Interaction between the C-terminal peptides of tubulin and tubulin S detected with the fluorescent probe 4',6-diamidino-2-phenylindole.
The digestion of tubulin with subtilisin and the reassociation of the digestion products was followed by means of the fluorescent probe 4',6-diamidino-2-phenylindole (DAPI). The fluorescence spectra of DAPI bound to chicken brain tubulin and to the main products of tubulin digested with subtilisin-agarose (tubulin S and C-terminal peptides) were analyzed. The corrected emission spectrum of DAPI in the presence of tubulin showed an enhancement of fluorescence intensity with a maximum at 452 nm. The digestion reaction was followed by the diminution of the area of DAPI-tubulin emission spectra, which showed biphasic pseudo-first-order kinetics. The values for the rate constants were 1.2 x 10(-2) min-1 and 3.5 x 10(-2) min-1 for the alpha and beta subunits, respectively, and were similar to those determined from the undigested subunits using polyacrylamide gel electrophoresis. Tubulin S and the C-terminal peptides were purified by means of a Bio-Gel P-60 column. The C-terminal peptides obtained from this column were analyzed by urea-sodium dodecyl sulfate polyacrylamide gel electrophoresis, and an apparent molecular weight around 3000 was determined. The corrected emission spectrum of DAPI in the presence of tubulin S showed a maximum shifted to 460 nm and a lower enhancement of fluorescence than the emission spectrum of the DAPI-tubulin complex. Titration of purified tubulin S with the C-terminal peptides of tubulin showed, after the addition of DAPI, an increase in the fluorescence intensity at 460 nm with a saturation function dependent on the concentration of peptides added. On the other hand, the emission spectrum of DAPI in the presence of the C-terminal peptides was unchanged from that of free DAPI in the solution. From these results we propose that the DAPI binding site is located on tubulin S and that the C-terminal peptides interact with tubulin S after digestion with subtilisin.
Ortiz
M
M
Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago.
Lagos
R
R
Monasterio
O
O
D005456:Fluorescent Dyes; D007211:Indoles; D010446:Peptide Fragments / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013381:Subtilisins / Q000494:pharmacology; D014404:Tubulin / Q000737:chemistry*
D005456:Fluorescent Dyes; D007211:Indoles; D010446:Peptide Fragments; D014404:Tubulin; C007293:DAPI; D013381:Subtilisins