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

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Abstract
The association of [rabbit skeletal muscle] myosin L chains [LC] with H chains [HC], i.e., the intact oligomeric structure, profoundly affects the Ca2+-binding properties of the LC. The Ca2+-binding affinity of the LC is more than 2 magnitudes higher in the presence of HC than in its absence. Modification of the reactive SH2 thiol of myosin results in an alteration in the conformation of HC of the molecule that influences the Ca2+-binding properties of LC and generation of tension. When the SH2 moiety is blocked with N-ethylmaleimide the influence of the HC on the Ca2+-binding properties of LC2 [Ca2+-binding L chain-2] is lost; under these conditions the Ca2+-binding affinity value of SH2-N-ethylmaleimide-blocked myosin (3.3 .times. 104 M-1) decreases to near that expressed with the dissociated LC2 (0.7 .times. 104 M-1). 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 6 M urea with either native myosin or the dissociated LC. With SH2-N-ethylmaleimide-blocked myosin normal Ca2+- and (Mg2+ + actin)-stimulated ATPase activities are expressed. There is a loss in K+-stimulated ATPase activity, and the synthetic actomyosin threads of such myosin express no isometric tension. There are 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 pH 7.4 (site 1: 1.2 .times. 106 M-1 and site 2: 0.4 .times. 106 M-1) as compared with values obtained at pH 6.5 (site 1: 0.64 .times. 106 M-1 and site 2: 0.2 .times. 106 M-1). The Ca2+-binding affinity of LC2 and S1 [myosin subfragment head], where the (S-1).sbd.(S-2) junction was absent, were not influenced by changes in pH values. Both expressed a low Ca2+-binding affinity, .apprx. 0.7 .times. 104 M-1. 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. It is important to indicate that in preparation of S1 myosin subfragment LC2 was lost and was added back to the purified S1 fraction. LC2 was not added to the heavy meromyosin fraction because it was not lost during preparation of the heavy meromyosin subfragment. The (S-1).sbd.(S-2) junction apparently is needed for the positioning of LC2 and influences its essential conformation for Ca2+-binding.

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