on the Sedimentation Coefficient of Ribosomes from Rat Liver

Transcription

1 Eur. J. Biochem. 19 (1971) The Effect of the Mg2+ Concentration on the Sedimentation Coefficient of Ribosomes from Rat Liver Willy S. BONT, Martha DE VRIES, Janny GEELS, and Anneke HUIZINGA Laboratorium voor Biochemie, Antoni van Leeuwenhoek-Huis, Het Nederlands Kankerinstituut, Amsterdam (Received September 16/November 11, 1970) 1. Polyribosomes were broken down into 77 S monomers either by RNAase action (RNAase ribosomes) or by incubation under conditions of amino acid incorporation at 5 mm Mg2+ ( run-off ribosomes ). Various properties of these two types of monomers were compared. 2. Run-off ribosomes showed a high degree of poly(u)-directed phenylalanine incorporation. At 0.5 mm Mg2+ they contained a large number of particles with a sedimentation coefficient of 59 s. 3. When run-off ribosomes were incubated under conditions favourable for poly(u)-directed [14C]phenylalanine incorporation and then examined at 0.5 mm Mg2+, radioactivity was associated with 77 S monomers and not with 59 S particles. 4. A preparation of run-off ribosomes was isolated which contained a substantial amount of 77 S particles at 0.5 mm Mg2+ after the phenylalanine incorporation directed by poly(u). Before incubation these run-off ribosomes contained a negligible amount of 77 S ribosomes at 0.5 mm Mg It was concluded that before run-off ribosomes had reacted with poly(u), the sedimentation coefficient could be converted from 77 S into 59 S at 0.5 mm Mg2+. After [14C]phenylalanine incorporation directed by poly(u), however, the sedimentation coefficient remained 77 S at 0.5 mm Mg2+. It has been demonstrated [1,2] that monomers can be obtained from polyribosomes either by the action of RNAase or by incubation under conditions of amino acid incorporation. Studies with free polyribosomes, isolated from rat liver without the utilization of detergent, indicated that breakdown of these structures under conditions of amino acid incorporation only took place if the Mg2+ concentration was lower than 7 mm. The resulting monomers showed much higher poly(u)- directed incorporation of phenylalanine than monomers obtained by breakdown with RNAase [3]. On the other hand the conversion of the sedimentation coefficient of ribosomal monomers from 80 S into 55 S when suspended in a medium containing 0.1 mm Mg2+ was attributed by Bielka and coworkers [4] to a configurational change of these particles. In order to obtain more information about the monomers, the conversion of the sedimentation coefficient described by Bielka et al. 141 was studied for both types of monomers at 5 mm Mg2+, and at 0.5 mm Mg2+. It was fonud that run-off ribosomes in addition to giving a high poly(u)-dependent phenylalanine incorporation also contained a large number of 77 S particles which were converted into 59 S particles at 0.5 mm Mg2f. We therefore investigated whether a correlation existed between phenylalanine incorporation and the occurrence of 59 S particles. MATERIALS AND METHODS Incubations Unless stated otherwise all incubations were performed in a medium containing 0.07 M KC1, 0.05 M Tris (ph 7.6) and 8 mm MgC1, (= medium A) in a final volume of 1 ml at 37 C for 30 min. Preparation of RNAase Ribosomes Free polyribosomes (1 mg ribosomal RNA) were obtained as previously described [3] and were broken down by incubation for 30 min in medium A only, without any further additions. The resulting monomers will be referred to as RNAase ribosomes. Preincubation for the Formation of Run- 0 f f Ribosomes The incubation was performed in a medium containing 0.5 pmoles ATP, 0.25 pmole GTP, 5 pmoles P-pyruvate, 25 pg pyruvate kinase, x g super-

