ISSN:
1573-2657
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Medicine
Notes:
Summary To examine the mechanism by which aluminiofluoride, a tightly binding analogue of inorganic phosphate, inhibits force in single, chemically skinned fibres from rabbit psoas muscle, we measured the Ca2+-dependence of the kinetics of inhibitor dissociation and the kinetics of actomyosin interactions when aluminiofluoride was bound to the crossbridges. The relation between stiffness and the speed of stretch during small amplitude ramp stretches (〈 5 nm per h.s.) was used to characterize the kinetic properties of crossbridges attached to actin; sarcomere length was assessed with HeNe laser diffraction. During maximum Ca2+-activation at physiological ionic strength (pCa 4.0, 0.2 m Γ/2), stiffness exhibited a steep dependence on the rate of stretch; aluminiofluoride inhibition at pCa 4.0 (0.2 m Γ/2) resulted in an overall decrease in stiffness, with stiffness at high rates of stretch (103–104 nm per h.s. per s) being disproportionately reduced. Thus the slope of the stiffness-speed relation was reduced during aluminiofluoride inhibition of activated fibres. Relaxation of inhibited fibres (pCa 9.2, 0.2 m Γ/2) resulted in aluminiofluoride being ‘trapped’ and was accompanied by a further decrease in stiffness at all rates of stretch which was comparable to that found in control relaxed fibres. In relaxed, low ionic strength conditions (pCa 9.2, 0.02 m Γ/2) which promote weak crossbridge binding, stiffness at all rates of stretch was significantly inhibited by aluminiofluoride ‘trapped’ in the fibre. To determine the Ca2+-dependence of inhibitor dissociation, force was regulated independent of Ca2+ using an activating tropinin C (aTnC). Results obtained with aTnC-activated fibres confirmed that there is no absolute requirement for Ca2+ for recovery from force inhibition by inorganic phosphate analogues in skinned fibres; the only requirement is thin filament activation which enables active crossbridge cycling. These results indicate that aluminiofluoride preferentially inhibits rapid equilibrium or weak crossbridge attachment to actin, that aluminiofluoride-bound crossbridges attach tightly to the activated thin filament, and that, at maximal (or near-maximal) activation, crossbridge attachment to actin prior to inorganic phosphate analogue dissociation is the primary event regulated by Ca2+.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00130423
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