The effect of phosphorylation of the myosin light chains (LC-2) on cross-bridge conformation in synthetic myosin filaments from vertebrate skeletal muscle was studied by using chemical cross-linking and chymotryptic digestion methods. Phosphorylated and dephosphorylated myosin filaments, which were used in these experiments, had similar sedimentation coefficients, turbidities, and rates of growth from the respective minifilament structures. The proteolytic susceptibility at the heavy meromyosin-light meromyosin (HMM-LMM) junction was somewhat greater in the phosphorylated than in the dephosphorylated filaments at both pH 7.0 and pH 8.0. At the same time, the normalized rate of subfragment 2 (S-2) cross-linking to the filament surface, kS-2/kLMM, was reduced by phosphorylation of myosin. These results are consistent with partial release of cross-bridges from the thick filament surface in phosphorylated myosin filaments.
The interaction of alkali light chains of vertebrate skeletal myosin with nucleotides and KCl has been examined by chemical modifications of these proteins, by direct binding measurements, and in circular dichroism studies. The reactivities of the single thiol groups in the isolated alkali light chains A1 and A2 have been studied by reacting these proteins with 5,5′‐dithiobis(2‐nitrobenzoic acid) (Nbs2). MgATP and MgADP reduced the reactivities of thiol groups while high concentrations of KCl increased them. Subsequent equilibrium dialysis experiments verified the presence of a low‐affinity nucleotide binding site per each alkali subunit. Circular dichroism measurements revealed that KG induced local (around phenylalanines) and overall (α‐helical content) conformational changes of equal magnitude in the two alkali light chains. However, salt induced different conformational changes in the subfragment 1 isoenzymes, S‐1(A1) and S‐1(A2). This differential salt effect on the S‐1 isoenzymes was confirmed by comparing their thermal stability in different salt conditions. At low KCl concentrations (5 mM), S‐1(A1) was found to be considerably more heat labile than S‐1(A2); at higher salt levels (50 mM KCl) the stability of S‐1(A1) approached that of S‐1(A2). These experiments are discussed in terms of the relationship between the alkali subunits and the ATP and the actin‐binding sites of myosin.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.