28 



MICROSOMAL PARTICLES 



quickly frozen in Dry Ice within 3 minutes of slaughter, and thawed immedi- 

 ately before proceeding with the preparation. The liver was thoroughly chopped 

 while thawing and the cell walls were broken by blending in an Osterizer 

 Blendor intermittently for 3 to 4 minutes at about half speed. The suspending 

 medium for this operation was ice-cold 0.25 M sucrose (2 cc/g liver). 



The resulting suspension was centrifuged twice for 30 minutes at 1500^ in 

 the cold. The supernatant solution, containing the microsomes, glycogen, and 

 soluble liver proteins, was removed by pipet after each centrifugation. The 

 microsomal particles were then separated from the lipoprotein portion of the 

 microsomes by emulsifying the microsomes with sodium deoxycholate [1]. A 

 pellet of microsomal particles can then be obtained by ultracentrifugation of 

 the deoxycholate-treated microsome suspension. To the supernate from the 

 second low-speed centrifugation, 1/9 volume 5 per cent sodium deoxycholate 

 (in 0.05 M Tris buffer, pH 8.2) was added with stirring. Stirring was con- 

 tinued for 15 minutes at 0° C. The microsomal particles were sedimented by 

 centrifugation for 5 hours at 29,000 rpm in the no. 30 rotor of a Spinco model L 

 ultracentrifuge. The dark red supernatant solution was removed from the 

 microsomal-particle pellet by decantation. The pellet was used without further 

 purification for preparing RNA. For studies on the microsomal particles, fur- 

 ther centrifugation, both high- and low-speed, was employed to obtain micro- 

 somal particles free from contaminating proteins. 



Properties of the Microsomal Particles. The degree of homogeneity of the 

 particles is revealed by the sedimentation diagram shown in figure 1. Extra- 

 polation of numerous measurements at various concentrations in the 0.025 M 



Fig. 1. Sedimentation diagram of microsomal particles in 0.025 M NaHCO s , 0.004 M 

 MgCl 2 , pli 8.5. Picture taken at bar angle of 32° after 24 minutes at 27,690 rpm. ^ 20 = 78.7 

 at this concentration (1.2 g/dl) ; ^° 20 = 81.3. 



