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Mitsuaki Moriguchi - One of the best experts on this subject based on the ideXlab platform.
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Characterization of salt-tolerant Glutaminase from Stenotrophomonas maltophilia NYW-81 and its application in Japanese soy sauce fermentation
Journal of Industrial Microbiology and Biotechnology, 2005Co-Authors: Mamoru Wakayama, Kazuaki Yoshimune, Tomohiro Yamagata, Aki Kamemura, Nitaya Bootim, Shigekazu Yano, Takashi Tachiki, Mitsuaki MoriguchiAbstract:Glutaminase from Stenotrophomonas maltophilia NYW-81 was purified to homogeneity with a final specific activity of 325 U/mg. The molecular mass of the native enzyme was estimated to be 41 kDa by gel filtration. A subunit molecular mass of 36 kDa was measured with SDS-PAGE, thus indicating that the native enzyme is a monomer. The N-terminal amino acid sequence of the enzyme was determined to be KEAETQQKLANVVILATGGTIA. Besides l -glutamine, which was hydrolyzed with the highest specific activity (100%), l -asparagine (74%), d -glutamine (75%), and d -asparagine (67%) were also hydrolyzed. The pH and temperature optima were 9.0 and approximately 60°C, respectively. The enzyme was most stable at pH 8.0 and was highly stable (relative activities from 60 to 80%) over a wide pH range (5.0–10.0). About 70 and 50% of enzyme activity was retained even after treatment at 60 and 70°C, respectively, for 10 min. The enzyme showed high activity (86% of the original activity) in the presence of 16% NaCl. These results indicate that this enzyme has a higher salt tolerance and thermal stability than bacterial Glutaminases that have been reported so far. In a model reaction of Japanese soy sauce fermentation, Glutaminase from S. maltophilia exhibited high ability in the production of glutamic acid compared with Glutaminases from Aspergillus oryzae , Escherichia coli , Pseudomonas citronellolis , and Micrococcus luteus , indicating that this enzyme is suitable for application in Japanese soy sauce fermentation.
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Micrococcus luteus K-3-type Glutaminase from Aspergillus oryzae RIB40 is salt-tolerant.
Journal of bioscience and bioengineering, 2005Co-Authors: Naohisa Masuo, Yasuji Koyama, Kotaro Ito, Kazuaki Yoshimune, Kenichiro Matsushima, Mitsuaki MoriguchiAbstract:Aspergillus oryzae RIB40 possesses the gene of Glutaminase (Micrococcus luteus K-3-type Glutaminase; AoGls), which has 40% homology with the salt-tolerant Glutaminase from M. luteus K-3 (Micrococcus Glutaminase). It was found that AoGls is a salt-tolerant enzyme, and its properties are similar to those of Micrococcus Glutaminase.
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Digestion by serine proteases enhances salt tolerance of Glutaminase in the marine bacterium Micrococcus luteus K-3
Extremophiles, 2004Co-Authors: Kazuaki Yoshimune, Naohisa Masuo, Mamoru Wakayama, Ryoko Yamashita, Mitsuaki MoriguchiAbstract:Salt-tolerant Glutaminase ( Micrococcus Glutaminase, with an apparent molecular mass of 48.3 kDa, intact Glutaminase) from the marine bacterium Micrococcus luteus K-3 was digested using protease derived from M. luteus K-3. The digestion products were a large fragment (apparent molecular mass of 38.5 kDa, the Glutaminase fragment) and small fragments (apparent molecular mass of 8 kDa). The digestion was inhibited by phenylmethanesulfonyl fluoride (PMSF). Digestion of intact Glutaminase by serine proteases including trypsin, elastase, lysyl endopeptidase, and arginylendopeptidase also produced the Glutaminase fragment. The N-terminus of the Glutaminase fragment was the same as that of intact Glutaminase. The N-termini of two small fragments were Ala394 and Ala396, respectively. The enzymological and kinetic properties of the Glutaminase fragment were almost the same as those of intact Glutaminase except for salt-tolerant behavior. The Glutaminase fragment was a higher salt-tolerant enzyme than the intact Glutaminase, suggesting that Micrococcus Glutaminase is digested in the C-terminal region by serine protease from M. luteus K-3 to confer salt tolerance on Glutaminase.
