Rotational motions of myosin heads in myofibril studied by phosphorescence anisotropy decay measurements.

S Ishiwata,K Kinosita,H Yoshimura,A Ikegami

doi:10.1016/s0021-9258(18)47565-5

Abstract

We studied the rotational Brownian motions of myosin heads, of which the sulfhydryl group was selectively labeled with the triplet probe 5-eosinylmaleimide, in myofibril by using flash-induced phosphorescence anisotropy decay measurements. The anisotropy decay curve under relaxing conditions consisted of a fast (submicrosecond) and a slow (a few microseconds) component and a small constant part as in the synthetic myosin filaments in solution. The decay curves could be analyzed by assuming that a head part, i.e. subfragment 1 (S1), wobbles in the first cone and a part connecting S1 and the tail of a myosin molecule of which the length is shorter than subfragment 2 (S2) wobbles in the second cone (a double-cone model); the semiangles of the former and the latter cones were about 30 degrees and 50 degrees, respectively. The rotational freedom of myosin heads was only slightly restricted by the limited space of the filament lattice in myofibrils. Under rigor conditions, no motion of myosin heads was observed in the 10-microseconds time scale.

Highlights

We studiedtherotationaBl rownianmotionsof examined by changing the osmotic pressure outside the mymyosin heads, of which the sulfhydryl group was se- ofibrils
Of which the lengthis shorter than subfragmen2t(S2) The specific labeling of myosin heads in myofibrils was performed wobbles in the second cone(a double-cone model); the according to the previous work with slight modifications [7,8]
Probaextent due to the SH labeling [12].) we expect that the bly, dissociatedheads were present at the H zone of myofibrils mobility of labeled heads will wellsimulate that of unlabeled ones at and bound tothin filaments after contraction

Summary

Introduction

We studiedtherotationaBl rownianmotionsof examined by changing the osmotic pressure outside the mymyosin heads, of which the sulfhydryl group was se- ofibrils. Glycerol was removed by repeated centrifugation a t 600 X g in the rigor solution (solution A 60 mM KC1, 5 mM MgCI,, 10 mM Tris maleate buffer (pH 6.8), and 1 mM EGTA), myofibrils (10 mg/ml; the concentration of myosin heads was estimated to be about 20 p ~ ) were incubated in solution A in the presence of 1mM N-ethylmaleimide (NEM; Sigma) for 10 min.

Results

Conclusion