Effects of preparative procedures on the sedimentation behaviour of 40-S ribosomal subunits

The sedimentation characteristics on sucrose gradients of derived 40-S subunits from rat liver depend on the ionic conditions used in their preparation. Following the procedure of Wettenhall, R.E.H., Wool, I.G. and Sherton, C.C. (Biochemistry 12 (1973) 2403-2411) in which 12.5 mM Mg2+ is present throughout the preparation of the subunits, sucrose gradients of the 40-S fraction show two peaks consistent with the presence of a mixed and non-interchanging population of monomers and dimers. By contrast, when ribosomes and subunits are prepared in 2 mM Mg2+ it is shown here that 40-S subunits sediment as a single peak at a rate intermediate between that of monomers and dimers as established by prior fixation with glutaraldehyde or formaldehyde - a phenomenon observed by Spirin, A.S., Belitsina, N.V. and Lishnevskaya, E.B. (FEBS Lett. 24 (1972) 219-224) for the Escherichia coli 30-S--50-S couple but not previously shown for subunits of eukaryotic origin. Under conditions of low [Mg2+] it is suggested that the subunits are of a uniform population capable of dimerization and that equilibrium can continue during sedimentation. In the present experiments it was not possible to find tRNA in 40-S subunits as shown by Wettenhall et al. By fixation before sedimentation the relative proportions of monomers and dimers of 40 S incubated under a variety of conditions was determined. Warming from 0 to 37 degrees C increased the proportion of monomers markedly, but variation of [Mg2+] had little effect. Incubation with albumin or tRNA or spermine also had little effect, but addition of ribosomal salt-wash increased the proportion of monomers. It seems most likely that many of the 40-S subunits prepared in 12.5 mM Mg2+ have initiation factors or other proteins bound to them which prevent dimerization, but that subunits prepared at any stage with buffer containing only 2 mM Mg2+ have lost these proteins and therefore are able to dimerize to a degree depending on concentration. Different relative proportions of monomers and dimers were observed depending on whether glutaraldehyde or formaldehyde was used as fixative. Reasons are suggested for believing that formaldehyde is to be preferred to glutaraldehyde for fixation in experiments of this nature.