G. REVERBERI 325 



egg gives a very intensive reaction to the Nadi reagent. This 

 reaction is negative if the egg was treated before by sodium azide. 

 It also seems likely that the benzidinperoxidase and the oxido- 

 reductase are localized in the mitochondria. 



Another enzyme which is also supposed to be localized in the 

 mitochondria is the "succinodehydrogenase" which has been 

 studied in the ascidian egg by Mancuso ( 1952). Mancuso showed 

 that this enzyme presents the same pattern of distribution and 

 segregation that is typical for the cytochiome oxidase. We pro- 

 pose to check all these data on cell-free mitochondria. We plan 

 to investigate also the locahzation of these enzymes in the mito- 

 chondrion and their relative quantity. At the moment we can only 

 say, judging from the intensity and rapidity of the Nadi reaction, 

 that the quantity of cytochrome oxidase is very high. 



Inhibition of Mitochondrial Enzymes in the Ascidian Egg 



It is well known that the activity of an enzyme can be specifi- 

 cally inhibited by the use of appropriate chemical substances. In 

 some conditions the inhibition can be reversed. Much research 

 has been done by the biochemists on enzyme inhibition; but the 

 methods and the conclusions deduced from their studies cannot 

 be used without reservation for the mitochondria inside the cells. 

 In fact, strong chemical solutions could not be used in the devel- 

 oping eggs without seriously impairing development. Also specific 

 substances acting only on one enzyme and not on other cellular 

 components are scarcely known. In other words, great care must 

 be employed when using inhibitors on living and developing 

 eggs. 



A specific inhibitor of cytochrome oxidase (CO.) is sodium 

 azide. At appropriate concentrations in vitro it inhibits 80% of 

 the enzyme activity. The sodium azide is not absolutely specific; 

 in fact, it influences also the phosphorylating system. In the 

 ascidian egg the inhibition of the CO. can be easily and at any 

 moment checked by the Nadi reaction, which is negative or re- 

 tarded. Solutions of sodium azide at O.IM inhibit the enzyme in 

 fifteen minutes, but at that concentration the fertilized egg does 

 not develop, polocytes are not emitted, and the egg does not 



