Functional effects of LC1-reassociation with cardiac papain Mg � S1

Abstract

The effect of LC1 on cardiac myosin structure and activity was investigated using as a model S1 prepared by papain digestion in the presence of Mg2+. The resulting S1 contained LC2 but a part of the N-terminal region of LC1 was cleaved. Sequencing the N-terminal part of the band migrating below LC1 on SDS gels revealed it to consist of alternating alanyl and prolyl residues thus establishing LC1 as the origin of this band. However, Western blots did not reveal any LC1 while radioimmunoassays indicated it to be present at the 5% level suggesting the anti-LC1 antibody used in these experiments did not recognize the C-terminal portion of LC1 still attached to Mg.S1. Mixing a 10-15 M excess of isolated light chains with Mg.S1 in the presence of 10 mM ATP, 12 mM MgCl2, 4.7 M NH4Cl allowed LC1 to recombine with LC1-deficient Mg.S1. Equilibrium ultracentrifugation analysis revealed a highly heterogeneous LC1-deficient S1 which upon recombination with intact LC1 became monodisperse as indicated by the superimposition of molecular weight averages all across the centrifuge cell. LC1-deficient Mg.S1 had a Vm of 0.4 s-1, Ka of 30 microM and a Kbind of 28 microM. In the presence of intact LC1, Vm rose to 0.8 s-1 while Ka and Kbind were reduced to 7.5 and 12 microM, respectively. The fourfold decrease in Ka strongly indicated an increased affinity for actin by Mg.S1 in the presence of uncleaved LC1. Also, Ca(2+)-regulation of dog heart myofibrils was suppressed when Ca(2+)-activated MgATPase assays, as a function of Ca2+, were performed in the presence of anti-LC1 antibodies. These observations suggest the presence of intact, uncleaved LC1 in S1 is required for the stability of S1 heavy chains and proper Ca(2+)-regulation.

The role of the skeletal muscle myosin light chains N‐terminal fragments

“…The removal of the N-terminus of A1 in myosin leads to destabilization of the head with time as observed by reduced K ÷ ATPase activity [38]. The destabilization could result from dimerization of heads observed by Margossian et al [36] for the cardiac papain S 1 and by us for the rabbit cardiac and skeletal myosin (unpublished observations).…”

“…Margossian et al [36] found that, in S 1 preparations obtained by papain digestion of cardiac myosin (with the N-terminus of A1 cleaved off, referred to as AI'), reassociation of intact A1 with the A1 deficient S1 caused increased affinity and increased Vm,x in comparison with preparations of SIAl' (with shorter A1). However, in the experiments with the rabbit cardiac myosin which had been treated with papain to obtain preparations with the N-terminus of A1 digested off (but with the preserved intact heavy chain) Moczarska and Kgkol [37] observed an increase by one order of magnitude of the myosin affinity to actin.…”

“…An antibody directed against the N-terminal part of A1 caused a four-fold decrease of the cardiac myofibrillar ATPase activity [36]. Boey et al [39] using an antibody against the N-terminus of AI in skeletal myosin achieved a complete inhibition of the S 1 (A 1) actin-activated ATPase whilst the activity of S I(A2) was not affected; however, the antibody bound also to A2.…”

Breakdown and Release of Myofilament Proteins During Ischemia and Ischemia/Reperfusion in Rat Hearts

“…45 This suggested that MLC1 fragment 20 to 199 could alter function. However, since the MLC fragment is released from the cell during severe ischemia (Fig 3), it would make only a small contribution toward the myofilament dysfunction observed with 60 minutes of ischemia and 45 minutes of reperfusion.…”

Cleavage points of rabbit skeletal myosin light chains selectively modified in situ by limited proteolysis: structural characteristics of the neoformed isozymes

“…The whole LC2 has been removed either chemically or by proteolysis, tH NMR and proteolytic investigations suggested that the N-terminus particularly was involved in the movement of the heads to and away from the filament backbone [1 I]. A recent study showing functional effects of LCI reassociation with cardiac (papain-Mg)-Sl suggested that intact, uncleaved LC1 is required ['or the stability of S1 heavy chains and correct Ca > regulation [12].…”