Photo-crosslinking of proteins in intact cells reveals a dimeric structure of cyclooxygenase-2 and an inhibitor-sensitive oligomeric structure of microsomal prostaglandin E2 synthase-1
| Autor: | Joel Robichaud, Chidambaram Ramachandran, Jean-Pierre Falgueyret, Robert Zamboni, Pierre-Olivier Hétu, Denis Riendeau, Marc Ouellet |
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| Rok vydání: | 2008 |
| Předmět: |
Photochemistry
Protein Conformation Ultraviolet Rays Stereochemistry Dimer Blotting Western Biophysics Disuccinimidyl suberate Trimer Biochemistry Dinoprostone Cell Line chemistry.chemical_compound Humans Enzyme Inhibitors Molecular Biology Prostaglandin-E Synthases A549 cell chemistry.chemical_classification Molecular mass ATP synthase biology Chemistry Intramolecular Oxidoreductases Enzyme Cyclooxygenase 2 Microsome biology.protein Electrophoresis Polyacrylamide Gel Dimerization |
| Zdroj: | Archives of Biochemistry and Biophysics. 477:155-162 |
| ISSN: | 0003-9861 |
| Popis: | We have characterized the structures of cyclooxygenase-2 (COX-2) and microsomal prostaglandin E2 synthase-1 (mPGES-1) in intact cells using bifunctional and photo-activatable crosslinking agents. A dimeric complex was detected for COX-2 by both crosslinking approaches, consistent with the crystal structure of the enzyme. For mPGES-1, treatment of A549 cells with disuccinimidyl suberate yielded immunoreactive protein bands corresponding to a dimer (33 kDa) and a trimer (45 kDa), as observed for the isolated enzyme. Photo-crosslinking with photoactivatable methionine in intact cells generated complexes with molecular weights corresponding to the dimer (33 kDa) and two putative trimer forms (50 and 55 kDa). Treatment with the selective mPGES-1 inhibitor MF63 prevented the formation of the 50 and 55 kDa crosslinked complexes, while an inactive structural analogue had no effect. Our data indicate that COX-2 forms a dimer in intact cells and that mPGES-1 has an oligomeric structure that can be disrupted by a selective inhibitor. |
| Databáze: | OpenAIRE |
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