166 MICROSOMAL PARTICLES 



elude, therefore, that more than 80 per cent of the divalent ions are bound by 

 RNA. Experiments with pancreatic ribonuclease further support this con- 

 clusion. By the action of ribonuclease, a partial separation of protein and 

 degraded nucleic acid of the particles is achieved. Most of the protein precipi- 

 tates from solution, leaving behind a mixture of nucleotides and polynucleo- 

 tides. The distribution of magnesium between supernatant and precipitate 

 should provide information concerning the binding sites of the magnesium 

 ions. It was found that, after RNAase had acted on the particles for 2 hours, 

 85 per cent of the magnesium had been liberated into the supernatant, which 

 contains 65 per cent of the nucleotide phosphate and only 10 to 15 per cent of 

 the protein. If we assume that 80 per cent of the magnesium is associated with 

 RNA in the particle, then half of the phosphate groups in the RNA exist in 

 the form of magnesium salts. 



Attempts to study the protein of the particle have been made with RNAase. 

 After 3 hours' incubation with 7 ug/ml of pancreatic RNAase in a 1 per cent 

 solution of particles at 27° C, the solution becomes turbid. Between 85 and 

 90 per cent of the total protein and 8 per cent of the total phosphorus are sedi- 

 mentable by low-speed centrifugation, leaving 10 to 15 per cent of the protein 

 and 90 per cent of the phosphorus in the form of mono- or oligonucleotides in 

 the supernatant. That the protein aggregation is related to RNAase action and 

 not to the presence of nucleotides was shown by experiments in which large 

 amounts of 2,3'-phosphate nucleotides were added to particle preparations with- 

 out aggregating effect. The protein aggregate is insoluble in buffer at pH 4 to 

 11, in strong salt solution, in acid, in urea, or in performic acid, but does dissolve 

 in alkali at pH 12 to 13 as well as in 80 per cent saturated guanidium chlo- 

 ride at pH 8 to 9. If the guanidium ions are removed by dialysis or if the pH 

 is lowered to 10 to 11, the protein again precipitates. 



The action of RNAase on the particles is enhanced by addition of potassium 

 chloride or phosphate buffer and is suppressed by addition of magnesium. It 

 was also found that there is a small amount of contaminating RNAase in our 

 preparation of particles. Incubation of particles in phosphate buffer (0.05 u, 

 pH 6.5) for only 2 hours at room temperature hydrolyzes 4 to 5 per cent of the 

 RNA. This rate can be increased by 8 times by addition of potassium chloride 

 (0.15 to 0.7 M), and it can be suppressed back to 1.5 to 2 times by addition of 

 magnesium chloride (0.001 M) with the potassium chloride. 



The present studies shed little light on the nature of the binding between 

 RNA and protein. Electrostatic forces cannot be the only ones involved, since 

 at least half of the phosphate groups of the RNA are present in the form of 

 salt linkages with magnesium and calcium. In addition, RNA does not sepa- 

 rate from protein in the ultracentrifuge in the presence of 0.7 to 1.0 M KCl, 

 nor can protein be separated from nucleic acid by alcohol precipitation in such 

 solutions. Agents which denature proteins, such as phenol or detergent, have 

 been successfully applied to separate RNA from the protein of the particle. 

 The protein, however, aggregates as the RNA is removed, as shown by the 



