increase in hepatic activity. http://jcp.bmj.com/ the liver, and raised serum activities may reflect We have studied the effect of recent heavy con- damage to other organs. Gamma-glutamyl trans- sumption of alcohol on the activity of GDH in both peptidase (EC 2.3.2.2.) (yGT) has been suggested as serum and liver in unselected alcoholic patients a useful marker of alcoholic liver disease,3 but the undergoing diagnostic liver biopsy, and tested the correlation between raised serum yGT activities and usefulness of serum GDH measurement in dis- liver cell necrosis is poor,4 and increases due to other criminating between those with and those without hepatic causes are common. alcoholic hepatitis. The level of glutamate dehydrogenase (GDH) in on September 23, 2021 by guest. Protected serum might be expected to provide a more reliable Patients and methods index of liver damage in alcoholics, since its activity in the liver is very much higher than in other organs.5 PATIENTS Also, it is concentrated in the centrilobular zones of Seventy-three inpatients (56 men, 17 women) were the liver, where alcohol exerts its greatest toxic studied. They all had a history of alcohol abuse, and effects,6 and within the hepatocytes it is found all had either symptoms, physical signs, or bio- exclusively in mitochondria, which are thought to be chemical evidence of liver disease. Other possible damaged early in the course of alcoholic liver causes of liver disease besides alcohol were excluded disease.7 In a study of 100 alcoholic patients, by the relevant investigations. The reasons for Van Waes and Lieber claimed that the serum activity admission to hospital varied. Some patients were admitted for diagnostic liver biopsy, some for Accepted for publication 3 July 1981 detoxification, and some had the clinical syndrome *Present address: Postgraduate Medical School, Ist Depart- of overt alcoholic hepatitis. All had a percutaneous ment of Medicine, 1135 Budapest, Hungary. liver biopsy performed. Venous blood was sampled 207 J Clin Pathol: first published as 10.1136/jcp.35.2.207 on 1 February 1982. Downloaded from 208 Jenkins, Rosalki, Foo, Scheuer, Nemesanszky, Sherlock for serum enzyme analysis thirty minutes before the with, or without fat or cirrhosis; and eight had biopsy. cirrhosis without evidence of alcoholic hepatitis. A detailed history of alcohol drinking was taken from each patient. The patients were divided arbi- ENZYME ASSAYS trarily into those who had consumed > 80 g of Aspartate aminotransferase (AST), GDH and yGT ethanol/day immediately before liver biopsy, were assayed in serum by standard laboratory designated the "recent alcohol excess" group, and techniques.10-12 Serum AST was determined immedi- those who had consumed <80 g of ethanol/day in ately, and the samples of serum were stored at the preceding week, designated the "no recent - 20°C before GDH and yGT were assayed. Most alcohol excess" group. In practice many of the samples were examined within two weeks of col- patients admitted specifically for liver biopsy had lection, and all within two months. Stability studies considerably reduced their alcohol intake before confirmed that the activities of these enzymes were admission, and others, particularly those with unaltered during storage for this period. alcoholic hepatitis, had been in hospital without In 49 patients the amount of liver tissue obtained alcohol for more than a week before liver biopsy by biopsy exceeded that judged necessary for was performed. histological assessment. The remaining portions of Twelve non-alcoholic patients admitted for liver these biopsies were disrupted in 0-25 mol/l sucrose biopsy for suspected liver disease, but who had containing 3 mmol/l imidazole, pH 7-4, at 4°C in normal serum liver function tests at the time, and a glass Dounce homogeniser. GDH activity and whose liver