Investigation of the temperature dependence of the cross bridge parameters for attachment, force generation and detachment as deduced from mechano-chemical studies in glycerinated single fibres from the dorsal longitudinal muscle of Lethocerus maximus.

Abstract

1) A study is presented on the effect of temperature on the mechanochemical states involved in the cross bridge cycle of single glycerinated dorsal longitudinal fibres from Lethocerus. Contraction was induced by immersing the fibre in a MgATP-salt solution at Ca2+∼10 ΜM (pH 6.7). 2) Rising the temperature increases the rates of isometric tension generation following an increase in the [Ca2+] from 0.01 to 10 ΜM as well as the steady state levels of isometric tension and the rates of ATP splitting. 3) Tension transients following stretches of rise times 250 Μs and amplitudes up to 0.4% L i comprise at least four phases: an elastic phase the amplitude of which decreases by raising the temperature; a biphasic quick phase of tension decay with a mean Q10=2; a delayed tension rise (Q10∼5). 4) Tension transients following releases of fall time 250 Μs and amplitudes up to 0.3% L i also comprise four phases: an elastic phase comparable to that observed following stretches; a deactivation phase composed of a single exponential and a slow recovery phase. 5) The number of cross bridges attached to the actin at any moment is not changed during the elastic and quick recovery phase following a release as well as during the elastic and fast quick phase following a stretch. However, the number of attached cross bridges decreases during the deactivation phase. 6) The early phases of tension adjustment (T curves) which were recorded during the releases showed a marked dependence on temperature. The T curves fitted with high accuracy the Huxley and Simmons (1971) predictions of cross bridge rotation. 7) Analysis of the T curves in terms of the Huxley and Simmons (1971) model shows that a) the stiffness of a single cross bridge (D=1.2 104− N/m) obeys Hook's law; b) the number of myosin heads attached to actin (24% of the total number) is not altered during releases; c) rotation of myosin heads from a perpendicular to an acute angled position extends the elastic element of a cross bridge by 11 nm; d) at 25‡ C the rate constants for rotation from the perpendicular position to the acute angled position and vice versa are 8300 s−1 (Q10=3.5) and 3600 s−1 (Q10=1.5). 8) Thermodynamics applied to cross bridge rotation predicts that during sudden releases the temperature within the fibre should fall and the driving force for tension generation is an increase in entropy of rotated bridges. 9) Rate constant of detachment of cross bridges from the actin is determined to k2=500 s−1 (25‡ C; Q10=2.3). 10) The values of steady state rate of ATP splitting in conjunction with estimates of the number of attached cross bridges indicate that rate limiting steps of ATP cleavage occur while myosin heads are detached and while they are attached to the actin: Rate limiting for the attachment is the decay of the refractory myosin-product (Eisenberg and Kielley, 1973; k4=1.7 s−1: 25‡ C; Q10=2.5). Rate limiting for detachment is the concentration ratio of actomyosin-ATP to actomyosin-product K1=0.018 (25‡ C; Q10=2.7).