ISOLATION AND COMPOSITION OF NUCLEI AND NUCLEOLI 135 



damage to a given enzyme depending upon its location in the cell, such 

 data would be very desirable. The writer is inclined not to go so far as 

 Mirsky et al. in condemning procedures for isolating nuclei in aqueous 

 media, and believes that even in the case of soluble enzymes there is still 

 room for considerable work of a comparative nature, utilizing different 

 methods of isolation, both aqueous and nonaqueous. This belief is founded 

 in part on an uncertainty as to the purity of the Behrens-type nuclei, and 

 in part on the possibility of unequal damage to an enzyme in nuclei and 

 cytoplasm which might be caused by the Behrens technique. In addition, 

 it has not yet been demonstrated that the Behrens-type procedure is always 

 free from artifacts caused by adsorption, or some other type of enzyme 

 translocation. 



Although only two of the enzymes of the glycolytic system can be termed 

 oxidative enzymes, the glycolytic system as a whole will be considered at 

 this point for convenience, and certain of the individual enzymes of glycol- 

 ysis will then be discussed. The glycolytic enzymes can all be brought into 

 solution in aqueous media \A'ithout difficulty, and they are apparently lack- 

 ing in mitochondria, so that, even if nuclei are contaminated by adsorption 

 of mitochondrial fragments, these fragments cannot be expected to cause 

 misleading results in analyses for glycolytic enzymes. 



G. T. Beyer and Bounce found in nuclei isolated in very dilute citric 

 acid at pH 6^^'^^*'^^^ reasonably high concentrations (about half those for 

 whole homogenate) of several glycolytic enzymes, viz., aldolase, 3-phos- 

 phoglyceraldehyde dehydogenase, enolase, and lactic dehydrogenase. 

 Phosphorylase activity was also demonstratable after breaking the nuclei 

 by grinding in sand. In demonstrating lactic dehydrogenase by the Thun- 

 berg technique with methylene blue, it was necessary to add diaphorase, 

 since this enzyme (or more properly diphosphopyridine nucleotide-cyto- 

 chrome C reductase) was not present in the nuclei. The necessity for adding 

 diaphorase has been overlooked in earlier work. 



Lang and Siebert^*'^^* claim to have demonstrated weak anerobic 

 glycolysis in their isolated nuclei starting from fructose diphosphate, but 

 have concluded that the degree of glycolysis is probably too low to be of 

 significance from the standpoint of nuclear metabolism. However, in view 

 of the probable loss of intermediate substrates, cofactors, and to some ex- 

 tent the apo-enzymes themselves, this work cannot be considered to be of 

 more than qualitative significance. 



Stern and Mirsky,^^^ studying plant cell nuclei, have found several glyco- 

 lytic enzymes in reasonably high concentrations, and have concluded that 



'24 A. L. Bounce and G. T. Beyer, J. Biol. Chem. 173, 159 (1948). 



1" A. L. Bounce, S. R. Barnett, and G. T. Beyer, J. Biol. Chem. 185, 769 (1950). 



'" K. Lang and G. Siebert, Biochem. Z. 322, 196 (1951). 



•" H. Stern and A. E. Mirsky, J. Gen. Physiol. 36, 181 (1952). 