biopsies were subsequently found to be protein content were assayed in the resulting normal, were used as controls in the study of GDH homogenates. activities in the liver. The results were analysed statistically by the Wilcoxon rank sum test. HISTOLOGY Histology of the liver biopsies was interpreted Results independently without knowledge of the serum copyright. enzyme analyses. Sections were stained by standard Most of our alcoholic patients had raised activities methods and examined for the presence of fat, of one or more of the serum enzymes assayed: inflammation, fibrosis, necrosis and Mallory bodies. 58 had raised serum AST; 59 had raised yGT; and Eight patients had histologically normal livers; 37 had raised GDH. Fig. 1 shows the individual 38 had alcoholic steatosis; 19 had alcoholic hepatitis values in patients with and without recent excessive http://jcp.bmj.com/ 4, Glutamate V Glutamate Aspartate dehydrogenase transpeptidase aminotransferase , 40 E 1 1I 0 a a30 0 lI -aE Wo 0, c 20 0 a 0 on September 23, 2021 by guest. Protected x ._ 0 a E10.- 0 000. 4.. :.0 0 e -> _ 42 St 0 *0 Q. 45 0 a : . : S0*. 3 *^J .% %0- 0@ 2 000o0 0. E 0 000 0 4 1 CA* IS i i - N i CiN 1 %k c 0 w 0 .- I I &' <. N.'z *\' ,X\. 0ro&N' Fig. 1 Serum GDH, yGT and AST in alcoholic patients grouped by liver biopsy histology. (0 > 80 g ethanol/day before biopsy; 0 < 80 g ethanol/day before biopsy) J Clin Pathol: first published as 10.1136/jcp.35.2.207 on 1 February 1982. Downloaded from .Serum glutamate dehydrogenase is not a reliable marker of liver cell necrosis in alcoholics 209 consumption of alcohol grouped according to the criminating between those with and those without histological diagnosis. Clearly the individual values alcoholic hepatitis. However, the serum GDH of serum enzyme activities show a considerable activity in those patients who were drinking heavily overlap among the various histological groups, and immediately before liver biopsy is significantly serum GDH is no better than AST or yGT in dis- higher (p < 0-01) than in those who were not, irrespective of the underlying histological diagnosis .121 (Fig. 2). p<O-O1 Fig. 3 shows the specific activities of GDH in 0 49 liver biopsies from alcoholic patients, and in the 10. 12 control patients. The activities of GDH in the livers of those alcoholic patients, who had a history 0 of recent alcohol excess and whose liver biopsies 81 showed fatty change only, are significantly higher (p < 0-01) than in the normal controls. In contrast the hepatic activities of GDH are lower (p < 0 01) 0 in those patients with alcoholic hepatitis presumably Enzyme activity bl expressed as multiple 0 because of liver cell necrosis. 0@ upper limit of normal 0 90 Discussion 4. 0S 0 Our results clearly contrast with those of a similar previous study of alcoholics, in which the activity of 2 GDH in plasma was reported to be a reliable marker *% of liver cell injury.8 We found that the serum GDH activity was considerably raised in many of our 0 Recent alcohol No recent alcohol alcoholic patients whose liver biopsies showed fatty copyright. excess excess change alone, whereas other patients with histo- severe alcoholic Fig. 2 Serum GIDH in alcoholic patients (0 > 80 g logically proven and often hepatitis ethanol/day before liver biopsy; 0 < 80 g ethanol/day had only slightly raised serum GDH activities. Thus, before biopsy) a serum GDH activity two and a half times the normal value does not discriminate between those alcoholic patients who had alcoholic hepatitis, and others who had not, as was previously claimed. However, we have shown that recent heavy http://jcp.bmj.com/ alcohol consumption increases the activity of GDH .c 2500 in both serum and liver. This is compatible with 0 induction of the enzyme by alcohol, and limits the use of serum GDH measurement as an indicator of 8 2000 pathological change.
