Open AccessN ‐(7‐Nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yI)colcemid, a probe for different classes of colchincine‐binding site on tubulin
Author(s) -
SENGUPTA Suparna,
PURI Kamal Deep,
SUROLIA Avadhesha,
ROY Siddhartha,
BHATTACHARYYA Bhabatarak
Publication year - 1993
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1993.tb17673.x
Subject(s) - colcemid , colchicine , binding site , tubulin , microtubule , chemistry , biophysics , dissociation constant , stereochemistry , biology , biochemistry , cell , microbiology and biotechnology , receptor , genetics
The nature of binding of 7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl‐colcemid (NBD‐colcemid), an environment‐sensitive fluorescent analogue of colchicine, to tubulin was tested. This article reports the first fluorometric study where two types of binding site of colchincine analogue on tubulin were detected. Binding of NBD‐colcemid to one of these sites equilibrates slsowly. NBD‐colcemid competes with colchicine for this site. Binding of NBD‐colcemid to this site also causes inhibition of tubulin self‐assembly. In contrast, NBD‐colcemid binding to the other site is characterised by rapid equilibration and lack of competition with colchicine. Nevertheless, binding to this site is highly specific for the cholchicine nucleus, as alkyl‐NBD analogues have no significant binding activity. Fast‐reaction‐kinetic studies gave 1.76 × 10 5 M –1 s –1 for the association and 0.79 s –1 for the dissociation rate constants for the binding of NBD‐colcemid to the fast site of tubulin. The association rate constants for the two phases of the slow site are 0.016 × 10 –4 M –1 s –1 and 3.5 × 10 –4 M –1 respectively. These two sites may be related to the two sites of colchicine reported earlier, with binding characteristics altered by the increased hydrophobic nature of NBD‐colcemid.