Assessment of benzimidazole binding to individual recombinant tubulin isotypes from Haemonchus contortus.
One a- and 2 beta-tubulin isotypes (isotypes 1 and 2) from the parasitic nematode Haemonchus contortus were artificially expressed in E. coli and purified to obtain tubulin that was capable of polymerizing into microtubules. Binding of [14C] mebendazole (MBZ), a benzimidazole compound, to each individual unpolymerized isotype and to microtubules polymerized from recombinant alpha- and beta-tubulin was assessed and Kd and Bmax values determined. Mebendazole bound to the individual tubulin isotypes with a stoichiometry of 1:1. Binding occurred with highest affinity to alpha-tubulin followed by beta-tubulin isotype 2 and beta-tubulin isotype 1 indicating that alpha-tubulin may play a role in benzimidazole binding to microtubules. Upon polymerization of alpha- and beta-tubulin isotype 2 into microtubules the stoichiometry of binding increased to 2:1 (mebendazole : tubulin) while binding affinity remained the same. Mebendazole binding to alpha/beta-isotype 1 microtubules remained unchanged following polymerization. The increase in the number of benzimidazole receptors on alpha/beta-isotype 2 microtubules suggests the formation of a new benzimidazole receptor upon polymerization.
Oxberry
M E
ME
Molecular Immunology Laboratory, School of Biomedical Sciences, Curtin University, Perth, Western Australia. moxberry@yahoo.com
Gear
T G
TG
Prichard
R K
RK
D000818:Animals; D000871:Anthelmintics / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006188:Haemonchiasis / Q000188:drug therapy; D006189:Haemonchus / Q000187:drug effects* / Q000378:metabolism; D015801:Helminth Proteins / Q000378:metabolism*; D007700:Kinetics; D008463:Mebendazole / Q000378:metabolism* / Q000494:pharmacology; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism*
D000871:Anthelmintics; D015801:Helminth Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D014404:Tubulin; D008463:Mebendazole