Effect of ethylene glycol and Ca2+ on the binding of Mg2+ x adenyl-5'-yl imidodiphosphate to rabbit skeletal myofibrils.
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.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives*; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D019855:Ethylene Glycol; D005026:Ethylene Glycols / Q000494:pharmacology*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D008433:Mathematics; D009132:Muscles / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism; D009218:Myosins / Q000378:metabolism; D011817:Rabbits
D005026:Ethylene Glycols; D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D009218:Myosins; D019855:Ethylene Glycol; D008274:Magnesium; D002118:Calcium