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Nucleic Acids Research, 1978, Vol. 5, No. 11 4305-4316
© 1978

Articles

Separation of ribosomal subunits of Escherichia coli by Sepharose chromatography using reverse salt gradient

S.V. Kirillov, V.I. Makhno, N.N. Peshin and Yu.P. Semenkov

Leningrad Institute of Nuclear Physics of Academy of Sciences of USSR Gatchina, Leningrad district 188350, USSR

Received August 18, 1978. A mixture of 30 S and 50 S subunits quantitatively adsorbs 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 Kurland1.


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