2 16 ~ Effect of Mg2+ Concentration on the Sedimentation of Ribosomes Ear. J. Biochem. natant (4 mg protein) polyribosomes (1 mg ribosomal RNA), 50 pmoles Tris buffer ph 7.6, 70 pmoles KC1 and 5 pmoles MgCl,. Incubation for Poly( U)-Directed Phenylalanine Incorporation The incubation was performed in medium A containing 0.5 pmole ATP, 0.25 pmole GTP, 5 pmoles P-pyruvate, 25 pg pyruvate kinase, x g supernatant (4 mg protein), 200 pg poly(u), [14C]- phenylalariine (0.045 $3; specific activity 3.5 mci/ mmole) and run-off ribosomes. Density Gradient Centrifugation The preparations were layered on top of a linear sucrose density gradient (15-45O/,) in medium A and centrifuged for 2 h at rev./min in a Spinco swing-out rotor SW 50. Determination of Xedimentation Coefficients Analytical centrifugation was performed in a Spinco Model E analytical ultracentrifuge at 260 nm, by using absorption optics in combination with an automatic scanning device, developed in this laboratory [5\ ; either the absorbance or the differentiated absorbance was recorded. Assay for Radioactivity Radioactivity was assayed in a Packard Tri- Carb liquid scintillation spectrometer model 5312, with a counting efficiency of 500/,. RESULTS The Effect of Mg2+ Concentration on the Sedimentation Coefficient of Ribosomes Two types of monomers were prepared as described under Materials and Methods. One type, the RNA- ase ribosomes, was obtained by incubation of polyribosomes in medium A only, while the run-off ribosomes were obtained by incubation, under conditions of amino acid incorporation in 5 mm Mg2+ [3]. These preparations were subjected to analytical centrifugation. All experiments performed with the analytical ultracentrifuge were carried out in medium, A at 0.5 mm MgZi and at 20 C unless stated otherwise. To this end, the Mg2-t concentration was lowered by three methods, which gave identical results, i.e. dialysis, gel filtration or dilution. Only in the latter case was it possible to test a sample in the analytical ultracentrifuge within 30 min after the Mg2+ concentration was changed to 0.5 mm. The results are summarized in the Table. In free polyribosomes (Expt A) and RNAase ribosomes (Expt B) the content of 59 S particles was about 16 O/,. Run-off ribosomes contained much more 59 S particles (approx. 4S0/,; Expt. D). The amount of 59 S particles was relatively low (about 23O/,) when the run-off ribosomes were examined within 15 min at 5 C (Expt F). The 48 S particles were always present but only after prolonged periods of time this compound appeared in relatively high concentrations (Expts C and E). When run-off ribosomes were examined in the analytical ultracentrifuge at 5 mm Mg2+, keeping other conditions constant, no detectable amount of material with a sedimentation coefficient of either 59 S or 70 S was present. In Expt F of the Table the Mg2+ concentration change was brought about by dilution. The background absorbance in these experiments was rather high (Fig. 1) because non-sedimenting ultraviolet-absorbing material had not been removed by gel filtration as was done in most other experiments. In Fig. 2 an experiment at 20 C is shown that serves as a control for the Expt F in Fig. 1. Except for the difference in temperature all other conditions (including the batch) were the same in Table. Percentages of various ribosomal components at 0.5 mm Mg2+ Ultracentrifugal experiments, except Expt F, were performed at 20 C. Material with a sedimentation coefficient higher than 77 S was present but is not specified in this table. The number of experiments is given in parentheses. Two different batches were used in Expt A; the same two batches were used in Expts B and C. Three different batches were used in Expt E and three others in Expt F. The nine different batches used in D included those of E and F Espt I regaration Time of standing - in 0.5 mm Mgp+ 48 S 0 0 OI O1. ~ _ A _ Fresh polyribosomes (2) 4 h a ~~ & 1 -Th * 2 ~ a I I3 RNase ribosomes (2) 30 min & 3 di C Rh ase ribosomes (2) one night 10 * 3 14 f ~ ~~ ~ a D - ltun-off ribosomes (9) 30 min E Run-off ribosomes (3) one night 18 & 5 31 & 8 8* F Run-off ribosomes (3) 10 min (at 5 C in analytical centrifuge) a 23& i10 a Prcsent iii ncgligible miounts (< 3O/,).

