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Analysis of ribosomal proteins from adult Drosophila melanogaster in relation to age
Mechanisms of Ageing and Development, 11 (1979) 105-112
105
©Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
ANALYSIS OF RIBOSOMAL PROTEINS FROM ADULT DROSOPHILA MELANOGASTER IN RELATION TO AGE
T. SCHMIDT and G. T. BAKER* Zoologisch-vergl. anatomisches Institut der Universitat Zurich, CH-8806 (Switzerland] and (G. T.B.) Department of Biological Sciences, Drexel University, Philadelphia, PA, 19104 (U.S.A.)
(R~ceived March 5, 1979)
SUMMARY Analysis of high salt washed ribosomal proteins by two-dimensional polyacrylamide electrophoresis revealed no detectable qualitative differences in ribosomal proteins from young (4-day) and old (30-day) male flies.
INTRODUCTION A decline in the capacity for net protein synthesis is an apparent widespread correlate of the aging process, having been demonstrated in a number of invertebrates [1-4] and vertebrate species [5-9], including man [10]. Ribosomes, although occupying a central and integral position in translational processes, have been little investigated with regard to possible structural alterations which could effect the rate or fidelity of protein synthesis with advancing age. Some evidence suggesting possible age-related alterations in ribosomal structure and/or function has been presented. Ogrodnik et al. [11] reported a decrease in the discrimination ratio of methionine vs. ethionine in the synthesis of ribosomal proteins and rRNA in liver from aging rats. However, these authors reported no change in the number of ribosomal protein bands on one-dimensional SDS-polyacrylamide gels with age. Recently, direct evidence of altered structural integrity of the rRNA-protein complex was reported by Baker and Schmidt [12] wherein a five-fold increase in the amount of protein disassociated from high salt washed (0.5 M) ribosomes was observed between young and old flies in the presence of 2.0 M KC1. As ribosomes from adult Drosophila apparently turn over with half-life of approximately 10 days [ 13], this change could be the result of a loss of the fidelity of newly synthesized ribosomal protein for the rRNA, or vice versa, or both, through any number of mechanisms which alter the strength *To whom all inquiries should be addressed at Department of Biological Sciences, Drexel University,Philadelphia,PA, 19104, U.S.A.
106 of ionic bonding within the ribosomal complex. The decrease in thermal stability of ribosomal monomers reported for nematodes [ 14] and Drosophila [ 15 ] with age, could also reflect altered physicochemical interactions within the ribosomal complex with advancing age. The present studies were undertaken to evaluate the possibility of qualitative changes in ribosomal proteins which might account for the previously reported alterations in some physicochemical properties of ribosomes with age.
MATERIALS AND METHODS
Drosophila melanogaster (Sevelen strain) were reared from 4-6-day-old parents and maintained on a corn-sugar--agar--yeast medium in plexiglass cages (60 × 30 × 40 cm) under constant environmental conditions, 25 + 0.5 °C, 60 + 5% relative humidity and a 12:12 h light-dark cycle, as previously described [ 16]. Under these conditions males of this strain exhibit 50% and 90% mortalities of 29 and 40 days, respectively. Ribosomes were routinely extracted from 8 g (wet weight) of 4- and 30-day-old flies, or 4 g each of 4- and 30-day-old male flies, according to the methods of Lambertsson [17]. Optical density determinations of the high salt washed ribosomes revealed a high degree of accepted purity for eukaryotic ribosomes [18, 19] for all preparations; that is, OD26o/OD2so of 1.88 -+ 0.08 (n = 12), 1.86 + 0.09 (n = 11), and 1.87 -+ 0.07 (n = 4) for 4-day, 30-day, and 4- and 30-day-old ribosomal preparations, respectively. Total ribosomal protein from the high salt extracted ribosomes was recovered essentially according to the methods of Lambertsson [20, 21]. The ribosomal pellet was suspended in a small amount of deionized double-distilled H20 containing 6 mM mercaptoethanol and gently homogenized in a ground-glass homogenizer. The suspension was