Abstract
The incorporation of various radioactive reagents into myosin and sarcoplasmic proteins of resting and electrically stimulated frog muscles was studied. N-Ethylmaleimide and iodoacetamide showed a decreased incorporation into myosin during contraction, whereas benzenediazonium chloride could not differentiate between myosin of contracting and resting muscle. None of these reagents showed any specificity as far as the sarcoplasmic proteins were concerned. On account of its high incorporation into myosin, N-ethylmaleimide was used to determine the distribution of label in the substructure of myosin. Most of the label was found in the globular head part (heavy meromyosin subfragment 1) of the myosin molecule and only a small amount of label was in its rod-like part (heavy meromyosin subfragment 2 and light meromyosin). The entire difference in incorporation between contracting and resting muscle (0.5 to 0.7 mole of label per mole of myosin) occurred in the globular head. In terms of muscle structure, these results indicate that the change in reactivity during contraction appears in the cross-bridges, whereas the backbone of the filament and its linkage with the cross-bridge remain unaffected. The decreased incorporation was not caused by a protecting effect of actin or ATP, or both, on the active site of myosin. This was evidenced by the identical actin-binding ability and ATPase activity of myosin from contracting or resting muscle. Dowex 50 chromatography of peptic, tryptic, peptictryptic-chymotryptic, and subtilisin digests of myosin from contracting and resting muscle yielded a large number of radioactive peptides. Most of the peptides were similar but a number of them were different. The prevalent difference was that the peptides derived from myosin of contracting muscle contained less radioactivity than the corresponding ones from myosin of resting muscle. These results are compatible with the idea that during contraction a conformational change occurs in a certain area of the cross-bridges rendering this area inaccessible for reaction with N-ethylmaleimide.
Highlights
We have found previously that FDNB is rather specific in chemically differentiating myosin of resting and contracting muscle or of relaxed and contracting myofibrils [1]
The reagent had no effect on the contractility of frog muscles
NO difference in incorporation was found between myosin of resting and contracting muscle (Table I)
Summary
IncorporationStudies with Living il4uscles-These muscles were dissected from large frogs (Rana pipiens). In addition to the rectus muscle used previously (I), we have used the gastrocnemius muscle in the present work to increase the yield of myosin. The gastrocnemius spent 16% of the total time in the shortening and 43 74 of t#he tota,l time in the combined shortening and subsequent lengthening phase of the mechanical activity (the corresponding data for rectus are 17 and 36%, respectively). Treatment of the muscle with various reagents was performed following the same procedure as described for the treatment with [3H]FDNB [1]. The concentration of the reagent and the time of treatment is given in the legends of the appropriate tables. The removal of unbound reagent from the muscles by washing with Ringer’s solution, and the determinat,ion of incorporation [14C]NES, N-ethyl-[1-‘4C] succinimide; [3H]BDCl, [3H]benzenediazonium chloride; HMM, heavy meromyosin; LMM, light meromyosin; HMM
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
