208 GEORGE H. HOGEBOOM AND WALTER C. SCHNEIDER 



scope. '^'''' The number of unbroken liver cells can also be determined by direct count. 

 The relatively high PNA content of liver microsomes makes it possible to determine 

 the degree of separation of these particles from mitochondria, which have a low PNA 

 content. 



The question of yield is also of importance. To isolate 5% of the mitochondria or 

 microsomes of a tissue and assume that analysis of these preparations are representa- 

 tive of the whole tissue is not a sound procedure. This is particularly true in the case 

 of microsomes, which are a heterogeneous group of particles with respect to both size 

 and function and, therefore, might readily be fractionated in a procedure leading to 

 low yields. It is also possible that the components of different cell types within a single 

 tissue vary sufficiently in their properties to lead to similar difficulties when poor 

 yields are obtained. 



Another important point arises from the fact that a high yield makes it possible 

 to determine the proportion of the enzyme activity of the whole tissue recovered in 

 each cell fraction. Recovery values can be very enlightening. Many enzymes that are 

 said to reside in the liver cell nucleus, for example, have been found in isolated prep- 

 arations in a concentration of 40 to 100% of that in the whole tissue.' '^ When it is 

 realized that the nucleus of the liver cell accounts for about 10% of the total mass 

 of the cell, it is apparent that these concentration values of 40 to 100% would have 

 represented enzyme recoveries of only 4 to 10% if the yield of nuclei had been complete, 

 and in these experiments the yield of nuclei was far from complete. It is apparent that 

 cell fractionation procedures are fraught with too many uncertainties to permit 

 definitive conclusions in the face of enzyme recoveries of this order. 



Attention to yield and recovery also makes it possible to draw up a balance sheet, 

 a point that for some reason has had to be defended on numerous occasions^ '^ ■*■''* 

 (cf. footnotes 32, 50). It is not enough to determine the enzyme activity of a single 

 fraction with the object of interpreting that analysis in terms of the whole tissue. 

 The original whole tissue and all fractions must be analyzed. If the sum of the ac- 

 tivity of the fractions equals within reasonable limits the activity of the whole tissue, 

 then the method of enzyme assaj^ is likely to be adequate, at least for cytochemical 

 purposes. If not, something is wrong with the enzyme determination. The necessity 

 for drawing up balance sheets becomes obvious when one considers the inherent 

 difficulties involved in enzyme assays, especially in dealing with complicated mix- 

 tures of unknown biochemical composition. Enzyme assays are based on determina- 

 tions of reaction rates. Reaction rates can be markedly affected by a number of fac- 

 tors, including inhibitors, activators, interference or competition as a result of side 

 reactions, relatively slight changes in the pH or ionic strength of reaction mixtures, 

 and permeability barriers set up by membranes. Unfortunately, one of the most 

 common sources of misleading data, particularly in certain investigations of complex 

 enzyme systems, has been the probability that the enzyme under study was not the 

 rate-limiting component of the reaction. This situation has arisen a number of times 

 from measurements of oxygen uptake when various oxidizable substrates are added 

 either to isolated mitochondria or to heterogeneous particulate preparations con- 

 taining mitochondria, e.g., "cyclophorase."" When it is remembered, as will be 

 demonstrated later, that cytochrome oxidase is exclusively localized in the mito- 

 chondrion, it becomes obvious that this is the only cell structure capable of taking up 

 oxygen in any reaction leading to the reduction of cytochrome c. Oxygen uptake will 

 therefore occur even if the enzyme supposedly under study (e.g., a dehydrogenase) 



" E. Shelton, W. C. Schneider, and M. J. Striebich, Exptl. Cell Research 4, 32 (1953). 

 ^« H. Stern, V. G. Allfrey, A. E. Mirsky, and H. Saetren, /. Gen. Physiol. 35, 559 (1952). 



