Identification of Residues Critical for Enzymatic Activity in the Domain Encoded by Exons 8 and 9 of the Human Inducible Nitric Oxide Synthase
| Autor: | N. Tony Eissa, Walter A. Patton, Joel Moss, Celeste D. Palmer, Cynthia M. Haggerty |
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| Rok vydání: | 2001 |
| Předmět: |
Pulmonary and Respiratory Medicine
Blotting Western Molecular Sequence Data Clinical Biochemistry Mutant Nitric Oxide Synthase Type II Kidney Transfection Cell Line Nitric oxide Structure-Activity Relationship chemistry.chemical_compound Humans Molecular Biology Polyacrylamide gel electrophoresis Conserved Sequence Alanine chemistry.chemical_classification Binding Sites Sequence Homology Amino Acid biology Chemistry Mutagenesis HEK 293 cells Exons Cell Biology Molecular biology Protein Structure Tertiary Amino acid Enzyme Activation Nitric oxide synthase Amino Acid Substitution Biochemistry Mutagenesis Site-Directed biology.protein Electrophoresis Polyacrylamide Gel Nitric Oxide Synthase Dimerization |
| Zdroj: | American Journal of Respiratory Cell and Molecular Biology. 24:616-620 |
| ISSN: | 1535-4989 1044-1549 |
| Popis: | Overproduction of nitric oxide (NO) by inducible NO synthase (iNOS) has been implicated in the pathogenesis of several diseases including airway inflammation of asthma. iNOS is active only as a homodimer. We previously demonstrated that the region encoded by exons 8 and 9 is critical for dimerization. In this study, alanine-scanning mutagenesis was used to identify critical amino acids in that region by expression of mutant proteins in human embryonic kidney 293 cells. All iNOS mutants yielded iNOS protein as detected by Western analysis. Four iNOS mutants with alanine replacing Trp260, Asn261, Tyr267, or Asp280 did not generate NO. Dimer formation was tested by sodium dodecyl sulfate polyacrylamide gel electrophoresis at 4 degrees C, followed by immunoblotting. Wild-type iNOS migrated both as monomers and dimers. iNOS mutants with alanine replacing Trp260, Asn261, or Tyr267, however, migrated only as monomers, suggesting that their inability to produce NO is related to a defect in dimer formation. Interestingly, the Asp280 mutant retained the ability to dimerize, indicating that it represents an inactive form of an iNOS dimer. These data identify four amino acids in exons 8 and 9 critical for iNOS activity, three of which also influence dimerization. These residues are strictly conserved among all NOS isforms and across species. Thus all NOS isoforms share general structural similarities, including specific amino acids critical for dimerization and catalytic activity. These data increase our understanding of the structural elements critical for NO synthesis and lay the groundwork for future studies aimed at downregulation of iNOS activity. |
| Databáze: | OpenAIRE |
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