DETERMINATION OF FREE AND BOUND MICROTUBULAR PROTEIN AND GUANINE-NUCLEOTIDE UNDER EQUILIBRIUM CONDITIONS

被引:76
作者
ZEEBERG, B [1 ]
CAPLOW, M [1 ]
机构
[1] UNIV N CAROLINA,DEPT BIOCHEM,CHAPEL HILL,NC 27514
关键词
D O I
10.1021/bi00585a007
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
The dissociation constant for GDP binding to the E site of tubulin isolated by chromatography on Sepharose 6B is equal to 6.1 × 10-8 M, as determined by the Hummel-Dryer procedure. This is smaller than any previously reported value, and the discrepancy with earlier results is analyzed. By use of a recently described column centrifugation procedure [Penefsky, H. S. (1977) J. Biol. Chem. 252, 2891-2899], it was established that GDP and GTP bind to the same site. GTP is bound 2.8-fold tighter than GDP, and the dissociation constant is 2.2 × 10-8 M. A new method for the determination of dissociation constants for a protein-bound ligand, based on a quantitative analysis of the loss of ligand during exclusion chromatography, is presented. This has been used to determine that the dissociation constant for GDP bound to tubulin is equal to 5.5 X 10~8 M, in excellent agreement with that determined independently from the Hummel-Dryer method. A previous theoretical treatment [Dixon, H. B. F. (1976) Biochem. J. 159, 161-162] of ligand loss during exclusion chromatography is discussed. © 1979, American Chemical Society. All rights reserved.
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页码:3880 / 3886
页数:7
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