Macromolecular accessibility of fluorescent taxoids bound at a paclitaxel binding site in the microtubule surface.
The macromolecular accessibility of the paclitaxel binding site in microtubules has been investigated using a fluorescent taxoid and antibodies against fluorescein, which cannot diffuse into the microtubule lumen. The formation of a specific ternary complex of microtubules, Hexaflutax (7-O-{N-[6-(fluorescein-4'-carboxamido)-n-hexanoyl]-l-alanyl}paclitaxel) and 4-4-20 IgG (a monoclonal antibody against fluorescein) has been observed by means of sedimentation and electron microscopy methods. The kinetics of binding of the antibody to microtubule-bound Hexaflutax has been measured. The quenching of the observed fluorescence is fast (k+ 2.26 +/- 0.25 x 10(6) m(-1) s(-1) at 37 degrees C), indicating that the fluorescein groups of Hexaflutax are exposed to the outer solvent. The velocity of the reaction is linearly dependent on the antibody concentration, indicating that a bimolecular reaction is being observed. Another fluorescent taxoid (Flutax-2) bound to microtubules has also been shown to be rapidly accessible to polyclonal antibodies directed against fluorescein. A reduced rate of Hexaflutax quenching by the antibody is observed in microtubule-associated proteins containing microtubules or in native cellular cytoskeletons. It can be concluded that the fluorescent taxoids bind to an outer site on the microtubules that is shared with paclitaxel. Paclitaxel would be internalized in a further step of binding to reach the known luminal site, this step being blocked in the case of the fluorescent taxoids. Because the fluorescent ligands are able to induce microtubule assembly, binding to the outer site should be enough to induce assembly by a preferential binding mechanism.
Díaz
José Fernando
JF
Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, Madrid 28040, Spain. fer@cib.csic.es
Barasoain
Isabel
I
Souto
André A
AA
Amat-Guerri
Francisco
F
Andreu
José Manuel
JM
D000818:Animals; D000906:Antibodies / Q000378:metabolism; D000972:Antineoplastic Agents, Phytogenic / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D001665:Binding Sites; D002417:Cattle; D019793:Fluorescein; D005456:Fluorescent Dyes; D007700:Kinetics; D008854:Microscopy, Electron; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D017239:Paclitaxel / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D011487:Protein Conformation; D043823:Taxoids / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D000972:Antineoplastic Agents, Phytogenic; D005456:Fluorescent Dyes; C413450:Flutax 2; D043823:Taxoids; D017239:Paclitaxel; D019793:Fluorescein