then brought to 3 ml with ice-cold I N HC1 added to a final concentration of 0.25 N. The mixture was stirred gently for 2 h at 4 °C and centrifuged at 48 000 g for 15 min, at 4 °C. The resulting pellet was then washed with 0.25 N HCI, centrifuged again as above, and the supernatants pooled. Ribosomal proteins were precipitated with 5 volumes of acetone at --27 °C overnight. After centrifugation (20 000 g for 20 min, at 4 °C), the protein precipitate was taken up in 0.3 ml of ice-cold formic acid, lyophilized, and stored at -27°C until use. Protein concentration was determined by the Folin procedure using recrystallized bovine serum albumin as a standard. Two-dimensional electrophoresis was carried out with some modifications according to the methods of Kaltschmidt and Wittman [22]. The modifications consisted of a decrease in electrophoresis time and reduction in the amount of protein which facilitated better separation and resolution, as described by Howard and Traut [23], as well as the use of a specially constructed L-shaped plexiglass gel former for the second dimension. All solutions except where noted were those used by Kaltschmidt and Wittmann [22]. One-dimensional electrophoresis was performed in 8% urea-polyacrylamide disc gels (0.3 × 13 cm) at pH 8.6, as previously described [23] except that the protein sample was not polymerized between two gels but applied in 25/21 of the electro-
107 phoresis buffer containing 8 M urea, 20% sucrose, and 10/al of 1% Pyronin G as a tracking dye. For analysis of basic proteins a series of concentrations (approx. 100, 250, 400, and 500/ag) of total ribosomal protein were subjected to standard electrophoresis. For good resolution of the acidic proteins 1.0-1.5 mg of the total ribosomal protein were required for the electrophoresis. Electrophoresis was performed at 4 °C. A constant voltage of 80 V was applied for the first 30 rain and thereafter 190 V for 6 h. For the two-dimensional electrophoresis, the first-dimension disc gel was first equilibrated against the starting buffer for 1 h at 25 °C, then polymerized on to an 18% urea-acrylamide slab gel (2.5 × 124 × 160 mm). The voltage was held constant at I00 V for 30 min and thereafter at 250 V for 13-15 h. Following electrophoresis, proteins were visualized by staining with 0.1% Coomassie Brilliant Blue dissolved in a 7.5% acetic acid-50% methanol solution for 12 h at 25 °C. The gels were destained at room temperature in a 5:5:1 mixture of methanol, water, and acetic acid for 48 h, and immediately analyzed and photographed.
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Fig. la-d. Two-dimensional electrophoreto~Fam of ribosomal proteins extracted from high salt washed ribosomes from 30-day-old male 1). melanogaster. First dimension: 8% acrylamide (pH 8.6) run at a
constant voltage (190 V) for 6.5 h at 4 °C. Second dimension: 18% acrylamide (pH 4.6) run at a constant voltage (250 V) for 13-15 h at 4 °C. Fig. l a - d are the electrophoretograms of increasing
concentrations of ribosomal proteins of approximately 80, 250, 400, and 500 ag, respectively.
108 RESULTS AND DISCUSSION
Figure la-d presents photographs of a typical series of two-dimensional electrophoretograms performed for ribosomal proteins extracted from 30-day-old male flies. This series is representative of the results obtained for all ages studied. Due to a number of factors, a series of protein concentrations is necessary to visualize all proteins and to be certain of their ribosomal origin; for example, lightly stained spots could be attributed to e
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Fig. 2. Schematic representation of the 80 S ribosomal proteins extracted from 4-day-old male D.
melanogaster. Total ribosomal protein was extracted from 0 . 2 5 N HC1 as described by Lambertsson [21]. Two-dimensional eleetrophoresis was performed essentially according to the methods of Kaltsehmidt and Wittmann [22]. First dimension: 8% acrylamide (pH 8.6) run at a constant voltage (190 V) for 6.5 h at 4 °C. Second dimension: 18% acrylamide (pH 4.6) run at constant voltage (250 V) for 13-15 h at 4 °C. The intensities of the 56 basic and 11 acidic proteins are indicated by • < • < o.
