ASCORBATE-IXDUCED LYSIS OF ISOLATED MITOCHONDRIA 57 



the cytochrome c. Some persists for a period of time, possibly feeding 

 electrons to cytochrome b. It is clear that rapid transfer of electrons from 

 ascorbate to added cytochrome c to O2 via the electron transport chain, 

 as must be occurring, is not capable of producing swelling in the presence 

 of 15 niM ascorbate. If electron transfer through some carrier like cyto- 

 chrome b is of special importance, one possible mechanism for the blocking 

 action of high concentrations of ascorbate would be that cytochrome c 

 is kept so completely reduced that electron transfer through cytochrome b 

 is not possible. 



DNP at ID"'* M, which blocks phosphate + substrate induced swelling 

 under the conditions used here, does not influence the action of ascorbate 



0.5 



0.4 



o 0,3 - 



0,2 - 



^ lOi^M CYT0CHR0MEC+15mM ASCORBATE 



0.3m M ASCORBATE \^\ 

 + CYT0CHR0ME C -^X \ 



l5mM ASCORBATE 



10 



20 30 40 



MINUTES 



Fig. 5. Failure of added cytochrome c to modify the effects of either low or 

 high concentrations of ascorbate on mitochondrial suspensions. 



(Fig. 6). Lower concentrations (lO"'' m), which hasten substrate-induced 

 swelling, do not shorten the lag period with ascorbate. 



The chelating agent EDTA (10 ' to io~^ m) blocks ascorbate induced 

 lysis at least as easily as it blocks virtually all other swelling inducing 

 agents (Fig. 6). Other chelating compounds are \ery effective in preventing 

 ascorbate lysis, even though they may be much less effective or ineffective 

 in blocking phosphate type swelling. Complete inhibition of the ascorbate 

 effect was seen with i mM penicillamine, 2 mM o-phenanthroline, o • i mM 

 8-hydroxyquinoline, 2 mM citrate, i mM inorganic pyrophosphate, i mM 

 inorganic triphosphate, 10 m.M oxalate, and 0-2 mM diethyldithiocar- 

 bamate (Fig. 6). In each case the concentration is roughly the minimum 

 for complete inhibition lasting an hour or more. Lower concentrations 

 cause partial block or markedly prolong the lag period. 



Oxaloacetate, pyruvate, and phenvlpyruvate block ascorbate (Fig. 7) 



