N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yI)colcemid, a probe for different classes of colchincine-binding site on tubulin

Sengupta, Suparna and Puri, Kamal Deep and Surolia, Avadhesha and Roy, Siddhartha and Bhattacharya, Bhabatarak (1993) N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yI)colcemid, a probe for different classes of colchincine-binding site on tubulin. In: European Journal of Biochemistry, 212 (2). pp. 387-393.

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Abstract

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 × 105 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.

Item Type:	Journal Article
Publication:	European Journal of Biochemistry
Publisher:	John Wiley and Sons
Additional Information:	Copyright of this article belongs to John Wiley and Sons.
Department/Centre:	Division of Biological Sciences > Molecular Biophysics Unit
Date Deposited:	03 Jun 2010 08:21
Last Modified:	19 Sep 2010 06:01
URI:	http://eprints.iisc.ac.in/id/eprint/27515

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