109 supernatant proteins or protein synthesis factors which bind to ribosomes during isolation despite the high salt wash. Also, varying protein concentration allows one to determine if a given spot contains more than one protein. For instance, in Fig. ld the stained areas corresponding to proteins 5 and 7 and 27 and 28 cannot be ascribed to two proteins; however, in Fig. l b - d it becomes clear that each of these stained areas contains two individual proteins. Even with these precautions it is impossible to be certain with these techniques that only one protein is located in each stained area. Indeed, two proteins having different amino acid sequences may co-migrate provided they have the same molecular size and net charge. A schematic representation of the total ribosomal protein complement is presented in Fig. 2. Fifty.six basic and 11 acidic proteins were reproducibly observed. No qualitative differences were observed between male and female flies or as a function of age. With some exceptions, particularly with regards to the acidic proteins, these results are in good agreement with those previously reported for D. melanogaster by Lambertsson [17, 20, 24], for other Drosophila species [19, 25, 26], and with estimates of the number of proteins in eukaryotic ribosomes in general [27, 28]. The electrophoretic molibilities of the basic proteins in both dimensions were calculated from a series of 3-4 electrophoretograms for each age preparation relative to protein No. 48 which was taken as unity in each direction (Table I). The percentage error for each set of numbers at any age for any protein was generally less than +2% and in no case more than -+5%. Between each of the different preparations of ribosomal proteins from young, old, or young and old combined, no statistically significant differences in Rf values in either direction were observed. Due to the amount of total protein required for reproducible analysis, acidic proteins were not systematically examined in this study. It is doubtful, however, given the high degree of similarity in R e values for the basic proteins between young and old flies, that significant differences would be observed for the acidic proteins. This finding would indicate that the previously observed increase in the amounts of ribosomal protein dissociated from the high salt ribosomes from older flies' [12] and the decreased thermal stability of ribosomal monomers [15] is not due to alteration of ribosomal proteins but rather must reflect either changes in rRNA or the presence of other factors in the internal milieu which affect ribosomal stability in this species with advancing age.
ACKNOWLEDGEMENTS
We thank Drs. Kubli and Christian for their critical reading, of the manuscript, Ms P. C. Snelling for her skillful preparation thereof, and Mr. R. Sulzer for construction of the two-dimensional PAA gel apparatus. This work was supported in part ,by the KarlHerscheler-Stiftung, Drexel Research Scholar Award to G.T.B., and the Paul Glenn Foundation for Medical Research.
110 TABLE I RELATIVE MOB1LIT1ES OF BASIC RIBOSOMAL PROTEINS FROM MALE D. MELANOGASTER IN RELATION TO AGE The two numbers represent for each protein an Rf value in the first and second dimension relative to protein No. 48. The migration of protein No. 48 was taken as 1.0 in each dimension. Protein no.
4-Day-old
30-Day-oM
4-Day. + 30-day-old
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
0.30--0.18 0.51-0.20 0.34-0.26 0.48-0.27 0.62-0.31 0.46-0.32 0.67-0.32 0.79--0.36 0.65-0.38 0.73-0.36 0.21-0.35 0.55-0.36 0.13-0.39 0.35-0.39 0.69-0.40 0.80-0.41 0.69-0.46 0.45-0.45 0.57-0.47 0.12-0.50 0.70-0.51 0.13-0.51 0.27-0.55 0.08-0.55 0.70-0.54 0.70-0.56 0.77-0.56 0.84-0.59 0.51-0.59 0.32-0.61 0.61-0.61 0.41-0.63 0.73-0.61 0.69-0.65 0.76-0.66 0.88-0.65 1.07-0.68 0.28-0.65 0.90-0.70 0.28-0.70 0.97-0.72 0.82-0.77 1.08-0.76 0.31-0.78