Load more

Recommended publications
  • Comparison of Control Materials Containing Animal and Human Enzymes 579
    Gruber, Hundt, Klarweinf and Möllfering: Comparison of control materials containing animal and human enzymes 579 J. Clin. Chem. Clin. Biochem. Vol. 15,1977, pp. 579-582 Comparison of Control Materials Containing Animal and Human Enzymes Comparison of Enzymes of Human and Animal Origin, III By W. Gruber, D. Hundt, M. Klarweinf and A Mollering Boehringer Mannheim GmbH, Biochemica Werk Tutzing (Received February 7/May 31,1977) Summary: Highly purified enzymes of diagnostic interest from human and animal organs, dissolved in pooled human serum and in bovine serum albumin solution, were compared with respect to their response to alterations in routine clinical chemical assay conditions. Their response to changes in temperature, substrate concentration and pH-value was the same. In addition, the storage stability in each matrix was identical in the lyophilized and the reconstituted state, whereas some enzymes were remarkably less stable in the pooled human serum than in bovine serum albumin. This better stability, the better availability and decreased infectious nature of the material lead to the conclusion that animal enzymes in bovine serum albumin matrix are the material of choice for the quality control of enzyme activity determinations in clinical chemistry. Vergleichende Untersuchungen an Kontrollproben, aufgestockt mit tierischen und humanen Enzymen. Vergleich humaner und tierischer Enzyme, III. Mitteilung Zusammenfassung: Hoch gereinigte humane und tierische Enzyme von diagnostischem Interesse, gelöst in gepooltem Humanserum und in Rinderserumalbumin-Lösung wurden in Bezug auf ihr Verhalten gegenüber Änderungen der Reaktionsbedingungen bei klinisch-chemischen Routine-Methoden verglichen. Ihre Aktivitätsänderung bei Ver- änderung der Reaktionstemperatur, der Substrat-Konzentrationen und des pH-Wertes waren gleich.
    [Show full text]
  • Normalization of Γ-Glutamyl Transferase Levels Is Associated With
    Ma et al. BMC Gastroenterol (2021) 21:215 https://doi.org/10.1186/s12876-021-01790-w RESEARCH ARTICLE Open Access Normalization of γ-glutamyl transferase levels is associated with better metabolic control in individuals with nonalcoholic fatty liver disease Qianqian Ma1, Xianhua Liao1, Congxiang Shao1, Yansong Lin1, Tingfeng Wu1, Yanhong Sun2, Shi‑Ting Feng3, Junzhao Ye1* and Bihui Zhong1* Abstract Background: The normalization of liver biochemical parameters usually refects the histological response to treat‑ ment for nonalcoholic fatty liver disease (NAFLD). Researchers have not clearly determined whether diferent liver enzymes exhibit various metabolic changes during the follow‑up period in patients with NAFLD. Methods: We performed a retrospective analysis of patients with NAFLD who were receiving therapy from January 2011 to December 2019. Metabolism indexes, including glucose levels, lipid profles, uric acid levels and liver bio‑ chemical parameters, were measured. Magnetic resonance imaging‑based proton density fat fraction (MRI‑PDFF) and liver ultrasound were used to evaluate steatosis. All patients received recommendations for lifestyle modifcations and guideline‑recommended pharmacological treatments with indications for drug therapy for metabolic abnormalities. Results: Overall, 1048 patients with NAFLD were included and received lifestyle modifcation recommendations and pharmaceutical interventions, including 637 (60.7%) patients with abnormal GGT levels and 767 (73.2%) patients with abnormal ALT levels. Patients with concurrent ALT and GGT abnormalities presented higher levels of metabo‑ lism indexes and higher liver fat content than those in patients with single or no abnormalities. After 12 months of follow‑up, the cumulative normalization rate of GGT was considerably lower than that of ALT (38% vs.
