Chromatography of Escherichia coli Ribosomes on Diethylaminoethyl Cellulose

From the ¹ From the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland 20014

Biologically active ribosomes of Escherichia coli can be purified by chromatography on diethylaminoethyl cellulose. Such ribosomes (DEAE-ribosomes) contain about 30% less protein than ribosomes washed by repeated centrifugation. In spite of the loss in protein, the DEAE-ribosomes have the same sedimentation values as washed ribosomes, are active in polypeptide synthesis, and are able to bind phenylalanyl soluble ribonucleic acid in the presence of polyuridylic acid but without guanosine triphosphate or any component of the cell sap. The loss of protein from the DEAE-ribosomes is concomitant with a loss of about 80% of the latent ribosomal DNase but none of the latent ribosomal RNase. Removal of the protein does not alter the response of the DEAE-ribosomes to various inhibitors of protein synthesis, including streptomycin.

The DEAE-ribosomes differ from the washed ribosomes in their response to pH, NH₄Cl, and magnesium. The activity of the DEAE-ribosomes changes little if at all over the range of pH from 6.9 to 8.5 whereas the washed ribosomes exhibit a broad optimum between pH 7.8 and 8.2. High concentrations (0.12 m) of NH₄Cl produce a parallel increase in the binding of phenylalanyl soluble RNA and polypeptide synthesis with the DEAE-ribosomes but not with the washed ribosomes. The optimal magnesium concentration for polypeptide synthesis is 0.012 m with the DEAE-ribosomes as compared to 0.016 m with the washed ribosomes. Electrophoretic patterns of the ribosomal protein obtained from the DEAE-ribosomes were largely the same as those obtained from the washed ribosomes except for the absence of one protein band. These differences indicate that all of the ribosomes chromatographed on DEAE-cellulose have lost protein.

The electrophoretic patterns of the ribosomal protein obtained from DEAE-ribosomes and washed ribosomes of E. coli K-12 was compared to ribosomal protein from similar ribosomal preparations of E. coli B.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?