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Microbial Glutaminase: biochemistry, molecular approaches and applications in the food industry
Journal of Molecular Catalysis B-enzymatic, 2003Co-Authors: R. Nandakumar, Kazuaki Yoshimune, Mamoru Wakayama, Mitsuaki MoriguchiAbstract:Abstract Glutaminase is widely distributed in microorganisms including bacteria, yeast and fungi. The enzyme mainly catalyzes the hydrolysis of γ-amido bond of l -glutamine. In addition, some enzymes also catalyze γ-glutamyl transfer reaction. A highly savory amino acid, l -glutamic acid and a taste-enhancing amino acid of infused green tea, theanine can be synthesized by employing hydrolytic or transfer reaction catalyzed by Glutaminase. Therefore, Glutaminase is one of the most important flavor-enhancing enzymes in food industries. In this review, subsequent to a discussion on the definition of Glutaminase, the enzymatic properties, applications of Glutaminase in the food industry, and occurrence and distribution of the enzyme are described. We then illustrate the gene cloning, primary structure, and 3D-structure of Glutaminase. Finally, to facilitate the future applications of Glutaminase in food fermentations, the mechanisms of action of salt-tolerant Glutaminase are briefly discussed.
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isolation and characterization of salt tolerant Glutaminases from marine micrococcus luteus k 3
Journal of Fermentation and Bioengineering, 1994Co-Authors: Mitsuaki Moriguchi, Kenji Sakai, Ryoji Tateyama, Yoichi Furuta, Mamoru WakayamaAbstract:Marine Micrococcus luteus K-3 constitutively produced two salt-tolerant Glutaminases, designated Glutaminase I and II. Glutaminase I was homogeneously purified about approximately, 1620-fold with a 4% yield, and was a dimer with a molecular weight of about 86,000. Glutaminase II was partially purified about 190-fold with a 0.04% yield. The molecular weight of Glutaminase II was also 86,000. Maximum activity of Glutaminase I was observed at pH 8.0, 50°C and 8–16% NaCl. The optimal pH and temperature of Glutaminase II were 8.5 and 50°C. The activity of Glutaminase II was not affected by the presence of 8 to 16% NaCl. The presence of 10% NaCl enhanced thermal stability of Glutaminase I. Both enzymes catalyzed the hydrolysis of l-glutamine, but not its hydroxylaminolysis. The Km values for l-glutamine were 4.4 (Glutaminase I) and 6.5 mM (Glutaminase II). Neither of the Glutaminases were activated by the addition of 2 mM phosphate or 2 mM sulfate. p-Chloromercuribenzoate (0.01 mM) significantly inhibited Glutaminase I, but not Glutaminase II. The conserved sequences LA**V and V**GGT*A were observed in the N-terminal amino acid sequences of Glutaminase I, similar to that for other Glutaminases.
Javier Márquez - One of the best experts on this subject based on the ideXlab platform.
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Metabolic Reprogramming of Cancer by Chemicals that Target Glutaminase Isoenzymes.
Current medicinal chemistry, 2020Co-Authors: José M. Matés, José A. Campos-sandoval, Juan De Los Santos-jiménez, Juan A. Segura, Francisco J. Alonso, Javier MárquezAbstract:Background Metabolic reprogramming of tumours is a hallmark of cancer. Among the changes in the metabolic network of cancer cells, glutaminolysis is a key reaction altered in neoplasms. Glutaminase proteins control the first step in glutamine metabolism and their expression correlates with malignancy and growth rate of a great variety of cancers. The two types of Glutaminase isoenzymes, GLS and GLS2, differ in their expression patterns and functional roles: GLS has oncogenic properties and GLS2 has been described as a tumour suppressor factor. Results We have focused on Glutaminase connections with key oncogenes and tumour suppressor genes. Targeting Glutaminase isoenzymes includes different strategies aimed at deactivating the rewiring of cancer metabolism. In addition, we found a long list of metabolic enzymes, transcription factors and signalling pathways dealing with Glutaminase. On the other hand, a number of chemicals have been described as isoenzyme-specific inhibitors of GLS and/or GLS2 isoforms. These molecules are being characterized as synergic and therapeutic agents in many types of tumours. Conclusion This review states the metabolic pathways that are rewired in cancer, the roles of Glutaminase isoforms in cancer, as well as the metabolic circuits regulated by Glutaminases. We also show the plethora of anticancer drugs that specifically inhibit Glutaminase isoenzymes for treating several sets of cancer.
