TS'O 



161 



0.1 M) partly suppresses but does not completely eliminate the dissociation (fig. 

 4d). The effects of potassium chloride are, therefore, probably twofold. Potas- 

 sium ions appear to replace magnesium within the particle. The further effect 

 of potassium which cannot be suppressed by addition of magnesium is probably 

 due to ionic effects upon charged groups. An additional complication sets in 

 when the particles are exposed to potassium chloride solution at room tem- 

 perature. As will be described in more detail later, the RNA in the dissociated 

 particles is now hydrolyzed by the contaminating RNAase in the preparation 

 with a rate eight times higher than the rate constant for attack of the RNA 

 ot the nondissociated particles. The effect of the contaminating RNAase on 

 the particles in 0.7 M KCl can be observed by comparing figures 4a and \b. 

 There is much more material of very low sedimentation coefficient in the 

 room-temperature runs. Furthermore, the area ratio of the 80 S : 60 S : 40 S 

 components in the low-temperature run is 1.3 : 1.8 : 1.0, whereas in the run af- 

 fected by RNAase it is about 1:1:3. The 40 S component, therefore, may be 

 more resistant to RNAase than the others. 



Magnesium ions can be further removed by EDTA. At neutral pH, the sys- 

 tem treated with EDTA aggregates at room temperature. Thus, this system 

 can be analyzed only at low temperature or at alkaline pH. In pH 6.5 phos- 

 phate buffer, and in the presence of 2.5 X 10~ 2 M EDTA, the particles disso- 

 ciate to yield components of 40 S (64 per cent), 26 S (30 per cent) and 3 to 6 S 

 (6 per cent) (fig. 5b). Lower concentrations of versene, such as 5 X 10" 3 M, 

 yielded poor resolution of the 40 and 26 S components (fig. 5a). At higher 

 pH (9.0), higher concentrations of EDTA (5x10"' M), and room tempera- 

 ture, the particles dissociate into the 40 and 26 S units together with increased 

 amounts of material of 3 to 6 S(fig. 5c). Dialysis to remove EDTA and to 

 replenish magnesium causes aggregation of the system. It should be noted 



(c) 



Fig. 5. Ultracentrifuge patterns of particles in EDTA. Speed 42,040 rpm. (a) In 

 5 X 10- 3 M EDTA, pH 6.6, 0.025 [x, K-P0 4 0° C, for 1 hour. Centrifuged at 4.6-6.0° C. 

 (b) In 2.5 X 10" 2 M EDTA, pH 6.6, 0.025 ^ K-P0 4 , 0° C, for 1 hour. Centrifuged at 

 6° C. (c) In 5 X 10" 2 M EDTA, pH 9.0, 0.02 M K-CO s , 0° C, for 3 hours. Centrifuged 

 at 20°C. 



