Local Conformational Change of Myosin Detected by Tryptic Digestibility

I. Monovalent and Divalent Ion-dependent Conformation of the Active Site of Myosin A ATPase

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The tryptic inactivation of EDTA-ATPase activity of myosin A was studied in 0.5M monovalent ion media at pH 7.6 and 20°.
1) Trypsin activity itself was affected by monovalent ions in the absence of divalent ions, but not by nucleotide regardless of the presence of monovalent and divalent ions.
2) Only in the absence of divalent ions were remarkable differences in the digestibility pattern observed between K⁺ and Na⁺ ions; in the K⁺ medium the inactivation rate was hardly promoted by ITP but was slightly increased by ATP and ADP. In the Na⁺ medium, on the other hand, these nucleotides, including ITP, markedly increased the rate of inactivation. Pyrophosphate showed almost the same potent effect as ATP in both media. In the presence of divalent ions, however, nucleotides strongly accelerated the inactivation of ATPase activity and no differences between K⁺ and Na⁺ were observed in the digestibility pattern. The mode of inactivation in the absence of divalent cations was first order, while the semilogarithmic plot of the inactivation rate in the presence of Mg²⁺ was biphasic in the presence of ATP.
3) A plot of the degree of tryptic inactivation of ATPase [EC 3. 6. 1. 3] activity in the presence of ATP and ITP against the crystal ionic radius of monovalent ions from 0.6 (Li⁺) to 1.7 Å (Cs⁺) was bell-shaped, with a minimum in K⁺ medium (1.33 Å). The accelerating effect of ITP was not observed in K⁺ and NH₄⁺ media, where ATP was still effective.
4) The actions of K⁺ and Na⁺ on the tryptic digestibility were independent of each other but were competitive on the catalytic activity.
5) When a specific sulfhydryl group, S_I, was blocked with N-ethylmaleimide, myosin became more susceptible to trypsin. The typical effect of nucleotides seen in intact myosin was hardly observed in K⁺ medium, though the nucleotides were still effective in Na⁺ medium.
From the experimental data obtained, the ion-dependent conformations of the active site of myosin ATPase were discussed.