Cooperative multiple binding of bisANS and daunomycin to tubulin.
The binding of daunomycin and bisANS to tubulin was studied by direct equilibrium techniques. Both ligands generated abnormal Scatchard plots. Their concave-downward nature indicated positive cooperativity. The data conform to tubulin possessing ca. 35 daunomycin binding sites with a binding constant of 570-1430 M-1. The binding of bisANS is characterized by 1 strong binding site (KA = 4.5 x 10(5) M-1) and 40-50 lower affinity sites. Hill plots of both showed low degrees of cooperativity (m = 1.8 for daunomycin and 2.3 for bisANS). A detailed analysis was carried out of the cooperativity of binding of daunomycin to tubulin. Concentration differences spectra and sedimentation velocity analysis of daunomycin showed that this molecule undergoes self-association in the drug concentration range used in the binding study. The low level of polymerization (approximately tetramer), however, indicated that this could not be the source of the observed cooperativity between 35 molecules. Both the shape and concentration dependence of the daunomycin concentration difference spectra were strikingly similar to those generated on the binding of daunomycin to tubulin, which indicates the stacking of daunomycin in both cases. The observed Scatchard plot of the binding was found to be consistent with a process that involves in part ligand-ligand interactions when complexed to tubulin. Examination of the binding of bisANS in the presence of daunomycin revealed a strong increase of bisANS binding to tubulin, which suggests a loosening of tubulin structure with the exposure of new sites as these ligands bind. The mutual interaction between the two ligands in dilute solution was demonstrated by difference spectroscopy.(ABSTRACT TRUNCATED AT 250 WORDS)
Ward
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LD
Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254-9110.
Timasheff
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D000494:Allosteric Regulation; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001921:Brain; D002417:Cattle; D003630:Daunorubicin / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D008024:Ligands; D008956:Models, Chemical; D015335:Molecular Probes; D013053:Spectrophotometry; D014404:Tubulin / Q000378:metabolism*
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D008024:Ligands; D015335:Molecular Probes; D014404:Tubulin; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D003630:Daunorubicin