The aim of this study was to compare the effects of increased concentrations; of MgADP, inorganic phosphate (P(i)) and H+ ([MgADP], [Pi] and [H+], respectively) on the rate of relaxation in two different muscle types: skinned muscle fibres from the frog Rana temporaria and myofibrillar bundles from the giant Pacific acorn barnacle Balanus nubilus. Relaxation transients are produced by the photolysis of diazo-2 and are well fitted with a double exponential curve, giving two rate constants: k(1) [5.6±0.1 s-1 for barnacle, n=30; 26.3±0.7 s-1 for frog, n=14 (mean±SEM)] and k2 [0.6±0.1 s-1 in barnacle, n=30; 10.4±1.0 s-1 in frog, n=14 (mean±SEM)], at 10°C. Decreasing the pH by 0.5 pH units did not significantly affect k(1) for barnacle relaxation [5.6±0.1 s-1 (mean±SEM), n=15] compared to the decrease in k1 of 40% seen in frog. Use of the Ca2+-sensitive fluorescent label acrylodan on barnacle wild-type troponin C demonstrated that decreasing the pH from 7.0 to 6.6 only alters the pCa50 value by 0.23 in the cuvette, while stopped-flow experiments with acrylodan revealed no significant change in k(off) from the labelled protein [322±32 s-1 at pH 7.0 and 381±24 s-1 (mean±SEM) at pH 6.6]. Increasing [MgADP] by 20 μM (50 μM added ADP) from control values of 50 μM in frog decreased k1 to 12.3±0.4 s-1 (mean±SEM, n=8), and at 400 μM MgADP, k1=9.6±0.1 s-1 (mean±SEM, n=12). In barnacle, 500 μM MgADP had a much smaller effect on k1 (4.0±0.9 s-1, mean±SEM, n=8). Increasing the free [P(i)] from the contaminant level of 0.36 mM to 1.9 mM slowed k1 by ≃ 15% in barnacle [4.8±0.8 s-1, mean±SEM, n=7], compared to a ≃ 30% reduction seen in frog. We conclude that the differences between barnacle and frog seen here are most probably due to different isomers of the contractile proteins, and that events underlying the crossbridge cycle are the same or similar. We interpret our results according to a model of crossbridge transitions during relaxation.
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