116 ALEXANDER L. BOUNCE 



A device recently reported by Behrens"' ^' which might be very useful in the 

 final stages of the isolation procedure is the addition of a small amount of lecithin to 

 the suspended nuclei. This is said to prevent agglutination which occurs when the 

 nuclei become quite free from lipid, and thus facilitates the separation of clean nuclei. 



Mirsky et al^^ state that they were unable to isolate satisfactory cell nuclei from 

 normal rat liver by anj' procedure. Behrens stated^^ that livers of fasted animals 

 should be used, owing to their low glycogen content. The animals chosen by him were 

 guinea pigs and rabbits. It does not seem likely to the writer that the glycogen con- 

 tent in itself has much to do with ease in isolating nuclei from liver by the Behrens 

 type of procedure. At the worst, the nuclei might be contaminated with particulate 

 glycogen if the latter were present in high concentration. It seems likely that the 

 specific gravity of the nuclei themselves as well as that of the cytoplasm may vary 

 with the glycogen content of the liver, so that nuclear specific gravity might well be 

 the determining factor in ease of obtaining a satisfactory product. Mirsky et al.*^ 

 admit that nuclei of fair quality can be obtained from the livers of fasted rats. It is 

 evident that the diet of the animal could be of importance as well as the frequency of 

 feeding and it is likely that the large slaughterhouse animals from which organs were 

 obtained by Mirsky et al. were always fasted to some degree. 



In conclusion it should be stated that careful and patient work is required for the 

 isolation of cell nuclei by any modification of the Behrens technique and that great 

 attention must be paid to adjustments of the specific gravities of the solvent mix- 

 tures . It may be necessary to use slightly different procedures for each batch of 

 nuclei, as can be inferred from what has been said concerning the effect of diet and 

 fasting on the ease of production of the nuclei. The directions of Behrens et al. and 

 the directions given in this Chapter for rat liver nuclei, as well as the more elaborate 

 directions of Mirskj- et al. for a number of tissues all can furnish guides to the proper 

 procedure in a given instance, but the experimenter must be left to revise the details 

 according to the particular tissue being used. It should be added that it is generally 

 impossible to complete the isolation of nuclei in one day, but the procedure can be 

 stopped at any point where the material is in the form of a sediment. The latter is 

 air-dried and stored in a desiccator in the icebox or preferably the deep-freeze cabi- 

 net. When work is resumed, great care must be taken to get the dry powder com- 

 pletely dispersed in the solvent before proceeding with the isolation. 



3. Comparison of Results Obtained Using the Various Methods 



Described Above, and Choice of Method for the Particular 



Study Being Undertaken 



a. Overall Comparison of Results 



Several series of results of analyses for a given substance in nuclei iso- 

 lated by different procedures have now accumulated. Some of these results 

 will be given in detail in Section II of this chapter. In some cases the results 

 are in agreement, in others there is 100% disagreement, and occasionally 

 there is partial agreement. For instance, the enzyme arginase has been 

 found in liver cell nuclei prepared by a variety of methods.^'^^'^'^^-*'' On 



6' M. Behrens and G. Seydl, Arzneimittel-Forsch. 1, 228 (1951). 



" K. Lang, G. Siebert, S. Lucius, and H. Lang, Biochem. Z. 321, 538 (1951). 



" S. Ludewig and A. Chanutin, Arch. Biochem. 29, 441 (1950). 



" A. H. Schein and E. Young, Exptl. Cell. Research 3, 383 (1952). 



