Phosphorylation and the Binding of Calcium and Magnesium to Skeletal Myosin

Abstract

It has previously been shown that the binding of calcium and magnesium ions to the isolated metalbinding light chains, i.e. those dissociable by 5,5'-dithiobis(2-nitrobenzoate), of rabbit skeletal muscle myosin is moderated by phosphorylation and is accompanied by a sizeable conformational change. As judged by circular dichroism in the region of the aromatic Cotton effects, this conformational change occurs when calcium ions bind to the light chain in situ on the myosin head. Moreover the affinity for calcium is again changed by phosphorylation. The change in chymotryptic digestion patterns, in particular the protection of the head-rod junction in insoluble myosin, by divalent cations, has been used to obtain binding profiles. The results are consistent with the presence of a single class of independent sites, showing no cooperativity. The affinity of the site for both calcium and magnesium ions is enhanced by 1-2 orders of magnitude when the light chain is incorporated in the myosin heads. The effect of phosphorylation on the affinity persists in these circumstances, being marked for calcium and small for magnesium. On phosphorylation the calcium binding constant falls from 8 x lo6 M-' to 4 x lo6 M-' at physiological ionic strength, compared with 2.5 x lo5 M-' and 5 x lo4 M -for the isolated light chains. The sensitivity of the proteolytic cleavage sites is affected by phosphorylation. Thus in the absence of calcium ions the yield of subfragment 1 at a low chymotrypsin concentration is substantially greater in dephosphorylated than phosphorylated myosin, whereas at saturating concentrations of calcium ions attack at the light meromyosin/heavy meromyosin junction is favoured by phosphorylation. These observations may signify a structural effect of phosphorylation on the prevailing interactions within the myosin filament in physiological solvent conditions. It has become clear that the calcium ions, which are released into the sarcoplasm from the sarcoplasmic reticulum when a muscle is stimulated, have multifarious effects. In vertebrate striated muscle, calcium ions are captured by troponin C, with relief of the inhibition (exerted by the regulatory complex on the thin filaments) of the actomyosin interaction. In molluscan muscle, a homologous protein is present as a subunit (regulatory light chain) on the myosin heads, and this is the site of the primary control mechanism; in some other muscles both forms of regulation coexist (see Lehman and Szent-Gyorgyi [l] and Perry [2] for reviews). In smooth muscle, moreover, the ATPase activity is generally held to be regulated by phosphorylation of the calcium-binding myosin light Abbreviations. Nbsz light chains, the metal-binding light chains which can be dissociated from myosin by treatment with 53'-dithiobis(2-nitrobenzoate); Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulphonic acid; EPR, electron paramagnetic resonances. chain, through the action of a kinase, which is itself activated by calcium ions. A similar phosphorylation reaction operates in striated muscle...

Light-chain phosphorylation and cross-bridge conformation in myosin from vertebrate skeletal muscle

“…The major effect of LC-2 phosphorylation was, as previously noted (Kardami et al, 1980;Ritz-Gold et al, 1980), the protection of this subunit from proteolytic degradation (Figure 1). The increase in the stability of the phosphorylated light chain could be detected even in the presence of 1 mM Ca2+;…”

“…The rate of proteolytic cleavage at the HMM-LMM junction is about 40% faster in the phosphorylated myosin than in the dephosphorylated protein. Although this phosphorylation effect is rather small, in particular when compared to that caused by the increase in pH from 7.0 to 8.0, it has been noted in previous reports as well (Kardami et al, 1980;Ritz-Gold et al, 1980). The increased rate of digestion at the HMM-LMM hinge does not result from the phosphorylation-induced stabilization of the head-rod junction against proteolysis.…”

“…In this 1 Abbreviations: HMM, heavy meromyosin; LMM, light meromyosin; LC-2, Ca2+ binding 19 000 molecular weight subunit of myosin; S-l, subfragment 1; S-2, subfragment 2; EDTA, ethylenediaminetetraacetic acid; BSA, bovine serum albumin. context, the observation of increased chymotryptic susceptibility at the HMM-LMM junction in phosphorylated filaments (Kardami et al, 1980, Ritz-Gold et al, 1980) merits special attention. The aim of this work was to examine the effect of light-chain phosphorylation on cross-bridge conformation.…”

Effect of calcium on synthetic myosin minifilaments

“…Earlier work has shown that the high-affinity binding sites for Ca2+ on myosin are fully saturated at pCa = 5 (Weeds & Pope, 1977;Kardami et al" 1980;Morimoto & Harrington, 1974). Accordingly, the changes in hydrodynamic properties (Morimoto & Harrington, 1974) and proteolytic digestion of myosin filaments (Weeds & Pope, 1977) occur between pCa = 7 and pCa = 5.…”

Conformational effects of cation binding to myosin and their relation to phosphorylation

“…It does not change the myosin ATPase (Morgan et al, 1976), though an enhancement of ATPase activation by regulated actin filaments has been reported (Pemrick, 1980). Phosphorylation causes a small but readily measurable diminution in the affinity of the light chain for calcium, though not for magnesium, in both the isolated state (Alexis & Gratzer, 1978) and in situ (Kardami et al, 1980). The functional consequences of these small effects of phosphorylation have yet to be established.…”