Preparation and characterization of des-C-terminal tubulin.
Tubulin, from which the C-terminal peptide had been removed by limited proteolysis was compared to intact native tubulin. Des-C-terminal tubulin (with a nominal molecular weight of 48,000) was prepared by digestion with 1% subtilisin carlsberg at 25 degrees C for 16 min, and the product was purified by ion-exchange chromatography on cellulose DE-52 followed by Sephadex G-50 chromatography. The purified product was composed of the cores of both the alpha- and beta-subunits of tubulin and was free from other proteins and peptides containing the COOH-terminal moiety as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Sephadex G-50 and ion exchange DE-52 cellulose chromatographies, and ultracentrifugation analysis. The ultraviolet (UV) absorption and fluorescence spectra of des-C-terminal tubulin were the same as those of native tubulin. The sedimentation coefficient of des-C-terminal tubulin (5.9S) was slightly higher than that of native tubulin reflecting a decrease in axial ratio. The change in circular dichroism in the far UV indicated a decrease of alpha-helical contents by 10-15%. These optical properties of des-C-terminal tubulin indicate that the elimination of the COOH-terminal region from tubulin did not change the conformation of the core tubulin molecule significantly, the decrease in alpha-helix being due to the elimination of the C-terminal peptide. des-C-terminal tubulin bound 2 moles/mole of GTP and 1 mole/mole of cholchicine, just as intact tubulin, but its binding ability of ruthenium red was reduced.
Kanazawa
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Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254.
Timasheff
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D000818:Animals; D001704:Biopolymers / Q000032:analysis; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D003078:Colchicine / Q000032:analysis; D006868:Hydrolysis; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013381:Subtilisins; D014404:Tubulin / Q000032:analysis / Q000302:isolation & purification*
D001704:Biopolymers; D010446:Peptide Fragments; D014404:Tubulin; D013381:Subtilisins; D003078:Colchicine