90 MICROSOMAL PARTICLES 



TABLE 1. Chemical Fractionation of coli Components 



Total 100 20 30 50 



obtained from the swinging bucket give a constant ratio indicating two amino 

 acids per nucleotide (NA/P = 60/40 measured by absorption at 260 mu and 

 Folin [12] test for protein). 



The protein of ribosomes differs from other proteins of the cell. Purified 

 ribosomes were obtained from cells grown with C 14 glucose as the sole carbon 

 source. The protein, after hydrolysis and chromatography, showed an amino 

 acid distribution in which glutamic acid, alanine, glycine, and lysine were pro- 

 portionately higher than in the whole cell, whereas methionine and aspartic acid 

 were lower. In this protein neither cysteine nor cystine seems to be present. 



The absence of cystine can best be shown by growing the cells in the presence 

 of S 35 Oi to label cystine and methionine. After hydrolysis and chromatography 

 the radioactivity of methionine and cystine can be measured. In the protein of 

 the whole cell there is approximately twice as much methionine as cystine [13]. 

 In ribosomes purified in the swinging bucket centrifuge this ratio is 10:1. In 

 nucleoprotein eluted from the column and sedimented the ratio is greater than 

 100:1. 



Alternatively the lack of cystine can be demonstrated without hydrolysis and 

 chromatography. Cells containing S 35 cystine and S 32 methionine were grown 

 by adding S 35 Oi and S 32 methionine to the medium. To prevent even a slight 

 leakage of S 35 into methionine, a methionine-requiring mutant was used [13]. 

 The sulfur radioactivity per unit protein of the nucleoprotein (obtained by col- 

 umn analysis of a microsome pellet and sedimentation of the nucleoprotein frac- 

 tion of the eluate) was 50 times lower than that of the whole cell. Since the 

 usual occurrence of cystine is only 1 per 60 residues, its occurrence in the nucleo- 

 protein is less than 1 per 3000. 



Kinetic studies of the fractions obtained from the column are also in progress. 

 S 35 has been used to follow incorporation into protein. Exponentially growing 

 cells were exposed to the tracer for varying periods of time and then broken 

 and their constituents analyzed. The specific radioactivity of the protein frac- 

 tions was measured by TCA-precipitable S 35 and Folin reaction color. When the 

 cells are exposed to the tracer for a prolonged period (steady state) the specific 

 radioactivity varies throughout the elution pattern by a factor of roughly 3, 

 being lowest in the nucleoprotein fraction. These variations are simply due to 

 variations in the sulfur content. Other cells were grown for three generations in 



