Photoaffinity Labeling of Avermectin Binding Sites from Caenorhabditis elegans and Drosophila melanogaster

• Published in: Proceedings of the National Academy of Sciences - PNAS 1992-05, Vol.89 (9), p.4168-4172
• Main Authors: Rohrer, Susan P., Meinke, Peter T., Hayes, Edward C., Mrozik, Helmut, Schaeffer, James M.
• Format: Article
• Language: English
• Subjects: Affinity Labels; analogs; Animals; Antibiotics. Antiinfectious agents. Antiparasitic agents; Antiparasitic agents; avermectin; binding; Binding Sites; Biochemistry; Biological and medical sciences; Brain; Caenorhabditis - chemistry; Caenorhabditis elegans; Chloride Channels; Crosslinking; Drosophila; Drosophila melanogaster; Drosophila melanogaster - chemistry; Gels; Insects; Ivermectin - analogs & derivatives; Ivermectin - chemistry; Ivermectin - metabolism; Medical sciences; Membrane proteins; Membrane Proteins - isolation & purification; Nematodes; Nervous system; P branes; Pharmacology. Drug treatments; photoaffinity labelling; Photochemistry; Proteins - chemistry; Proteins - isolation & purification; Receptors; Rodents; sites
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.89.9.4168

An azido-avermectin analog [4"α-(4-azidosalicylamido-ε-caproylamido-β- alanylamido)-4"-deoxyavermectin B1a; azido-AVM] was synthesized and used to photoaffinity label avermectin binding sites present in the membranes of Caenorhabditis elegans and Drosophila melanogaster. Azido-AVM was biologically active and behaved like a competitive inhibitor of3H]ivermectin binding to C. elegans membranes (Ki= 0.2 nM). Radiolabeled azido-AVM bound specifically and with high affinity to C. elegans membranes (Kd= 0.14 nM) and, upon photoactivation, became covalently linked to three C. elegans polypeptides of 53, 47, and 8 kDa. Photoaffinity labeling of a membrane preparation from D. melanogaster heads resulted in labeling of a single major polypeptide of ≈47 kDa. The proteins that were covalently tagged in these experiments are believed to be associated with avermectin-sensitive chloride channels present in the neuromuscular systems of C. elegans and D. melanogaster. Azido-AVM did not bind to rat brain membranes and therefore was selective for the nematode and insect receptors.