Pressure-Induced Dissociation of Sedimenting Ribosomes: Effect on Sedimentation Patterns

Abstract

Anomalous sedimentation patterns arise when free ribosomes from sea urchin eggs are centrifuged at high speeds. Pressure-induced dissociation of the ribosomes during sedimentation can explain the peculiar behavior; the assumption of such dissociation also yields estimates of the equilibrium constant (as a function of KCI concentration) and the change in molecular volume (500 i 100 ml/mol) in the reaction: subunits :± ribosome. Such dissociation during centrifugation may explain many experiments in which apparent reduced sedimentation coefficients for ribosomes, and increased coefficients for the subunits, have been ascribed to conformational changes.Anomalies in the sedimentation characteristics of ribosomes and subunits under various ionic conditions have been reported in many studies. These results have been generally interpreted in terms of conformational changes in the ribosomes and ribosomal subunits (1-7). However, a recent study (8) showed that the sedimentation pattern of free ribosomes from sea urchins is strongly dependent not only on the ionic conditions but also on the gravitational field and the concentration of ribosomes. When these factors are appropriately varied, the ribosomes display an apparently reduced sedimentation velocity or dissociate into subunits of apparently increased velocities. The results were interpreted in terms of (a) the dissociation of ribosomes at a rather distinct point during ultracentrifugation, and (b) the notion that the dissociation is due to a shift in the equilibrium between subunits and ribosomes in the sucrose gradient.The cause of this dissociation is suggested by the results of studies of protein dissociation, which have shown that the high pressure experienced by particles in the analytical ultracentrifuge can induce dissociation of multimers into subunits if thejnolecular volume decreases upon dissociation (for a general treatment of this subject, see refs. 9 and 10). Some of these ideas were formulated by Josephs and Harrington (11,12) arudwere used to explain the anomalous sedimentation of myosin. In this report, we examine the effect of hydrostatic pressure on the dissociation of ribosomes during zonal centrifugation in sucrose gradients. We conclude that pressure does induce the 75S free ribosomes to dissociate into their 56S and 35S subunits, and that this behavior can explain various peculiar sedimentation patterns of both eukaryotic and prokaryotic ribosomes that had previously been ascribed to conformational changes. MATERIALS AND METHODSUnfertilized eggs of the sea urchin, Strongylocentrotus purpuratus, were used in these studies, since the bulk (>95%) of the ribosomes from this source are not complexed with mRNA and peptidyl-tRNA (8). The procedures used to obtain cell-free homogenates are described in detail elsewhere (13,8). The eggs were homogenized in TM buffer containing 0.25 M sucrose and the indicated concentrations of KCl. (TM buffer contains 5 mM MgCl2-50 mM triethanolamine, pH 7.8.) A 15,000 X g supernatant (S15) was prepared and ana...

Run off ribosomes of Escherichia coli: their accumulation in response to cold treatment and their dissociation during centrifugation

“…Although we cannot exclude the possibility that some stable 63 S particles exist, most of the material which resediments as a peak in the 63 S region ( fig. 3a) presumably represents a reassociation of subunits which were dissociated but not separated by the first centrifugation [7] . Parallel to the recentrifugations, the content of selected fractions in 16 S and 23 S ribosomal RNA was determined by polyacrylamide gel electrophoresis.…”

“…There have been several reports of bacterial ribosomes with apparently reduced sedimentation coefficients . However, studies of the sedimentation properties of free ribosomes from the sea urchin egg have led Infante and Baierlein [7] to conclude that dissociation during centrifugation provoked by high hydrostatic pressure can produce sucrose gradient profiles in which ribosomes appear to sediment with a lower sedimentation constant. In this report we show that during prolonged centrifugation the 70 S ribosomes produced by low temperature-induced run off in E. coli appear to sediment more slowly than complexed 70 S ribosomes, and that this effect is due in part at least to dissociation during centrifugation.…”

Regulation of Turnover GTPase Activity of Elongation Factor G: the 30‐S‐Coupled and 30‐S‐Uncoupled Reactions

“…4A -C) ; addition of glutaraldehyde (Fig. 4 D -F) revealed the presence of small amounts of multimers in the original subunit solutions, and the 62-S material now appeared in the 70-S position, as expected since it represents an artefact of the centrifugation method [21,22]. In the absence of monovalent cations, 30-S subunits displayed several peaks without glutaraldehyde treatment, whereas the 50-S subunit appeared as a single species (Fig.4G, H).…”

Rapid Exchange of Subunits between Free Ribosomes in Extracts of Escherichia coli

“…It therefore seemed that the "hybrid" peak might not consist of true hybrids, but might owe its altered sedimentation rate to some other mechanism. An explanation was provided by the recent demonstration that the high hydrostatic pressure in the gradient can cause dissociation of free ribosomes, resulting in a decreased apparent sedimentation constant (10). In the mixtures used here, the free HH ribosomes, which run ahead of the LL ribosomes, would be at least 100-fold more dilute and would also encounter higher pressures.…”

“…Hydrostatic pressure promotes dissociation of free ribosomes as they advance in a gradient, as Infante (10) showed with ribosomes from seaurchin eggs: the resulting greater slowing of heavy ribosomes, which advance ahead of the light ribosomes, can mimic formation of hybrid ribosomes. This artefact is eliminated by fixation with glutaraldehyde before sedimentation analysis.…”