SUBTILISIN CLEAVAGE OF TUBULIN HETERODIMERS AND POLYMERS

被引:22
作者
LOBERT, S
CORREIA, JJ
机构
[1] UNIV MISSISSIPPI, MED CTR, DEPT BIOCHEM, 2500 N STATE ST, JACKSON, MS 39216 USA
[2] UNIV MISSISSIPPI, MED CTR, SCH NURSING, JACKSON, MS 39216 USA
关键词
D O I
10.1016/0003-9861(92)90557-D
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Native pig brain tubulin in heterodimer or polymer form was subjected to limited proteolysis by subtilisin, which is known to cleave at accessible sites within the last 50 amino acids of the highly variable carboxyl-termini of the α and β subunits. Heterodimeric tubulin or tubulin polymerized in the presence of 4 m glycerol or taxol was used in these experiments. Digested tubulin was purified by cycles of polymerization and depolymerization, ammonium sulfate precipitation, or ion-exchange chromatography in the absence or presence of nonionic detergent; however, smaller cleaved products of about 34,000 to 40,000 MW remained associated with the major cleaved subunits, α′ and β′, under all purification conditions. In order to determine the effect of subtilisin cleavage on tubulin heterogeneity, purified native or subtilisin-cleaved tubulin was subjected to isoelectric focusing, followed by SDS-PAGE. The total number of isotypes was reduced from 17-22 for native α,β tubulin to 7-9 for subtilisin-cleaved α′,β′ tubulin. When tubulin heterodimers were cleaved, a single major β′ isotype was evident; however, when tubulin polymerized in 4 m glycerol was cleaved, two major β′ isotypes were found. Monoclonal antibodies that recognize a β carboxyl-tertninal peptide, residues 410-430, reacted with both major β′ isotypes, indicating that subtilisin cleavage occurred within the last 20 of the 450 amino acids. In order to establish whether this difference was in fact associated with polymer or heterodimer forms of tubulin, digestion was carried out in the presence of taxol, which stabilizes tubulin polymers. A single major β′ isotype different from the cleaved heterodimer, but coincident with one of the bands of the cleaved glycerol-induced polymers, was found when taxol-treated tubulin was digested. This result suggests the presence of more than one subtilisin site in the β subunit, near residues 430-435, with different accessibility to the enzyme in the heterodimer and polymer form. © 1992.
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页码:152 / 160
页数:9
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