Aging of tubulin monomers using 5,5'-bis(8-anilino-1-naphthalenesulfonate) as a probe.
The fluorescent probe bis(8-anilino-1-naphthalenesulfonate) (bis-ANS) has been used to monitor the time- and temperature-dependent aging of tubulin, whereby new hydrophobic binding sites of lower affinity are generated on the protein [Prasad, A. R. S., et al. (1986a) Biochemistry 25, 739-742]. We carried out a detailed analysis of this phenomenon and found that, in addition to antimitotic drugs like colchicine or vinblastine, other parameters, viz., low temperature and protein stabilizers (e.g., glycerol and sucrose), inhibit the extent of enhanced binding of bis-ANS. Moreover, the generation of additional bis-ANS binding sites are also suppressed at high concentrations of tubulin. Cleavage of the carboxy-termini of tubulin (bound to bis-ANS) by subtilisin causes a significant reduction in the enhanced fluorescence, but has no effect on the high-affinity binding site of bis-ANS. All of these observations can be explained by the correlation of the presence of additional binding with the dissociation of heterodimeric tubulin into monomers. Enhanced binding of bis-ANS is due to tubulin dimers that have undergone dissociation, resulting in a loosening of its tertiary structure with the generation of a plethora of hydrophobic sites.
Sarkar
N
N
Department of Biochemistry, Bose Institute, Calcutta, India.
Mukhopadhyay
K
K
Parrack
P K
PK
Bhattacharyya
B
B
D000817:Anilino Naphthalenesulfonates; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D003078:Colchicine / Q000494:pharmacology; D005456:Fluorescent Dyes; D006041:Goats; D007700:Kinetics; D011480:Protease Inhibitors / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013381:Subtilisins / Q000378:metabolism; D014404:Tubulin / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification; D014747:Vinblastine / Q000494:pharmacology
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D011480:Protease Inhibitors; D014404:Tubulin; D014747:Vinblastine; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D013381:Subtilisins; D003078:Colchicine