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Cloning, expression and characterization of human serine racemase mutants
Nováková, Ilona ; Konvalinka, Jan (advisor) ; Brynda, Jiří (referee)
AAbbssttrraacctt Human serine racemase (hSR) is a cytosolic pyridoxal-5'-phosphate dependent enzyme localized in the central nervous system. It synthesizes D-serine, which is an endogenous coagonist for the N-methyl-D-aspartate (NMDA) receptors and plays a key role in excitatory neurotransmission in the brain. Thus, human serine racemase is a promising target for the treatment of neurodegenerative diseases connected with NMDA receptors. However, few specific inhibitors have been identified to date and the crystal structure of hSR has become available only very recently. We decided to perform a random mutagenesis to determine the amino acid residues critical for the enzyme activity. Ser 84 was reported as a catalytic residue along with Lys 56. After analysis of a double mutant S84G/P111L which retained its capability to convert L-serine to pyruvate, we prepared and characterized the single mutant S84G in order to exclude potential effect of the P111L mutation.on the activity of the analyzed enzyme. KKeeyy wwoorrddss:: D-serine; Serine racemase; PLP-dependent enzymes; Random mutagenesis; Racemases
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Analysis of serine racemase expression in the CNS of epileptic patients
Vorlová, Barbora ; Konvalinka, Jan (advisor) ; Maloy Řezáčová, Pavlína (referee)
Serine racemase is a pyridoxal-5'-phosphate dependent enzyme that converts L-serine to D-serine. D-serine is a recognized physiological co-agonist of N-methyl-D-aspartate type of glutamate receptors - key receptors that participate in the neurotransmission in the mammalian brain. Dysfunction of these receptors has been implicated in several neuropathologies, including schizophrenia, brain ischemia, neurodegenerative disorders and epilepsy. Serine racemase is thus a promising pharmaceutical target in these diseases. In this study, three anti-human serine racemase monoclonal antibodies were characterized and the best one was used for the Western blot detection of the enzyme in resected human epileptic tissues. For better interpretation of the results, accuracy of the tissue processing, the protein concentration determination and the Western blot quantification were verified. Finally, the activity of human serine racemase was determined with the L-serine-O-sulfate, the substrate with the highest-affinity to this enzyme. (Thesis in Czech)
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The role of D-amino acids in central nervous system
Pangrácová, Marie ; Konvalinka, Jan (advisor) ; Balík, Aleš (referee)
Only recently, the presence of D-amino acids in the mammalian central nervous system has been confirmed and their biological functions revealed. D-serine and D-aspartate, the best described D-amino acids, have been found to be the co-agonists activating NMDA receptors. In this way D-serine and D-aspartate, among other functions, affect synaptic plasticity which is the basic cellular mechanism for learning and memory. Pathological changes in the levels of these D-amino acids and their metabolical enzymes can lead to the development of epilepsy, schizophrenia, and neurodegenerative diseases such as amyotrophic lateral sclerosis, Huntington disease or Alzheimer disease. The main role in the D-serine synthesis is played by serin racemase while D-aspartate is synthetised with the help of aspartate racemase. The key enzymes for the degradation of D-amino acids are DAAO (D-amino acid oxidase) and DAspO (D-aspartate oxidase). This thesis presents an overview of available knowledge on the individual amino acids and their respective metabolical enzymes in the mammalian central nervous system, i.e. their distribution, cell localizations, metabolism and functions. Furthermore, the emphasis is put on the possibilities of inhibition and activation of the metabolical enzymes and their importance with respect to...
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Cloning, expression and characterization of human serine racemase mutants
Nováková, Ilona ; Brynda, Jiří (referee) ; Konvalinka, Jan (advisor)
AAbbssttrraacctt Human serine racemase (hSR) is a cytosolic pyridoxal-5'-phosphate dependent enzyme localized in the central nervous system. It synthesizes D-serine, which is an endogenous coagonist for the N-methyl-D-aspartate (NMDA) receptors and plays a key role in excitatory neurotransmission in the brain. Thus, human serine racemase is a promising target for the treatment of neurodegenerative diseases connected with NMDA receptors. However, few specific inhibitors have been identified to date and the crystal structure of hSR has become available only very recently. We decided to perform a random mutagenesis to determine the amino acid residues critical for the enzyme activity. Ser 84 was reported as a catalytic residue along with Lys 56. After analysis of a double mutant S84G/P111L which retained its capability to convert L-serine to pyruvate, we prepared and characterized the single mutant S84G in order to exclude potential effect of the P111L mutation.on the activity of the analyzed enzyme. KKeeyy wwoorrddss:: D-serine; Serine racemase; PLP-dependent enzymes; Random mutagenesis; Racemases
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Analysis of serine racemase expression in the CNS of epileptic patients
Vorlová, Barbora ; Maloy Řezáčová, Pavlína (referee) ; Konvalinka, Jan (advisor)
Serine racemase is a pyridoxal-5'-phosphate dependent enzyme that converts L-serine to D-serine. D-serine is a recognized physiological co-agonist of N-methyl-D-aspartate type of glutamate receptors - key receptors that participate in the neurotransmission in the mammalian brain. Dysfunction of these receptors has been implicated in several neuropathologies, including schizophrenia, brain ischemia, neurodegenerative disorders and epilepsy. Serine racemase is thus a promising pharmaceutical target in these diseases. In this study, three anti-human serine racemase monoclonal antibodies were characterized and the best one was used for the Western blot detection of the enzyme in resected human epileptic tissues. For better interpretation of the results, accuracy of the tissue processing, the protein concentration determination and the Western blot quantification were verified. Finally, the activity of human serine racemase was determined with the L-serine-O-sulfate, the substrate with the highest-affinity to this enzyme. (Thesis in Czech)
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