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Interaction between the C-terminal peptides of tubulin and tubulin S detected with the fluorescent probe 4',6-diamidino-2-phenylindole.

Abstract：

: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.

journal_name

Arch Biochem Biophys

journal_title

Archives of biochemistry and biophysics

authors

Ortiz M,Lagos R,Monasterio O

doi

10.1006/abbi.1993.1267

subject

Has Abstract

pub_date

1993-05-15 00:00:00

pages

159-64

issue

eissn

0003-9861

issn

1096-0384

pii

S0003-9861(83)71267-1

journal_volume

303

pub_type

杂志文章

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Interaction between the C-terminal peptides of tubulin and tubulin S detected with the fluorescent probe 4',6-diamidino-2-phenylindole.