274 A SYMPOSIUM ON RESPIRATORY ENZYMES 



and in hypotonic media occurs to some extent with certain tissues 

 other than brain, but not with all tissues. PreHminary studies would 

 have to be made on each tissue to which the method of isotonic 

 suspensions is to be applied before it could be assumed that the 

 behavior of suspensions would be comparable to that of slices of the 

 particular tissue. 



REFERENCES 



1. Elliott, K. A. C, and Libet, B., in press. 



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



THE HOMOGENIZED TISSUE TECHNIQUE, THE DILUTION 

 EFFECT AND ION EFFECTS 



VAN R. POTTER 

 McArdle Me7norial Laboratory, University of Wisconsin 



We have developed the homogenized tissue technique for the 

 study of isolated phases of metabolic activity. Since the enzymes 

 that catalyze biological oxidations are in many cases extremely la- 

 bile, we have used the device of homogenization to effect a physical 

 isolation of a particular enzyme system by dilution. We believe that 

 by so doing we can retain the original activity of the tissue and thus 

 develop assay methods for specific enzymes, where separation by 

 chemical treatment, such as fractional precipitation, could not result 

 in 100 per cent yields and hence would be useless for assay purposes. 

 Since certain components of various enzyme systems are readily 

 soluble and are capable of diffusing away from each other or from 

 solid phases, such as cytochrome oxidase, it is necessary to fortify 

 the homogenate with these diffusible components. Whether it is 

 necessary to add these accessoiy factors is determined by measur- 

 ing the enz)Tne activity at various dilutions. If the measured effect is 

 proportional to the amount of enzyme used, fortification is unneces- 

 sary. With regard to choice of buffer, it should be pointed out that 

 since we are now dealing in tenns of intracellular components, buf- 

 fers based on extracellular fluids (such as serum) may not necessarily 

 be optimum. 



A system which may illustrate these points is the succinoxidase 

 system. It is inhibited by chloride ions, hence these are omitted from 

 the buffer medium. It requires at least three soluble components for 

 maximal activity, namely, cytochrome c, calcium ions, and aluminum 

 ions. The dehydrogenase and the cytochrome oxidase appear to be 

 associated with solid particles of protoplasm. When the dissociable 



