N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)colcemid, a probe for different classes of colchicine-binding site on tubulin.
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 a colchicine analogue on tubulin were detected. Binding of NBD-colcemid to one of these sites equilibrates slowly. 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 colchicine nucleus, as alkyl-NBD analogues have no significant binding activity. Fast-reaction-kinetic studies gave 1.76 x 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 444.4 M-1 s-1 and 11.67 M-1 s-1 [corrected], 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.
Sengupta
S
S
Department of Biochemistry, Bose Institute, Calcutta, India.
Puri
K D
KD
Surolia
A
A
Roy
S
S
Bhattacharyya
B
B
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D003078:Colchicine / Q000378:metabolism*; D003703:Demecolcine / Q000031:analogs & derivatives* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D006041:Goats; D007700:Kinetics; D014404:Tubulin / Q000378:metabolism*
D005456:Fluorescent Dyes; D014404:Tubulin; C052280:NBD-colcemid; D009327:4-Chloro-7-nitrobenzofurazan; D003078:Colchicine; D003703:Demecolcine