Influence of myosin heavy chains on the Ca2+-binding properties of light chain, LC2

Abstract

The association of myosin light chains with heavy chains, i.e. the intact oligomeric structure, profoundly affects the Ca2+-binding properties of the light chains. The Ca2+-binding affinity of the light chains is more than two magnitudes higher in the presence of heavy chains than in its absence. Modification of the reactive SH2 thiol of myosin results in an alteration in the conformation of heavy chains of the molecule that influences the Ca2+-binding properties of light chains and generation of tension. When the SH2 moiety is blocked with N-ethylmaleimide the influence of the heavy chains on the Ca2+-binding properties of light chain LC2 is lost; under these conditions the Ca2+-binding affinity value of SH2-N-ethylmaleimide-blocked myosin (3.3×104m−1) decreases to near that expressed with the dissociated light chain LC2 (0.7×104m−1). Conversely, the presence of actin, nucleotides or modification of either the reactive lysyl residue or SH2 thiol does not affect Ca2+ binding. The native secondary and tertiary structure of myosin seem to be required for Ca2+ binding; binding does not occur in the presence of 6m-urea with either native myosin or the dissociated light chains. With SH2-N-ethylmaleimide-blocked myosin normal Ca2+- and (Mg2++actin)-stimulated ATPase activities are expressed; however, there is a loss in K+-stimulated ATPase activity and the synthetic actomyosin threads of such myosin express no isometric tension. There are also variances in the binding of Ca2+ with alterations in pH values. In the absence of Ca2+/EGTA buffer the biphasic Ca2+-binding affinity of myosin is twice as high at pH7.4 (site one: 1.2×106m−1 and site two: 0.4×106m−1) as compared with values obtained at pH6.5 (site one: 0.64×106m−1 and site two: 0.2×106m−1). The Ca2+-binding affinity of light chain LC2 and S1, where the (S-1)–(S-2) junction was absent, were not influenced by changes in pH values. Both expressed a low Ca2+-binding affinity, approx. 0.7×104m−1, whereas heavy meromyosin, where both (S-1) and (S-2) myosin subfragments were present, expressed a Ca2+-binding affinity value similar to that of native myosin, but was not biphasic. However, it is important to point out than in preparation of S1 myosin subfragment light chain LC2 was lost and thus was added back to the purified S1 fraction. Light chain LC2 was not, however, added to the heavy meromyosin fraction because it was not lost during preparation of the heavy meromyosin subfragment. In conclusion, it appears that the (S-1)–(S-2) junction is needed for the positioning of light chain LC2 and thus influences its essential conformation for Ca2+ binding.