Gel electrophoresis of ribosomal components from seeds of Pisum sativum L

Copyright AN rights

0 1973 by Academic Press, Inc. of reproduction in any form reserved

Experimental Cell Research 77 (1973) 298-302

GEL ELECTROPHORESIS OF RIBOSOMAL COMPONENTS FROM SEEDS OF PISUM SATZVUM L H. THOMAS Welsh Plant Breeding Station, Aberystwyth, Wales

SUMMARY The ribosomes of dry pea seeds were analysed by polyacrylamide gel electrophoresis. ribosomal subunits, rRNA and ribosomal proteins were separated by variations basic technique. Pea seed ribosomes were shown to have a subunit structure, rRNA and ribosomal protein distribution similar to other eukaryotic ribosomes. A total of proteins were identified, 24 on the small and 28 on the large RSU. The molecular mostly in the range 10-35 x 10s.

The ribosomes of seeds become active in protein synthesis during germination [l]. There has been much interest in the mechanism of ribosome activation and consequently a great deal is known about the proteinsynthesising systems of seeds [2]. In this respect seeds of Pisum sativum have been well studied: ribosomes active in in vitro amino acid incorporation have been isolated from cotyledons and embryonic axes [3-61 and the physical properties of pea seed ribosomes have also been described [7, 81. We have studied pea seed ribosomes using variations of the technique of polyacrylamide gel electrophoresis that give rapid and sensitive separations of ribosomal components. The value of gel electrophoresis in the fractionation of proteins and RNA has been demonstrated repeatedly in recent years [9, lo]. Moreover there have been a number of reports of gel electrophoresis used in the fractionation of ribosomal subunits [ 1 l-l 31 ribosomes and polysomes [14, 151 but this Exptl Cell Res 77 (1973)

Ribosomes, of this same complement 52 ribosomal weights were

application of the technique has yet to be employed widely. In this paper we describe the characterization of the ribosomes of dry peas using gel electrophoresis. MATERIALS

AND METHODS

Extraction of riboJomes All buffers used in the extraction of ribosomes contained tris HCl 0.05 M. MgCl, 5 mM. KC1 15 mM and mercaptoethanol 5 m-M at pH ‘7.6 (‘TMK’). Dry peas (P. satiuum var ‘Alaska’) were milled to a fine powder and mixed with TMK containing 0.25 M sucrose in the nronortion 3 ml buffer ner a of tissue. The brei was filtered through two iayers of muslin and centrifuaed for 15 min at 20 000 g. The pellet was discarded and 5 % sodium deoxycholate solution was added to the sunernatant to give a final concentration of 0.5 %. Aliquots of 2.0 ml were layered over discontinuous gradients of 2.5 ml 1.5 M sucrose and 1 ml 0.5 M sucrose, both in TMK, and centrifuged for 3 h at 105 000 g in an MSE Superspeed 50 centrifuge. The supernatants were discarded-and the walls of each tube wiped to remove lipid and protein. The ribosomal pellet, colourless or pale yellow in appearance was resuspended by gentle stroking with a glass rod and the suspension was spun for 5 min at 1 Ooo g. Resuspension buffer was either ribosome gel running buffer containing 10% sucrose or subunit gel buffer containing 10 % sucrose. All operations were carried out at O&C.

