Cryoenzymological Studies on Myosin Subfragment 1

The ATPase activities of myosin subfragment 1 were studied under subzero conditions with ethylene glycol as the antifreeze. The pH profile of the Ca ATPase activity was affected by both ethylene glycol and the temperature but the pH profile of the Mg ATPase activity was only slightly affected. At 20°C the Mg ATPase activity was not affected by the solvent. The Ca and acto‐Mg ATPase activities decreased to similar extents as the solvent concentration was increased. These results are discussed in terms of the accessibility of the active site. The Mg, Ca and acto‐Mg ATPase activities were studied down to −20°C, in 40% ethylene glycol. All three systems showed an increase in activation energy at low temperatures. These results were compared to previous work and they were analysed in terms of a change in the rate‐limiting step. When myosin S1 is mixed with ATP, difference spectra are obtained whose amplitudes decrease with time and which are a manifestation of the intermediate M ** · ADP · P i (the product complex). The results obtained show that the disappearance of this complex occurs in the post‐steady‐state phase at 20°C but in the pre‐steady‐state phase at –20°C. These studies provide the necessary data for planning work on the stabilization of the reaction intermediates on, in particular, the actomyosin pathway. By working at –20°C in 40% ethylene glycol a 1000‐fold decrease in this activity is obtained.