    [Show full text]
  • Convergent Electron Transfer at the Q-Junction and Additive Effect of Substrate Combinations
    O2k-Protocols Mitochondrial Physiology Network 12.12: 1-13 (2012) 2007-2012 OROBOROS Version 6: 2012-11-04 Mitochondrial Pathways through Complexes I+II: Convergent Electron transfer at the Q-Junction and Additive Effect of Substrate Combinations Erich Gnaiger Medical University of Innsbruck D. Swarovski Research Laboratory 6020 Innsbruck, Austria OROBOROS INSTRUMENTS Corp, high-resolution respirometry Schöpfstr 18, A-6020 Innsbruck, Austria [email protected]; www.oroboros.at ‘It is not at all easy to draw a sharp line between cases where what is happening could be called “addition”, and where some other word is wanted.’ Douglas R. Hofstadter (1979) Gödel, Escher, Bach: An Eternal Golden Braid. A metaphorical fugue on minds and machines in the spirit of Lewis Carroll. Penguin Books. Section 1. Electron Transfer System and ET Chain 1 Page 2. Historical Perspectives 4 3. Pyruvate+Glutamate+Malate: PMG 9 4. Pyruvate+Malate+Succinate: PMS 9 5. Glutamate+Malate+Succinate: GMS 10 6. Pyruvate+Malate+Glutamate+Succinate: PMGS 11 7. Glycerophosphate Dehydrogenase and ETF 11 8. Implications 13 1. Electron Transfer System and ET Chain The term ‘electron transfer chain’ (or electron transport chain, ETC) is a misnomer. Understanding mitochondrial respiratory control has suffered greatly from this inappropriate terminology, although textbooks using the term ETC (Lehninger 1970; Nicholls & Ferguson 2002) make it sufficiently clear that electron transfer systems are not arranged as a chain: the ‘ETC’ is in fact not a simple chain but an arrangement of electron transfer complexes in a non-linear, convergent electron transfer system (Hatefi et al 1962; ETS; Fig. 1). [email protected] www.oroboros.at MiPNet12.12 MitoPathways to Complexes I+II 2 O2 Linear NADH CI CIII CIV ETC H2O GpDH CI Q-junction O2 Convergent Q ETS CII H2O ETF Figure 1.
    [Show full text]
  • Is Gdh a Marker for Mitochondria in Brain? / James C
    Fordham University Masthead Logo DigitalResearch@Fordham Chemistry Faculty Publications Chemistry 1986 The ubs cellular localization of glutamate dehydrogenase (gdh): is gdh a marker for mitochondria in brain? / James C. K. Lai, Kwan-Fu Rex Sheu, Young Tai Kim, Donald D. Clarke, and John P. Blass Department of Neurology, Cornell University Medical College and Altschul Laboratory for Dementia Research Burke Rehabilitation Center White Plains, NY 10605 and Department of Medicine Cornell University Medical College New York, NY 10021 James C. K. Lai Cornell University. Department of Neurology and Neuroscience Kwan-Fu Rex Sheu Burke Rehabilitation Center Recommended Citation Lai, James C. K.; Sheu, Kwan-Fu Rex; Kim, Young Tai; and Clarke, Donald Dudley PhD, "The ubcs ellular localization of glutamate dehydrogenase (gdh): is gdh a marker for mitochondria in brain? / James C. K. Lai, Kwan-Fu Rex Sheu, Young Tai Kim, Donald D. Clarke, and John P. Blass Department of Neurology, Cornell University Medical College and Altschul Laboratory for Dementia Research Burke Rehabilitation Center White Plains, NY 10605 and Department of Medicine Cornell University Medical College New York, NY 10021" (1986). Chemistry Faculty Publications. 21. https://fordham.bepress.com/chem_facultypubs/21 This Article is brought to you for free and open access by the Chemistry at DigitalResearch@Fordham. It has been accepted for inclusion in Chemistry Faculty Publications by an authorized administrator of DigitalResearch@Fordham. For more information, please contact [email protected]. Young Tai Kim Cornell University. Medical College Donald Dudley Clarke PhD Fordham University, [email protected] Follow this and additional works at: https://fordham.bepress.com/chem_facultypubs Part of the Biochemistry Commons • Neurochemical Research, Vol.
