Solution of ribosomal proteins under mild conditions

Solution of ribosomal proteins under mild conditions

421 Biochimica et Bioph ysica Acta, 383 (1975) 421--426 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 98263...

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421

Biochimica et Bioph ysica Acta, 383 (1975) 421--426

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98263 S O LUTI ON OF RIBOSOMAL PR O T E I N S UNDER MILD CONDITIONS

PIROSKA HUVOSa and ROBERT A.

COX b

aDepartmenl of Biophysics, King's College, 26--29 Drury Lane, London, W.C.2. and bNational Institute of Medical Research, The Ridgeway, London, N.W. 7. 1AA, (U.K.)

(Received August 26th, 1974) (Revised manuscript received November 29th, 1974)

Summary Ribosomal proteins f r om two eucaryotic species, prepared by either the guanidine • HC1 or LiC1 • urea m e t h o d and subsequently dissolved in 8 M urea were f o u n d to be largely retained in solution after removal of the urea by dialysis against a solution of low ionic strength (0.05 M Tris • HC1, pH 7.6, 0.025 M KC1, 0.005 M magnesium acetate) and centrifugation at 100 000 X g. The protein composition of this preparation was virtually identical to that of the original urea-containing solution as determined by two-dimensional polyacrylamide gel electrophoresis. Thus, there exists a very simple m e t h o d for obtaining the bulk of the ribosomal proteins in solution under conditions where ribosomes themselves are stable.

Ribosomal proteins are generally regarded as rather insoluble in nondenaturing solvents. T hey are known to be soluble in urea or detergent solutions and at e x t r e m e pH values when separated from ribosomal RNA [1, 2]. The proteins have also been solubilised unde r conditions closer to those occurring physiologically, i.e. at neutral pH, but still at high ionic strength [3, 4]. Here we would like to report that most if n o t all ribosomal proteins of two eucaryotic species can be brought into solution at neutral pH at low ionic strength by dialysis. Rabbit reticulocyte ribosomal subparticles were prepared as described by Blobel and Sabatini [ 5 ] . Ribosomes from X e n o p u s laevis oocyt es were prepared essentially as described by Pratt and Cox [ 6 ] . Preparation o f the ribosomal proteins was p e r f o r m e d either with the guanidine • HC1 m e t h o d [ 7 ] , or with the LiC1 • urea m e t h o d by keeping the ribosomes overnight in 2.5 M LiC1 and 5 M urea (see ref. 8). Quantitative protein determination was done by the colorimetric m e t h o d of Bramhall et al [9]. Two-dimensional polyacrylamide gel electrophoresis of the ribosomal proteins was d o n e according to Martini and Gould [10] : at pH 4.3 in the presence of

423 urea in the first dimension and in the presence of sodium dodecylsulphate in the second dimension. When proteins from the larger ribosomal subparticle of rabbit reticulocytes were dialysed as described in the legend to Fig. 1 and centrifuged in the MSE 4b centrifuge for 0.5 h at 2500 X g, a slightly opalescent solution was obtained which contained 80---85% protein of the original urea-containing solution. About 20% of the protein of this solution was removed on further centrifugation for 1 h at 100 000 × g. This high speed supernatant, as can be seen in Fig. 1A, contains all the proteins present in the original urea-containing solution (three spots are missing when the pattern is compared to Plate II in ref. 10, but these are also absent on the original electropherogram, probably owing to degradation and/or loading of the proteins). The relative densities of the spots, as far as can be judged by inspection by eye, are very similar indeed, showing that no specific loss of any proteins occurred during dialysis. Similar experiments were performed with the smaller subparticle of rabbit reticulocytes, i.e. it was shown by two-dimensional electrophoresis that the solution dialysed against standard buffer contained the same proteins as the original urea-containing solution (Fig. 1B), with the exception of one protein (arrow) which was partially lost during dialysis. The recovery of the proteins was, however, in this case only 2 0 - 3 0 % after centrifugation at 100 000 X g. We investigated whether small differences in the dialysis procedure would give differences in the yield of the protein after dialysis and centrifugation. For these experiments proteins from the whole ribosome of X. laevis were used. The proteins were either prepared by the guanidine • HC1 or by the LiC1 • urea method. The a m o u n t of insoluble material formed during the dialysis against standard buffer varied according to the method used initially to extract the proteins from the ribosomes. With the guanidine • HC1 method 15% of the total protein was lost on centrifugation at 2500 X g for 30 min. When the proteins had been extracted with LiC1 • urea, then directly dialysed against the buffer, the recovery from centrifugation of the dialysed sample was 55%; but if the proteins in the LiC1 • urea were first dialysed against 8 M urea before being dialysed against the buffer, 75% of the proteins remained in solution after centrifugation. The material remaining in solution in the buffer after centrifugation retained the composition of the starting material in all cases, as judged by one-dimensional electrophoresis at pH 4.3 in 6 M urea (data not shown). Thus, the highest yield was obtained when the proteins had originally been prepared with guanidine • HC1 method.

