TAILIEUCHUNG - Báo cáo khoa học: Amino acids at the N- and C-termini of human glutamate carboxypeptidase II are required for enzymatic activity and proper folding

Human glutamate carboxypeptidase II (GCPII) is a co-catalytic metallopeptidase and its putative catalytic domain is homologous to the aminopeptidases fromVibrio proteolyticus and Streptomyces humans, the enzyme is expressed predominantly in the nervous system and form, termedprostate-specific membraneantigen, is overexpressed inprostate cancer and is used as a diagnostic marker of the | Eur. J. Biochem. 271 2782-2790 2004 FEBS 2004 doi Amino acids at the N- and C-termini of human glutamate carboxypeptidase II are required for enzymatic activity and proper folding Cvril Barinka1. Petra Mlcochová1 2 Pavel Sacha1 2 Ivan Hilaert3 Pavel Majer4 Barbara S. Slusher4 B Vaclav HorejSi3 and Jan Konvalinka1 2 institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic Prague the Czech Republic department of Biochemistry Faculty of Natural Science Charles University Prague the Czech Republic 3Institute of Molecular Genetics Academy of Sciences of the Czech Republic Prague the Czech Republic 4Guilford Pharmaceuticals Inc. Baltimore MD USA Human glutamate carboxypeptidase II GCPII is a co-catalytic metallopeptidase and its putative catalytic domain is homologous to the aminopeptidases from Vibrio proteolyticus and Streptomyces griseus. In humans the enzyme is expressed predominantly in the nervous system and the prostate. The prostate form termed prostate-specific membrane antigen is overexpressed in prostate cancer and is used as a diagnostic marker of the disease. Inhibition of the form of GCPII expressed in the central nervous system has been shown to protect against ischemic injury in experimental animal models. Human GCPII consists of 750 amino acids and six individual domains were predicted to constitute the protein structure. Here we report the analysis of the contribution of these putative domains to the structure function of recombinant human GCPII. We cloned 13 mutants of human GCPII that are truncated or extended at one or both the N- and C-termini of the GCPII sequence. The clones were used to generate stably transfected Drosophila Schneider s cells and the expression and carboxypeptidase activities of the individual protein products were determined. The extreme C-terminal region of human GCPII was found to be critical for the hydrolytic activity of the enzyme. The deletion of as few as .

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