    [Show full text]
  • The Comparative Enzymology and Cell Origin of Rat Hepatomas II
    The Comparative Enzymology and Cell Origin of Rat Hepatomas II. Glutamate Dehydrogenase, Choline Oxidase, and Glucose-6-phosphatase* HENRY C. PITOT~ (McArdle Memorial Laboratory, The Medical School, University of Wisconsin, Madison, Wis.) SUMMARY The activities of glucose-6-phosphatase, glutamate dehydrogcnase, and choline ox[- dase were determined in some or all of ten rat hepatomas, including the Novikoff, Dunning L-C18, McCoy MDAB, and the Morris 3683, 39524A, and 51~3 hepatomas, together with primary hepatomas produced by feeding ethionine or 3%nethyl-4- dimethylaminoazobenzene, and transplanted hepatomas derived from the primary tumors induced with ethionine. Of these neoplasms, only the Morris hepatoma 51~3, the primary and transplanted ethionine-induced hepatomas, and one of the 3'-methyl-4-dimethylaminoazobenzene- induced tumors possessed significant glucose-6-phosphatase activity. These same tu- mors in addition to the Dunning L-C18 hepatoma had demonstrable glutamate dehydro- genase activity, whereas the other neoplasms tested failed to show significant activity of this enzyme. With the exception of the primary dye-induced neoplasm, which was not tested, only those neoplasms having significant glucose-6-phosphatase activities showed any choline oxidase activity. Of those neoplasms tested for tryptophan peroxidase activity only the Morris hepa- toma 51~3, the primary ethionine-induced hepatoma, and some of the Dunning L-C18 hepatomas had any demonstrable activity of this enzyme. In contrast to most of the enzymatic activities reported here, the threonine dehydrase activity of the Morris hepatoma 51r was of the order of 40 times the level of this enzyme in the livers of animals bearing this tumor.
    [Show full text]
  • Amino Acid Degradation
    BI/CH 422/622 OUTLINE: OUTLINE: Protein Degradation (Catabolism) Digestion Amino-Acid Degradation Inside of cells Protein turnover Dealing with the carbon Ubiquitin Fates of the 29 Activation-E1 Seven Families Conjugation-E2 nitrogen atoms in 20 1. ADENQ Ligation-E3 AA: Proteosome 2. RPH 9 ammonia oxidase Amino-Acid Degradation 18 transamination Ammonia 2 urea one-carbon metabolism free transamination-mechanism to know THF Urea Cycle – dealing with the nitrogen SAM 5 Steps Carbamoyl-phosphate synthetase 3. GSC Ornithine transcarbamylase PLP uses Arginino-succinate synthetase Arginino-succinase 4. MT – one carbon metabolism Arginase 5. FY – oxidase vs oxygenase Energetics Urea Bi-cycle 6. KW – Urea Cycle – dealing with the nitrogen 7. BCAA – VIL Feeding the Urea Cycle Glucose-Alanine Cycle Convergence with Fatty acid-odd chain Free Ammonia Overview Glutamine Glutamate dehydrogenase Overall energetics Amino Acid A. Concepts 1. ConvergentDegradation 2. ketogenic/glucogenic 3. Reactions seen before The SEVEN (7) Families B. Transaminase (A,D,E) / Deaminase (Q,N) Family C. Related to biosynthesis (R,P,H; C,G,S; M,T) 1.Glu Family a. Introduce oxidases/oxygenases b. Introduce one-carbon metabolism (1C) 2.Pyruvate Family a. PLP reactions 3. a-Ketobutyric Family (M,T) a. 1-C metabolism D. Dedicated 1. Aromatic Family (F,Y) a. oxidases/oxygenases 2. a-Ketoadipic Family (K,W) 3. Branched-chain Family (V,I,L) E. Convergence with Fatty Acids: propionyl-CoA 29 N 1 Amino Acid Degradation • Intermediates of the central metabolic pathway • Some amino acids result in more than one intermediate. • Ketogenic amino acids can be converted to ketone bodies.