0.32-0.18 0.48-0.20 0.35-0.26 0.47-0.26 0.61-0.31 0.48-0.32 0.69--0.30 0.76-0.36 0.64-0.36 0.70-0.35 0.24-0.37 0.56-0.37 0.12-0.38 0.35-0.38 0.68-0.39 0.78-0.41 0.67-0.43 0.44-0.45 0.56-0.46 0.13-0.50 0.69-0.50 0.14-0.51 0.26-0.54 0.08-0.55 0.68-0.55 0.68-0.57 0.77-0.57 0.82-0.58 0.50--0.58 0.33-0.60 0.6 0-0.60 0.40-0.62 0.71)-0.61 0.66-0.65 0.75-0.65 0.87-0.65 1.06-0.68 0.29-0.68 0.88-0.69 0.28-0.71 0.94-0.72 0.79-0.76 1.07-0.76 0.29-0.79
0.31--0.17 0.46-0.21 0.33-0.31 0.48-0.25 0.60-0.31 0.47-0.33 0.70-0.31 0.77-0.35 0.64-0.36 0.70-0.34 0.23-0.35 0.58-0.38 0.12-0.39 0.32-0.38 0.68-0.40 0.77-0.42 0.66-0.45 0.46-0.47 0.56-0.47 0.10-0.48 0.68-0.51 0.09-0.48 0.25-0.53 0.08-0.56 0.68-0.56 0.67-0.58 0.78-0.58 0.82-0.60 0.51-0.60 0.32-0.61 0.61-0.62 0.41-0.63 0.72-0.62 0.67-0.65 0.75-0.66 0.87-0.65 1.07-0.68 0.28-0.68 0.89-0.69 0.27-0.73 0.95-0.71 0.80--0.76 1.07-0.77 0.29-0.78 (Continued on facing page)
111 T A B L E I [Continued) 45 46 47 48 49 50 51 52 53 54 55 56
0.89-0.78 0.81-0.93 1.05-0.91 1 1.13-1.12 1.07-1.16 1.27-1.23 0.66-1.36 0.97-1.50 1.18-1.56 1.36-1.72 1.56-1.91
0.86-0.77 0.80-0.92 1.03-0.90 1 1.12-1.12 1.06-1.16 1.29-1.22 0.62-1.34 0.97-1.54 1.19-1.55 1.38-1.74 1.57-1.90
0.87-0.78 0.80-0.91 1.05-0.91 1 1.13-1.13 1.06-1.17 1.27-1.23 0.65-1.33 0.98-1.52 1.17-1.56 1.35-1.71 1.57-1.90
REFERENCES 1 P. A. Baumann and P. S. Chen, Alterung und Proteinsynthese bei Drosophila melanogaster. Rev. Suisse Zool., 75 (1968) 1051-1055. 2 L. Levenbook and I. Krishna, Effect of aging on amino acid turnover and rate of protein synthesis in the blow fly, Ph ormia regina. J. In sect Physiol., 17 ( 1971 ) 9-12. 3 C. Lang and Smith, Protein metabolism in the aging adult mosquito. Fed. Proc., 31 (1972) 878. 4 M. Rockstein and G. T. Baker, Effects of X-irradiation of pupae on aging of the thoracic flight muscle of the adult fly Musca Domestica L. Mech. AgeingDev., 3 (1974) 271-278. 5 W. I. P. Mainwaring, The effect of age on protein synthesis in mouse liver. Biochem. J., 113 (1969) 869--878. 6 J. P. Hrachovec, The effect of age on tissue protein synthesis. I. Age changes in amino acid incorporation by rat liver purified microsomes. Gerontologia, 17 (1971) 75-86. 7 G. W. Britton and F. G. Sherman, Altered regulation of protein synthesis during aging as determined by in vitro ribosomal assays. Exp. GerontoL, 10 (1975) 67-77. 8 J. R. Florini and R. N. Sorrentino, Protein metabolism during aging. In M. F. Elias, B. E. Elftheriou and P. K. Elias (eds.), Special Review of Experimental Aging Research. Progress in Biology. EAR, Inc., Bar Harbor, Maine, 1976. 9 J. T. Du, T. A. Beyer and C. A. Lang, Protein biosynthesis in aging mouse tissues. Exp. Gerontol, 12 (1977) 181-191. 10 J. C. Winterer, W. P. Steffee, W. Davy, A. Perera, R. Yang, N. S. Scrimshaw and V. R. Young, Whole body protein turnover in aging man. Exp. Gerontol., 11 (1976) 79-87. 11 J. P. Ogrodnik, J. H. Wolf and R. G. Cutler, Altered protein hypothesis of mammalian aging processes II. Discrimination ratio of methionine vs. ethionine in the synthesis of ribosomal protein and RNA of C57BI/6J mouse liver. Exp. Gerontol, 10 (1975) 119-136. 12 G. T. Baker and T. Schmidt, Changes in 80S ribosomes from Drosophila melanogaster with age. Experientia, 32 (1976) 1505-1506. 13 J. Maynard Smith, A. N. Bozcuk, and S. Tebbutt, Protein turnover in adult Drosophila. J. Insect Physiol., 16 (1970) 601-613. 14 Z. Wallach and D. Gershon, Altered ribosomal particles in senescent nematodes. Mech. Ageing Dev., 3 (1974) 225-234. 15 G. T. Baker, R. Zschunke and E. Podgorski, Analysis of ribosomal proteins from adult Drosophila melanogaster with age. Experientia, in press. 16 G. T. Baker, Age-dependent arginine phosphokinase activity changes in male vestigial and wildtype Drosophila melanogaster. Gerontologia, 21 ( 1975) 203-210. 17 A. G. Lambertsson, The ribosomal proteins of Drosophila melanogaster. III. Further studies on the ribosomal composition during development. Mol. Gen. Genet., 128 (1974) 241-247.