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Glutaminases regulate glutathione and oxidative stress in cancer
Archives of Toxicology, 2020Co-Authors: José M. Matés, Juan De Los Santos-jiménez, José A. Campos-sandoval, Javier MárquezAbstract:Targeted therapies against cancer have improved both survival and quality of life of patients. However, metabolic rewiring evokes cellular mechanisms that reduce therapeutic mightiness. Resistant cells generate more glutathione, elicit nuclear factor erythroid 2-related factor 2 (NRF2) activation, and overexpress many anti-oxidative genes such as superoxide dismutase, catalase, glutathione peroxidase, and thioredoxin reductase, providing stronger antioxidant capacity to survive in a more oxidative environment due to the sharp rise in oxidative metabolism and reactive oxygen species generation. These changes dramatically alter tumour microenvironment and cellular metabolism itself. A rational design of therapeutic combination strategies is needed to flatten cellular homeostasis and accomplish a drop in cancer development. Context-dependent Glutaminase isoenzymes show oncogenic and tumour suppressor properties, being mainly associated to MYC and p53, respectively. Glutaminases catalyze glutaminolysis in mitochondria, regulating oxidative phosphorylation, redox status and cell metabolism for tumour growth. In addition, the substrate and product of Glutaminase reaction, glutamine and glutamate, respectively, can work as signalling molecules moderating redox and bioenergetic pathways in cancer. Novel synergistic approaches combining Glutaminase inhibition and redox-dependent modulation are described in this review. Pharmacological or genetic Glutaminase regulation along with oxidative chemotherapy can help to improve the design of combination strategies that escalate the rate of therapeutic success in cancer patients.
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Cocaine modulates both Glutaminase gene expression and Glutaminase activity in the brain of cocaine-sensitized mice
Psychopharmacology, 2012Co-Authors: Eduardo Blanco, José A. Campos-sandoval, Ana Palomino, María Jesús Luque-rojas, Ainhoa Bilbao, Juan Suárez, Javier Márquez, Fernando Rodríguez De FonsecaAbstract:Rationale Glutaminase is considered the main glutamate (Glu)-producing enzyme. Two isoforms, liver (LGA)- and kidney (KGA)-type Glutaminases, have been identified in neurons. The role of both enzymes in psychopharmacological responses to cocaine remains unknown. Objectives We examined both mRNA and protein expression of KGA and LGA in the brain of mice sensitized to cocaine. Additionally, total Glutaminase activity was also measured. Methods Total Glutaminase activity and mRNA and protein expression of KGA and LGA were measured on the dorsal striatum, prefrontal cortex, hippocampus and cerebellum of cocaine-sensitized mice. Results Cocaine-sensitized animals (20 mg/kg × 5 days, followed by 5 drug-free days) exhibited a decrease of total Glutaminase activity in both the dorsal striatum and the prefrontal cortex. This was associated with an increase in KGA mRNA expression in both brain areas that was not observed when protein KGA levels were measured by western blot. LGA mRNA expression was increased as results of acute cocaine administration in sensitized animals, although protein levels were only enhanced in the prefrontal cortex of sensitized mice. These findings suggest that chronic cocaine administration modulates glutamate production through the regulation of Glutaminase expression and activity. These actions are mainly observed in the prefrontal cortex–dorsal striatum circuit, the neuroanatomical target for the psychostimulant sensitization properties of cocaine. Conclusions The present results indicate that Glutaminase enzymes (mainly KGA) are modulated by cocaine in both the prefrontal cortex and the dorsal striatum, as part of the neuroadaptions associated with behavioural sensitization to this drug of abuse.