    [Show full text]
  • Synthetic Analogues of 2-Oxo Acids Discriminate Metabolic Contribution of the 2-Oxoglutarate and 2-Oxoadipate Dehydrogenases in Mammalian Cells and Tissues Artem V
    www.nature.com/scientificreports OPEN Synthetic analogues of 2-oxo acids discriminate metabolic contribution of the 2-oxoglutarate and 2-oxoadipate dehydrogenases in mammalian cells and tissues Artem V. Artiukhov1,2, Aneta Grabarska3, Ewelina Gumbarewicz3, Vasily A. Aleshin1,2, Thilo Kähne4, Toshihiro Obata5,7, Alexey V. Kazantsev6, Nikolay V. Lukashev6, Andrzej Stepulak3, Alisdair R. Fernie5 & Victoria I. Bunik1,2* The biological signifcance of the DHTKD1-encoded 2-oxoadipate dehydrogenase (OADH) remains obscure due to its catalytic redundancy with the ubiquitous OGDH-encoded 2-oxoglutarate dehydrogenase (OGDH). In this work, metabolic contributions of OADH and OGDH are discriminated by exposure of cells/tissues with diferent DHTKD1 expression to the synthesized phosphonate analogues of homologous 2-oxodicarboxylates. The saccharopine pathway intermediates and phosphorylated sugars are abundant when cellular expressions of DHTKD1 and OGDH are comparable, while nicotinate and non-phosphorylated sugars are when DHTKD1 expression is order(s) of magnitude lower than that of OGDH. Using succinyl, glutaryl and adipoyl phosphonates on the enzyme preparations from tissues with varied DHTKD1 expression reveals the contributions of OADH and OGDH to oxidation of 2-oxoadipate and 2-oxoglutarate in vitro. In the phosphonates-treated cells with the high and low DHTKD1 expression, adipate or glutarate, correspondingly, are the most afected metabolites. The marker of fatty acid β-oxidation, adipate, is mostly decreased by the shorter, OGDH-preferring, phosphonate, in agreement with the known OGDH dependence of β-oxidation. The longest, OADH- preferring, phosphonate mostly afects the glutarate level. Coupled decreases in sugars and nicotinate upon the OADH inhibition link the perturbation in glucose homeostasis, known in OADH mutants, to the nicotinate-dependent NAD metabolism.
    [Show full text]
  • Glutaminase from Escherichia Coli (G5894)
    SIGMA QUALITY CONTROL TEST PROCEDURE ProductInformation Enzymatic Assay of GLUTAMINASE1 (EC 3.5.1.2) (From E. coli) PRINCIPLE: Glutaminase L-Glutamine + H2O > Glutamate + NH3 CONDITIONS: T = 37°C, pH = 4.9, A340nm, Light path = 1 cm METHOD: Spectrophotometric Stop Rate Determination REAGENTS: A. 100 mM Sodium Acetate Buffer, pH 4.9 at 37°C (Prepare 100 ml in deionized water using Sodium Acetate, Trihydrate, Sigma Prod. No. S-8625. Adjust to pH 4.9 at 25°C with 1 M HCl.) B. 5 mM Sodium Acetate Buffer, pH 6.0 at 37°C (Prepare 10 ml in deionized water using Sodium Acetate, Trihydrate, Sigma Prod. No. S-8625. Adjust to pH 6.0 at 25°C with 1 M HCl.) C. 80 mM L-Glutamine Solution (Prepare 10 ml in Reagent A using L-Glutamine, Sigma Prod. No. G-3126. PREPARE FRESH.) D. Glutaminase Solution (Immediately before use, prepare a solution containing 5 units/ml of Glutaminase in cold Reagent B.) E. Ammonia Diagnostic Kit (171-20) (Use Ammonia Reagent, Sigma Stock No. 171-20.) F. Ammonia Diagnostic Kit (170-4) (Use L-Glutamate Dehydrogenase, Sigma Stock No. 170-4) SSGLUT02 Page 1 of 3 03/01 Enzymatic Assay of GLUTAMINASE1 (EC 3.5.1.2) (From E. coli) PROCEDURE: Step 1: Pipette (in milliliters) the following reagents into suitable tubes: Test Blank Reagent A (Sodium Acetate Buffer) 0.4 0.4 Reagent C (L-Glutamine) 0.5 0.5 Equilibrate to 37°C. Then add: Reagent D (Glutaminase) 0.1 ------ Reagent B (Sodium Acetate Buffer) ------ 0.1 Immediately mix by swirling and incubate at 37°C for 15 minutes.