F i g . 1. E l e c t r o p h o r e t i c p a t t e r n o f t h e s o l u b i l i z e d p r o t e i n s o f t h e s e p a r a t e d s u b p a r t i c l e s o f r a b b i t r e t i c u l o cyte ribosomes. Proteins were obtained in a form of an ethanol precipitate from the subparticles of rabbit reticulocytes after extraction w i t h g u a n i d i n e • HC1. T h i s p r e c i p i t a t e w a s w a s h e d w i t h a m i x t u r e o f e t h a n o l / e t h e r (1 : 1, v / v ) c o n t a i n i n g 0.1 M m e r c a p t o e t h a n o l a n d t h e p r e c i p i t a t e w a s d i s s o l v e d in 8 M u r e a . The urea-containing protein solution was dialyzed against 3--4 changes of 100--150 vol. of 0.05 M Tris • HC1 p H 7 . 6 , 0 . 0 2 5 M KC1, 0 . 0 0 5 M m a g n e s i u m a c e t a t e ( s t a n d a r d b u f f e r ) f o r 1 6 2 4 h a t 4~C, t h e n c e n t r i f u g e d f o r 1 h a t 1 0 0 0 0 0 X ~. T h e s u p e r n a t a n t w a s p r e c i p i t a t e d w i t h h a l f a v o l u m e o f 2 0 % ( w / v ) trichloroacetie acid, or three volumes of ethanol, the precipitate was washed with ethanol/ether and d i s s o l v e d i n S M u r e a , as d e s c r i b e d a b o v e . E l e c t r o p h o r e s i s w a s p e r f o r m e d as d e s c r i b e d i n t h e t e x t . T h e f i r s t d i m e n s i o n a l gel r o d s w e r e e m b e d d e d i n t h e s e c o n d d i m e n s i o n a l gel s l a b i n a m i r r o r - l i k e f a s h i o n , w i t h the origins of the two one-dimensional gels near the left and right hand edges of the slab. (A) Larger subparticle: left hand side, control; right hand side, dialysed. (B) Smaller subparticle: left hand side, control; right hand side, dialysed.

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Fig. 2. S o l u b i l i t y of t h e p r o t e i n s of t h e l a r g e r s u b p a r t i c l e o f r a b b i t r e t i c u l o c y t e r i b o s o m e s u n d e r d i f f e r e n t c o n d i t i o n s o f d i a l y s i s . P r o t e i n s f r o m t h e l a r g e r s u b r i b o s o m a l p a r t i c l e w e r e p r e p a r e d by t h e g u a n i d i n e • HC1 m e t h o d a n d d i s s o l v e d in 8 M u r e a as d e s c r i b e d in t h e l e g e n d to Fig. 1. T h e u r e a - c o n t a i n i n g p r o t e i n s o l u t i o n s w e r e d i a l y s e d a g a i n s t 4 × 1 0 0 vol. o f : s t a n d a r d b u f f e r ( 0 . 0 5 M Tris " HCI, p H 7.6, 0 . 0 0 2 5 M KCI, 0 . 0 0 5 M m a g n e s i u m a c e t a t e } (AA); 0 . 0 5 M T r i s • HCI, p H 7.6, 0.3 M KCl, 0 . 0 0 5 M m a g n e s i u m acetate (o----o): 0 . 0 5 M Tris • tlCl, p H 7.6, 0 . 8 m KC1, 0 . 0 0 5 M m a g n e s i u m a c e t a t e ( e ~). A f t e r d i a l y s i s t h e s a m p l e s w e r e c e n t r i f u g e d f o r 4 0 r a i n at 1 0 0 0 0 0 X tL T h e p r o t e i n c o n c e n t r a t i o n o f t h e s u p e r n a t a n t w a s m e a s u r e d b y t h e m e t h o d o f L o w r y et al. [ 1 2 ] .

