Separation of tubulin subunits under nondenaturing conditions.
The dissociation and separation of the tubulin alpha- and beta-subunits have been achieved by binding alpha-subunits to an immunoadsorbent gel and selectively inducing release of free beta-subunits. The immunoadsorbent gel was prepared by coupling the monoclonal antibody YL1/2 to Sepharose 4B which specifically recognizes the C-terminal end of tyrosinated alpha-subunits. Extensive tubulin subunit dissociation and separation occurred in Tris buffer at neutral pH but was greatly enhanced at basic pHs (8. 0-8.5). The binding of colchicine to heterodimeric tubulin resulted in a marked protection against dissociation. The dissociation of tubulin subunits was accompanied by loss of colchicine binding capacity, and ability to polymerize into microtubules. As shown by circular dichroism, loss of functional properties was not due to extensive denaturation of tubulin, as tubulin retained most of its secondary structure. Neither of the separated alpha- or beta-subunits was able to bind colchicine, but functional tubulin that was able to bind colchicine could be reconstituted from the dissociated subunits by changing the buffer to a neutral mixture of Tris and Pipes. The yield of reconstitution, as estimated from kinetic measurements of colchicine binding capacity, amounted to about 25%. Such a yield can probably be improved with minor changes in experimental conditions. The quantitative dissociation of tubulin into separated "native" alpha- and beta-subunits should provide a powerful tool for further studies on the properties of the individual tubulin subunits and the structure-function relationships of the tubulins.
Giraudel
A
A
Département de Biologie Moléculaire et Structurale, CEA-Laboratoire du Cytosquelette, Institut National de la Santé et de la Recherche Médicale Unité no 366, Commissariat à l'Energie Atomique, 17 rue des Martyrs, 38054, Grenoble, France.
Lafanechère
L
L
Ronjat
M
M
Wehland
J
J
Garel
J R
JR
Wilson
L
L
Job
D
D
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D011487:Protein Conformation; D011489:Protein Denaturation; D013329:Structure-Activity Relationship; D014404:Tubulin / Q000737:chemistry* / Q000302:isolation & purification*
D014404:Tubulin