THE CYTOPLASM 233 



The probability that the intact mitochondrial membrane is relatively 

 impermeable to compounds of low molecular weight is also brought out by 

 the high recovery of citrate in the liver mitochondria of rats receiving 

 fiuoroacetate injections (Table IV). In this respect, it may also be noted 

 that the citrate was retained by the particles when they were isolated and 

 washed in isotonic or hypertonic sucrose solutions but was completely 

 extracted when the particles were washed in water. Bartley and Davies^^* 

 have recently reported observations somewhat along the present lines. 

 Thus isolated kidney mitochondria, when maintained under conditions of 

 active metabolism, were found to contain hydrogen, sodium, potassium, 

 magnesium, and phosphate ions in concentrations 2 to 26 times as great 

 as the corresponding concentrations in the surrounding fluid. The implica- 

 tions of these experiments in terms of renal physiology are, of course, of 

 great interest. 



As pointed out earlier, adenosinetriphosphatase is another example of 

 an enzyme that is inactive in intact mitochondria. When the mitochondria 

 are disrupted, however, adenosinetriphosphatase does not pass into solution 

 but remains attached to particles sedimentable by high-speed centrifuga- 

 tion.^'* The tremendous activation of adenosinetriphosphatase resulting 

 from damage to the mitochondria^^* is not readily explained on the ground 

 that the intact membrane is impermeable to ATP, since recent data have 

 indicated that ATP can penetrate the membrane. ^^^ It is surprising, how- 

 ever, that the membrane should be permeable to ATP and impermeable 

 to such compounds as /3-glycerophosphate and citrate. 



Several enzyme systems in addition to adenosinetriphosphatase are found 

 mainly in the pellet when preparations of mitochondria, disintegrated either 

 by sonic oscillations or by other means, are clarified by high-speed cen- 

 trifugation. Among these are succinic dehydrogenase, which is inactivated 

 to a considerable extent by sonic oscillations, cytochrome oxidase, and 

 DPN-cytochrome c reductase. ^^ Interestingly enough, cytochrome c, a 

 soluble protein of low molecular weight, is also attached to these sediment- 

 able particles. Table VI shows the distribution of cytochrome oxidase, 

 DPN-cytochrome c reductase, and cytochrome c in fractions obtained 

 from disrupted mitochondria.^" In these experiments, isolated rat liver mito- 

 chondria suspended in 0.25 M sucrose were disintegrated by sonic oscilla- 

 tions and centrifuged in the cold at 50,740 r.p.m. (148,000 g) for 30 minutes 

 in the Spinco Model E ultracentrifuge (Type A rotor). The pellet was re- 

 dispersed in 0.25 M sucrose and resedimented by 1 hour's centrifugation 

 at the same speed (rapidly sedimenting particles of Table VI). The super- 

 natant from the first centrifugation was centrifuged for 1 hour at 50,740 



"* J. Berthet, L. Berthet, F. Appelmans, and C. de Duve, Biochem. J. 50, 182 (1951). 

 1" W. Bartley and R. E. Davies, Biochem. J. 52, xx (1952). 



