Table 1.

Experimental solutions

	Relaxing solutionsa		Activating solutions
Component	Skinning	Low Mg2+, low EGTA	ATP	CTP	ITP	Mn2+	Ni2+
ATP	7	8	5	—	—	5.8	5 (10)
CTP	—	—	—	5	—	—	—
ITP	—	—	—	—	5 (10)	—	—
MgAc	8	0.01	7	—	7 (12)	—	—
MnCl2	—	—	—	—	—	6	—
NiAc	—	—	—	—	—	—	5
KAc	—	73.2	85	85	85 (59)	112.4	89.8 (47.9)
KProp	70	—	—	—	—	—	—
EGTA	5	0.5	—	—	—	—	—
CaEGTA	—	—	10	10	10	—	—
CaCl2	—	—	—	—	—	0.285	0.15
Imidazole	6	50	50	50	50	50	50
Phosphocreatine	—	20	10	10	10	10	20
CK (mg ml−1)	—	2.5	2.5	2.5	2.5	2.5	20 (40)

Ac, acetate; Prop, propionate; CK, creatine kinase. Concentrations are millimolar, except for CK which is in mg ml⁻¹ (125–250 units mg⁻¹). In some experiments using Ni·ATP, the concentration of CK added to the solution was raised to 40 mg ml⁻¹; the solubility of CK was measured to be 34 mg ml⁻¹. pH was 7.0 at 5°C and ionic strength 200 mm for all solutions except skinning, which was pH 7.0 at 0°C and ionic strength 126 mm. Proteases added to skinning solution: leupeptin (8 μg ml⁻¹), phenylmethylsulphonyl fluoride (0.1 mm), trypsin inhibitor (0.1 mg ml⁻¹).

^aThe low Mg²⁺, low EGTA relaxing solution was used before activations with Ni·ATP and Mn·ATP because of the strong affinity of Ni²⁺ and Mn²⁺ for EGTA, thus making it impractical to maintain low calcium and high metal–nucleotide concentrations; additionally EGTA was not used in the activation solutions for these two substrates; the concentration of EGTA in the relaxing solution was reduced as much as possible while still keeping the fibre relaxed. CTP, ITP and ATP relaxing solutions were identical to the corresponding activating solutions, but with EGTA substituted for Ca·EGTA. Values in parentheses indicate concentrations used in control experiments for Mg·ITP and Ni·ATP (see text). Free Mg²⁺ was usually ∼2 mm, except for activations comparing Mn·ATP with Mg·ATP in which 1 mm free Mn²⁺ or Mg²⁺ (6 mm total) were used. Free Mn²⁺ was minimized as it is known to bind the regulatory light chain with high affinity (Bagshaw, 1977) and has been reported to activate smooth muscle (Hoar & Kerrick, 1988). Ten millimolar free ATP was used with 5 mm NiAc in control experiments to minimize free Ni²⁺. pCa was 4 in the Mn·ATP and Ni·ATP activating solutions and about 4.5 otherwise. The concentration of the metal–nucleotide complexes was 4.9–5 mm except in control solutions using 10 mm Mg·ITP. Total and free concentrations were calculated from equations for multiple-binding equilibria using the program of Fabiato (1988) and the apparent stability constants therein, as well as from Godt & Lindley (1982), Sillen & Martell (1970) and Smith & Martell (1989). Variation in stability constants with temperature was taken into account. All chemicals were obtained from Sigma (UK).