190 S. Spiegelman 



to the action of enzymes. These observations may be related 

 to the recently published experiments of Thomas (1955) who 

 noted a periodic variation in permeability of Pneumococci to 

 such large molecules as DNA and DNAse. 



It was also found that consistent removal of RNA and DNA 

 by the corresponding enzymes can be achieved only if treat- 

 ment is instituted during the formation of the protoplasts. 

 Once protoplasts have been formed and have been incubated 

 for a while in the stabilizing medium, they become relatively 

 impervious to enzymatic resolution. It may be noted that in 

 addition they become more and more resistant to the dis- 

 ruptive effects of lipase, although never completely so. The 

 procedure employed for resolution may be outlined as follows. 

 The cells are suspended in the hypertonic medium containing 

 all supplements necessary for synthesis including amino acids 

 and HDP but lacking the inducer. Lysozyme is added at a 

 level of 200 {ig. per ml. to convert the cells into protoplasts. 

 In addition, at the same time, the enzyme to be tested for 

 ability to resolve the protoplasts is included. The incubation 

 is carried out for a period of 45 minutes at 30°C with constant 

 shaking, by which time the cells will have been converted 

 completely into protoplasts. The protoplasts are then re- 

 covered by centrifugation. An aliquot is removed for test of 

 enzyme-forming ability; the remainder is retained for chemical 

 analysis. Residual capacity to synthesize enzyme is examined 

 by suspending the treated protoplasts in hypertonic medium 

 containing amino acids, HDP, and inducer. The resulting 

 suspension is incubated on a roller-type device for 2-3 hours 

 with periodic sampling for enzyme assay. Controls are always 

 run in parallel. 



Table II summarizes a typical series of experiments in 

 which the effect of DNAse on protoplasts is examined in 

 terms of the percentage removal of DNA, RNA, and the 

 residual enzymxC-forming capacity. It will be noted that in 

 some cases the treatment with DNA has led to the removal 

 of some RNA. The reasons for this are still under investiga- 

 tion. The results, in so far as enzyme-forming abilities are 



