p-Azidosalicyl-5-amino-6-phenoxybenzimidazole photolabels the N-terminal 63-103 amino acids of Haemonchus contortus beta-tubulin 1.
Benzimidazoles (BZ) are broad spectrum anthelmintics thought to exert their effects by interacting with and disrupting the functions of microtubules. However, direct biochemical evidence for binding between BZ and tubulin has not been shown nor is it known what sequences in tubulin interact with BZ. In this study, a photoactive analogue of 2-acetamido-5-(3-aminophenoxy)benzimidaz ole that has biological activity similar to other benzimidazoles was synthesized and used to photoaffinity label cell lysates from the parasitic nematode of sheep Haemonchus contortus. The photoactive analogue, 2-acetamido-5-[3-(4-azido-3-125I-salicyl amido)phenoxy]benzimida zol e or 125I-ASA-BZ, was shown to photolabel a 54-kDa protein that was specifically immunoprecipitated with anti-tubulin monoclonal antibodies. Tubulin photoaffinity labeling by 125I-ASA-BZ was also inhibited with molar excess of various BZ analogues and colchicine. Interestingly, 125I-ASA-BZ photoaffinity-labeled the beta- and not the alpha-subunits of tubulin. Proteolytic digestion of 125I-ASA-BZ-labeled tubulin with Staphylococcus aureus V8 proteinase revealed one major peptide with an apparent molecular mass of 3.5 kDa. Exhaustive digestion of 125I-ASA-BZ-labeled beta-tubulin with trypsin resulted in two fractions containing radioactive peptides. Protein sequencing of the high performance liquid chromatography-purified tryptic ASA-BZ-photolabeled peptides identified the N-terminal 63-77 and 78-103 sequences as the BZ binding domain.
Nare
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Institute of Parasitology of McGill University, Macdonald Campus, Ste Anne de Bellevue, Quebec H9X 3V9, Canada.
Lubega
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Prichard
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RK
Georges
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E
D000345:Affinity Labels; D000818:Animals; D000871:Anthelmintics / Q000737:chemistry*; D001386:Azides / Q000737:chemistry; D001562:Benzimidazoles / Q000737:chemistry*; D003078:Colchicine / Q000737:chemistry; D003600:Cytosol / Q000737:chemistry; D006189:Haemonchus; D010449:Peptide Mapping; D010777:Photochemistry; D012459:Salicylates / Q000737:chemistry; D013329:Structure-Activity Relationship; D014404:Tubulin / Q000737:chemistry*
C098855:2-acetamido-5-(3-(4-azido-3-iodosalicylamido)phenoxy)benzimidazole; D000345:Affinity Labels; D000871:Anthelmintics; D001386:Azides; D001562:Benzimidazoles; D012459:Salicylates; D014404:Tubulin; D003078:Colchicine