56 F. EDMUND HUNTER, JR. 



chain inhibitors on ascorbate-induced lysis was investigated. These in- 

 hibitors have been shown to block the oxygen and substrate requiring 

 swelling induced by phosphate, etc. [i, 5, 6]. Figure 4 illustrates that 

 amytal does not block ascorbate lysis, while 2 mM NaCN, 4-6 /xM anti- 

 mycin A, or 4-6 [xM SN 5949 inhibit completely. These observations 

 suggest that inhibition of electron transport from cytochrome b and above 

 prevents the ascorbate effect. Somewhat puzzling is the fact that slightly 

 higher cyanide and antimycin concentrations are required to block 

 ascorbate than to block the phosphate type of swelling. Moreover, 10 mM 

 NaNg, which is moderately effective against phosphate, produces only 

 slight inhibition of ascorbate and GSH induced swelling. Possibly these 



0.5 



A + 6«M ANTIMYCIN^ A + 6z^M SN 5949 



A 



l^^^4^^::zz: ^ \ ' 



20 30 



Ml NUTES 



Fig. 4. Effect of electron transport chain inhibitors on the absorbancy change 

 associated with ascorbate lysis of mitochondria. 



reducing agents interfere with the action of azide. Perhaps an explanation 

 for some of the other differences will evolve from the work of Chappell 

 and Greville [7]. 



Ascorbate has long been known to feed electrons into the electron 

 transport chain via added cytochrome c [8, 9]. A much lower, but not 

 insignificant oxygen consumption occurs without added cytochrome c. 

 Just how much of this represents electron transfer via the cytochromes is 

 uncertain at the moment. If ascorbate lysis depends on ascorbate oxidation 

 (electron transfer from ascorbate), inhibition by antimycin A suggests 

 electrons entering the electron transport chain at cytochrome b, ubiquinone, 

 or lower, rather than cytochrome c. Figure 5 shows an experiment to test 

 whether added cytochrome c would change the effect of ascorbate. It does 

 not, but the experiment is inconclusive, as the concentration differences 

 make it impossible for all the ascorbate to be oxidized instantaneously by 