3 Vol.19, No.2, 1971 W. S. BONT, M. DE VRIES, J. GEELS, and A. HUIZINGA 213 Fig.1. Run-off ribosomes analyzed in medium A at 0.5 mm Mg2f after standing for 10 min at 5 C. The absorbance was traced 10 min after reaching maximum speed (= rev./ min) szo, values were corrected for viscosity at 5 C. r = reference point; m = meniscus Fig.2. Run-off ribosomes analyzed in medium A at 0.5 mm Mg2f after standing for 10 min at 20 C. The absorbance was traced 7 rnin after reaching maximum speed. The same batch as in Fig. 1 was used. Except for the temperature, conditions in Fig. 1 and Fig.2 were the same. r = reference point; m = meniscus these two experiments. At 5 C the concentration of 59 S was found to be lower whereas that of 70 S was higher than at 20 C. The relative concentrations of the various components differed considerably, however, at low temperature as indicated by the relatively high value for the standard deviation. From these experiments we concluded that run-off ribosomes could be converted at 0.5 mm Mg2+ into 59 S and 70 S particles (Fig.3). Within 30 min (Fig.4) after raising the Mg2+ concentration to 5 mm by addition of Mg2+ the transition of 77 S particles into 59 S and 70 S was reversed. Keeping all other conditions constant after raising the Mg2+-concentration, the 48 S component however was converted into 53 S and not into 77 S (Fig.4). It was therefore concluded that the transition 59 S + 48 S was not reversible. In order to test the possibility that the conversion of 77 S into 59 S particles was accompanied by removing part of the ribosomal protein, run-off ribosomes were submitted to gel filtration on a column of Sepharose 6B. By elution with medium A at 0.5mM Mg2+, three peaks were obtained. The 59 S particles in the first peak were converted quantitatively within 30 min into 77 S ribosomes by raising the Mg2+ concentration to 5 mm (compare Fig. 4). It was concluded that material with lower molecular weight in the second and third peak respectively was not essential for this conversion.

4 2 14 Effect of Mg2+ Concentration on the Sedimentation of Ribosomes Eur. J. Biochem. Fig. 3 Fig. 3. Sedimentation characteristics of a preparation of run-off ribosomes as described in Expt. E of the Table. At the left thc absorbance, traced 13 min after reaching maximum speed (= rev./min). At the right the differentiated absorbance traced 90 sec after the absorbance. r = reference point; b = bottom; m = meniscus Fig.4. Bun-off ribosomes analyzed at 0.5 mm My2+ under conditions described in Expt D of the Table with the My2+ concentration subsequently raised to 5 mm by addition of Mg2+, keeping all other conditions constant. In addition to 77 S ribosomes a small amount of 53 S particles was observed. The differentiated absorbance was traced about 8 min after reaching maximum speed. m = meniscus; r = reference point Fig.4 Stimulation of Phenylalanine Incorporation by Poly( U) The most pronounced biochemical difference between RNAase ribosomes and run-off ribosomes was the higher phenylalanine incorporation by the latter after the addition of poly(u) [3]. After poly(u)-directed [14C]phenylalanine incorporation, the incubation mixture was submitted to gel filtration on Sephadex G-25 in order to remove labelled amino acids. The first peak was analyzed by density gradient centrifugation and both the absorbance and radioactivity were measured (Fig. 5). With the analytical ultracentrifuge also the amounts of 59 S and 77 S particles were determined as a function of the number. The maximal radioactivity did not coincide with the maximal absorbance but with the maximal amount of 77 S particles. The PoEy( U)-Dependent Conversion of 59 S Ribosomes into 77 S Ribosomes In order to obtain evidence that ribosomes which could be converted into 59 S particles at 0.5 mm Mg2+ reacted preferentially with poly(u), another series of experiments was performed. In this series, in contrast to all experiments mentioned before,

5 Vol. 19, No. 2, 1971 w. S. BONT, M. DE VRIES, J. GEELS, and A. HUIZINGA 215 I E 0 w Tube no Fig.5. flucrose density gradient analysis in medium A at 0.5 mm Mg2+ of run-off ribosomes which are incubated with poly( U) and [14C]phenylalanine. For experimental details see Materials and Methods and text. The arrows indicate maximal concentrations of 59 S and 77 S, respectively. 0, absorbance at 260 nm; A, radioactivity E 5 2 N I m / + 77 Fig.7. Analysis of the peak fruction (see Fig.6) at 0.5 mm Mg2+ after sucrose had been removed as described in the text. M Practically all sedimenting material had a sedimentation coefficient lower than 77 S. The differentiated absorbance 1 was traced about 8 min after reaching maximum speed; the absorbance 90 sec later. r = reference point; m = meniscus /. I /.