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Cocaine modulates both Glutaminase gene expression and Glutaminase activity in the brain of cocaine-sensitized mice
Psychopharmacology, 2011Co-Authors: Eduardo Blanco, José A. Campos-sandoval, Ana Palomino, María Jesús Luque-rojas, Ainhoa Bilbao, Juan Suárez, Javier Márquez, Fernando Rodríguez De FonsecaAbstract:Rationale Glutaminase is considered the main glutamate (Glu)-producing enzyme. Two isoforms, liver (LGA)- and kidney (KGA)-type Glutaminases, have been identified in neurons. The role of both enzymes in psychopharmacological responses to cocaine remains unknown.
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A novel Glutaminase isoform in mammalian tissues.
Neurochemistry international, 2009Co-Authors: Vanessa De La Rosa, José M. Matés, Juan A. Segura, Francisco J. Alonso, José A. Campos-sandoval, Mercedes Martín-rufián, Carolina Cardona, Javier MárquezAbstract:The synthesis of neurotransmitter glutamate in brain is mainly carried out by Glutaminase enzymes. This synthesis must be exquisitely regulated because of its harmful potential giving rise to excitotoxic damage. It is noteworthy that two Glutaminase isozymes coded by different genes are expressed in the brain of mammals. The need for two genes and two isozymes to support the single process of glutamate synthesis is unexplained, and identifying the role of each Glutaminase is an important factor in understanding glutamate-mediated neurotransmission. Multiple transcripts for Glutaminase genes and simultaneous expression of Glutaminase isoforms have been reported in mammalian tissues and cells. The recent discovery of protein interacting partners widens the possibilities of regulatory mechanisms controlling these biosynthetic enzymes. The expression of distinct isozymes and binding partners may represent the biochemical and molecular basis to achieve fine-tuning control of glutamate synthesis in different cell types or developmental states. In this review, we will briefly summarize recent works on Glutaminase proteins in mammals, with particular emphasis on brain studies. We present convergent evidence supporting the existence of a novel Glutaminase isozyme in mammalian tissues.
Kazuaki Yoshimune - One of the best experts on this subject based on the ideXlab platform.
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Characterization of salt-tolerant Glutaminase from Stenotrophomonas maltophilia NYW-81 and its application in Japanese soy sauce fermentation
Journal of Industrial Microbiology and Biotechnology, 2005Co-Authors: Mamoru Wakayama, Kazuaki Yoshimune, Tomohiro Yamagata, Aki Kamemura, Nitaya Bootim, Shigekazu Yano, Takashi Tachiki, Mitsuaki MoriguchiAbstract:Glutaminase from Stenotrophomonas maltophilia NYW-81 was purified to homogeneity with a final specific activity of 325 U/mg. The molecular mass of the native enzyme was estimated to be 41 kDa by gel filtration. A subunit molecular mass of 36 kDa was measured with SDS-PAGE, thus indicating that the native enzyme is a monomer. The N-terminal amino acid sequence of the enzyme was determined to be KEAETQQKLANVVILATGGTIA. Besides l -glutamine, which was hydrolyzed with the highest specific activity (100%), l -asparagine (74%), d -glutamine (75%), and d -asparagine (67%) were also hydrolyzed. The pH and temperature optima were 9.0 and approximately 60°C, respectively. The enzyme was most stable at pH 8.0 and was highly stable (relative activities from 60 to 80%) over a wide pH range (5.0–10.0). About 70 and 50% of enzyme activity was retained even after treatment at 60 and 70°C, respectively, for 10 min. The enzyme showed high activity (86% of the original activity) in the presence of 16% NaCl. These results indicate that this enzyme has a higher salt tolerance and thermal stability than bacterial Glutaminases that have been reported so far. In a model reaction of Japanese soy sauce fermentation, Glutaminase from S. maltophilia exhibited high ability in the production of glutamic acid compared with Glutaminases from Aspergillus oryzae , Escherichia coli , Pseudomonas citronellolis , and Micrococcus luteus , indicating that this enzyme is suitable for application in Japanese soy sauce fermentation.
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Micrococcus luteus K-3-type Glutaminase from Aspergillus oryzae RIB40 is salt-tolerant.
Journal of bioscience and bioengineering, 2005Co-Authors: Naohisa Masuo, Yasuji Koyama, Kotaro Ito, Kazuaki Yoshimune, Kenichiro Matsushima, Mitsuaki MoriguchiAbstract:Aspergillus oryzae RIB40 possesses the gene of Glutaminase (Micrococcus luteus K-3-type Glutaminase; AoGls), which has 40% homology with the salt-tolerant Glutaminase from M. luteus K-3 (Micrococcus Glutaminase). It was found that AoGls is a salt-tolerant enzyme, and its properties are similar to those of Micrococcus Glutaminase.
