Regulation of Scallop Myosin by Calcium

Cooperativity and the “Off” State
  • Vassilios N. Kalabokis
  • Andrew G. Szent-Györgyi
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 453)


Scallop subfragment 1 (S1) is an unregulated molecule; it differs from heavy meromyosin (HMM) and myosin in that it has no “off” state, although it contains the full complement of light chains and the triggering calcium binding site. S1 differs from myosin by lacking the head-rod junction and being single-headed. The contribution of the head-rod junction was evaluated by studying single-headed myosin. Isolated single-headed myosins show some regulation; their actin activated ATPase is stimulated about 3-fold by calcium. However, in contrast to HMM and myosin, the calcium dependence of ATPase activation of single-headed myosin is non-cooperative. The single ATP turnover rate of single-headed myosin in the absence of calcium is less than 30 seconds (our experimental resolution) compared to the approximately 5 minute turnover rate of myosin. HMM and myosin exhibit several cooperative features not shown by S1. Calcium binding becomes cooperative in the presence of nucleotide analogues in HMM and myosin, but not in S1. Nucleotide analogues are bound cooperatively by myosin and HMM in the absence of calcium; the introduction of calcium to the system reduces the affinity and abolishes the cooperative binding of nucleotide in the double headed molecules. Conversely, S1 shows normal binding curves for nucleotide analogues both in the presence and absence of calcium. Therefore, there is direct communication between the calcium binding sites and nucleotide binding sites in regulated molecules that is mediated by interaction between the two heads.

Molluscan muscles are activated by direct binding of calcium to myosin. The system is the simplest “on” and “off” switch of muscle contraction. The myosins of molluscs are regulated molecules, their regulatory subunits are their light chains. Scallop myosin is particularly well suited for study since its regulatory light chains (RLC) can be removed fully from the heavy chains in the absence of divalent cations (reviewed in ref. 1 and 2). The ease of RLC removal is due to its inability to form a salt link with the heavy chain; this occurs because this RLC has methionine at position 127 in place of glutamate present in other RLCs.

The RLC is the inhibitory subunit while the triggering calcium binding site is on the essential light chain (ELC). The system has several unusual properties. The calcium binding site is an unusual EF hand of domain I; the loop providing the liganding residues is nine residues long, five of which are found only in molluscs3,4. Calcium binding requires the presence of both light chains and the heavy chain. In the presence of millimolar concentrations of magnesium ions, none of these subunits bind calcium in isolation. Upon removal of the RLC, calcium binding is lost2, although the RLC does not provide liganding residues; its role in calcium binding is to stabilize the calcium binding loop of the ELC4.

The changes associated with regulation take place on myosin in the absence of actin5. Calcium stimulates ATPase activity of heavy meromyosin (HMM) about one hundred-fold, with approximately seven-fold further activation by actin6. Therefore, regulatory events can be studied on myosin and HMM alone in the absence of actin. The propensity of the scallop myosin fragments to crystallize4, and the fact that the myosins of catch and striated adductor muscles are isoforms produced by alternative RNA splicing of a single gene7,8 further demonstrate the advantages of studying these muscles. Significantly, S1 is unregulated, although this myosin fragment contains both the calcium binding and nucleotide binding sites. S1 does not require calcium for activity; it is fully turned on in its absence9. In S1, communication seems to flow in one direction, from the motor domain towards the regulatory domain. The atomic structure of the regulatory domain has been determined4,10. However, this fragment lacks the nucleotide binding site and is not regulated. As of now, there is no direct evidence what the structure of the “off” state may be. Even the elucidation of the atomic structure of S1 will not produce direct information as to how activity is blocked in the absence of calcium. It has been proposed that the structural transition upon removal of calcium may confer flexibility to the bend of the long helix between the RLC and ELC, which induces the N-terminal end of the helix to occupy a stable blocking position at the converter region10. In the absence of direct structural information, we have focused our attention on differences between S1 and HMM which pertain to regulation. We have also explored the properties of single-headed myosins to evaluate the possible contribution of the head-rod junction. Activation of tension development and activation of ATPase activity are highly cooperative processes. This cooperative activation characterizes both molluscan and skeletal muscles, therefore it is independent of the manner in which the particular regulatory system functions. Our studies on cooperative behavior were made possible by the development of a highly calcium sensitive HMM preparation which showed 90–95% calcium sensitivity of its Mg-ATPase activity, and was activated 10-20 fold by calcium even in the absence of actin11.


Light Chain ATPase Activity Calcium Binding Nucleotide Binding Site Myosin Head 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



adenosine triphosphate


adenylyl imidodiphosphate; Vi, vanadate ion


ethylene glycol bis (ß-aminoethyl ether)-N,N,N,N’-tetraacetic acid


3-(N-morpholine) propanesulfonic acid


subfragment 1


heavy meromyosin


regulatory light chain


essential light chain


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Copyright information

© Plenum Press, New York 1998

Authors and Affiliations

  • Vassilios N. Kalabokis
    • 1
  • Andrew G. Szent-Györgyi
    • 1
  1. 1.Department of BiologyBrandeis UniversityWalthamUSA

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