Single-headed scallop myosin (shM) was prepared by papain digestion of filamentous scallop myosin and purified by hydrophobic interaction chromatography. The shM preparation consisted of equimolar amounts of polypeptides corresponding to an intact heavy chain, rod chain, essential light chain, and regulatory light chain. In electron micrographs the shape of shM showed the presence of a single head domain to which a normal looking rod was attached. Myosin , which is highly cooperative. Single turnover experiments of myosin with radioactive ATP gave a halflife for the ATPase cycle of ϳ3 min in the presence of EGTA, whereas that of single-headed myosin was shorter than ϳ30 s, which was the resolution time of these measurements. The results suggest that the presence of two heads, as well as the attachment of the head to the coiled coil rod, contribute to the regulation of scallop myosin by Ca 2؉ .Muscle myosins belong to class II of the myosin family (1). These proteins consist of a pair of heavy chains (HCs), 1 two ELCs, and two RLCs. Muscle myosins can be divided into three categories depending on the mode of activation by Ca 2ϩ . In skeletal muscles contraction occurs after the binding of Ca 2ϩ to the thin filaments, which changes the interactions of the myosin cross-bridges with the actin filaments. In contrast to skeletal muscle myosin, smooth muscle and molluscan myosins are regulated molecules (cf. Ref. 2). Smooth muscle myosin is regulated by phosphorylation of its RLC by a Ca 2ϩ -and calmodulindependent myosin light chain kinase. Contraction of molluscan muscles is regulated by binding of Ca 2ϩ to myosin. The Ca 2ϩ binding site of molluscan myosins is located in domain I of the ELC and is stabilized by specific contacts between the ELC and both the RLC and the HC (3). The affinity for Ca 2ϩ is preserved in the proteolytic fragments of myosin, regulatory domain, subfragment 1 (S1), and heavy meromyosin (HMM) (4). The structural requirements for the ELC, RLC, and HC that are needed for the formation of the specific Ca 2ϩ binding site have been studied by the construction of hybrids between naturally occurring and mutant LCs and HCs (5-8).The structural features of the regulated smooth and molluscan muscle myosins that place the molecules in the off state have been studied by examining the properties of three proteolytic fragments of these myosins: S1, HMM, and shM. The single-headed S1 and the double-headed HMM are soluble, whereas shM forms filaments in conditions similar to those of the parent myosin. In contrast to HMMs, S1s derived from smooth and molluscan myosins are not regulated, suggesting a role for the presence of the two heads and/or the coiled coil rod in the regulation of these myosins (9 -11). The activating effect of Ca 2ϩ on the ATPase activity of mixtures of single-and double-headed scallop myosin (prepared by papain digestion of scallop myosin) indicated that single-headed scallop myosin was regulated by Ca 2ϩ , since the increase of the ATPase activity of the preparations could not be at...

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