Classical ligands interact with native and recombinant tubulin from Onchocerca volvulus with similar rank order of magnitude.
The alpha- and beta-tubulin genes from Onchocerca volvulus were individually expressed for the first time in Escherichia coli (DH5alpha). The recombinant tubulins were purified, renatured and reconstituted into oligomers, probably dimers, which were competent to bind three classical tubulin ligands: mebendazole (MBZ), taxol (TAX) and vinblastine (VBN). A new charcoal-dependent binding assay allowed accurate discrimination between specific and non-specific ligand binding in crude cell extracts. To compare the magnitude of binding of both native and recombinant forms of tubulin, we developed an ELISA assay for estimating the amount of tubulin in soluble protein extracts of O. volvulus. Binding assays were performed; both the maximum binding at saturating ligand concentrations (B(max)) and the equilibrium dissociation constants (K(d)) were determined. The B(max) values of the different ligands were significantly different from one another (P<0.05), but the order of the B(max) and K(d) for each drug were VBN > TAX > MBZ for both native and recombinant tubulin. Indeed, B(max) values for MBZ with native and recombinant tubulins were similar. On average, native tubulin had higher or similar binding capacity (B(max)) but a consistently higher affinity (lower K(d)) than the recombinant tubulin. We conclude that at least some of the recombinant molecules form receptors that are similar to those in native tubulin dimers. These data suggest that recombinant tubulin can be used to develop a molecular screen for novel anti-tubulin ligands to develop into drugs against onchocerciasis.
Department of Parasitology and Microbiology, Faculty of Veterinary Medicine, Makerere University P.O. Box 7062, Kampala, Uganda.
Eddie M
EM
Wampande
J
J
Richard McIntosh
George W
GW
Lubega
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D008024:Ligands; D009854:Onchocerca / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D014404:Tubulin / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D018076:DNA, Complementary; D008024:Ligands; D011994:Recombinant Proteins; D014404:Tubulin