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Digestion by serine proteases enhances salt tolerance of Glutaminase in the marine bacterium Micrococcus luteus K-3
Extremophiles, 2004Co-Authors: Kazuaki Yoshimune, Naohisa Masuo, Mamoru Wakayama, Ryoko Yamashita, Mitsuaki MoriguchiAbstract:Salt-tolerant Glutaminase ( Micrococcus Glutaminase, with an apparent molecular mass of 48.3 kDa, intact Glutaminase) from the marine bacterium Micrococcus luteus K-3 was digested using protease derived from M. luteus K-3. The digestion products were a large fragment (apparent molecular mass of 38.5 kDa, the Glutaminase fragment) and small fragments (apparent molecular mass of 8 kDa). The digestion was inhibited by phenylmethanesulfonyl fluoride (PMSF). Digestion of intact Glutaminase by serine proteases including trypsin, elastase, lysyl endopeptidase, and arginylendopeptidase also produced the Glutaminase fragment. The N-terminus of the Glutaminase fragment was the same as that of intact Glutaminase. The N-termini of two small fragments were Ala394 and Ala396, respectively. The enzymological and kinetic properties of the Glutaminase fragment were almost the same as those of intact Glutaminase except for salt-tolerant behavior. The Glutaminase fragment was a higher salt-tolerant enzyme than the intact Glutaminase, suggesting that Micrococcus Glutaminase is digested in the C-terminal region by serine protease from M. luteus K-3 to confer salt tolerance on Glutaminase.
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Microbial Glutaminase: biochemistry, molecular approaches and applications in the food industry
Journal of Molecular Catalysis B-enzymatic, 2003Co-Authors: R. Nandakumar, Kazuaki Yoshimune, Mamoru Wakayama, Mitsuaki MoriguchiAbstract:Abstract Glutaminase is widely distributed in microorganisms including bacteria, yeast and fungi. The enzyme mainly catalyzes the hydrolysis of γ-amido bond of l -glutamine. In addition, some enzymes also catalyze γ-glutamyl transfer reaction. A highly savory amino acid, l -glutamic acid and a taste-enhancing amino acid of infused green tea, theanine can be synthesized by employing hydrolytic or transfer reaction catalyzed by Glutaminase. Therefore, Glutaminase is one of the most important flavor-enhancing enzymes in food industries. In this review, subsequent to a discussion on the definition of Glutaminase, the enzymatic properties, applications of Glutaminase in the food industry, and occurrence and distribution of the enzyme are described. We then illustrate the gene cloning, primary structure, and 3D-structure of Glutaminase. Finally, to facilitate the future applications of Glutaminase in food fermentations, the mechanisms of action of salt-tolerant Glutaminase are briefly discussed.
Mamoru Wakayama - One of the best experts on this subject based on the ideXlab platform.
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Identification of a Glutaminase-producing bacterial strain isolated from Thai fermented pork sausage and characterisation of Glutaminase production
Annals of Microbiology, 2008Co-Authors: Jaruwan Thongsanit, Takashi Tachiki, Shigekazu Yano, Mamoru WakayamaAbstract:Glutaminase is one of the most important flavour-enhancing enzymes used in the food industry. Among 108 Glutaminase producing bacteria isolated from fermented foods in Thailand, one isolate (JRT-7) that exhibited the highest Glutaminase activity was selected for further study. Morphological and physiological characteristics, and 16S rDNA sequence-based phylogenetic analysis identified strain JRT-7 asKurthia gibsonii. Moreover, the highest specific activity of Glutaminase (0.057 U/mg) was obtained when JRT-7 was grown in a medium containing 2.5% succinate and 2.5% L-glutamine.