112 18 T. E. Martin, F. S. RoUeston, R. B. Low and I. G. Wool, Dissociation and reassociation of skeletal muscle ribosomes. J. Mol. Biol., 43 (1969) 135-149. 19 E. Berger, The ribosomes of Drosophila. I. Subunit and protein composition. Mol. Gen. Genet., 128 (1974) 1-9. 20 A. G. Lambertsson, S. B. Rau'nuson and G. D. Bloom, The ribosomal proteins of Drosophila melanogaster I. Characterization in polyacrylamide gels of proteins from larval, adult, and ammonium chloride treated ribosomes. Mol. Gen. Genet., 128 (1970) 241-247. 21 A. G. Lambertsson, The ribosomal proteins of Drosophila melanogaster. 1I. Comparison of protein patterns of ribosomes from larvae, pupae, and adult flies by two-dimensional polyacrylamide gel electrophoresis. Mol. Gen. Genet., 118 (1972) 215-222. 22 E. Kaltschmidt and H. G. Wittmann, Ribosomal proteins. VII. Two-dimensional polyacrylamide gel electrophoresis for fingerprinting of ribosomal proteins. Anal Biochem., 36 (1970) 401--412. 23 R. Howard and R. R, Traut, Separation and radioautography of microgram quanties of ribosomal proteins by two-dimensional PAA-gel electrophoresis. FEBS Lett., 29 (1973) 177-180. 24 A. G. Lambertsson, The ribosomal proteins of Drosophila melanogaster IV. Characterization by two-dimensional gel electrophoresis of the ribosomal proteins from nine postembryonic developmental stages. Mol. Gen. Genet., 129 (1975) 133-144. 25 E. Berger and L. Weber, The ribosomes of Drosophila II. Intraspecifie protein variation. Genetics, 78 (1974) 1173-1183. 26 L. Weber, E. Berger, C. Vaslet and B. Yedvobnick, The ribosomes of Drosophila III. RNA and protein homology between D. melanogaster and D. virilis. Genetics, 84 (1976) 573-585. 27 C. C. Sherton and I. G. Wool, Determination of the number of proteins in liver ribosomes and subunits by two-dimensional gel electrophoresis. J. Biol. Chem., 247 (1972) 4460--4467. 28 D. E. Leister and I. B. Dawid, Physical properties and protein constituents of cytoplasm and mitochondrial ribosomes of X~nopus laevis. J. Biol. Chem., 249 (1974) 5108-5118.
105
©Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
ANALYSIS OF RIBOSOMAL PROTEINS FROM ADULT DROSOPHILA MELANOGASTER IN RELATION TO AGE
T. SCHMIDT and G. T. BAKER* Zoologisch-vergl. anatomisches Institut der Universitat Zurich, CH-8806 (Switzerland] and (G. T.B.) Department of Biological Sciences, Drexel University, Philadelphia, PA, 19104 (U.S.A.)
(R~ceived March 5, 1979)
SUMMARY Analysis of high salt washed ribosomal proteins by two-dimensional polyacrylamide electrophoresis revealed no detectable qualitative differences in ribosomal proteins from young (4-day) and old (30-day) male flies.
INTRODUCTION A decline in the capacity for net protein synthesis is an apparent widespread correlate of the aging process, having been demonstrated in a number of invertebrates [1-4] and vertebrate species [5-9], including man [10]. Ribosomes, although occupying a central and integral position in translational processes, have been little investigated with regard to possible structural alterations which could effect the rate or fidelity of protein synthesis with advancing age. Some evidence suggesting possible age-related alterations in ribosomal structure and/or function has been presented. Ogrodnik et al. [11] reported a decrease in the discrimination ratio of methionine vs. ethionine in the synthesis of ribosomal proteins and rRNA in liver from aging rats. However, these authors reported no change in the number of ribosomal protein bands on one-dimensional SDS-polyacrylamide gels with age. Recently, direct evidence of altered structural integrity of the rRNA-protein complex was reported by Baker and Schmidt [12] wherein a five-fold increase in the amount of protein disassociated from high salt washed (0.5 M) ribosomes was observed between young and old flies in the presence of 2.0 M KC1. As ribosomes from adult Drosophila apparently turn over with half-life of approximately 10 days [ 13], this change could be the result of a loss of the fidelity of newly synthesized ribosomal protein for the rRNA, or vice versa, or both, through any number of mechanisms which alter the strength *To whom all inquiries should be addressed at Department of Biological Sciences, Drexel University,Philadelphia,PA, 19104, U.S.A.