-- r \ i L I I Tube no. Fig. 6. Sucrose density gradient analysis of run-off ribosomes at 5 mm Mg2+. Centrifugation was for 2.5 h in a SW 25 rotor at rev./min. The peak fractions with 77 S ribosomes were used. M = meniscus run-off ribosomes were first fractionated by means of sucrose gradient centrifugation in medium A (Fig.6). The 77 S peak fractions from the gradient were submitted to gel filtration on Sephadex G-25 in order to remove sucrose and eluted either with medium A at 5 mm Mg2+ or with medium A at 0.5 mm Mg2+. When these two samples were analyzed in the analytical ultracentrifuge, the following results were obtained. At 5 mm Mg2+ no ribosomes with a sedimentation coefficient smaller than 77 S could be detected. At 0.5 mm Mg2+ in 11 experiments 85 & 6O/, of the particles had a sedimentation coefficient smaller than 77 S. Most of these were 59 S particles (Fig.7). After incubation with poly(u), 37 & 2O//, of the original 59 S particles remained present as 59 S particles and 36 & 2O//, were converted into 77 S in three experiments with different batches (Fig. 8). When poly(u) was omitted from the incubation mixture the amount of ribosomes with a sedimentation coefficient of 77 S was negligible. From Figs.7 and 8 it is evident that the presence of poly(u) was required for the conversion of run-off ribosomes into particles which remained 77 S in medium A at 0.5 mm Mg2+. When either ATP, GTP or supernatant fraction was omitted the sedimentation patterns were similar to that shown in Fig.7. In order to keep background absorbance in the analytical ultracentrifuge as low as

6 216 Effect of Mg2+ Concentration on the Sedimentation of Ribosomes Eur. J. Biochem. Pig.8. The analysis of the peak fraction of Pig.6 at 0.5 mm &Ig2+ after poly( U) directed phenylalanine incorporation as described under Materials and Methods. A substantial amount of the particles was not converted into 59 S particles but remained 77 S. The differentiated absorbance at the left was traced about 8 min after reaching maximum speed; the absorbance 90 sec later. r = reference point; m = meniscus possible, the incubation mixture had to be submitted to gel filtration on Sepharose 6B. The first peak eluted with medium A at 0.5 mm Mg2+ containing the ribosomes was used for the sedimentation velocity analysis. Other ultraviolet absorbing material, e.g. ATP, GTP and supernatant fraction was removed in this way. Therefore all samples in this series of experiments were analyzed in the ultracentrifuge 60 min after their application to the Sepharose column. DISCUSSION Dissociation of hepatic ribosomes has been described by a number of investigators. Hamilton and Petermann [6] found that at low Mg2d- concentration and high salt concentration, monomers were dissociated into particles of 63 S, 46 S and 30 S. Tashiro and Siekevitz [7] found subunits at low EDTA concentration with sedimentation coefficients of 60 S and 50 S ; at high EDTA concentration 47 S and 32 S subunits were observed. Martin and Wool [8] and Lawford [9] were able to dissociate the monomers into subunits of 60 S and 40 S which could be recombined into active ribosomes. Bielka et al. [4] stated that at low Mg2+ concentration the first step in the dissociation of 80 S particles into 48 S and 28 S subunits is a structural change which is accompanied by a drop in the sedimentation coefficient of the monomers to 55 S. Our results are in agreement with those of Bielka et al. The amount of particles, of which the sedimentation coefficient was changed from 77 S into 70 S and 59 S, depended on experimental conditions. After polyribosomes were incubated under conditions of amino acid incorporation in 5 mlcz Mg2+, more 59 S particles were present than in a preparation of monomers obtained by RNAase treatment of polyribosomes. As prior to incubation, part of the total untreated ribosomal pellet was converted to 59 S at 0.5 mm Mg2+, we concluded that part of the monomers present in vivo were also of the run-off type of ribosomes. All 59 S particles could be restored to 77 S particles by raising the Mg2+ concentration to 5 mm. The transition of 77 S monomers into 70 S and 59 S particles and vice versa could be accomplished by respectively lowering or raising the Mg2+ concentration. However, the process of conversion of 59 S into 48 S was irreversible under our experimental conditions, since by raising the Mg2+ concentration 48 S changed into 53 S. The 48 S component might be a subunit of the 59 S particle. Experiments with Sepharose BE indicated that the drop in sedimentation at 0.5 mm was not accompanied by a loss of material of low molecular weight which could be required for the reconversion into 77 S particles. Therefore we concluded that this reversible transition depended solely on the Mg2+ concentration. Bielka et al. [4] were able to prove that the 55 S component was not a large subunit as it contained both 33 S and 18 S RNA at a mass ratio of 2 : 1. Even when large amounts of 59 S were present in our experiments, no evidence for a small subunit of about 40 S was obtained, while all 59 S particles could be restored to 77 S particles by raising the Mg2+ concentration. We therefore concluded that configurational change, as proposed by Bielka etal. [4] is the most probable explanation for the drop in the sedimentation coefficient. Direct evidence that those particles which could be transformed into 59 S particles, were responsible for the high poly(u)-directed phenylalanine incorporation was obtained as follows.