    [Show full text]
  • Role of Glutamate Dehydrogenase and Glutamine Synthetase In
    J. Phycol. 44, 1188–1196 (2008) Ó 2008 Phycological Society of America DOI: 10.1111/j.1529-8817.2008.00572.x ROLE OF GLUTAMATE DEHYDROGENASE AND GLUTAMINE SYNTHETASE IN CHLORELLA VULGARIS DURING ASSIMILATION OF AMMONIUM WHEN JOINTLY IMMOBILIZED WITH THE MICROALGAE-GROWTH-PROMOTING BACTERIUM AZOSPIRILLUM BRASILENSE1 Luz E. de-Bashan Environmental Microbiology Group, Northwestern Center for Biological Research (CIBNOR), La Paz, B.C.S., Mexico De´partement des Sols et de Ge´nie Agroalimentaire, Universite´ Laval, Que´bec City, Que´bec, Canada Department of Soil, Water and Environmental Science, University of Arizona, Tucson, Arizona, USA Paola Magallon Environmental Microbiology Group, Northwestern Center for Biological Research (CIBNOR), La Paz, B.C.S., Mexico Hani Antoun De´partement des Sols et de Ge´nie Agroalimentaire, Universite´ Laval, Que´bec City, Que´bec, Canada and Yoav Bashan2 Environmental Microbiology Group, Northwestern Center for Biological Research (CIBNOR), La Paz, B.C.S., Mexico De´partement des Sols et de Ge´nie Agroalimentaire, Universite´ Laval, Que´bec City, Que´bec, Canada Department of Soil, Water and Environmental Science, University of Arizona, Tucson, Arizona, USA Enzymatic activities of glutamate dehydrogenase Key index words: Azospirillum; Chlorella; enzymes; (GDH) and glutamine synthetase (GS) participating glutamate dehydrogenase; glutamine synthetase; in the nitrogen metabolism and related ammo- microalgae; nitrogen metabolism; PGPB; PGPR; nium absorption were assayed after the microalga plant-growth-promoting bacteria Chlorella vulgaris Beij. was jointly immobilized with Abbreviations: GDH, glutamate dehydrogenase; the microalgae-growth-promoting bacterium Azospir- GOGAT, glutamate synthase; GS, glutamine syn- illum brasilense. At initial concentrations of 3, 6, and )1 + thetase; SGM, synthetic growth medium 10 mg Æ L NH4 , joint immobilization enhances growth of C.
    [Show full text]
  • Respiration with Pyruvate, Glutamate and Malate
    O2k-Protocols Mitochondrial Physiology Network 11.04: 1-9 (2011) 2007-2011 OROBOROS Version 6: 2011-12-11 Mitochondrial Pathways to Complex I: Respiration with Pyruvate, Glutamate and Malate Erich Gnaiger Medical University of Innsbruck D. Swarovski Research Laboratory A-6020 Innsbruck, Austria OROBOROS INSTRUMENTS Corp, high-resolution respirometry Schöpfstr 18, A-6020 Innsbruck, Austria [email protected]; www.oroboros.at Section 1. Malate ......................................................... 2 Page 2. Pyruvate+Malate: PM ..................................... 3 3. Glutamate ...................................................... 4 4. Glutamate+Malate: GM .................................. 5 5. Boundary conditions ..................................... 8 6. Notes - Pitfalls .............................................. 9 Introduction Mitochondrial respiration depends on a continuous flow of substrates across the mitochondrial membranes into the matrix space. Glutamate and malate are anions which cannot permeate through the lipid bilayer of membranes and hence require carriers, which is also true for pyruvate. Various anion carriers in the inner mitochondrial membrane are involved in the transport of mitochondrial metabolites. Their distribution across the mitochondrial membrane varies mainly with ΔpH and not Δψ, since most carriers (but not the glutamate-aspartate carrier) operate non- electrogenic by anion exchange or co-transport of protons. Depending on the concentration gradients, these carriers also allow for the transport of mitochondrial metabolites from the mitochondria into the cytosol, or for the loss of intermediary metabolites into the incubation medium. Export of intermediates of the tricarboxylic acid (TCA) cycle plays an important metabolic role in the intact cell. This must be considered when interpreting [email protected] www.oroboros.at MiPNet11.04 MitoPathways to CI 2 the effect on respiration of specific substrates used in studies of mitochondrial preparations (Gnaiger 2009).
