THE CYTOPLASM 209 



TABLE I 

 Intracellular Distribution of Isocitric Dehydrogenase** 



Per cent of original activity 



Isocitric Oxidation of 



Preparation dehydrogenase" D-isocitrate* 



Homogenate 100 100 



Nuclear fraction 3 7 



Mitochondria 12 23 



Microsomes 0.9 1 



Supernatant 82 1 Ca. 



" Activity determined by following spectrophotometrically the rate of reduction of TPN on addition of 

 D-isocitrate. 



* Activity determined by measuring the rate of oxygen uptake on addition of D-isocitrate in the presence 

 of TPN, cytochrome c, and ATP. 



is present in the mitochondrial fraction in only trace amounts. An excellent example 

 of this phenomenon was reported in a study of the intracellular distribution of iso- 

 citric dehydrogenase.^' As is shown in Table I, when the dehydrogenase was deter- 

 mined directly by following the rate of reduction of TPN on addition of D-isocitrate, 

 the recovery of enzyme activity was essentially complete, 82% being in the super- 

 natant or soluble fraction and only 12% in the mitochondrial fraction. When D-iso- 

 citrate was added as an oxidizable substrate in the presence of TPN and cytochrome 

 c, however, only the mitochondrial and nuclear fractions (the latter because of its 

 mitochondrial content) took up significant amounts of oxygen, and a very low re- 

 covery was obtained. It can be seen that the oxygen uptake determinations gave a 

 completely false picture of the distribution of isocitric dehydrogenase and that the 

 inadequacy of the enzyme assay was indicated by a low recovery. The results of 

 further experiments indicated, in fact, that TPN-cytochrome c reductase, rather 

 than isocitric dehydrogenase, was the rate-limiting enzyme in the oxidation of iso- 

 citrate. The value of drawing up balance sheets and the necessity for direct deter- 

 minations of enzyme activities are thus clearly demonstrated. 



c. Method of Cell Disruption 



The general plan followed in the isolation of cellular components is to disrupt the 

 cells in a suitable medium and to segregate the liberated cell structures by means of 

 differential centrifugation. The method used for the disruption of cells is of primary 

 importance, since it must stop short of damage to the nucleus, mitochondria, and 

 other cell structures, while producing almost quantitative breakage of cells. The 

 Potter-Elvehjem homogenizer,*' which has been mentioned in Chapter 18, has satis- 

 factorily met these requirements. It has been found in the writers' laboratory, for 

 example, that a homogenizer of this type, consisting of a pestle machined from the 

 plastic Kel-F (a monochlorotrifluoroethylene polymer) to fit a smooth-walled pyrex 

 test tube and rotated at 600 to 1000 r.p.m., is ideally suited for the disruption of the 

 cells of soft tissues (e.g., liver, kidney, and certain tumors). Furthermore, a higher 

 percentage of cells are disrupted if there is a slight clearance rather than a tight fit 



" V. R. Potter and C. A. Elvehjem, J. Biol. Chem. 114, 495 (1936). 



