THE CYTOPLASM 235 



r.p.m. and the transparent pellet redispersed in 0.25 M sucrose and resedi- 

 mented. Since the resedimentation of the latter slowly sedimenting particles 

 was incomplete, the supernatant from the last centrifugation was also 

 analyzed. It can be seen from the data of Table VI that the rate of sedimen- 

 tation of the particles containing DPN-cytochrome c reductase was con- 

 siderably lower than that of particles containing cytochrome oxidase. The 

 distribution of cytochrome c among the fractions was generally similar to 

 that of cytochrome oxidase. The experiments thus suggest that the mito- 

 chondrial cytochrome c is tightly bound in its native state to particulate 

 material that contains cytochrome oxidase. Additional evidence for a close 

 structural proximity of cytochrome c, cytochrome oxidase, and succinic 

 dehydrogenase is provided by the finding that the mitochondrial fragments 

 show a high rate of oxygen uptake in the presence of succinate and in the 

 absence of added cytochrome c.^** 



The experiments of Table VI also demonstrate the inadequacy of rela- 

 tively low speed centrifugation (e.g., 20,000 to 25,000 g for 1 to 2 hours) 

 as a means of testing for the "solubilization" of such particulate enzymes 

 as cytochrome oxidase (cf. footnotes 176, 177). Although 43% of the origi- 

 nal cytochrome oxidase activity remained in the supernatant after centrifu- 

 gation at 148,000 g for 30 minutes (Table VI), the experiments^" indicate 

 that the enzyme system was not in true solution but was still bound to 

 complex, polydisperse fragments of mitochondria. 



Damage to or disruption of the mitochondrial membranes results in the 

 complete inactivation of certain complex enzyme systems, including those 

 capable of the oxidation of fatty acids*^ and of oxidative phosphorylation.^^* 

 Whether this inactivation can be fully explained by the destruction of ATP 

 through the activation of adenosinetriphosphatase is not entirely clear. In 

 view of the fact that the succinoxidase system, which is not dependent on 

 ATP, is partially inactivated, the possibility remains that the inactivation 

 results from actual physical separation of the components of the multi- 

 enzyme systems. 



It is evident from these experiments that the biochemical organization 

 of the mitochondrion is certainly no less complex than its internal structure. 

 The general picture is that of an osmotically active system, protected from 

 its environment by a relatively impermeable membrane, and containing a 

 high concentration of proteins (including enzymes) and metabolites in a 

 diffusible state. In addition, a number of enzymes appear to be firmly 

 bound to the structural framework of the mitochondrion. 



176 w. W. Wainio, S. J. Cooperstein, S. Kollen, and B. Eichel, J. Biol. Chem. 173, 



145 (1948). 

 1" B. Eichel, W. W. Wainio, P. Person, and S. J. Cooperstein, J. Biol. Chem. 183, 



89 (1950). 



