Abstract
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.
Full text
PDF
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Arai K. I., Kawakita M., Kaziro Y. Studies on polypeptide elongation factors from Escherichia coli. II. Purification of factors Tu-guanosine diphosphate, Ts, and Tu-Ts, and crystallization of Tu-guanosine diphosphate and Tu-Ts. J Biol Chem. 1972 Nov 10;247(21):7029–7037. [PubMed] [Google Scholar]
- Barritault D., Expert-Bezancon A., Guérin M. F., Hayes D. The use of acetone precipitation in the isolation of ribosomal proteins. Eur J Biochem. 1976 Mar 16;63(1):131–135. doi: 10.1111/j.1432-1033.1976.tb10215.x. [DOI] [PubMed] [Google Scholar]
- Eikenberry E. F., Bickle T. A., Traut R. R., Price C. A. Separation of large quantities of ribosomal subunits by zonal ultracentrifugation. Eur J Biochem. 1970 Jan;12(1):113–116. doi: 10.1111/j.1432-1033.1970.tb00827.x. [DOI] [PubMed] [Google Scholar]
- Gavrilova L. P., Ivanov D. A., Spirin A. S. Studies on the structure of ribosomes. 3. Stepwise unfolding of the 50 s particles without loss of ribosomal protein. J Mol Biol. 1966 Apr;16(2):473–489. doi: 10.1016/s0022-2836(66)80186-9. [DOI] [PubMed] [Google Scholar]
- Hardy S. J., Kurland C. G., Voynow P., Mora G. The ribosomal proteins of Escherichia coli. I. Purification of the 30S ribosomal proteins. Biochemistry. 1969 Jul;8(7):2897–2905. doi: 10.1021/bi00835a031. [DOI] [PubMed] [Google Scholar]
- Holmes W. M., Hurd R. E., Reid B. R., Rimerman R. A., Hatfield G. W. Separation of transfer ribonucleic acid by sepharose chromatography using reverse salt gradients. Proc Natl Acad Sci U S A. 1975 Mar;72(3):1068–1071. doi: 10.1073/pnas.72.3.1068. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kirillov S. V., Makhno V. I., Peshin N. N., Semenkov Iu P. Razdelenie 30 S i 50 S subchastits ribosom Escherichia coli gidrofobnoi khromatografiei na sefaroze-4B. Dokl Akad Nauk SSSR. 1977;237(3):725–727. [PubMed] [Google Scholar]
- Kirillov S. V., Makhno V. I., Semenkov Iu P. Vliianie molekuliarnogo vesa poliuridilovoi kisloty i prisutstviia ribosomnogo belka S1 na stabil'nost' kompleksa transportnoi RNK s maloi subchastitsei ribosom. Dokl Akad Nauk SSSR. 1976;229(2):488–491. [PubMed] [Google Scholar]
- Staehelin T., Maglott D. M., Monro R. E. On the catalytic center of peptidyl transfer: a part of the 50 S ribosome structure. Cold Spring Harb Symp Quant Biol. 1969;34:39–48. doi: 10.1101/sqb.1969.034.01.008. [DOI] [PubMed] [Google Scholar]
- Welfle H. Studies on proteins of animal ribosomes. XI. A simple method of two-dimensional polyacrylamide gel electrophoresis of ribosomal proteins of rat liver. Acta Biol Med Ger. 1971;27(3):547–551. [PubMed] [Google Scholar]