72 INFLUENCE OF TEMPERATURE ON BIOLOGICAL SYSTEMS 



the molecule and temperature to unfold it, the two variables should have 

 opposite effects. Their effects on bioluminescence cannot be explained as 

 effects on a simple rate process, but show that the enzyme is also re- 

 versibly denatured. As in any other equilibrium, pressure favors the form 

 with less volume, the native form. A decrease in the rate of bioluminescence 

 may be due either to direct inhibition of the rate process or to the removal 

 of active enzyme by reversible denaturation. 



Although myosin ATP-ase activity has generally been regarded as re- 

 sponding to temperature and pressure like a simple rate process (12), 

 these two factors do not have opposite effects. Instead, they both increase 

 the activity of the enzyme. This anomalous behavior is caused by the 

 presence of reversible denaturation (3), as will be shown. Such a con- 

 figurational change or deformation has previously been proposed in several 

 reaction schemes devised to explain other types of evidence on myosin 

 (16, 19, 24). 



HYDROGEN ION EQUILIBRIUM 



The experiments are simple. For most of them, myosin was extracted 

 from rabbit skeletal muscle with a dilute salt solution at pH 7.4 (Weber- 

 Edsall solution) , giving myosin B. This myosin was then washed twice with 

 pH 6 buffer, which converted it to myosin A, as judged by the loss of mag- 

 nesium activation. For the experiments on the pH dependence of pressure 

 activation, myosin was extracted with a salt solution at pH 6.3 (Guba- 

 Straub solution). A buffered ATP solution was added to the buffered 

 myosin solution, and the ATP-ase activity was measured by the amount 

 of inorganic phosphate recovered after a certain reaction time. The re- 

 sults were calculated as micromoles phosphate split by one milligram of 

 myosin in one minute. Ionic strength was supplied by sodium rather than 

 the customary potassium salts. ATP concentrations were always above 

 3 mM, more than ten times the concentration usually needed to saturate 

 the enzyme. This simplifies the theoretical interpretation of the result, 

 since combination of substrate with enzyme was not rate-limiting. No 

 inhibition by excess substrate was noted. 



The effect of pressure on ATP-ase activity depends on the pH of the 

 solution (fig. 1). It decreases the activity at a pH slightly below 7, and 

 increases it at alkaline pH. This suggests that pressure affects two different 

 processes in the two pH regions. High pH may reasonably be expected 

 to favor unfolding (reversible denaturation) of the molecule, which pres- 

 sure will oppose, thus increasing the observed rate at high pH. Near 

 neutrality, reversible denaturation should be small, so that most en- 

 zymatic sites are active. The inliibiting effect of pressure at this pH is 

 then a direct effect on the rate of breakdown of the enzyme-substrate 



