Vol.
187,
September
No.
2, 1992
BIOCHEMICAL
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16, 1992
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Pages 1071-1076
POLYPEPTIDE COMPOSITION OF THE 8s FORM OF PROLYL-tRNA SYNTHETASE FROM RAT LIVER Cristina Bianchi, Roberto Peregoand Ugo Del Monte Institute of General Pathology, University of Milan0 and C.N.R. Center for Researchin Cellular Pathology, Via L. Mangiagalli 31,20133 Milano, Italy Received
August
3, 1992
SUMMARY - Rat liver Fraction X containing the 24s complex of nine aminoacyl-tRNA synthetases,including prolyl-tRNA synthetase, was centrifuged on a 1535% sucrosedensity gradient to obtain the 8s form of prolyl-tRNA synthetase. The enzyme was purified on a prolyldiaminohexyl-Sepharose4B affinity column, specifically binding prolyl-tRNA synthetaseto Sepharose-boundproline. After SDS-polyacrylamide gel electrophoresis,two peptidesof 58 and 61 kDa were detected in the peak of prolyl-tRNA synthetaseactivity eluted from the affinity column. The 58 and 61 kDa peptides were also present in the 24s complex containing prolyltRNA synthetaseactivity isolatedon the sucrosedensity gradient. z 199~~~~~~~~~ ~~~~~~ inc.
The aminoacyl-tRNA synthetases(EC 6.1.1), a ubiquitous class of enzymes essential to aminoacidactivation andtRNA aminoacylationduring protein synthesis,may be obtainedfrom the higher eukaryotic cell cytosol either asmultiple high molecular weight complexesor asfree forms or both. Different-sized complexescontaining severalaminoacyl-tRNA synthetaseactivities were isolated from different tissues(1,2). The largest stablecomplex contains nine synthetases(the specific ones for Lys, Arg, Ile, Leu, Met, Gln, Glu, Asp, Pro) and sedimentsat 24s (3-5). Prolyl-tRNA synthetaseis bounded to the 24s complex in a labile way and a stable complex containing prolyl-tRNA synthetaseactivity wasonly isolatedin a few cases(3,4,6-g). Under mild conditions of homogenization, we constantly isolated a stable 24s complex of nine aminoacyltRNA synthetasesfrom both Yoshida hepatomacells (6) and rat liver. Furthermore, we observed that the prolyl-tRNA
synthetasepartition between the noncomplex-bound form (KS) and the
complex-bound form (24s) shifts towards the latter in growing tissues(5). In the literature, different methodshave beenusedto identify the peptide composition of the 24s complex (2). It hasrecently been publishedthat the largestpolypeptide in sheepliver complex and in Drosophila complex is a bifunctional protein encoding both glutamyl-tRNA synthetase and prolyl-tRNA synthetaseactivity (9,lO). We report here a rapid and simpleprocedureto identify and isolate the peptidesassociatedwith the 8s foml of rat liver prolyl-tRNA synthetaseactivity. The procedure, based upon density gradient centrifugation followed by affinity chromatography on prolyldiaminohexyl-Sepharose4B, takes advantageof the specific interaction betweenSepharose-
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bound proline and the functional forms of prolyl-tRNA synthetase in the presence of ATP (11). A preliminary account of this paper has already been published as an abstract (12).