The recovery of the protein after dialysis was also measured as a function of both the initial protein concentration (0.5--8 mg protein/ml) and the KC1 concentration (0.25---0.8 M) of the buffer used for dialysis (Tris • HC1 and magnesium acetate concentrations were both kept constant). The results obtained with the larger subparticle of rabbit reticulocyte ribosomes are shown in Fig. 2. Similar results to those shown in Fig. 2 were also found with the total ribosomal proteins of rabbit reticulocytes, i.e. recovery diminished as the protein concentration was increased, or if the KC1 concentration in the buffer used for dialysis was increased. The differences in recovery between samples dialysed against buffers of different ionic strength were less pronounced when solutions of low protein concentration were used for dialysis. The combination of high protein concentration and high KC1 concentration reduced the yield approximately three times. The recovery of the proteins of the smaller subparticle was lower (10--30%) than that of the larger subparticle proteins under all conditions, as already mentioned in results from experiments using standard buffer; and the yield was less sensitive to changes in protein concentration and in KC1 concentration. The greater tendency of the proteins of the smaller subparticle to aggregate and their decreased sensitivity to changes in the conditions of dialysis may reflect differences in the intermolecular interactions among the proteins in preparations derived from the two subparticles. It should be noted that the proteins of both subparticles could be fully recovered when urea was included in the buffer in a final concentration of 2 M. To examine the extent of aggregation of the proteins in dialyzed prepara-

425

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Fig. 3. E l e c t r o p h o r e t i c m o b i l i t y o f t h e s o l u b f l i z e d r i b o s o m a l p r o t e i n s at p H 7. R i b o s o m a l p r o t e i n s f r o m t h e s m a l l e r s u b p a r t i c l e o f r a b b i t r e t i c u l o c y t e s w e r e p r e p a r e d a n d d i a l y s e d as d e s c r i b e d in Fig. 1. T h e d i a l y z e d m a t e r i a l ( a f t e r c e n t r i f u g a t i o n at 1 0 0 0 0 0 X g) w a s e l e c t r o p h o r e s e d in 6% p o l y a c r y l a m i d e gels c o n t a i n i n g : A, 0.1 M p h o s p h a t e b u f f e r , p H 7.0; B, as A + 5 M u r e a . T h e gels w e r e e m b e d d e d in a 10% gel slab c o n t a i n i n g s o d i u m d o d e c y l s u l p h a t e a n d e l e c t r o p h o r e s i s w a s d o n e in t h e s e c o n d d i m e n s i o n a c c o r d i n g to ref. 10.

tions, these, in standard buffer, were subjected to electrophoresis at pH 7 in gels either containing or n o t containing urea. To facilitate comparison of the two electropherograms the samples were run in the second dimension as well, in the presence of sodium dodecylsulphate. As can be seen in Fig. 3, the pattern obtained by electrophoresis on phosphate buffer-containing gels is very similar to that obtained on phosphate buf f er + urea-containing gels. This shows that the proteins are n o t extensively aggregated at low ionic strength, or the intermolecular complexes f or m e d are rather unstable and dissociate during electrophoresis. The protein solution in standard buffer was stable for at least a week when kept at 4°C, i.e. it did n o t b e c o m e turbid as shown by spect rophot ometric measurements at 330 nm. Protein solutions that were kept for a week at 4°C before electrophoresis in the absence of urea showed little evidence of

