10 



Fractionation of Escherichia coli for 

 Kinetic Studies 



RICHARD B. ROBERTS ROY J. BRITTEN ELLIS T. BOLTON 



Department of Terrestrial Magnetism 

 Carnegie Institution of Washington 



A single cell of the bacterium Escherichia coli contains roughly 10,000 ribo- 

 somes (microsomal particles). If the cells are broken open and their contents 

 are examined, the analytical centrifuge shows a series of peaks with sedimenta- 

 tion constants of roughly 20, 30, 40, 60, and 80 S [1-8]. The existence of these 

 particles in such variety and in such large numbers immediately provokes a 

 number of questions. Is the rapid growth rate of E. coli a consequence of the 

 high proportion (25 per cent) of the cellular material that is organized into ribo- 

 somes? In other words, are ribosomes the sites of protein synthesis in E. coli? 

 If so, what size of particle is active in protein synthesis ? How are the particles 

 themselves synthesized? Do the different sizes of particles represent different 

 stages in the growth of a particle ? 



Eventually answers will be found for these questions, but not easily. It will 

 be necessary to know the composition of the particles— the composition of the 

 individual classes of particles, not just the composition of a pellet containing an 

 assortment of particles plus other material. Also the kinetics of isotope incor- 

 poration will have to be studied. It will not be sufficient simply to deal with 

 the microsomal fraction, the 100,000g- pellet; rather, the individual groups of 

 particles will have to be sorted out and measured. Suppose, for example, that 

 one size of particle is the precursor of another. Kinetic measurements will show 

 this clearly if the two groups can be resolved; kinetic measurements of both 

 groups lumped together in a pellet will show nothing. 



Chemical fractionation of E. coli gives good separation between the different 

 classes of compounds, and it is easy to show by kinetic measurements of the 

 incorporation of radioactive compounds that the small molecules serve as 



84 