7 Vol.19, No.2,1971 W. S. BONT, M. DE VRIES, J. GEELS, and A. HUIZINGA 217 In order to correlate the high phenylalanine incorporation shown by run-off ribosomes with the transition of the sedimentation coefficient, the 59 S particles were partly separated from 77 S particles by means of density gradient centrifugation. The separation was sufficient to draw conclusions from the relative stimulations exhibited by these two types of monomers. When run-off ribosomes were analyzed at 0.5 mm Mg2+ following poly(u)-directed [T!]phenylalanine incorporation, the radioactivity appeared to be attached to 77 S particles. On the other hand, preparations of run-off ribosomes which had been enriched by gradient centrifugation and which contained predominantly 59 S and negligible amounts of 77 S particles at 0.5 mm Mga+, did contain a high percentage of 77 S ribosomes at 0.5 mm Mg2+ after incubation under conditions of phenylalanine incorporation, provided poly(u), ATP, GTP and supernatant fraction were added to the incubation mixture. From these experiments we concluded that part ofthose particles which were converted at 0.5 mm Mg2+ to 59 S could react with poly(u). The total amounts and the relative concentrations of monomers (59 S, 70 S and 77 S particles) were not the same in all experiments summarized in Table 1 and were a function of time spent standing in 0.5mM Mg2+ and of temperature. Some of the monomers could have been present as dimers. Components with sedimentation coefficients of 94 S, 104 S and 113 S were observed, especially in those preparations where the total amount of monomers was relatively low. These sedimentation coefficients could correlate with dimers of 59 S, 70 S and 77 S particles or of hybrids between two of these particles. The total amount of monomers may have increased with time by dissociation of the dimers. The authors wish to thank Prof. Dr. P.Emmelot for fruitful discussions and Dr. M. Sluyser for reading the manuscript. REFERENCES 1. Warner, J. R., Rich, A., and Hall, C. E., Science (Washington), 138 (1962) Watson, J. D., ij cience (Washington), 140 (1963) Bont, W. S., Rezelman, G., Meisner, J., and Bloemendal, H., Arch. Biochem. Biophys. 119 (1967) Bielka, H., Welfle, H., Bottger, M., and Forster, W., Eur. J. Biochem. 5 (1968) Van Es, W. L., and Bont, W. S., Anal. Biochem. 17 (1966) Hamilton, M. G., and Petermann, M. L., J. BWZ. Chem. 234 (1959) Tashiro, Y., and Yphantis, D., J. Mol. Biol. 11 (1965) Martin, T. E., and Wool, J. G., Proc. Nut. Ad. 8ci. U. 8. A. 60 (1968) Lawford, G. R., Biochem. Biophys. Res. Cmmun. 37 (1969) 143. W. S. Bont, M. de Vries, J. Geels, and A. Huizinga Laboratorium voor Bioohemie, Antoni van Leeuwenhoek-Huis Sarphatistraat 108, Amsterdam C, The Netherlands 16 Eur. J. Biochem., Vol.19