Ascorbate-Induced Lysis of Isolated Mitochondria — 



A Phenomenon Different from Swelling Induced 



by Phosphate and Other Agents* 



F. Edmund Hunter, jR.f 



The Edzcard Mallinckrodt Department of Pharmacology, Washington 

 University School of Medicine, St. Louis, Mo., U.S.A. 



Many substances induce swelling in isolated liver mitochondria. 

 Relatively few substances other than surface active agents such as the 

 detergents produce a lytic type of effect. In the course of earlier work [i] 

 we observed that low concentrations of ascorbate have a characteristic 

 lytic effect. This phenomenon has been studied to establish the nature of 

 the reaction, to obtain clues on the key groups in the mitochondrial 

 membrane, and to explore the possibility of preparing submitochondrial 

 particles or units and soluble proteins in this way. 



Glutathione and cysteine produce effects which appear to be similar 

 to but not identical with those seen with ascorbate. Feldott, Johnson, and 

 Lardy [2] mention a lytic effect of cysteine, and Lehninger [3] has ex- 

 tensively studied swelling induced by 10 mM glutathione. 



In the present work the swelling of isolated liver mitochondria [i] was 

 followed by light scattering or absorbancy changes of dilute suspensions 

 in 0-33 M sucrose containing 0-025 ^^ ^"s buffer, pH 7-4. Routinelv the 

 temperature was 22-25" ^^^ 5^0 m/x light was used. To minimize inter- 

 ference by certain additions such as dyes, 775 m^u, w^as used in some 

 experiments. Protein was measured by the method of Lowry et al. [4]. 



Figure i illustrates the striking differences between the absorbancv 

 changes occurring with low^ concentrations of ascorbate and those ac- 

 companying swelling induced by phosphate or /S-hydroxybutyrate. Charac- 

 teristically ascorbate induced swelling or lysis has a lag period averaging 

 20 min. This is followed by a rapid fall of the optical density to very low 



* Abbreviations used in this report are : DNP for 2,4 dinitrophenol, EDTA 

 for ethylenediaminetetraacetate, P-P for inorganic pyrophosphate, P-P-P for 

 inorganic triphosphate, DEDTC for diethyldithiosemicarbazide, /)CMB for 

 /)-chloromercuribenzoate, DHF for dihydrox^-fumarate, DHM for dihydroxy- 

 maleate, and DHA for dehydroascorbate. 



t The work in this communication was carried out with the collaboration and 

 assistance of Francisco Guerra, Beverly Schutz, Joan Fink, Lillian Ford, Audrey 

 Scott, and Ellen Smith. 