106 of ionic bonding within the ribosomal complex. The decrease in thermal stability of ribosomal monomers reported for nematodes [ 14] and Drosophila [ 15 ] with age, could also reflect altered physicochemical interactions within the ribosomal complex with advancing age. The present studies were undertaken to evaluate the possibility of qualitative changes in ribosomal proteins which might account for the previously reported alterations in some physicochemical properties of ribosomes with age.
MATERIALS AND METHODS
Drosophila melanogaster (Sevelen strain) were reared from 4-6-day-old parents and maintained on a corn-sugar--agar--yeast medium in plexiglass cages (60 × 30 × 40 cm) under constant environmental conditions, 25 + 0.5 °C, 60 + 5% relative humidity and a 12:12 h light-dark cycle, as previously described [ 16]. Under these conditions males of this strain exhibit 50% and 90% mortalities of 29 and 40 days, respectively. Ribosomes were routinely extracted from 8 g (wet weight) of 4- and 30-day-old flies, or 4 g each of 4- and 30-day-old male flies, according to the methods of Lambertsson [17]. Optical density determinations of the high salt washed ribosomes revealed a high degree of accepted purity for eukaryotic ribosomes [18, 19] for all preparations; that is, OD26o/OD2so of 1.88 -+ 0.08 (n = 12), 1.86 + 0.09 (n = 11), and 1.87 -+ 0.07 (n = 4) for 4-day, 30-day, and 4- and 30-day-old ribosomal preparations, respectively. Total ribosomal protein from the high salt extracted ribosomes was recovered essentially according to the methods of Lambertsson [20, 21]. The ribosomal pellet was suspended in a small amount of deionized double-distilled H20 containing 6 mM mercaptoethanol and gently homogenized in a ground-glass homogenizer. The suspension was then brought to 3 ml with ice-cold I N HC1 added to a final concentration of 0.25 N. The mixture was stirred gently for 2 h at 4 °C and centrifuged at 48 000 g for 15 min, at 4 °C. The resulting pellet was then washed with 0.25 N HCI, centrifuged again as above, and the supernatants pooled. Ribosomal proteins were precipitated with 5 volumes of acetone at --27 °C overnight. After centrifugation (20 000 g for 20 min, at 4 °C), the protein precipitate was taken up in 0.3 ml of ice-cold formic acid, lyophilized, and stored at -27°C until use. Protein concentration was determined by the Folin procedure using recrystallized bovine serum albumin as a standard. Two-dimensional electrophoresis was carried out with some modifications according to the methods of Kaltschmidt and Wittman [22]. The modifications consisted of a decrease in electrophoresis time and reduction in the amount of protein which facilitated better separation and resolution, as described by Howard and Traut [23], as well as the use of a specially constructed L-shaped plexiglass gel former for the second dimension. All solutions except where noted were those used by Kaltschmidt and Wittmann [22]. One-dimensional electrophoresis was performed in 8% urea-polyacrylamide disc gels (0.3 × 13 cm) at pH 8.6, as previously described [23] except that the protein sample was not polymerized between two gels but applied in 25/21 of the electro-
107 phoresis buffer containing 8 M urea, 20% sucrose, and 10/al of 1% Pyronin G as a tracking dye. For analysis of basic proteins a series of concentrations (approx. 100, 250, 400, and 500/ag) of total ribosomal protein were subjected to standard electrophoresis. For good resolution of the acidic proteins 1.0-1.5 mg of the total ribosomal protein were required for the electrophoresis. Electrophoresis was performed at 4 °C. A constant voltage of 80 V was applied for the first 30 rain and thereafter 190 V for 6 h. For the two-dimensional electrophoresis, the first-dimension disc gel was first equilibrated against the starting buffer for 1 h at 25 °C, then polymerized on to an 18% urea-acrylamide slab gel (2.5 × 124 × 160 mm). The voltage was held constant at I00 V for 30 min and thereafter at 250 V for 13-15 h. Following electrophoresis, proteins were visualized by staining with 0.1% Coomassie Brilliant Blue dissolved in a 7.5% acetic acid-50% methanol solution for 12 h at 25 °C. The gels were destained at room temperature in a 5:5:1 mixture of methanol, water, and acetic acid for 48 h, and immediately analyzed and photographed.
