Probing the coupling of Ca2+ and rigor activation of rabbit psoas myofibrillar ATPase with ethylene glycol.

We have exploited solvent perturbation to probe the coupling of Ca2+ and rigor activation of the ATPase of myofibrils from rabbit psoas. Three techniques were used: overall myofibrillar ATPases by the rapid-flow quench method; kinetics of the interaction of ATP with myofibrils by fluorescence stopped-flow; and myofibrillar shortening by optical microscopy. Because of its extensive use with muscle systems, ranging from myosin subfragment-1 to muscle fibres, we chose 40% ethylene glycol as the relaxing agent. At 4 degrees C, the glycol had little effect on the myofibrillar ATPase at low [Ca2+], but at high [Ca2+] the activity was reduced 50-fold, close to the level found under relaxing conditions, and there was no shortening. However, the ATPase of chemically cross-linked myofibrils (permanently activated even without Ca2+) was reduced only 3-4-fold. The lesser reduction of the ATPase of permanently activated myofibrils was also observed in single turnover experiments in which activation occurs by a few heads in the rigor state activating the remaining heads. The addition of ADP, which also promotes strong head-thin filament interactions, also activated the ATPase but only in the presence of Ca2+. Further experiments revealed that in 40% ethylene glycol, Ca2+ does initiate shortening but only with the aid of strong interactions and at temperatures above 15 degrees C. This confirms that in the organized and intact myofibril, Ca2+ and rigor activation are coupled, as proposed previously for regulated actomyosin subfragment-1.