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aggregation but it was noticed that some of the spots were elongated. This result also indicates low protease activity. The dialyzed proteins, in a concentration of 0.2--1 mg/ml, were kept at 37°C for up to 4 h in standard buffer to test the stability of the protein solution. After this treatment no more than 5% of the protein prepared from either the smaller or the larger subparticle sedimented on centrifugation for 30 min at 100 000 X g to remove aggregates. No more than 10% of the protein could be removed by similar centrifugation when the samples were kept at 50°C for up to 3 h. The ultraviolet absorption spectrum showed that the dialysed solution contained no more than 5% RNA (probably mostly 5 S RNA). Most of this small a m o u n t of RNA was pelleted after centrifugation at 100 000 × g for 1 h, The circular dichroism, [022 L - r e s s ] of the proteins decreased from 5700 degree • cm -2 • dmo1-1 in the intact ribosome to 750 after extraction with guanidine • HC1 in the presence of 8 M urea, whereas after dialysis against standard buffer the circular dichroism returned to approx. 3600 which is 65% of the original value, i.e. that found for ribosomal proteins within the intact ribosome. (Similar values have been reported for rat liver ribosomal proteins in sodium acetate buffer [11] ). The solution of ribosomal proteins in standard buffer, prepared as described above, should provide a useful starting preparation for the study of the properties of these proteins when uncomplexed with RNA, and since they are in solution in 0.05 M Tris • HC1, pH 7.6, 0.025 M KC1, 0.005 M magnesium acetate, their physicochemical properties and availability for chemical or enzymatic modifications can be compared directly with those of the proteins of the intact ribosome. Low ionic strength solutions of individual ribosomal proteins have been obtained [11]. The relative simplicity of the procedure presented here may be advantageous for some studies. Acknowledgements We thank the Wellcome Foundation for the award of a fellowship to P.H. during initial stages of work in N.I.M.R., and Dr H.J. Gould for reading the manuscript. References 1 2 3 4 5 6 7 8 9 10 11 12

S p a h r , P.F. ( 1 9 6 2 ) J. Mol. Biol. 4, 3 9 5 - - 4 0 6 Waller, J.P. a n d Harris, J.I. ( 1 9 6 1 ) Proc. Natl. A c a d . Sci. U.S. 47, 1 8 - - 2 3 T r a u b , P., M i z u s h i m a , S., L o w r y , C.V. a n d N o m u r a , M. ( 1 9 7 1 ) Meth. E n z y m o l . XX, 3 9 1 - - 4 0 7 S p i t n i k - E l s o n , P. ( 1 9 6 2 ) B i o c h i m . Biophys. A c t a 55, 7 4 1 - - 7 4 7 Blobel, G. a n d Sabatini, D. ( 1 9 7 1 ) Proc. Natl. A c a d . Sci. U.S. 68, 3 9 0 - - 3 9 4 P r a t t , H. a n d Cox, R . A . ( 1 9 7 1 ) B i o c h e m . J. 124, 8 9 7 - - 9 0 3 Cox, R . A . ( 1 9 6 8 ) Meth. E n z y m o l . , XII B, 1 2 0 - - 1 2 9 N o m u r a , M., T r a u b , P. a n d B e c h m a n n , H. ( 1 9 6 8 ) N a t u r e 219, 7 9 3 - - 7 9 9 B r a m h a l l , S., N o a c k , N.W.M. a n d L o e w e n b e r g , J.R. ( 1 9 6 9 ) Anal. B i o c h e m . 31, 1 4 6 - - 1 4 8 Martini, O.H.W. a n d G o u l d , H.J. ( 1 9 7 1 ) J. Mol. Biol. 62, 4 0 3 - - 4 0 5 W e s t e r m a n , P., Bielka, H. and B o t t g e r , M. ( 1 9 7 1 ) Mol. Gen. G e n e t . 1 1 1 , 2 2 4 - - 2 3 4 L o w r y , O.H., R o s e b r o u g h , N.J., Farr, A . L . and Randall, R.J. ( 1 9 5 1 ) J. Biol. C h e m . 193, 2 6 5 2 7 5