Dissociation of Light Chains from Cardiac Myosin

Abstract

The substrate, ATP, protects the active site of cardiac myosin during a 10-min treatment at 37 "C and neutral pH in the absence of divalent cations; under these conditions there is an approximate 20 % dissociation of light chain C1 and 60 % loss of light chain CZ with no corresponding decrease in myosin ATPase activity. Higher temperatures, the absence of divalent cations, increased treatment time, and low protein concentration were conducive to light chain dissociation; loss of light chains did not appear to be influenced by increments of pH from 7.0 to 9.0. On the other hand, a decrease in protein concentration and an increase in both duration of exposure at 37°C and an alkaline pH caused a decrease in myosin ATPase activity. Concomitant with the dissociation of cardiac myosin light chains, particularly light chain C2, there was a corresponding loss in the number of high-affinity calcium binding sites. When the dissociated light chains were recombined with lightchain-deficient myosin, reassociation took place and the number of high-affinity calcium binding sites were regained. Reassociation of light chains with light-chain-deficient myosin did not restore diminished light-chain-deficient myosin ATPase activity. There is a shift in the pH optimum of myosin at 37 "C in the absence of divalent cations, where conditions used for light chain dissociation are employed. The presence of calcium prevents light chain dissociation, helps retain myosin ATPase activity at 37 "C and prevents a shift in the pH optimum.

Stability and substructure of cardiac myosin subfragment 1 and isolation and properties of its heavy-chain subunit

“…The results of the thermal ion-exchange chromatography presented in Figure 2 indicate a high degree of similarity between the cardiac and the skeletal proteins in terms of the stability of the subunit interactions (Sivaramakrishnan & Burke, 1982). The fact that a portion of the cardiac protein can be isolated as the free heavy chain under conditions where the ATPase activity is not lost (Higuchi et al, 1978) suggests that it exists in a rapid, reversible equilibrium with its dissociated subunits under conditions which approach the physio-logical state. A further similarity in the two forms of SF1 is that prior cleavage at the two protease-sensitive sites destabilizes the subunit interactions (Burke & Kamalakannan, 1985).…”

“…In this regard, the cardiac protein appears to be more stable to thermal dissociation. A substantial dissociation of its light chains without loss in ATPase activity has been shown to occur in the presence of ATP at 37 °C in the absence of divalent cations while, on the other hand, the presence of divalent cations was found to prevent subunit dissociation (Higuchi et al, 1978).…”

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

“…4) is either the result of the Ca2+-binding light chain not reassociating with the S-1 subfragment lacking the (S-1)-(S-2) junction or partial reassociation occurred but the Ca2+-binding properties were no longer influenced by the heavy chains. Native myosin, conversely reassociated under these conditions Higuchi et al, 1978;Wikman-Coffelt, 1980;Hollosi et al, 1980) and the high Ca2+-binding affinity value was restored. The Ca2+-binding light chain has a different affinity for Ca2+ when complexed to the heavy chains as compared with when it is in the dissociated state (Wikman-Coffelt et al, 1979) and only binds at Ca2+ concentrations required to induce a conformational change in free light chains (Ca2+ 10-510-3 M) (Morakovcic et al, 1979;Yamamoto etal., 1980).…”

“…Gel electrophoresis patterns (5-20% polyacrylamide gradient containing SDS) of (a) rabbit skeletal and (b) sheep cardiac myosins I and II before and after treatment with N-ethylmaleimide respectively. The dye, Coomassie Blue, was eluted from the stained protein bands and quantified as described in earlier reports (Higuchi et al, 1978). The percentage of protein present was: (a) (I) 88.2% HC; 3.8% LCG; 6.1% LC2; 1.9% LC3.…”

“…The light chains are not denatured with dissociation because reassociation again promotes a high Ca2+binding affinity (Wikman-Coffelt et al., 1979;Okamoto & Yagi, 1977). Likewise, increases in temperature that induce light chain dissociation in the presence of chelating agents (Stafford et al, 1979;Higuchi et al, 1978) also promote conformational changes in myosin heavy chains (Mendelson & Cheung, 1976; Chiano & Harrington, 1979; Ishigami & Morita, 1977), but not the light chains (Chantler & Szent-Gyorgyi, 1978;Stafford & Szent-Gyorgyi, 1978).…”