92 



MICROSOMAL PARTICLES 



cules have the average specific radioactivity of the TCA-soluble pool. Second, 

 since the persistent and steady-state runs show all the macromolecular ultra- 

 violet-absorbing material to be uniformly labeled, assume that it is the end 

 product and that its specific radioactivity is that of the nucleoprotein. Analyzed 

 on this basis the data point to an intermediate containing roughly 10 per cent 

 of the nucleic acid. 



Some other characteristics of this intermediate have been determined. Very 

 short exposures to the tracer were used to prepare cell juices which were shown 

 by column analysis to have most of the P 32 in the intermediate and little in the 

 end products. Most of the low-molecular-weight materials were removed by 

 washing the cells with water before breaking. This material was analyzed in 

 the swinging bucket centrifuge. The results (fig. 7) show that the TCA-pre- 

 cipitable radioactivity sediments at less than half the rate of the ultraviolet- 

 absorbing material, which is mostly in 80 S ribosomes. Incubation with 

 RNAase showed the usual rate of release of nucleotides. 



■o 



c 

 o 



<u 

 a. 



o 

 O 



17 



Fraction no. 



Fig. 7. Growing cells were exposed to P 32 for 3 minutes, then broken, and the micro- 

 some pellet was analyzed in the swinging bucket centrifuge. Note that the maximum 

 radioactivity does not correspond to the maximum of the ultraviolet absorption. Cf. figure 1. 



Those findings, together with its column elution pattern, suggest that the 

 intermediate is RNA of high molecular weight, either free or associated with 

 less protein than the bulk of the nucleoprotein. It should be emphasized that 

 neither lipids nor fragments of cell wall or cell membrane are eluted from the 

 column, and it is observed that a large part of the P 32 incorporated in short ex- 

 posures is irreversibly bound to the column. An important part of the kinetics 

 may thereby be missed. 



DISCUSSION 



To interpret the detailed workings of the cell in terms of its structural com- 

 ponents, fractionation procedures are needed to separate those components. The 



