ROBERTS, BRITTEN, AND BOLTON 



85 



precursors of the large ones. In contrast, the simple separation into cell wall, 

 microsome, and soluble fractions is not sufficient to reveal clearly any precursors 

 or products among the macromolecules. A further fractionation of the micro- 

 some pellet is required. 



Pellets of somewhat greater homogeneity can be obtained by choosing an 

 appropriate centrifuging schedule. The material that sediments in 15 minutes at 

 100,000g- is richer in the large particles than the pellet obtained by centrifuging 

 down (2 hours at 100,000^) material which stayed in suspension during three 

 successive 15-minute periods at 100,000^. The composition of the pellet also 

 varies; the early pellet contains nearly twice the lipid and protein per unit of 

 nucleic acid. This approach, however, shows no promise of giving adequate 

 fractionation. 



A somewhat better fractionation can be obtained by using the swinging 

 bucket head for the Spinco Model L centrifuge. Microsome pellets are resus- 

 pended and layered on top of a sucrose gradient. After a period of centrifuga- 

 tion, layers are taken off with a pipet. This technique is adequate to show 

 marked differences in the distributions, depending on the initial material. Fig- 

 ure 1 shows one curve for a resuspended pellet composed mostly of large (80 S) 

 particles; another for the smaller particles (20 to 40 S) that result if magnesium 

 is lacking [8]; and a third, for the nonsedimenting material. The analytical 



7 9 



Fraction no 



Fig. 1. Fractionation of particle preparations using the swinging bucket centrifuge. 

 Five-tenths milliliter of suspension is placed on top of 4.5 ml sucrose gradient in the cen- 

 trifuge tube. After 45 minutes at 100,000^, 0.3-ml fractions are taken off from the top with 

 a pipet. 