A"
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~,
0 ~
I-D
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e
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i i i i ~i~!ii,i i ~: ~
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Fig. la-d. Two-dimensional electrophoreto~Fam of ribosomal proteins extracted from high salt washed ribosomes from 30-day-old male 1). melanogaster. First dimension: 8% acrylamide (pH 8.6) run at a
constant voltage (190 V) for 6.5 h at 4 °C. Second dimension: 18% acrylamide (pH 4.6) run at a constant voltage (250 V) for 13-15 h at 4 °C. Fig. l a - d are the electrophoretograms of increasing
concentrations of ribosomal proteins of approximately 80, 250, 400, and 500 ag, respectively.
108 RESULTS AND DISCUSSION
Figure la-d presents photographs of a typical series of two-dimensional electrophoretograms performed for ribosomal proteins extracted from 30-day-old male flies. This series is representative of the results obtained for all ages studied. Due to a number of factors, a series of protein concentrations is necessary to visualize all proteins and to be certain of their ribosomal origin; for example, lightly stained spots could be attributed to e
1-D
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Fig. 2. Schematic representation of the 80 S ribosomal proteins extracted from 4-day-old male D.
melanogaster. Total ribosomal protein was extracted from 0 . 2 5 N HC1 as described by Lambertsson [21]. Two-dimensional eleetrophoresis was performed essentially according to the methods of Kaltsehmidt and Wittmann [22]. First dimension: 8% acrylamide (pH 8.6) run at a constant voltage (190 V) for 6.5 h at 4 °C. Second dimension: 18% acrylamide (pH 4.6) run at constant voltage (250 V) for 13-15 h at 4 °C. The intensities of the 56 basic and 11 acidic proteins are indicated by • < • < o.
109 supernatant proteins or protein synthesis factors which bind to ribosomes during isolation despite the high salt wash. Also, varying protein concentration allows one to determine if a given spot contains more than one protein. For instance, in Fig. ld the stained areas corresponding to proteins 5 and 7 and 27 and 28 cannot be ascribed to two proteins; however, in Fig. l b - d it becomes clear that each of these stained areas contains two individual proteins. Even with these precautions it is impossible to be certain with these techniques that only one protein is located in each stained area. Indeed, two proteins having different amino acid sequences may co-migrate provided they have the same molecular size and net charge. A schematic representation of the total ribosomal protein complement is presented in Fig. 2. Fifty.six basic and 11 acidic proteins were reproducibly observed. No qualitative differences were observed between male and female flies or as a function of age. With some exceptions, particularly with regards to the acidic proteins, these results are in good agreement with those previously reported for D. melanogaster by Lambertsson [17, 20, 24], for other Drosophila species [19, 25, 26], and with estimates of the number of proteins in eukaryotic ribosomes in general [27, 28]. The electrophoretic molibilities of the basic proteins in both dimensions were calculated from a series of 3-4 electrophoretograms for each age preparation relative to protein No. 48 which was taken as unity in each direction (Table I). The percentage error for each set of numbers at any age for any protein was generally less than +2% and in no case more than -+5%. Between each of the different preparations of ribosomal proteins from young, old, or young and old combined, no statistically significant differences in Rf values in either direction were observed. Due to the amount of total protein required for reproducible analysis, acidic proteins were not systematically examined in this study. It is doubtful, however, given the high degree of similarity in R e values for the basic proteins between young and old flies, that significant differences would be observed for the acidic proteins. This finding would indicate that the previously observed increase in the amounts of ribosomal protein dissociated from the high salt ribosomes from older flies' [12] and the decreased thermal stability of ribosomal monomers [15] is not due to alteration of ribosomal proteins but rather must reflect either changes in rRNA or the presence of other factors in the internal milieu which affect ribosomal stability in this species with advancing age.
