Search

Ten Experimental Medical Care Review Organizations are developing and evaluating methods of conducting areawide and statewide review of physicians' services. The review is based on criteri...

Publisher Summary This chapter discusses the preparation of Escherchia coil ribosomal subunits active in polypeptide synthesis. All ribosomes are prepared from the RNase I deficient E.coli strain from Gesteland, which is a derivative of E. coli strain AB 301 (K12, Hfr, met - ). All manipulations for ribosome preparation are done at 0°-4°. At the end of centrifugation the 50 S subunits should be completely pelleted, and if, the 50 S boundary is still visible above the bottom of the tube, centrifugation should be continued. Upon complete pelleting of the 50 S particles, the clearly visible 30 S ribosome band in the lower one-third of the tube is withdrawn with a syringe and a stainless steel needle with the point filed or cut off. The aggregation of 70 S ribosomes, particularly at later times of incubation, seems to be due to the long hydrophobic polyphenylalanine chains rather than to poly(U) connecting them, because RNase will not cause disaggregation.

Publisher Summary This chapter describes the fractionation of Escherichia coli 50 S ribosomes into various protein-deficient cores and split protein fractions by CsCl density gradient centrifugation and reconstitution of active particles. In CsCl equilibrium density gradient centrifugation, 50 S ribosomes, in the presence of high Mg 2+ ion concentration, initially form three bands in CsCI density gradients with the approximate buoyant densities of 1.62 g/cm 3 (=α-core), 1.65 g/cm 3 (=β-core), and 1.68 g/cm 3 (=γ-core). The chapter shows that the presence of a commercial cell batch of heavy metals in the form of rust and iron granules and possibly traces of copper is detrimental to the stability of ribosomal subunits in CsCl density gradients; therefore, strongly recommends washing of cells several times in an EDTA-containing buffer before lysis. The physiological state of cells best suited for ribosome subunit preparation seems to be one in which all ribosomes are vacant (runoff) couples and free subunits.