J. Biol. Chem., Vol. 261, Issue 2, 728-732, 01, 1986
Effect of ethylene glycol and Ca2+ on the binding of Mg2+ x adenyl-5'- yl imidodiphosphate to rabbit skeletal myofibrils
RE Johnson
The binding of Mg2+ X adenyl-5'-yl imidodiphosphate (Mg2+ X AMP-PNP) to
rabbit skeletal myofibrils has been measured in aqueous solution and in 50%
ethylene glycol in the presence and absence of Ca2+. In water, the observed
binding was weak with less than half the calculated myosin active sites
filled even at 1 mM Mg2+ X AMP-PNP. In 50% ethylene glycol, the binding is
at least 100-fold tighter and extrapolates to the expected number of
binding sites. This is contrasted to the small change seen for Mg2+ X ADP
binding between the same sets of conditions. This difference between Mg2+ X
AMP-PNP and Mg2+ X ADP is attributed to the strong coupling of Mg2+ X
AMP-PNP binding to dissociation of myosin cross-bridges. The Ca2+
sensitivity of Mg2+ X AMP-PNP binding in 50% ethylene glycol is taken as
further evidence of the thermodynamic coupling of Mg2+ X AMP-PNP binding to
cross-bridge dissociation. In addition, the binding of Mg2+ X AMP-PNP in
50% ethylene glycol is biphasic while Mg2+ X ADP binding under the same
conditions is not. The biphasic Mg2+ X AMP-PNP binding could be caused by
either the presence of two or more classes of cross-bridges or by negative
cooperativity, but the presence of only a single class of Mg2+ X
ADP-binding sites implies that if multiple classes of sites are involved,
they do not simply differ in steric hindrance or accessibility of the
binding site as a whole. The importance of using purified AMP-PNP in the
study of actomyosin X AMP-PNP complexes is discussed.
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