MATERIALS
AND METHODS
[U-t4C]-L-proline (264 Ci/mol) was from Amersham, Bucks, U.K. Diorthonitrophenylsulfenyl proline (di-ONPS-Pro) was from SIGMA, St. Louis, MO, USA. Diaminohexyl-Sepharose 4B (AH-Sepharose 4B; 6-10 l.rmoles/ml capacity) was from Pharmacia, Uppsala, Sweden. DEAEcellulose (DE-52; 1 mequiv/g capacity) was from Whatman, Maidstone, U.K. Molecular weight protein standards were from BioRad, Richmond, CA, USA. Gradient calibration markers were as described (5). All other chemicals were analytical grade, in the purest form available. tRNA was prepared from rat liver as reported (5). Aminoacylation assays described in ref. 5 (Method B) were performed at 37” C with a reaction time of 2 or 10 minutes in a volume of 100 ~1 containing 5.5 mM (U-t4C)-L-proline (0.15 PCi) and 9.5 mM L-proline. One unit of enzyme activity is the amount of enzyme required to form 1 nmol of aminoacyl-tRNA/min. Direct coupling of di-ONPS-proline to AH-Sepharose 4B was carried out according to Robert-Gero and Waller (11) and a 3 ml affinity column was prepared and equilibrated with buffer A (0.2 M potassium phosphate, pH 7.5,25 mM KCl, 5 mM Mg Acetate, 2 mM dithiothreitol and 1 mM ATP). SDSpolyacrylamide gel electrophoresis was performed either according to Laemmli (13) (T= 10%; C=2.6%) or Fairbanks et al. (14) (T=5.6%; C=3.6%; SDS=l%). Densitometric tracings of gel patterns were obtained with an LKB 2202 Ultroscan Laser Densitometer. Protein was assayed according to Bradford et al. (15). All the following procedures were performed at 0-4°C unless otherwise mentioned. Male Wistar rats (2-3 months old), housed and fed as reported (5), were killed by decapitation after 12-h starvation, livers were dissected and homogenized (16). The homogenization medium was 0.35 M sucrose containing 50 mM Tris/HCl, pH 7.5, 25 mM KCl, 5 mM magnesium acetate, 2 mM dithiothreitol, 0.44 mM phenylmethylsulfonylfluoride. Fraction X (17) (20 mg) obtained from cell sap was layered on the top of a 25 ml exponential density gradient (15-35% w/v sucrose containing all other components of the homogenization medium) (16). Centrifugation was at 22500 rpm for 18 h at 2°C in a Beckman-Spinco rotor SW27. 2 ml fractions were collected and assayed for aminoacyl-tRNA synthetase activities. The gradient was calibrated with several markers covering the 4-288 range (5). The prolyl-tRNA synthetase fraction (0.8 mg) from 8s sucrose gradient region (fraction 3) was Ioaded on the affinity coIumn. Elution was carried out at 10°C with Buffer A (20 ml) at first and later with 20 r&l L-proline in Buffer A (30 ml) to elute the purified prolyl-tRNA synthetase. 1 ml fractions were collected and assayed for aminoacyl-tRNA synthetase activities. 2 ug of affinity-column purified prolyl-tRNA synthetase (fraction 23) was analyzed according to Laemmli (13) and 14 ug of 24s aminoacyl-tRNA synthetase complex from the sucrose density gradient (fraction 9) according to Fairbanks et al. (14).
RESULTS
AND
DISCUSSION
A rapid and simple method was used to identify on an analytical scale the peptides associated with prolyl-tRNA synthetase activity. Rat livers were homogenized under very mild procedures (16) in order to obtain the 24s complex containing nine aminoacyl-tRNA synthetases. A “Fraction X” (17) enriched with 24s complex, prepared from cell sap by ultracentrifugation, was sedimented on a 15-35% sucrose density gradient (Fig. l), to obtain roughly-purified synthetase. The sedimentation pattern of prolyl-tRNA synthetase activity on
8s prolyl-tRNA the density gradient
shows two major peaks. The first one, sedimenting around 8S, contains both part of the complex bound form of prolyl-tRNA synthetase detached from 24s complex during the gradient sedimentation (18) and the free form, present in the cytosol and pelleted in Fraction X during its preparation. The second peak contains the prolyl-tRNA synthetase bound to the 24s complex. Prolyl-tRNA synthetase sedimented in fraction 3 of the sucrose gradient was purified on the 1072
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NUMBER
Fig. Sucrose density gradient sedimentation of prolyl-tRNA synthetase activity. 20 mg of Fraction X was layered on a 1535% w/v sucrose density gradient. 2 ml fractions were collected and 16 pl assayed for 2 minutes at 37O C. Arrows indicate sedimentation markers. 0-a ProlyltRNA synthetase activity; gsz Protein concentration.
affinity column (Fig. 2). This steptakesadvantageof the specificity of the aminoacylationreaction and of the ability of this affinity chromatography to separatefunctional from non-functiona enzyme (11). The column eluate assayedfor prolyl-tRNA synthetaseactivity showsa main peak
5
10
15
20
25
30
35
40
45
50
FRACTION NUMBER
Fig. 2. Purification of prolyl-tRNA synthetase by affinity chromatography on prolyldiaminohexylSepharose column. Protein (0.8 mg) from fraction 3 of the density gradient (Fig. 1) was loaded on affinity column (3.8 x 1 cm). After washing the column with buffer A (20 ml), the enzyme was eluted with 20 mh4 L-proline in Buffer A (30 ml, 10 ml/In). 1 ml fractions were collected and 16 pl assayed for 10 min. at 37’ C. 0-O Prolyl-tRNA synthetase activity.
