Repriming the actomyosin crossbridge cycle.
The central features of the mechanical cycle that drives the contraction of muscle are two translational steps: the working stroke, whereby an attached myosin crossbridge moves relative to the actin filament, and the repriming step, in which the crossbridge returns to its original orientation. Although the mechanism of the first of these is understood in some detail, that of the second has received less attention. Here, we show that repriming occurs after detachment of the crossbridge from the actin, rather than intervening between two actomyosin states with ATP bound [Eisenberg, E. & Greene, L. E. (1980) Annu. Rev. Physiol. 42, 293-309]. To discriminate between these two models we investigated the single-molecule mechanics of the myosin-actin interaction in the presence of ATP analogues such as GTP, for which the hydrolytic step itself limits the actomyosin GTPase rate to a much lower rate than for ATP. The lifetimes of bound states was proportional to 1/[GTP], indicating that during the bound period myosin was in the actomyosin rigor configuration. Moreover, despite the very low actomyosin GTPase, the rate of actin binding and formation of the rigor state was higher than with ATP; it follows that most interactions with actin result in the release of GTP and not of the products, GDP and phosphate. There was no significant movement of the actin during this interaction, so repriming must occur while myosin is dissociated, as in the original Lymn-Taylor scheme [Lymn, R. W. & Taylor, E. W. (1971) Biochemistry 10, 4617-4624].
Randall Centre, King's College, London SE1 1UL, United Kingdom.
Walter
W
Steffen
John
J
Sleep
D000199:Actins / Q000378:metabolism; D000205:Actomyosin / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D006801:Humans; D007293:Inosine Triphosphate / Q000378:metabolism; D009119:Muscle Contraction / Q000502:physiology*; D009218:Myosins / Q000378:metabolism; D013997:Time Factors
D000199:Actins; D007293:Inosine Triphosphate; C036863:magnesium ITP; D000255:Adenosine Triphosphate; D000205:Actomyosin; D009218:Myosins