ACKNOWLEDGEMENTS
We thank Drs. Kubli and Christian for their critical reading, of the manuscript, Ms P. C. Snelling for her skillful preparation thereof, and Mr. R. Sulzer for construction of the two-dimensional PAA gel apparatus. This work was supported in part ,by the KarlHerscheler-Stiftung, Drexel Research Scholar Award to G.T.B., and the Paul Glenn Foundation for Medical Research.
110 TABLE I RELATIVE MOB1LIT1ES OF BASIC RIBOSOMAL PROTEINS FROM MALE D. MELANOGASTER IN RELATION TO AGE The two numbers represent for each protein an Rf value in the first and second dimension relative to protein No. 48. The migration of protein No. 48 was taken as 1.0 in each dimension. Protein no.
4-Day-old
30-Day-oM
4-Day. + 30-day-old
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
0.30--0.18 0.51-0.20 0.34-0.26 0.48-0.27 0.62-0.31 0.46-0.32 0.67-0.32 0.79--0.36 0.65-0.38 0.73-0.36 0.21-0.35 0.55-0.36 0.13-0.39 0.35-0.39 0.69-0.40 0.80-0.41 0.69-0.46 0.45-0.45 0.57-0.47 0.12-0.50 0.70-0.51 0.13-0.51 0.27-0.55 0.08-0.55 0.70-0.54 0.70-0.56 0.77-0.56 0.84-0.59 0.51-0.59 0.32-0.61 0.61-0.61 0.41-0.63 0.73-0.61 0.69-0.65 0.76-0.66 0.88-0.65 1.07-0.68 0.28-0.65 0.90-0.70 0.28-0.70 0.97-0.72 0.82-0.77 1.08-0.76 0.31-0.78
0.32-0.18 0.48-0.20 0.35-0.26 0.47-0.26 0.61-0.31 0.48-0.32 0.69--0.30 0.76-0.36 0.64-0.36 0.70-0.35 0.24-0.37 0.56-0.37 0.12-0.38 0.35-0.38 0.68-0.39 0.78-0.41 0.67-0.43 0.44-0.45 0.56-0.46 0.13-0.50 0.69-0.50 0.14-0.51 0.26-0.54 0.08-0.55 0.68-0.55 0.68-0.57 0.77-0.57 0.82-0.58 0.50--0.58 0.33-0.60 0.6 0-0.60 0.40-0.62 0.71)-0.61 0.66-0.65 0.75-0.65 0.87-0.65 1.06-0.68 0.29-0.68 0.88-0.69 0.28-0.71 0.94-0.72 0.79-0.76 1.07-0.76 0.29-0.79
0.31--0.17 0.46-0.21 0.33-0.31 0.48-0.25 0.60-0.31 0.47-0.33 0.70-0.31 0.77-0.35 0.64-0.36 0.70-0.34 0.23-0.35 0.58-0.38 0.12-0.39 0.32-0.38 0.68-0.40 0.77-0.42 0.66-0.45 0.46-0.47 0.56-0.47 0.10-0.48 0.68-0.51 0.09-0.48 0.25-0.53 0.08-0.56 0.68-0.56 0.67-0.58 0.78-0.58 0.82-0.60 0.51-0.60 0.32-0.61 0.61-0.62 0.41-0.63 0.72-0.62 0.67-0.65 0.75-0.66 0.87-0.65 1.07-0.68 0.28-0.68 0.89-0.69 0.27-0.73 0.95-0.71 0.80--0.76 1.07-0.77 0.29-0.78 (Continued on facing page)
111 T A B L E I [Continued) 45 46 47 48 49 50 51 52 53 54 55 56
0.89-0.78 0.81-0.93 1.05-0.91 1 1.13-1.12 1.07-1.16 1.27-1.23 0.66-1.36 0.97-1.50 1.18-1.56 1.36-1.72 1.56-1.91
0.86-0.77 0.80-0.92 1.03-0.90 1 1.12-1.12 1.06-1.16 1.29-1.22 0.62-1.34 0.97-1.54 1.19-1.55 1.38-1.74 1.57-1.90
0.87-0.78 0.80-0.91 1.05-0.91 1 1.13-1.13 1.06-1.17 1.27-1.23 0.65-1.33 0.98-1.52 1.17-1.56 1.35-1.71 1.57-1.90
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