222 A. TissiERES 



difference: it amounted to about 8% (dry weight) of the preparation before 

 centrifugation in sucrose. 



5. E. coli 'Ghosts' Containing the Bulk of the Cytochromes of the Cells 



According to Repaske (1956), E. coli cells are lysed by lysozyme, at 

 pH 8-0, in the presence of ethylenediaminetetraacetate (EDTA). 7 g of cells 

 harvested in the exponential phase of growth and washed twice with 20 vol. 

 of 0-1% NaCl, were suspended in 150 ml of 0-2 m sucrose, containing 

 0-01 M tris buifer, pH 8-0, and 0-01 m Mg++. Lysozyme (3 mg) and 3 ml 

 neutralized 0-1 m EDTA were then added. The mixture was left at room 

 temperature for 30 min, then kept 12 hr at 4°. By that time 98-99% of the 

 cells had undergone the characteristic transformation into spheroplasts. 

 These spheroplasts were washed twice in sucrose-tris-magnesium mixture 

 containing 1 //g/ml deoxyribonuclease. Finally, they were lysed by re- 

 suspending in 20 ml of 0-005 M tris, pH 7-3, containing 0-01 m Mg++ and 

 1 //g/ml deoxyribonuclease. Weibull (1956) has shown that when protoplasts 

 from Bacillus megatherium were lysed in the presence of 0-01 m Mg++, the 

 resulting 'ghosts' represented morphologically undamaged cytoplasmic 

 membranes: they were shghtly larger than the protoplasts and there was 

 approximately one ghost per protoplast. In the experiment described here, 

 the number of ghosts obtained was about the same as the number of sphero- 

 plasts from which they had derived. They appeared somewhat larger. 



The lysate was centrifuged at 10,000 rev/min for 15 min. A large pellet of 

 ghosts was formed, which was washed twice in the centrifuge with tris- 

 magnesium mixture. Spectroscopic examination of the washed ghosts showed 

 strong absorption bands of cytochromes a^, a^ and b^ on addition of succinate 

 or dithionite. No absorption band of cytochrome was seen in the washings. 

 The ratio of RNA to protein in the washed ghosts was found to be 5/100, 

 while it was 50/100 in the supernatant after centrifugation at 10,000 rev/min. 

 This supernatant was examined under the spectroscope in a 70 mm layer, in 

 presence of DPNH, succinate or dithionite. The absorption bands of 

 cytochrome were not visible. 



The supernatant was next centrifuged for 120 min at 100,000 x ^. A 

 characteristic RNP particle pellet was thus formed, slightly yellow in colour. 

 This pellet, from two 1 1 ml centrifuge tubes was resuspended in 5 ml of 

 sucrose solution with a density of 1-30, and centrifuged for 15 hr at 100,000 x g 

 in the swinging bucket rotor. A very thin yellow layer collected at the top of 

 the tube. It was carefully removed and examined for cytochromes in the 

 presence of dithionite, and also after addition of pyridine. No absorption 

 band could be detected. The colourless solution below the top layer, and the 

 RNP particle pellet were also examined under the spectroscope in the 

 presence of reducing agents, as well as after addition of pyridine. The cyto- 

 chrome components were not detected. 



