J 60 THE BIOLOGY OF HYDRA : 1961 



yet, we have not detected any other class of compounds. Next, we 

 thought it of interest to determine whether the succinoxidase in- 

 hibitory activity is associated with the integrity of the protein. This 

 was studied by incubating the purified inhibitor with the proteolytic 

 enzyme trypsin to see whether digestion by this enzyme reduced 

 the inhibitor's activity. After the incubation, soybean trypsin inhib- 

 itor was added to destroy the trypsin activity. The purified inhibitor, 

 after such treatment, lost virtually all of its activity against mouse 

 liver succinoxidase (6). From these studies we concluded that the 

 inhibitor is a protein and that a high degree of the integrity of the 

 protein is required for its activity. 



At this stage of the study we became interested in the nature 

 and mechanism of the succinoxidase inhibition produced by the 

 Hydra protein. The next few experiments describe some of our find- 

 ings in this area (7). The main parts of the succinoxidase chain, 

 as it is now believed to exist, is shown in Figure 3. It is possible 



SUCCINATE ->► SUCCINIC DEHYDROGENASE- 



-^CYT. c, ->► CYT. c > 



Fig. 3. A pathway for the oxidation of succinate. Not shown are the 

 fat-soluble factors implicated in succinate oxidation, e.g., coenzyme Q, 

 tocopherol and vitamin K. 



by the use of specific assays to locate in this chain the general and 

 perhaps the exact site at which an inhibitor acts. The quantitative 

 effect of the purified inhibitor on succinoxidase is presented in 

 Figure 4. These data show that the inhibition is linear to about the 

 50% level. The maximum level of inhibition is less than 100%, 

 although, in other experiments the 100% level has been reached. The 

 primary portion of the succinoxidase system, succinic dehydro- 

 genase, does not appear to be the area in which the Hydra inhibi- 

 tor operates ( Table 6 ) . Here, with 48 times the amount of material 

 needed to inhibit succinoxidase 50%, there is less than 20% inhibition. 

 The terminal portion of the succinoxidase chain, cytochrome oxi- 

 dase, is not inhibited at all by 22 times the 50% inhibitory level for 

 succinoxidase (Table 7). The next subsystem that we studied was 

 succinate-cytochrome-c reductase. This system probably includes a 



