ASCORBATE-INDUCED LYSIS OF ISOLATED MITOCHONDRIA 65 



testing various substances several striking alterations in the effect of 

 ascorbate have been observed. Two of these are shown in Fig. i8. Five niM 

 a-ketoglutarate usually causes a little swelling. In this experiment it caused 

 almost none, but it drastically shortened the lag period for ascorbate 

 lysis. Even more remarkable is the effect of lo mi\i gulonolactone. Alone 

 it never causes swelling, but it converted ascorbate-lysis into typical 

 phosphate type swelling. Only further work can add information on these 

 effects. 



We do not know much about the mechanism of the ascorbate lysis 

 phenomenon, but we can describe it in terms other than just light scattering 



0,5 



CONTROL 



20 30 



MINUTES 



Fig. 18. Effect of a-ketoglutarate and gulonolactone on the lag period and 

 absorbancy curve associated with ascorbate-induced lysis of mitochondria. 



changes. The experiments in Fig. 19 show the distribution of protein 

 recoveries on differential centrifugation after dilution of mitochondrial 

 suspensions and various experimental treatments. Control suspensions, 

 whether held at o or 25*^, yield about 8o"o of the protein in the regular 

 mitochondrial pellet at 8000 x g and 15-20% in the supernatant after 

 I hr. at 100 000 X g. Phosphate- and succinate-produced swelling alter 

 this distribution remarkably little. However, after treatment with ascorbate 

 75°,j of the protein appears in the "soluble" fraction, 3 to 7% in the 

 submitochondrial particle fraction (100 000 x g pellet). The 20 000 x g 

 pellet contains drastically swollen and damaged mitochondria. The sub- 

 mitochondrial particle fraction (100 000 x g pellet) is greatly increased by 

 ascorbate treatment. 



Electron microscopy has been used to examine the nature of the 

 morphological changes. After phosphate-induced swelling swollen and 

 unchanged mitochondria are clearly seen. With ascorbate treatment most 



