Cryoenzymic studies on an organized system: Myofibrillar ATPases and shortening

We have exploited cryoenzymology, first, to probe the product release steps of myofibrillar ATPase under relaxing conditions and, second, to define th e conditions for studying the contractile process in slow motion. Cryoenzym ology implies perturbation by temperature and by the antifreeze added to al low for work at subzero temperatures. Here, we studied myofibrillar shorten ing and ATPases by the rapid quench flow method over a wide temperature ran ge (-15 to 30 degrees C) in two antifreezes, 40% ethylene glycol and 20% me thanol. The choice of solvent and temperature was dictated by the purpose o f the experiment. Ethylene glycol (40%) is suitable for investigating the k inetics of the products release steps which is difficult in water. In this cryosolvent, the myofibrillar ATPase is not activated by Ca2+ nor is there shortening, except under special conditions, i.e., Ca2+ plus strong rigor b ridges [Stehle, R., Lionne, C., Travers, F., and Barman, T. (1998) J. Muscl . RES. Cell Motil. 19, 381-392]. By the use of the glycol, we show that at low Ca2+ the kinetics of the ADP release are much faster with myofibrils th :tn with S1. On the other hand, the kinetics of the Pi release were very si milar for the two materials. Therefore, we suggest that, upon Ca2+ activati on, only the Pi release kinetics an accelerated. In 20% methanol, in the pr esence of Ca2+, myofibrils shortened at temperatures above -2 degrees C but not below. At a given temperature above -2 degrees C, both the shortening and ATPase rates were reduced by the methanol. The temperature dependences of the myofibrillar ATPases (+/-Ca2+) converged with a decrease in temperat ure: at 20 degrees C, Ca2+ activated 30-fold, but at -15 degrees C, only ab out 5-fold. We suggest that studies in methanol may open the way for an inv estigation of muscle contraction in slow motion and, further, to obtain the rmodynamic information on the internal fords involved in the shortening pro cess.