96 MICROSOMAL PARTICLES 



proper incubation medium [4, 5]. During such incubation, hemoglobin ac- 

 counts for over 90 per cent of the protein produced. 



The microsomal fraction of rabbit reticulocytes has been shown to be very 

 active in incorporating radioactive amino acids [6]. The following work repre- 

 sents a beginning step toward an understanding of the relationship between 

 microsomal structure and hemoglobin synthesis in the sense described above. 



SOME PROPERTIES OF MICROSOMAL PARTICLES 



Preliminary experiments on isolation of the microsomal fraction showed that 

 the predominant component had a sedimentation coefficient of about 80 S. Vari- 

 ations in the method of breaking the cells and in the buffer used for isolating 

 the microsomal particles were explored before the following standard procedure 

 was developed. The reticulocytes were frozen, thawed, and mixed with 3 vol- 

 umes of cold buffer containing 0.14 M KCl, 0.001 M MgCl 2 , and 0.01 M tris- 

 chloride, pH 7.2. Cell walls and debris were spun out at low speed. Micro- 

 somes were then pelleted at 100,000g for 3 hours. The red pellet thus obtained 

 was then twice redissolved and respun in 100 volumes of buffer, giving a light 

 amber-colored pellet after the third centrifugation. 



Microsomal particles prepared in this way were found to give three compo- 

 nents in the ultracentrif uge : 82 per cent of 78 S, 9 per cent of 120 S, and 9 per 

 cent of 50 to 60 S. On electrophoresis in the same buffer the preparation showed 

 a negative charge, and migrated with only slight skewing in the descending 

 limb and a splitting into two components in the ascending limb. 



The intrinsic viscosity was found to be 0.08 dl/g in the buffer, and the partial 

 specific volume was found to be 0.63 ml/g. 



From these numbers it may be calculated that, if the particles are spherical, 

 the hydration is 2.6 g of water per gram of anhydrous particle, a very high 

 value indeed. Taking as a model a highly hydrated sphere, one calculates a 

 frictional coefficient of 1.72 and a molecular weight of 4.1 XlO 6 . 



That such a model cannot be very far wrong was indicated by light-scatter- 

 ing measurements on the same preparations. These showed a molecular weight 

 of 4 XlO 6 and a measured dissymmetry of 1.08 (45°/135°) with light of the 

 mercury blue line. Since a dissymmetry of 1.06 is to be expected for a hydrated 

 sphere of this molecular weight (diameter, 340 A), the axial ratio cannot be far 

 from unity, and shapes such as rods and random coils are definitely excluded. 



The protein/RNA ratio for these particles was found to be almost unity on 

 a weight basis. No lipid could be extracted. If the RNA from these particles 

 is banded in an equilibrium density gradient [7] in cesium formate, the molec- 

 ular weight of the RNA is found to be approximately 500,000. This is con- 

 sistent with a small integral number of RNA molecules per microsomal par- 

 ticle. If the above molecular weights are accepted, this integral number is 4. 



