Localization of the colchicine-binding site of tubulin.
We have previously shown that rat brain tubulin, a heterodimer consisting of an alpha and beta monomer, can be covalently labeled with [3H]colchicine by near UV irradiation. Most of the label appears in beta-tubulin. We show here that beta-tubulin can be separated and purified from SDS preparative gels and analyzed by proteolysis. Chymotrypsin yielded a labeled approximately 4-kDa band that contained two peptides. Tryptic digestion also yielded an approximately 4-kDa band containing two peptides. Sequence analysis revealed a peptide of residues 1-36 and 213-242 for chymotrypsin and a peptide of residues 1-46 and 214-241 for trypsin. To identify which peptide carried the label, limited hydrolysis of beta-tubulin was done with trypsin; this procedure yielded a labeled 16-kDa N-terminal peptide and a 35-kDa C-terminal peptide, as identified by antibodies. Isolation of these peptides and extensive digestion with trypsin yielded two labeled peptides corresponding to residues 1-46 from the 16-kDa N-terminal fragment and residues 214-241 from the 35-kDa C-terminal fragment. These results show that at least two regions in beta-tubulin are specifically involved in colchicine binding and that the span of the colchicine molecule, < or = 11 A, bridges these two regions in the native beta monomer.
Uppuluri
S
S
Laboratory of Biochemical Pharmacology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD 20892.
Knipling
L
L
Sackett
D L
DL
Wolff
J
J
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002918:Chymotrypsin; D003078:Colchicine / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D015151:Immunoblotting; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation; D051381:Rats; D014357:Trypsin; D014404:Tubulin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D046911:Macromolecular Substances; D010446:Peptide Fragments; D014404:Tubulin; D002918:Chymotrypsin; D014357:Trypsin; D003078:Colchicine