Binding specificities of purified porcine brain alpha- and beta-tubulin subunits and of microtubule-associated proteins 1 and 2 examined by electron microscopy and solid-phase binding assays.
In this study, the molecular interaction of separated alpha- and beta-tubulin with purified microtubule-associated protein 1 (MAP 1) and MAP 2 was studied using electron microscopy and solid-phase binding assays with 125I-radiolabeled proteins. Electron microscopy of proteins recovered from sodium dodecyl sulfate polyacrylamide gels and subsequently incubated in various combinations under conditions promoting tubulin polymer formation revealed that both subunits have binding sites for MAP 1 as well as MAP 2. Overlays of nitrocellulose-transblotted MAPs with electrophoretically separated tubulin subunits eluted from gels confirmed these results. In overlays of nitrocellulose-immobilized tubulin subunits with gel-eluted MAP 2, self-association of MAP 2, but no binding to tubulin was detected. However, overlays with MAP 1 and MAP 2 purified under nondenaturing conditions revealed binding of both MAPs to beta-tubulin. In addition, these experiments demonstrated binding of both MAPs to MAP 2 and to the neurofilament proteins NF 70, NF 150 and NF 200. It is concluded that both alpha- and beta-tubulin possess binding sites for MAP 1 as well as MAP 2, but that the accessibility and/or binding affinity of these sites are strongly dependent on the tertiary structure of proteins. The demonstrated in vitro binding of MAP 1 and MAP 2 to all three neurofilament proteins as well as to MAP 2 confirms their presumed role as cytoskeletal linking proteins.
Institute of Biochemistry, University of Vienna, Austria.
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Furtner
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Wiche
D000818:Animals; D001667:Binding, Competitive; D004591:Electrophoresis, Polyacrylamide Gel; D007457:Iodine Radioisotopes; D008854:Microscopy, Electron; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D013552:Swine; D014404:Tubulin / Q000378:metabolism*
D007457:Iodine Radioisotopes; D008869:Microtubule-Associated Proteins; D010446:Peptide Fragments; D014404:Tubulin