Characterization of homologous divalent metal ion binding sites of vertebrate and molluscan myosins using electron paramagnetic resonance spectroscopy

Abstract

The binding of Ca 2+, Mg 2+ and Mn 2+ to myosins from rabbit skeletal muscle, scallop striated adductor muscle and clam adductor muscle has been investigated. All three myosins bind two moles of divalent metal ion non-specifically and with high affinity (Mn 2+ > Ca 2+ > Mg 2+). In addition, the molluscan myosins bind about a further two moles of Ca 2+ specifically. Although rabbit myosin binds some Ca 2+ in the presence of an excess of free Mg 2+, this binding occurs at the nonspecific sites and should not be taken as evidence for a myosin-linked regulatory system of the type found in molluscan muscles. If such a system exists in vertebrate skeletal muscle, the homologous Ca 2+-specific sites must be lost during the early stages of the myosin preparation. The characteristic electron paramagnetic resonance spectrum of the bound Mn 2+ was utilized to confirm the homology of the non-specific sites in vertebrate and molluscan myosins. The sites are located on the “regulatory” class of light chain. Mn 2+ bound to scallop myosin has a broad electron paramagnetic resonance spectrum, in contrast to the well-resolved spectra that it gives when bound to many other myosin species. This situation was exploited to identify homologous nonspecific, divalent metal-ion sites on the regulatory light chains from a variety of muscle types, including frog skeletal, rabbit cardiac, chicken gizzard and molluscan adductor muscles. When these light chains are combined with desensitized scallop myofibrils the electron paramagnetic resonance spectra of Mn 2+ bound to the resultant hybrids are dominated by the signal from the non-specific site of the foreign regulatory light chain.