ISOLATION AND COMPOSITION OF NUCLEI AND NUCLEOLI 133 



chrome oxidase. More recently, Hogeboom and Schneider,'' ■"* using an 

 adaptation of the technique developed by R. M. Schneider and Peterman,^* 

 were able to isolate reasonably pure nuclei in sucrose solution, and showed 

 that the cytochrome oxidase activity of a number of preparations of nuclei 

 was strictly proportional to the number of mitochondria remaining with 

 the nuclei. This Avork, taken together with other work just mentioned, 

 furnished very convincing evidence of the absence of cytochrome oxidase 

 from cell nuclei. Nevertheless it was difficult to understand why the nuclei 

 obtained by Bounce at pH 6.0 in very dilute citric acid should have con- 

 tained appreciable cytochrome oxidase, since, in spite of the implication of 

 Schneider and Hogeboom that contaminating mictochondria must have 

 been present, no appreciable amounts of whole mitochondria could be de- 

 tected. It was eventually concluded that the cytochrome oxidase found in 

 these nuclei must have been present as the result of adsorption of very 

 finely divided pieces of mitochondria, too small to be noticeable by micro- 

 scopic examination."^* This was confirmed by displacing the adsorbed mito- 

 chondrial fragments with gum arable, and by developing the new method 

 of isolation of nuclei using calcium chloride (described above) in which the 

 mitochondria are very completely removed without being fragmented. It 

 has recently been possible to obtain by this procedure nuclei which show no 

 measureable cytochrome oxidase activity. 



The complete absence of succinic dehydrogenase from the nuclei isolated 

 by Bounce at pH 6.0 was undoubtedly due to a decay phenomenon,"^* 

 and not, as thought previously, to the absence of mitochondrial material. 



Uricase was found by T. H. Lan"^ in high concentration in nuclei iso- 

 lated at pH 6.0 from very dilute citric acid. But again this result was ap- 

 parently caused by adsorption of mitochondrial fragments, since Hoge- 

 boom and Schneider'' and Lang and Siebert,^® using nuclei isolated in aqueous 

 media, have found insufficient uricase to be of significance, and this result 

 has also been obtained by Mirsky et alP using Behrens-type nuclei. 



The results with cytochrome oxidase, uricase, and succinic dehydrogenase 

 can be considered as quite conclusive, owing to the insolubility of these 

 enzymes under the conditions of the isolation. There can be no question of 

 loss of the enzymes from the nuclei by extraction with the solvent, unless 

 it is postulated that nuclear oxidases are completely different in solubility 

 from cytoplasmic oxidases. However, it has been observed in this laboratory 

 that the cytochrome oxidase present in liver cell nuclei isolated at pH 6 in 

 very dilute citric acid solution appears to be just as insoluble as the mito- 

 chondrial cytochrome oxidase. 



D- Amino oxidase also is probably lacking in cell nuclei, in spite of the 



"* G. H. Hogeboom, W. C. Schneider, and M. J. Striebich, Cancer Research 13, 617 



(1953). 

 I's T. H. Lan, J. Biol. Chem. 151, 171 (1943). 



