The calcium concentration required for maximal activity of the reconstituted actomyosin ATPase depends on the troponin (TN) concentration. Reconstituted actomyosin was made from rabbit actin (A) and myosin and chicken tropomyosin (TM) and TN and the ATPase was measured in 50mM NaCl, 2mM MgCl2, 0.7mM ATP, 0.2mM (CaEGTA + EGTA), pH 7.5. The data were normalized by taking the ATPase at 1.4x10-9M Ca++ as 100% inhibition and that at 1.4x10-6M Ca++ as 0% inhibition, assuming the dissociation constant for CaEGTA at pH 7.5 is 2.7x10-8M. It is seen that the Ca++ concentration required for half maximal activity (normalized) increases from 3x10-8M to 1.5x10-7M with an 8 fold increase in TN concentration in a range approaching maximal inhibition (unnormalized). Half maximal calcium binding by free TN occurs at 6.5x10-8M Ca++ (pH 7.5). An explanation for the observed shift in Ca dependence of the ATPase is that TN without Ca has a higher affinity for A plus TM than TN with Ca. If the affinities were the same there would be no shift. This interpretation is supported by a mathematical analysis of this multiple equilibria system which predicts this shift. Preliminary experiments are consistent with the assumption that actin bound and free TN are in equilibrium.
|Original language||English (US)|
|Number of pages||1|
|Issue number||3 I|
|State||Published - 1973|
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