STRUCTURAL AND CHEMICAL ARCHITECTURE OF HOST CELLS 37 



constituents, such as proteins, coenzymes, and other metabohtes. However, 

 as might be expected, nuclei isolated by this method are altered morpho- 

 logically and certain enzymes, such as aldolase (Allfrey et al., 1953), are 

 inactivated by this procedure. 



In another early procedure, tissues were homogenized in citric acid. 

 Nuclei obtained in this fashion were clearly damaged. Dounce (1950) has 

 modified this technique by using citrate at pH6 to maintain enzymatic 

 activity. However, he has also shown that the protein to DNA ratio of these 

 nuclei is lower than those obtained by means of the Behrens procedure. In 

 addition, Kay et al. (1956) have recorded the extraction of an RNA fraction 

 of low metabolic activity from nuclei by the use of citrate. 



According to Schneider and Hogeboom (1951), these earlier procedures 

 gave poor yields, led to loss or denatuxation, or both, of the proteins, altered 

 the cytological appearance of the nuclei, and were poorly controlled for the 

 extent of contamination by cytoplasmic material. Some of these problems 

 have been resolved by homogenization and subsequent isolation in isotonic 

 or hypertonic sucrose solutions. Nuclei have been isolated in good yield in 

 this way and resemble hving nuclei in many respects; however, contamination 

 by intact cells and mitochondria has been a serious problem. 



As a result of osmotic lysis, the latter contaminant also tends to release 

 deoxyribonuclease (DNAase), which damages nuclei. Sucrose tends to reduce 

 this damage; the use of 0.3 M sucrose + 0.06 potassium glycerophosphate 

 more effectively minimizes this effect (Philpot and Stanier, 1955). It will be 

 recalled that glycerophosphate is also known to protect herpes virus from an 

 inactivating phosphatase during the isolation of the virus (Amos, 1953). 

 Novikoff (1956) has recommended a mixture of 0.25 % M sucrose — 7.3 % 

 polyvinylpyrollidone for homogenization and subsequent isolation steps 

 leading to the electron microscopy of nuclei. Nevertheless, cytoplasmic con- 

 taminants are still frequently observed in such preparations. 



Hogeboom and associates (1952) isolated cellular fractions from homo- 

 genates in 0.25 M sucrose containing 2 X 10~^M Ca++ and thereby reduced 

 mitochondria in nuclear preparations (70 to 90 % yield) to less than 0.5 % 

 of their number in liver cells. However, a small number of mtact cells and 

 collapsed cell membranes still contaminated such preparations. The isolated 

 nuclei were optically homogeneous and did not appear to be physically 

 altered. Liver cell nuclei isolated in this way, accounted for 12 % of the 

 total nitrogen of the hver, but the extraction of water-soluble compounds by 

 this technique was not ruled out. Nevertheless, these workers demonstrated 

 that the ability to synthesize diphosphopyridine nucleotide (DPN) was 

 completely retained by these nuclei. A modification of the Ca++-sucrose 

 procedure has recently been applied to the isolation of wheat germ nuclei 

 (Johnston et al, 1957), 



