ISOLATION AND COMPOSITION OF NUCLEI AND NUCLEOLI 117 



the other hand, Lang et al.,^^ using methods of isolation mth aqueous 

 solvents, find a high concentration of deoxyribonuclease with a pH opti- 

 mum of 5 in cell nuclei (nearly 100% of the total amount present in the cell 

 in kidney tissue) ; Laskowski et al.^^ finds deoxyribonuclease in all cell frac- 

 tions of thymus, with a lower specific activity for nuclei than for cytoplas- 

 mic particles but a firmly bound fraction in nuclei; and Schneider and 

 Hogeboom," using an aqueous medium with mouse liver, and Allfrey and 

 Mirsky,^* using a modified Behrens procedure, find that deoxyribonuclease 

 is predominantly a mitochondrial enzyme. The cause of this particular dis- 

 crepancy is not yet known. However, the finding of cytochrome oxidase in 

 liver cell nuclei isolated in very dilute citric acid at pH 6 is now known to 

 be due to the adsorption of very fine mitochondrial fragments, as has been 

 explained, and hence other difficulties of a similar nature may be anticipated 

 if the mitochondria are not removed while still intact. The difficulties that 

 may arise from permeability of the nuclear membrane to enzymes has al- 

 ready been discussed. 



If the test of gel formation with alkali or salt is used as a criterion of 

 intactness of nuclei, it is found that nuclei isolated at pH 6 in very dilute 

 citric acid, or, in general, nuclei isolated in sucrose solutions at pH 6.0 or 

 higher, do not form gels and hence have suffered degradation. However, the 

 addition of sufficient calcium chloride, as in the methods described above, 

 results in nuclei that do form gels. Nuclei of rat liver or chicken erythro- 

 cytes isolted in 0.9% NaCl, or liver cell nuclei isolated in 70% ethylene 

 glycol or glycerol, also will form gels. So do the Behrens-type nuclei and 

 nuclei isolated \\dth dilute citric acid at pH 4.0 or lower. 



b. Choice of Method for Studies of Lipids 



In carrying out studies of lipids, it is generally essential to obtain fairly 

 large quantities of nuclei, and the Behrens-type procedure, for obvious 

 reasons, cannot be employed. Hence the Waring Blendor or colloid mill 

 should be used to obtain homogenates in aqueous media from which nuclei 

 can be isolated. Nuclei isolated at pH 4.0 with dilute citric acid can be used 

 to advantage, but nuclei isolated at pH 6.0 probably should not be used 

 because of the presence of adsorbed mitochondrial frgments which may 

 contain lipid. It seems likely that the calcium chloride-sucrose method 

 described above would be suitable for isolating nuclei to be used in lipid 

 studies. This method would be particularly useful if a large-scale ball-type 

 homogenizer constructed of stainless steel could be used for homogenization. 



"K. Lang, G. Siebert, I. Baldus, and A. Corbet, Experientia 6, 59 (1950). 

 " K. D. Brown, G. Jacobs, and M. Laskowski, J. Biol. Chem. 194, 445 (1952). 

 " W. C. Schneider and G. H. Hogeboom, J. Biol. Chem. 198, 155 (1952). 

 " V. G. Allfrey and A. E. Mirsky, J. Gen. Physiol. 36, 227 (1952). 