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Characterization of salt-tolerant Glutaminase from Stenotrophomonas maltophilia NYW-81 and its application in Japanese soy sauce fermentation
Journal of Industrial Microbiology and Biotechnology, 2005Co-Authors: Mamoru Wakayama, Kazuaki Yoshimune, Tomohiro Yamagata, Aki Kamemura, Nitaya Bootim, Shigekazu Yano, Takashi Tachiki, Mitsuaki MoriguchiAbstract:Glutaminase from Stenotrophomonas maltophilia NYW-81 was purified to homogeneity with a final specific activity of 325 U/mg. The molecular mass of the native enzyme was estimated to be 41 kDa by gel filtration. A subunit molecular mass of 36 kDa was measured with SDS-PAGE, thus indicating that the native enzyme is a monomer. The N-terminal amino acid sequence of the enzyme was determined to be KEAETQQKLANVVILATGGTIA. Besides l -glutamine, which was hydrolyzed with the highest specific activity (100%), l -asparagine (74%), d -glutamine (75%), and d -asparagine (67%) were also hydrolyzed. The pH and temperature optima were 9.0 and approximately 60°C, respectively. The enzyme was most stable at pH 8.0 and was highly stable (relative activities from 60 to 80%) over a wide pH range (5.0–10.0). About 70 and 50% of enzyme activity was retained even after treatment at 60 and 70°C, respectively, for 10 min. The enzyme showed high activity (86% of the original activity) in the presence of 16% NaCl. These results indicate that this enzyme has a higher salt tolerance and thermal stability than bacterial Glutaminases that have been reported so far. In a model reaction of Japanese soy sauce fermentation, Glutaminase from S. maltophilia exhibited high ability in the production of glutamic acid compared with Glutaminases from Aspergillus oryzae , Escherichia coli , Pseudomonas citronellolis , and Micrococcus luteus , indicating that this enzyme is suitable for application in Japanese soy sauce fermentation.
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Digestion by serine proteases enhances salt tolerance of Glutaminase in the marine bacterium Micrococcus luteus K-3
Extremophiles, 2004Co-Authors: Kazuaki Yoshimune, Naohisa Masuo, Mamoru Wakayama, Ryoko Yamashita, Mitsuaki MoriguchiAbstract:Salt-tolerant Glutaminase ( Micrococcus Glutaminase, with an apparent molecular mass of 48.3 kDa, intact Glutaminase) from the marine bacterium Micrococcus luteus K-3 was digested using protease derived from M. luteus K-3. The digestion products were a large fragment (apparent molecular mass of 38.5 kDa, the Glutaminase fragment) and small fragments (apparent molecular mass of 8 kDa). The digestion was inhibited by phenylmethanesulfonyl fluoride (PMSF). Digestion of intact Glutaminase by serine proteases including trypsin, elastase, lysyl endopeptidase, and arginylendopeptidase also produced the Glutaminase fragment. The N-terminus of the Glutaminase fragment was the same as that of intact Glutaminase. The N-termini of two small fragments were Ala394 and Ala396, respectively. The enzymological and kinetic properties of the Glutaminase fragment were almost the same as those of intact Glutaminase except for salt-tolerant behavior. The Glutaminase fragment was a higher salt-tolerant enzyme than the intact Glutaminase, suggesting that Micrococcus Glutaminase is digested in the C-terminal region by serine protease from M. luteus K-3 to confer salt tolerance on Glutaminase.
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Microbial Glutaminase: biochemistry, molecular approaches and applications in the food industry
Journal of Molecular Catalysis B-enzymatic, 2003Co-Authors: R. Nandakumar, Kazuaki Yoshimune, Mamoru Wakayama, Mitsuaki MoriguchiAbstract:Abstract Glutaminase is widely distributed in microorganisms including bacteria, yeast and fungi. The enzyme mainly catalyzes the hydrolysis of γ-amido bond of l -glutamine. In addition, some enzymes also catalyze γ-glutamyl transfer reaction. A highly savory amino acid, l -glutamic acid and a taste-enhancing amino acid of infused green tea, theanine can be synthesized by employing hydrolytic or transfer reaction catalyzed by Glutaminase. Therefore, Glutaminase is one of the most important flavor-enhancing enzymes in food industries. In this review, subsequent to a discussion on the definition of Glutaminase, the enzymatic properties, applications of Glutaminase in the food industry, and occurrence and distribution of the enzyme are described. We then illustrate the gene cloning, primary structure, and 3D-structure of Glutaminase. Finally, to facilitate the future applications of Glutaminase in food fermentations, the mechanisms of action of salt-tolerant Glutaminase are briefly discussed.
