Separation of ribosomal subunits of Escherichia coli by Sepharose chromatography using reverse salt gradient.
A mixture of 30 S and 50 S subunits quantitatively absorbs on a column of Sepharose--4B from the buffer: 0.02 M Tris--HCl, pH 7.5, containing 1.5 M (NH4)2SO4. During elution by reverse gradient of ammonium sulphate (1.5--0.05 M) the subunits are eluted at different salt concentrations. Complete separation of subunits is attained in the absence of Mg2+ ions. The 30 S subunits prepared from 70 S ribosomes according to this procedure are fully active in the codon--dependent binding of a specific aminoacyl--tRNA. After their reassociation with 50 S subunits isolated by zonal centrifugation, the resulting 70 S ribosomes are active in polypeptide synthesis at the same degree as control 70 S ribosomes in which both types of subunits were prepared by zonal centrifugation. The initial 70 S ribosomes for the chromatographic separation into subunits can be obtained by their pelleting from a crude extract with subsequent washing with concentrated solutions of NH4Cl in the ultracentrifuge, or by salt fractionation of the crude extract according to a slightly modified procedure of Kurland.
D002458:Cell Fractionation / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism / Q000648:ultrastructure*; D007700:Kinetics; D008274:Magnesium; D009994:Osmolar Concentration; D012270:Ribosomes / Q000378:metabolism / Q000648:ultrastructure*; D014158:Transcription, Genetic
D008274:Magnesium