Use of Gel Filtration to Monitor Ribosome-Membrane ...

553rd MEETING, LONDON

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Use of Gel Filtration to Monitor Ribosome-Membrane Interaction ALAN L. IBBETSON and ROBERT B. FREEDMAN Biological Laboratory, University of Kent at Canterbury, Canterbury CT2 7NJ, U.K. In most eukaryotic cells, ribosomes may be found either free or bound to endoplasmic reticulum membranes. Proteins for export, and possibly other special classes of protein, may be synthesizedexclusively on membrane-boundribosomes, but the factors determining this attachment are not known (Rolleston, 1974). In differentiatingand regenerating tissues a high proportion of ribosomes is membrane-bound, whereas in some tumour cell lines, very few membrane-bound ribosomes are found. A large but incoherent body of evidence points to some role for ribosome-membrane interaction in the control of protein synthesis and its modulation in regeneration, differentiationand carcinogenesis. Investigation of the sites on ribosomes and endoplasmic reticulum membranes involved in their interaction may clarify these questions. As in all ‘binding-site’investigations, the primary task is the development of a reliable simple method for assaying binding. Several groups have used centrifugation methods (Rolleston, 1974) but these inevitably involve subjecting the system to unphysiological g values which may introduce artifacts. Escherichia coli ribosomes which bear no nascent polypeptide chain are dissociated by the high gravitational fields imposed by centrifugation: the dissociation is also affected by the presence of tRNA, poly(U) and elongation factors (No11etal., 1973). It is therefore possible that separation by centrifugation is not the most suitable method for assaying eukaryotic ribosome-membrane attachment. Agarose gels are available with exclusion limits in the range l o x 106-40xlo6 (Sepharose 2B and 4B). Tangen et al. (1973) showed that when a rat liver postmitochondrial supernatant is applied to a Sepharose 2B column, microsomal fractions are eluted at the void volume, well separated from soluble proteins. In principle, such gels should also separate microsomal membrane fragments (and large polyribosomes) from ribosomes and their subunits. We have used this principle to study the attachment of ribosomes to isolated rough microsomal fractions. Rat liver microsomalfractions were isolated as described previously (Blyth et al., 1971). Gel filtration was performed at 4°C on columns (1 cm x 38cm) of Sepharose 2B (Pharmacia, Uppsala, Sweden). Elution was by downward flow with a head height of 35cm, and initial flow rates wereapprox. 2.5ml/h. Fractionsofvolume 1.3ml werecollectedand assayed for RNA and protein. RNA was determined by the method of Schmidt & Thannhauser (1945) as modified by Blobel &Potter (1968) with the use of the extinction data of Munro & Fleck (1966). After precipitation with HClOd, protein was determined by the method of Lowry et al. (1951). RNA/protein ratios of isolated rough microsomal fractions were approx. 0.2. Fig. 1 shows the profile of untreated rough microsomal fractions eluted in 5 0 m ~ Tris-HC1, pH7.5, containing 25m~-KC1,5rn-MgCl, and 0.25 M-sucrose.There is a single peak of protein and RNA at the void volume (around fraction 15), and the RNA/ protein ratio of the material in this peak is close to 0.2. When rough microsomal fractions are dialysed against EDTA, degranulation occurs (Blyth et al., 1971). Rough microsomal fractionswere dialysed for 24h at 4°C against 5Om~-Tris-HCl,pH7.5, containing lorn-EDTA and 0.25 M-sucrose,and then eluted from Sepharose 2B in the same buffer minus EDTA; the elution profile was as in Fig. 2. The bulk of protein appears at the void volume, but RNA appears mainly in a broad peak around the total column volume. The material in the void peak has an RNAlprotein ratio of approx. 0.05, characteristic of smooth or degranulated microsomal fractions. Several difficultieshave to be overcome in order to extend these preliminary observations. The isolation of microsomal fractions involves fractionation on discontinuous gradients followed by further centrifugation to concentrate the sample. Isolated microsomal fractions have a tendency to aggregate, and this is apparently affected by slight variations in the history of samples. Further manipulations of a sample, such as EDTA

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treatment, exacerbate this tendency. As a result, microsomal samples are frequently eluted very slowly, or not at all, from Sepharose 2B columns, in which the spaces between beads should ideally range from 9 to 38pm in diameter (Tangen et al., 1973). A further limitation of present techniques is that lOmg of protein is the maximum loading for the column, and the elution then produces fractions in which protein and RNA concentrations are at the limit of what can be determined chemically. Blobel, G. & Potter, V. R. (1968)Biochim. Biophys. Acta 166,48-57 Blyth, C.A.,Freedman, R. B. & Rabin, B. R. (1971)Eur. J. Biochem. 20,58&586 Lowry, 0.H.,Rosebrough, N. J., Farr, A. L. &Randall,R. J. (1951)J. Biol. Chem. 193,265-275 Munro, H. N.& Fleck, A. (1966)Methods Biochem. Anal. 14,113-176 Noll, M.,Hapke, B., Schreier, M. H. & Noll, H. (1973)J. Mol. Biol. 75,281-294 Rolleston, F. S. (1974)Sub-cell. Biochem. 3,91-117 Schmidt, G . & Thannhauser, S. J. (1945)J. Biol. Chem. 161,83-89 Tangen, O., Jonsson, J. & Orrenius, S. (1973)Anal. Biochem. 54,597-603 1975