TS'O 167 



experiment with RNAase. These findings, coupled with the fact that urea does 

 demolish the particle, suggest that hydrogen bonding between RNA and pro- 

 tein may be an important factor in holding the two constituents together. 



Microsomal particles have been isolated from sections of pea seedlings that 

 have been incubated with C 14 -leucine for 1 hour. 3 The particles, after a series 

 of centrifugations and dialyses, were shown to be void of contaminating free 

 C 14 -leucine, by two methods. The specific activity (cpm/mg protein) of such 

 particles was the same as that of particles which were washed further by hot 

 TCA and 1 N NaOH. Moreover, acid hydrolysis of the microsomal protein 

 after treatment with dinitrofluorobenzene yields over 98 per cent of the C 14 - 

 leucine as free leucine, indicating that most of the C 14 -leucine is linked inside 

 the protein molecule. With such a preparation of labeled 80 S particles, we 

 wish to find answers to two specific questions. 



The first question is based on the idea that only about 1 to 5 per cent [7] of 

 the protein in the particle is being actively synthesized in the particles. It has 

 been suggested that this newly formed protein then passes into the cytoplasm. 

 In the dissociation studies, it is found that after treatment with EDTA, pH 

 6.5, most of the nucleoproteins aggregate and sediment out of solution, leaving 

 about 6 per cent of protein in the supernatant. After the action of RNAase, 

 there is also about 10 per cent of protein which stays in the solution with the 

 nucleotides, while 90 per cent of the protein precipitates out of solution. When 

 the labeled particles were subjected to the above two treatments, only a small 

 percentage of the total counts of the labeled protein remained in the super- 

 natant, while over 90 per cent of the counts precipitated with the aggregates. 

 The supernatant proteins obtained from these two treatments were precipitated 

 by TCA and were shown to have specific activities no higher than that of the 

 aggregated nucleoproteins. This experiment suggests that substantial removal 

 of magnesium and RNA does not liberate a large percentage of labeled proteins 

 or proteins of very high specific activity from the isolated particles. 



The second question is based on the idea that RNA is a template [8]. Thus, 

 is there a large amount of radioactive amino acids in the particle attached to 

 RNA through covalent bonds? The RNA was separated from protein by 

 phenol as well as by density gradient centrifugation in cesium chloride. In- 

 variably, very few if any counts could be found in the RNA. Furthermore, 

 the soluble nucleotide fraction of the supernatant after deproteinization by 

 TCA, obtained by RNAase treatment of the particles, also contained very few 

 counts. Therefore, if amino acids are attached to RNA through covalent bonds, 

 the total amount of such material at a given time is likely to be very small. 



In summary, microsomal particles from pea seedlings consist of smaller units 

 of nucleoproteins cemented together through linkage of magnesium ions and 

 the phosphate group of RNA. RNA and the protein (s) in these units are held 



3 Experiments with C 14 -amino acids were performed in cooperation with Dr. Clifford 

 Sato. 