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isolation and characterization of salt tolerant Glutaminases from marine micrococcus luteus k 3
Journal of Fermentation and Bioengineering, 1994Co-Authors: Mitsuaki Moriguchi, Kenji Sakai, Ryoji Tateyama, Yoichi Furuta, Mamoru WakayamaAbstract:Marine Micrococcus luteus K-3 constitutively produced two salt-tolerant Glutaminases, designated Glutaminase I and II. Glutaminase I was homogeneously purified about approximately, 1620-fold with a 4% yield, and was a dimer with a molecular weight of about 86,000. Glutaminase II was partially purified about 190-fold with a 0.04% yield. The molecular weight of Glutaminase II was also 86,000. Maximum activity of Glutaminase I was observed at pH 8.0, 50°C and 8–16% NaCl. The optimal pH and temperature of Glutaminase II were 8.5 and 50°C. The activity of Glutaminase II was not affected by the presence of 8 to 16% NaCl. The presence of 10% NaCl enhanced thermal stability of Glutaminase I. Both enzymes catalyzed the hydrolysis of l-glutamine, but not its hydroxylaminolysis. The Km values for l-glutamine were 4.4 (Glutaminase I) and 6.5 mM (Glutaminase II). Neither of the Glutaminases were activated by the addition of 2 mM phosphate or 2 mM sulfate. p-Chloromercuribenzoate (0.01 mM) significantly inhibited Glutaminase I, but not Glutaminase II. The conserved sequences LA**V and V**GGT*A were observed in the N-terminal amino acid sequences of Glutaminase I, similar to that for other Glutaminases.
Malcolm Watford - One of the best experts on this subject based on the ideXlab platform.
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Distribution of phosphate-activated Glutaminase isozymes in the chicken: absence from liver but presence of high activity in pectoralis muscle
Comparative biochemistry and physiology. Part B Biochemistry & molecular biology, 1998Co-Authors: Myung Il Chung-bok, Nadine Vincent, Timothy J. Kowalski, Yeon Hee Choi, Malcolm WatfordAbstract:The distribution of Glutaminase expression in a uricotelic species, the chicken, has been examined using cDNA probes to the rat isozymes. The results suggest that chickens do not possess a Glutaminase isozyme equivalent to the liver-type isozyme of mammalian liver. Measurements of enzymic activity also showed very low Glutaminase activity in chicken liver. Extra-hepatic tissues in the chicken do express a Glutaminase isozyme mRNA which is detected by rat kidney-type Glutaminase cDNA. The abundance of this mRNA was highest in kidney and breast muscle and relatively abundant in brain, spleen and adipose tissue. Chicken small intestine expressed relatively low levels of the mRNA. The high level of Glutaminase mRNA in chicken pectoralis muscle was accompanied by high Glutaminase enzymic activity. In contrast, in mixed leg muscle Glutaminase mRNA was barely detectable by Northern blot and Glutaminase activity was relatively low. Starvation for 48 h resulted in a slight decrease in the activity of Glutaminase in pectoralis muscle, but a large decrease in the relative abundance of the mRNA. The results suggest that in the chicken, hepatic glutamine hydrolysis is not quantitatively important, but skeletal muscle may be a major site of glutamine catabolism.
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Regulation of Glutaminase Activity and Glutamine Metabolism
Annual review of nutrition, 1995Co-Authors: Norman P Curthoys, Malcolm WatfordAbstract:Glutamine is synthesized primarily in skeletal muscle, lungs, and adipose tissue. Plasma glutamine plays an important role as a carrier of nitrogen, carbon, and energy between organs and is used for hepatic urea synthesis, for renal ammoniagenesis, for gluconeogenesis in both liver and kidney, and as a major respiratory fuel for many cells. The catabolism of glutamine is initiated by either of two isoforms of the mitochondrial Glutaminase. Liver-type Glutaminase is expressed only in periportal hepatocytes of the postnatal liver, where it effectively couples ammonia production with urea synthesis. Kidney-type Glutaminase is abundant in kidney, brain, intestine, fetal liver, lymphocytes, and transformed cells, where the resulting ammonia is released without further metabolism. The two isoenzymes have different structural and kinetic properties that contribute to their function and short-term regulation. Although there is a high degree of identity in amino acid sequences, the two Glutaminases are the products of different but related genes. The two isoenzymes are also subject to long-term regulation. Hepatic Glutaminase is increased during starvation, diabetes, and feeding a high-protein diet, whereas kidney-type Glutaminase is increased only in kidney in response to metabolic acidosis. The adaptations in hepatic Glutaminase are mediated by changes in the rate of transcription, whereas kidney-type Glutaminase is regulated at a posttranscriptional level.
