Interactions of microtubule-associated protein MAP2 with unpolymerized and polymerized tubulin and actin using a 96-well microtiter plate solid-phase immunoassay.
A solid-phase immunoassay is used to study the protein-protein interactions between microtubule-associated protein MAP2 and the cytoskeletal proteins tubulin and actin. The assay can be performed on 96-well microtiter plates and can be used to study the interactions with both subunit proteins and their respective polymers, microtubules and microfilaments. The microtiter format allows a large number of samples to be processed, and a number of conditions can be varied. In this solid-phase immunoassay MAP2 bound to microtubules/microfilaments and tubulin dimers/G-actin in a concentration-dependent manner. However, the bound MAP2 was not dissociated from the filaments even at high NaCl concentrations, while simultaneous addition of NaCl diminished MAP2 binding to these proteins. MgCl2 was 1 order of magnitude more efficient in decreasing MAP2 binding compared with NaCl, suggesting that MAP2 may act by "screening" the electrostatic repulsion between tubulin dimers. The role of MAP2 in cross-linking microfilaments and microtubules was also examined. Microtubule/tubulin-bound MAP2 showed a diminished ability to bind to both microfilaments and G-actin, while microfilament/G-actin-bound MAP2 was able to bind efficiently to both microtubules and tubulin dimers.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Biology, University of Milan, Italy.
B
B
Pedrotti
R
R
Colombo
K
K
Islam
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D066298:In Vitro Techniques; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D014404:Tubulin / Q000378:metabolism*
D000199:Actins; D008869:Microtubule-Associated Proteins; D014404:Tubulin