Table 1.

Composition of relaxing, activating, and preactivating solutions

Solution	MgCl2	Na2ATP	EGTA	HDTA	Ca-EGTA	KProp
Relaxing	6.41	5.95	10	—	—	50.25
Preactivating	6.25	5.95	0.25	9.75	—	50.51
Activating	6.20	6.08	—	—	10	29.98

Ca-EGTA was made by mixing equimolar amounts of CaCl₂ and EGTA. In addition, all solutions contained the following (mM): phosphocreatine 10, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) 100, leupeptin 0.1, phenylmethylsulfonyl fluoride (PMSF) 0.1, dithiothreitol (DTT) 1, and 4 u ml⁻¹ creatine phosphokinase. Free Mg²⁺ and Mg-ATP concentration was 1 and 5 mM, respectively. Relaxing and activating solutions was mixed to obtain the desired range of free [Ca²⁺] assuming an apparent stability constant of the Ca²⁺—EGTA complex of 10^6.39 at 15 °C. The preactivating solution with low Ca²⁺-buffering capacity containing 1,6-diaminohexane-N,N,N',N',-tetraacetic acid (HDTA) was used prior to the activating solution.