    [Show full text]
  • Determination of Glutamate Dehydrogenase Activity
    DETERMINATION OF GLUTAMATE DEHYDROGENASE ACTIVITY Glutamate dehydrogenase (GDH) (E.C. 1.4.1.3) is a hexameric enzyme that catalyzes the reversible conversion of L-glutamate to α-ketoglutarate and ammonia while reducing NAD(P)+ to NAD(P)H/H+ as coenzymes (Figure 1.). It is found in all living organisms serving both catabolic and anabolic reactions. In mammalian tissues, oxidative deamination of glutamate via GDH generates α-ketoglutarate, which enters into the Krebs cycle. The reaction is called as anaplerotic reaction, because it replenishes intermediates of the cycle that are used in biosynthetic processes. No extensive production of ammonia in peripheral tissue by GDH is observed. Thus, GDH contributes little to the blood level of ammonia, which is maintained at extremely low concentration (ammonia is higly toxic at concentration above 50 mole/L in blood). Therefore, ammonia is produced in the GDH reaction at sites where it is directly excreted as NH4+ (kidney), or incorporated in the non-toxic urea (liver). Reductive amination is carried out when α-ketoglutarate and ammonium ion are converted to glutamate by GDH with NAD(P)H/H+ consumption (Figure 1.). In mammals, the GDH equilibrium is shifted in favour of the production of ammonia and α-ketoglutarate (oxidative deamination) because of the high NAD/NADH ratio. The low rate of reductive amination probably relates also to the high Km of GDH for ammonia. Figure 1. Reaction catalyzed by GDH Isoenzymes Are Encoded by Different Genes Two isoforms of GDH have been identified in humans, GDH1 and GDH2, which are encoded by the genes GLUD1 and GLUD2, respectively.
    [Show full text]
  • Inhibition of Krebs Cycle Enzymes by Hydrogen Peroxide: a Key Role of ␣-Ketoglutarate Dehydrogenase in Limiting NADH Production Under Oxidative Stress
    The Journal of Neuroscience, December 15, 2000, 20(24):8972–8979 Inhibition of Krebs Cycle Enzymes by Hydrogen Peroxide: A Key Role of ␣-Ketoglutarate Dehydrogenase in Limiting NADH Production under Oxidative Stress Laszlo Tretter and Vera Adam-Vizi Department of Medical Biochemistry, Neurochemical Group, Semmelweis University of Medicine, Budapest, H-1444, Hungary In this study we addressed the function of the Krebs cycle to drogenase. Decrease in the glutamate content of nerve terminals determine which enzyme(s) limits the availability of reduced nic- was induced by H2O2 at concentrations inhibiting aconitase. It is otinamide adenine dinucleotide (NADH) for the respiratory chain concluded that (1) aconitase is the most sensitive enzyme in the under H2O2-induced oxidative stress, in intact isolated nerve Krebs cycle to inhibition by H2O2 , (2) at small H2O2 concentra- terminals. The enzyme that was most vulnerable to inhibition by tions (Յ50 ␮M) when aconitase is inactivated, glutamate fuels the ␮ H2O2 proved to be aconitase, being completely blocked at 50 M Krebs cycle and NADH generation is unaltered, (3) at higher H2O2 ␣ ␣ Ն ␮ ␣ H2O2. -Ketoglutarate dehydrogenase ( -KGDH) was also inhib- concentrations ( 100 M) inhibition of -ketoglutarate dehydro- Ն ␮ ited but only at higher H2O2 concentrations ( 100 M), and only genase limits the amount of NADH available for the respiratory partial inactivation was achieved. The rotenone-induced increase chain, and (4) increased consumption of NADPH makes a con- in reduced nicotinamide adenine dinucleotide
    [Show full text]