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hepatic Glutaminase mrna is confined to part of the urea cycle domain in the adult rodent liver lobule
FEBS Letters, 1994Co-Authors: Antoon F M Moorman, Pict A J De Boer, Malcolm Watford, Maria A Dingemanse, Wouter H LamersAbstract:Abstract This in situ hybridization study describes the developmental appearance of the lobular distribution of the mRNA encoding hepatic Glutaminase in normal rat liver. Glutaminase has been proposed to provide the urea cycle with ammonia [Hassinger and Gerok (1983) Eur. J. Biochem. 133, 269–275]. Hence, the (developmental) pattern of expression of the mRNA would be expected to be closely linked to that of the urea cycle enzymes. From embryonic day 20 onward, hepatic Glutaminase mRNA can be detected along the entire porto-central axis, with predominant expression in the portal area. In the adult phenotype, which is acquired at the end of the first postnatal week, Glutaminase mRNA is no longer present along the entire porto-central distance but has become confined to a relatively small periportal domain in which the expression decreases in a porto-central direction. Thus, in contrast to the large periportal domain, in which the urea cycle enzymes are expressed, the Glutaminase mRNA-expressing domain is much smaller and not contiguous with the glutamine synthase mRNA-expressing pericentral domain, leaving a midlobular area that is devoid of Glutaminase mRNA. A similar pattern of distribution was found in adult mouse liver. The significance of these observations is that, within the liver lobules, there is an area in which Glutaminase is not expressed and, hence, glutamine can not be the substrate for urea synthesis.
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Transcriptional regulation of the hepatic Glutaminase gene in the streptozotocin-diabetic rat.
The International journal of biochemistry, 1994Co-Authors: Ziran Zhan, Nadine Vincent, Malcolm WatfordAbstract:1. Liver possesses a unique isozyme of phosphate activated Glutaminase which is subject to long-term regulation. 2. In the rat streptozotocin-diabetes results in a 4-fold increase in the rate of transcription of the rat hepatic Glutaminase gene. 3. This is consistent with previous reports from this laboratory of increases, of similar magnitude, in the relative abundance of hepatic Glutaminase mRNA (Smith and Watford (1990) J. Biol. Chem. 265, 10631-10636), and enzyme activity (Watford, et al. (1984) Biochem. J. 224, 207-214). 4. The work establishes that, in contrast to the regulation of renal Glutaminase where mRNA stability plays an important role, the predominant site of long-term regulation of hepatic Glutaminase is at the level of gene transcription.
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Hepatic Glutaminase expression: relationship to kidney-type Glutaminase and to the urea cycle.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 1993Co-Authors: Malcolm WatfordAbstract:Glutamine functions as a major transport form of nitrogen and carbon within the body. In the liver, glutamine is hydrolyzed by a unique liver-type, phosphate-activated Glutaminase, and the end products of hepatic glutamine catabolism are glucose and urea. Other tissues possess a different, kidney-type, Glutaminase isozyme. The predicted amino acid sequences for the two Glutaminases show a high degree of identity, indicating that they are products of different but related genes. Hepatic Glutaminase activity is increased during diabetes, starvation, and on feeding high-protein diets, and decreased on feeding low-protein diets, whereas renal Glutaminase appears to be regulated only by changes in acid-base status. Changes in the rate of gene transcription are the principal mechanism responsible for the long-term regulation of hepatic Glutaminase, but the renal enzyme is regulated at the level of mRNA turnover. The pattern of regulation of hepatic Glutaminase parallels that seen for genes encoding key enzymes ...