Ribosomal components from pea seeds Fractionation subunits

299

of ribosomes and ribosomal

Ribosomes were fractionated by electrophoresis in rods of polyacrylamide gel. Gels 8 cm in length were prepared in plastic tubes 0.6 cm internal diameter from a stock solution containing 15 % acrylamide and 0.75 o,, N,N-methylene-bisacrylamide 1101. Gel concentration was 2.2% and elecirophoresis buffer was a 5-fold dilution of tris acetate 0.1 M, potassium acetate 75 mM and magnesium acetate 37.5 mM at pH 8.0. Gels were pre-run for 30 min before samples were applied. It is necessary to circulate fresh buffer through the gel apparatus continuously to prevent depletion of magnesium by accretion at the cathode (upper reservoir). Ribosome samples containing 10-25 pg rRNA were loaded and run at 2°C for 4.5-5 h at 5 mA/tube. Gels were washed in water for at least 1 h and scanned at 265 nm with a Joyce-Loebl Chromoscan. The location of rRNA on the gel was shown by immersing the gel for 1 h in a 0.02 % aqueous solution of toluidine blue followed bv destaining with several changes of water. Basic pro&n was located by staining with 0.6 l/o fast green in 7 % acetic acid and destaining with several changes of solvent. Gels were scanned at 595 nm in the Chromoscan. Ribosomal subunits (RSUs) were fractionated by electrophoresis in gel rods. Gel concentration was 3.4~ and electrophoresis buffer a 5-fold dilution of tris acetate 0.1 M, potassium acetate 0.25 M and tetrasodium pyrophosphate 25 mM at pH 8.0. After orerunning. the gels were loaded with ribosome suspension’ containing 10-25 pg rRNA and run at 2°C for 3-3.5 h at 5 mA/ael. Gels were washed and ‘scanned at 265 nm.

Fractionation of rR NA and ribosomal protein Ribosomes were dissociated into protein and RNA with guanidinium chloride [16]. Two volumes of 6 M guanidinium chloride were added to the ribosome suspension. After 15 min 0.5 vol of ethanol were added and the mixture was left for 30-60 min at - 20 The RNA precipitate was collected by centrifugation and converted to the sodium salt by several washes with 70 % ethanol containing 0.1 M sodium acetate. The final precipitate was washed with absolute ethanol, dried in vacua and electrophoresed in 2.4% polyacrylamide gels [lo]. Ribosomal protein was precipitated from the RNAfree supernatant by addition of an equal volume of 10 :;, trichloroacetic acid. The precipitate was collected by centrifugation, washed with absolute ethanol and dried in vacua. Protein was dissolved in 0.01 M phosphate buffer, pH 7.2, containing 0.1 % mercaptoethanol, 0.1 % sodium lauryl sulphate (SLS) and 10 % glycerol to give a concentration of 1 mg/ml and denatured by heating to 65°C for 15 min. The solution was allowed to stand overnight at room temoerature before electrophoresis in 1Ocm long polyacrylamide gels (10 % acrylamide, 0.27 % bis) containing SLS as described by Bickle & Traut [17] modified so that the concentration of phosphate buffer was 0.025 M and the running voltage i0 volts/cm. Gels were stained

h 1. Abscissa: migration; ordinate: absorbance. Polyacrylamide gel traces of dry pea ribosomes. (a) UV absorbance; (b) Ass5 of gels stained for RNA and protein. (Left) top of the gel. Fig. 2. Abscissa: migration; ordinate: A,,,. Polyacrylamide gel trace of dry pea RSUs. (Lefi) top of the gel. Fig.

with coomassie brilliant blue R 191 and scanned at 595 nm in the Chromoscan. Proteins from RSUs were fractionated in the following way. The subunits of a ribosome sample containing 50-60 pg rRNA were separated by electrophoresis and located by viewing the gel on a silica plate under ultraviolet light against a-fluorescent background. The absorbing bands were cut from the gel and each incubated for 1 h at 3540°C in 2 ml denaturation buffer. The liquid was poured away and the gel pieces heated to 65°C fo; 15 min. After standing-at room temperature overnight each slice was placed in contact with the top of a 10 “0 acrylamide-SLS gel and run as described.

RESULTS Electrophoresis of ribosomesand subunits

When electrophoresed in polyacrylamide gel the ribosomes of pea seeds migrate as a single band (fig. la). The UV-absorbing region of the gel is ribonucleoprotein in nature since it can be stained for both RNA and basic protein (fig. lb). There are no polysomal aggregates present. Fig. 2 shows RSUs separated by electrophoresis. Two 1aIge peaks are clearly visible, together with Exptl

Cell Res 77 (1973)

300

H. Thomas

Fig. 3. Abscissa: migration; ordinate: AEC5. Polyacrylamide gel trace of dry pea rRNA. (Left) top of the gel.

b

some minor peaks. It has been established [14] that there is a direct relationship between the logarithm of the weight of a ribonucleoprotein particle and its migration in polyacrylamide gel during electrophoresis. Taking the mol. wt of the large (60s) peak on the gel to be 2.44 x lo6 and of the smaller (40s) peak to be 1.5 x 106, the mol. wts of components a and b have been calculated as 1.751.80 x lo6 and 1.25-1.35 x lo6 respectively. Electrophoresis of rRNA

The high mol. wt RNAs of therribosomes are shown in fig. 3. The two major peaks represent the RNAs from the small and large RSU. I

I

Fig. 5. Polyacrylamide gel traces of protein from the RSUs of dry peas. (a) Protein from the 40s RSU (b) Protein from the 60s RSU. (Left) top of the gel.

