The effects of calcium and magnesium ions on the adenosine triphosphatase and inosine triphosphatase activities of myosin A

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Abstract
1. The effects of Ca2+ and Mg2+ on the enzymic activity of myosin were studied with myosin preparations treated by the ion-exchange resin Chelex-100. A reaction mixture containing 0·05m-potassium chloride was chosen in which the effects of univalent ions such as K+, Na+ and Cl− do not change significantly with small variations in their concentrations. 2. The relationship between the rate of hydrolysis of ATP or ITP and the concentration of Ca2+ suggests that a relatively weak binding of Ca2+ either to myosin or to the substrate nucleotide is responsible for the activation of the enzymic activity. According to the experiments with an ultrafiltration technique, the binding of Ca2+ to myosin proceeds in at least two steps, the first occurring at one site on every 500000 atomic mass units of myosin with an apparent association constant, Kapp., 1·3×106m−1, and the second seeming to be so weak that its binding parameters cannot be determined by the method used. The first type of Ca2+ binding is not observable with N-ethylmaleimide-modified myosin, yet this modified myosin shows activation by Ca2+ of its adenosine triphosphatase and inosine triphosphatase. 3. The inhibition by Mg2+ can be related to a binding reaction of Mg2+ with myosin having Kapp. ∼106m−1. Mg2+ replaces the Ca2+ bound tightly to myosin. The Kapp. for Mg2+–myosin binding calculated by assuming a competition between Ca2+ and Mg2+ for the same site is 2·1×105−3·0×105m−1. When myosin is modified with a thiol reagent (p-mercuribenzoate) at a certain ratio to myosin, the inhibition by Mg2+ becomes unobservable. 4. The behaviour of the hydrolytic activity of myosin on ATP or ITP in the presence of both Ca2+ and Mg2+ is consistent with the explanation that the inhibition by Mg2+ is due to the tight binding of Mg2+ to myosin, whereas the activation by Ca2+ is caused either by a weak binding of Ca2+ to myosin or by CaATP2− or by both.