Analysis of the interaction of rabbit skeletal muscle adenylate deaminase with myosin subfragments. A kinetically regulated system

B. A. Barshop, C. Frieden

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Abstract

The interaction of rabbit skeletal muscle adenylate deaminase with myosin fragments (heavy meromyosin and subfragment-2) has been studied by analytical centrifugation, gel chromatography, and stopped flow light scattering. Formation of the complex is highly cooperative with respect to addition of two molecules of adenylate deaminase/molecule of myosin fragment to form a ternary complex. Ternary complex formation is also highly pH-dependent with less complex formed at higher pH values, and the pH dependence is steeper with heavy meromyosin than with subfragment-2. At pH 6.5, the dissociation constant for the heavy meromyosin-deaminase complex is approximately 1.2 x 10-15 M2. Over the pH range 6.5-7.0, rate constants for the formation and dissociation of both the ternary and binary complexes of adenylate deaminase with heavy meromyosin have been determined. From analysis of the time course of stopped flow light scattering, the association steps are found to be extremely rapid, while the rate constant for dissociation of the first molecule of adenylate deaminase from the ternary complex is quite slow. This rate constant increases as the pH increased, but is sufficiently low that the interacting system does not equilibrate on the time scale of mass transport experiments (sedimentation velocity and gel chromatography), and thus displays apparent 'slow' behavior. The kinetic regulatory properties of adenylate deaminase are influenced by heavy meromyosin and subfragment-2, particularly with respect to inhibition by GTP. The association and dissociation of adenylate deaminase and myosin fragments and the resultant changes in kinetic properties of the adenylate deaminase can markedly alter the time course of the enzymatic reaction. The time scale over which this interaction is modulated by changes in pH may have significance in the metabolism of exercising muscle.

Original languageEnglish
Pages (from-to)60-66
Number of pages7
JournalJournal of Biological Chemistry
Volume259
Issue number1
StatePublished - 1984

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