There are also a number of breakdown products. The mol. wts of a and b were calculated from the weight-migration relationship described above [18]. They are: 0.97 x lo6 and 0.59 x 106. The guanidinium chloride method described here allows the rapid preparation of pure rRNA from ribosomes and has the advantage over the phenol method [18] that ribosomal protein can be recovered from the same extract in a state suitable for electrophoresis. Electrophoresis of ribosomal protein

Figs 4, 5. Abscissa: migration; ordinate:lA,,,. Fig. 4. Polyacrylamide gel trace of protein from dry pea ribosomes. (Left) top of the gel. Exptl CeN Res 77 (1973)

There are approx. 27 bands present in fractionations of protein from whole ribosomes (fig. 4). The 40s subunit yields 24 bands and the 60s subunit 28 bands (fig. Sa, b). This indicates that many of the bands in separations of protein from whole ribosomes represent at least two distinct proteins located on separate RSUs. The molecular weights of

RiboJomal componentA from pea Jeeds

301

Table 1. Molecular weights ( x 103) of ribosoma1proteins from peas

and not as polysomal aggregates.The absence of polyribosomes from non-germinated pea seedswas described by Barker & Rieber [4] Band number 40s RSU 60s RSU and seemsto be true of seedsin general (I]. Pea seedribosomes are dissociated by electro61.5 11.5 53.5 12.5 phoresis in the presence of pyrophosphate. 50.0 61.5 In addition to the two major RSUs there 43.5 51.5 40.5 56.0 are also minor particles present when ribo37.0 52.5 somesare dissociated in this way. The signif34.5 49.0 8 31.0 41.5 icance of these is not clear at present. Talens 9 29.0 42.5 et al. [ 131 reported that gel electrophoresis 10 25.0 41.5 II 24.0 37.0 resolved particles derived from E. coli ribo12 23.0 36.5 somes by dissociation that are not resolved 13 22.5 33.0 14 20.5 32.5 by density gradient centrifugation. 15 20.0 30.0 RNA extracted from pea seed ribosomes 16 18.0 21.0 17 16.5 22.5 comprises certain distinct degradation prod18 15.5 22.0 ucts in addition to the 1.29 y. 10” and 0.7 19 15.0 20.0 20 13.5 17.0 lo6 mol. wt speciesnormally present in plant 21 13.0 16.5 ribosomes. Their calcu!ated mol wts corre22 12.0 16.0 23 11.0 15.5 spond with degradation products describedby 24 10.0 14.5 Payne & Loening [18] in phenol-extracted 25 13.0 26 12.5 RNA from pea root ribosomes. 27 11.5 In addition to a molecule of high mol. 28 10.5 wt RNA, each RSU contains a number of Mol. wts determined by detergent gel electrophoresis. proteins of various mol. wts. The 60s subEach mol. wt is the mean of at least three independent determinations and is expressed to the nearest 500 D. unit contains at least 28 proteins with mol wts of 10.5-77.5 x 103, with most having mol. ribosomal proteins were determined by com- wts of IO-35 z 103. The 40s subunit also paring relative migration (distance moved contains a heterogeneous collection of at by protein/distance moved by bromophenol least 24 proteins with mol. wts ranging from 10 to 61.5 x 103, with most having mol. wts blue tracker dye) with that of standard proteins run under the same conditions using of 1040 x 103.Gumilevskaya et al. [7] found 32 separate bands in electrophoretograms of the relationship between relative migration and log mol. wt described by Weber & Osborn pea seed ribosomal protein. Since it is clear [19]. The standard proteins used were bovine that some of the bands resolved by detergent gel electrophoresis comprise at least two serum albumin (mol. wt 69 x 103) and cytochrome c (mol. wt 13.5 x 103). Table 1 is a distinct proteins of closely similar molecular list of the calculated mol. wts of proteins weight the total number of proteins present in the pea seed ribosome probably exceeds from the 60s and 40s subunits. the number resolved by this method. Martini & Gould [20] have shown this to be true for DISCUSSION reticulocyte ribosomal proteins, which numThe results described in this paper show that ber at least 61, by using a two-dimensional the ribosomes of dry peas exist as monosomes electrophoretic technique. There is little inExptl

