272 LuciLE Smith and Helen Conrad 



(2) The extent of the inhibitory effect of cytochrome c is different with 

 different kinds of preparations. The extent of the inhibition must be measured 

 with each kind of preparation. 



(3) Other proteins besides cytochrome c are also inhibitory to cytochrome 

 c oxidase activity, including some proteins found in tissue homogenates. 

 Sedimentation of the insoluble fraction of the homogenates and washing of 

 this fraction removes inhibitory substances. 



(4) In several rat organs examined, the same maximal cytochrome c 

 oxidase activity was observed with washed mitochondria treated in hypotonic 

 solution and with washed particles from a water homogenate. However, 

 with mitochondria there is a change in absorption spectrum due to changes in 

 light scattering which lasts for about 20 sec after addition of the mitochondria 

 to the buffer solution. Also the maximal cytochrome c oxidase activity is 

 obtained only after the mitochondria have stood in the hypotonic solution for 

 about 60 min. 



(5) The maximal cytochrome c oxidase activity that can be obtained using 

 a given cytochrome c concentration in the test mixture appears to be different 

 for different tissues, when expressed in terms of the content of cytochromes 

 a and a^. 



What can be deduced about the nature of the oxidase from this type of 

 observation? As might be suspected, since the oxidase is a particulate 

 enzyme, the experiments appear to show structural differences related to the 

 oxidase in different kinds of preparations, particularly regarding the extent of 

 'exposure' of the oxidase to reaction with cytochrome c in solution. 



(1) In relatively intact mitochondria, the oxidase does not react rapidly 

 with cytochrome c in solution. 



(2) The oxidase of swollen or ruptured mitochondria or of the insoluble 

 particles derived from the mitochondria is usually in a position to react with 

 cytochrome c in solution. However, in some preparations (Mackler and 

 Green, 1956) the structure of the particulate material is such that rapid reaction 

 with soluble cytochrome c is not observed. Our data indicate that, in addition, 

 cytochrome c or some other proteins can bind to the oxidase particles in such 

 a way that the oxidase becomes inaccessible to reaction with cytochrome c. 

 Some of these inhibitory proteins can be removed by washing. 



(3) The oxidase of a purified preparation is most susceptible of all to the 

 inhibitory binding of cytochrome c. 



The simplest explanation of our data seems to be that the inhibitory effect 

 of cytochrome c or salmine on the oxidase results from masking of the enzyme 

 by the binding of these proteins. After addition of salmine, the cytochrome c 

 has httle further inhibitory effect. We have found that the addition of salmine 

 has no effect on the steady-state levels of the cytochromes of heart muscle 

 particles oxidizing succinate with oxygen. This seems to show that the salmine 

 does not react specifically with one member of the oxidase system, but rather 



