64 



MICROSOMAL PARTICLES 



Fig. 1. Patterns at 32 minutes and 187,000^ for extracts from E. coli during growth in 

 lactose medium. Cells were harvested from: (a) a stationary-phase glucose mineral salts 

 culture; (b) lactose medium when in early logarithmic phase, 30 minutes after transfer 

 from (a); (c) 100 minutes after transfer; (d) after 160 minutes. Sedimentation is to the 

 left, and visible peaks have sedimentation coefficients of 29, 20, 13, and 8 S. In each pattern 

 the 40 S boundary has already sedimented. 



components might have some general significance in enzyme induction proc- 

 esses, or, alternatively, that the actual enzyme molecules are synthesized in 

 amounts sufficient to affect the ultracentrifuge patterns and that, by coincidence, 

 the molecules of those we have studied are all of the size (and shape) to sedi- 

 ment at about 13 S. 



Data tending to favor the second suggestion were obtained by following the 

 sedimentation of enzyme activities in extracts. For these measurements, the 

 rotor was allowed to come to rest after a field of 187,000g had been maintained 

 for a definite time interval, and the cell was carefully removed. By means of 

 a syringe it was possible to withdraw almost the whole of the supernatant 

 without disturbing the pellet deposited in the cell. The activity remaining in 

 the supernatant was then determined, and the sequence of operations was re- 

 peated for a different length of centrifugation so that a graph could be con- 

 structed to relate duration of spin to supernatant activity. 



The enzymes assayed were arginine, lysine, and glutamate decarboxylases 

 (Gale [7]) for extracts from cells grown in media containing 2 per cent glucose 

 and 1 per cent peptone with addition of the corresponding amino acids; cit- 

 ratase using the media and methods of Dagley and Sykes |6); and the 3-galac- 

 tosidase (Lederberg [8]) of cells grown at the expense of lactose. The consti- 

 tutive enzymes of the TCA cycle, malic and isocitric dehydrogenases, fumarase 

 and aconitase, present in extracts of cells grown in mineral salts media with 

 limiting glucose, were also assayed by the spectrophotometric methods used by 

 Englesberg and colleagues [9, 10]. 



In figure 2 it is seen that centrifuging for about 90 minutes removed all the 

 citratase, (3-galactosidase, and glutamic decarboxylase of extracts. This is the 

 time taken to sediment the 13 S boundary. Lysine and arginine decarboxylases 

 sedimented faster, at about the speed of the 20 S boundary. Malic and isocitric 

 dehydrogenase activity moved much more slowly, and fumarase and aconitase 

 appreciably slower than 13 S. The behavior of the four TCA-cycle enzymes 

 is in agreement with the view that they were present in these extracts as indi- 

 vidual molecules, since an approximate molecular weight of 40,000 has been 