Cell

Res 77 ( 1973)

302 H. Thomas formation concerning the molecular weights of higher plant ribosomal proteins with which these results may be compared, but the predominance of proteins with mol. wts of 10-30x lo3 agrees with results for other eukaryotes [21]. Some ways in which gel electrophoresis is superior to other techniques such as density gradient centrifugation may be mentioned. Mole samples may be electrophoresed at any one time than can be fractionated in conventional high-speed heads, and the gel is not destroyed during scanning as a density gradient is. Moleover, UV absorbing bands on the gel may be visualised by staining for RNA and protein. It is not always true that peaks on a density gradient that absorb UV are ribonucleoprotein in nature. It is also possible to fractionate polyribosomes using the method described for electrophoresing dry seed ribosomes (unpublished results) and the relationship between molecular weight or S value and migration [14, 221 holds true for particles fractionated in this way so that it is possible to obtain very accurate measurements of these parameters without recourse to the analytical ultracentrifuge. For these reasons alone the methods described here should be valuable, not only for the analysis of ribosomes but also other ribonucleoproteins such as ribosomal precursors and “informosomes”. The author is grateful to Professor P. F. Wareing for supervising, and the University of Wales for

Exptl

Cell Res 77 (1973)

financing, the course of study for a Ph.D. of which this work forms a part.

REFERENCES 1. Thomas, H, Viability of seeds (ed E H Roberts) p. 360. Chapman & Hall, London (1972). 2. Boulter, D, Ann rev plant physiol 21 (1970) 91. 3. Webster, G, Whitman, S L & Heintz, R L, Exptl cell res 26 (1962) 595. 4. Barker, G R & Rieber, M, Biochem j 10.5 (1967) 1195. 5. Swain, R R & Dekker, E E, Plant physiol 44 (1969) 319. 6. Chin, L T Y, Poulson, R & Beevers, L, Plant physiol 49 (1972) 482. 7. Gumilevskaya, N A, Kuvaeva, E B, Chumikina, L V & Kretovich, V L, Biokhimiya 36 (1971) 271. 8. Phillips, M & Hersh, R T, Exptl cell res 61 (1970) 365. 9. Smith, I, Chromatographic and electrophoretic techniques (ed I Smith) 2nd edn, vol. 2, p. 365. Heinemann, London (1968). 10. Loening, U E, Biochem j 113 (1969) 131. 11. von der Decken, A, Ashby, P, Mcllreavy, D & Campbell, P N, Biochem j 120 (1970) 815. 12. Hjerttn, S, Jerstedt, S & Tiselius, A, Anal biothem 11 (1965) 211. 13. Talens, J, Kalonsek, F & Bosch, L, FEBS letters 12 (1970) 4. 14. Dahlberg, A E, Dingman, C W & Peacock, A C, J mol biol 41 (1969) 139. 15. Mikhailova, I ‘Y &’ Bogdanov, A A, Biokhimiya 35 (1970) 403. 16. Cox, R k, Biochem prep 11 (1966) 104. 17. Bickle. T A & Traut. R R, J biol them 246 (1971) 6828. 18. Payne, P I & Loening, U E, Biochim biophys acta 224 (1970) 128. 19. Weber, K & Osborn, M, J biol them 244 (1969) 4406. 20. Martini. 0 H W & Gould. , H J. J mol biol 62 (1972) 403. 21. Wittmann, H G, Symp sot gen microbial 20 (1970) 55. 22. MacPhie, P, Hounsell, J & Gratzer, W B, Biochemistry 5 (1966) 988. Received September 1, 1972