BINDING SPECIFICITIES OF PURIFIED PORCINE BRAIN ALPHA-TUBULIN AND BETA-TUBULIN SUBUNITS AND OF MICROTUBULE-ASSOCIATED PROTEIN-1 AND PROTEIN-2 EXAMINED BY ELECTRON-MICROSCOPY AND SOLID-PHASE BINDING ASSAYS

  • 1 December 1987
    • journal article
    • research article
    • Vol. 45  (1) , 1-8
Abstract
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 MAP1 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.