Kinetic and equilibrium analysis of the interactions of actomyosin subfragment-1.ADP with beryllium fluoride

The hypothesis that the stable ternary complex formed between myosin subfragment-1, MgADP and beryllium fluoride (BeF3-), denoted S-1 not equal to .ADP.BeF3-, is an analog of the intermediate state S-1**.ADP.P(i) has been tested in this work by examining the interactions of S-1 not equal to .ADP.BeF...

Full description

Saved in:
Bibliographic Details
Published in:Biochemistry (Easton) 1993-08, Vol.32 (30), p.7712-7719
Main Authors: PHAN, B. C, FALLER, L. D, REISLER, E
Format: Article
Language:English
Subjects:
Actins - chemistry
Adenosine Diphosphate - chemistry
Adenosine Triphosphatases - metabolism
Analytical, structural and metabolic biochemistry
Animals
Beryllium - chemistry
Biological and medical sciences
Contractile proteins
Enzyme Activation
Fluorides - chemistry
Fundamental and applied biological sciences. Psychology
Holoproteins
Kinetics
Myosin Subfragments - chemistry
Proteins
Rabbits
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:The hypothesis that the stable ternary complex formed between myosin subfragment-1, MgADP and beryllium fluoride (BeF3-), denoted S-1 not equal to .ADP.BeF3-, is an analog of the intermediate state S-1**.ADP.P(i) has been tested in this work by examining the interactions of S-1 not equal to .ADP.BeF3- with actin. Equilibrium binding measurements revealed that actin bound weakly to the S-1 not equal to .ADP.BeF3- complex (Ka = 10(4) M-1) in the presence of 40 mM KCl. The stability of this complex was strongly salt-dependent. The association constant of BeF3- to the acto-S-1.ADP complex (KBe approximately 10(3) M-1) was 100-fold weaker than its binding to the S-1.ADP complex. While inhibiting the S-1 ATPase strongly, BeF3- had no effect on the Vmax value (10 +/- 1.0 s-1) of the actin-activated ATPase of S-1. The rates of BeF3- binding and dissociation from the acto-S-1.ADP.BeF3- complex were determined by stopped-flow measurements. The hyperbolic dependence of the rates of BeF3- binding to acto-S-1.ADP (kobs) on BeF3- concentrations suggested that the acto-S-1.ADP.BeF3- complex was formed in at least two steps: binding followed by isomerization. The binding constant was 1.2 x 10(3) M-1, and the maximum kobs was 2.5 s-1. The dissociation of BeF3- from the acto-S-1.ADP.BeF3- complex was monitored via decrease in the fluorescence of 1-N6-ethenoadenosine diphosphate (epsilon ADP). The fluorescence decrease fitted two exponential terms.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi00081a016