6 MICROSOMAL PARTICLES 



Ribose nucleic acid prepared from the particle by chloroform extraction of 

 the protein shows markedly the hyperchromic effect characteristic of poly- 

 merized nucleic acids. Immediately upon addition of the alkali the optical 

 density at 260 mu increases 15 per cent. After incubation for 16 hours at 30° C 

 with 0.5 N NaOH and adjusting the pH to 7.5, the final hyperchromic effect 

 is found to be 39.1 per cent. The nucleotides (table 1) arising upon alkaline 

 hydrolysis of the ribose nucleic acid have been chromatographed on Dowex-1- 

 formate ion-exchange columns developed with gradient elution. The unknown 

 nucleotide shows chromatographic behavior similar to that of the new ribonu- 

 cleotide reported by Cohn, but its acid, alkaline, and neutral ultraviolet ab- 

 sorption spectra are not identical to those of the fifth nucleotide which we have 

 isolated from yeast. 



The 86 S particle has been examined for its stability as a function of salt, 

 chelating agents, enzymatic attack, pH, and sucrose concentration (fig. 4) . The 

 results of these studies were fed back into improvements in the preparative pro- 

 cedure and are of utmost importance to the interpretation of labeled amino 

 acid incorporation studies in the particulate fractions of A. vinelandii. If the 

 86 S particle is suspended in a pH 7.05 buffer of 2xl0" 3 M phosphate, 10~ 3 

 M MgSO-i, with NaCl added to a total ionic strength of 0.03, or is dialyzed 

 against 2 X 10" 3 M K 2 HPC>4:KH 2 P04 (4:1) buffer, it dissociates to yield 58 and 

 39 S components. 



Our early studies showed that the 58 and 39 S components could be returned 

 to 10^ 3 M Mg ++ -containing solutions without re-forming the 86 S particle which 

 had previously been stable to that environment. Encouraged by our discussions 

 with Dr. Paul Ts'o at this conference, we explored further and found that in 

 5 X 10" 3 M Mg ++ the 58 and 39 S components recombined to form the 86 S par- 

 ticle, and that once formed this particle again was stable in 10" 3 Mg ++ . In all 

 these studies the buffer also contained 2 X 10" 3 M potassium phosphate buffer 

 of pH 7.05. 1 We also confirm Ts'o's observation that the area of the 39 S peak 

 is about one-half that of the 58 S peak, which suggests that one small 39 S 

 and one larger 58 S particle combine to form the 86 S particle. Upon addition 

 of 0.01 M, pH. 7, ethylenediaminetetraacetic acid, the particles further dissociated 

 to ribonucleoprotein of sedimentation coefficient less than 5 S (not extrapolated 

 to zero concentration). The particles are also rapidly degraded to small frag- 

 ments by ribonuclease but are not attacked by deoxyribonuclease. They are 

 precipitated by pH below 6.5 or above 7.5. 



Attempts to use sucrose for certain stages of the purification led to the ob- 

 servation that, if sucrose was added to the RNP buffer, the particles aggregated 

 and were readily removed by low-speed centrifugation. Sucrose concentrations 

 from 3 to 30 per cent were all found to have this effect. This finding necessi- 



1 Note added in proof: We recently reported at the 1958 meeting of the Federation of 

 American Societies for Experimental Biology that a buffer 5xl0~ 3 M in MgO and ad- 

 justed to p¥L 7.05 with cacodylic acid gives improved yield and excellent stability of the 

 80 S class of RNP from yeast, E. coli, and A. vinelandii. 