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Table 1 - Prolyl-tRNA synthetase activity from 2.9 g rat liver during purification Steps
Cellsap FractionX
Protein
Activity
Specific
Recovery (mg)
Recovery (units)
(units/mg)
180
56
20
Activity
0.31
9.88
0.49 0.24 2.65
DensityGradient region8s (fraction3) region24s(fraction9)
3.8 0.2
0.92 0.53
Affinity Chromatography (fraction23)
0.0033
8.10
2454
containingnearly all its activity, without other aminoacyl-tRNA synthetaseactivities. AS shownin Table 1, the specific activity of prolyl-tRNA synthetaseisolatedby this method is very high. The presencein the cytosol of someinhibitors that affect the enzymatic activity and are lost during the purification procedures, might explain these data. Inhibitors severely affecting prolyl-tRNA synthetaseactivity have recently beendescribed(19). Moreover, we shouldstressthat the reported activity values reflect the non-Iinear kinetic propertiesof the free form of prolyl-tRNA synthetase that interfere with accuratepurification measurement,asalsonoted by others(9). The SDS-polyacrylamide gel electrophoresisanalysisof the fraction containing the prolyl-tRNA synthetaseactivity peak eluted from the affinity column showedtwo peptidesof 58 and 61 kDa, with a molar ratio of about 1 (Fig. 3). In literature, it has beenreported as unpublisheddata that the same-sizedpeptides were presentin the purified prolyl-tRNA synthetasefrom rat and rabbit liver (7). In our preparation of the 24s complex, obtained by centrifugation on a 15-35% sucrose density gradient, the 58 and 61 kDa peptidesare also clearly represented.Our 24s complex also contains the bandscorrespondingto the other eight aminoacyl-tRNA synthetasesand somestill unidentified bandsdescribedin the literature (Fig. 4) (2). To characterize the 8s form of prolyltRNA synthetasebetter, the main peak eluted from the affinity column wascentrifuged on a linear 7-20% sucrosedensity gradient. The prolyl-tRNA synthetaseactivity sedimentedaround 8S, the sedimentationvalue typical of proteinsof about 150 kDa (20). In addition to this, the gel filtrate of rat liver cell sap showed an heterogeneousprofile of prolyl-tRNA synthetase activity whose smallest molecular weight peak was 120 kDa (data not shown). The sedimentation and gel filtration data suggestthat the 8s prolyl-tRNA synthetaseexists in a dimeric form. Our Iater preparationsof prolyl-tRNA synthetasefrom rat liver by a different method specifically aimed at purifying the free form only,revealed no heterodimeric structure (unpublisheddata), like E. cob. prolyl-tRNA synthetase(21). Perhapsthe 58 and 61 kDa peptides are analogousto the recently describedtwo different forms of arginyl-tRNA synthetase(22), one a complex-bound form (72 kDa> with a hydrophobic NH, terminal extension and the other a free form (60 l&a). Even in our purified preparation of prolyl-tRNA synthetase,the two peptidesmight representthe complex1074
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kDa kDa
109 98
iW9.S GlnPS
= -ZZ
“3; * 24
*
m SDS-polyacrylamide gel electrophoresis, according to Laemmli (13), of purified prolyltRNA synthetase. Lane 1: Protein standards. Lane 2: fraction 23 of Fig. 2 (2 pg loaded). The corresponding densitomemc pattern is shown. Fig,. 4. SDS-polyacrylamide gel electrophoresis, according to Fairbanks et al (14), of 24s aminoacyl-tRNA synthetase complex. Lane 1: protein standards. Lane 2: fraction 9 of Fig. 1 (14 pg loaded). The protein bands are assigned to synthetases according to the literature (2). The corresponding densitomemc pattern is shown. * Unidentified peptides.
bound form of the synthetase detached from the 24s complex during sucrose density gradient sedimentation (18) and the native free form of prolyl-tRNA
synthetase pelleted in the Fraction X
during its preparation. Recent reports suggest that during the preparation of the 24s complex from sheep liver, the complex-bound form of prolyl-tRNA synthetase is excised from a larger bifunctional
peptide encoding glutamyl-
and prolyl-tRNA
synthetase activity, by a proteolytic
reaction. As a matter of fact, in this peptide, the region joining the two catalytic domains can be a proteolytic target (9). If this should be the case in rat liver, too, the two different-sized could be the result of proteolytic
peptides
cleavage in different sites of the joining region, which might
bring about different functional forms of prolyl-tRNA
synthetase. The preliminary evidence that
the 24s complex is coeluted from an affinity column and cosediments on a density gradient with an ATP-independent protease activity (23, 24) is of particular interest. This tight association between the 24s complex and the proteases could also account for the low molecular weight peptides always present in the various preparations of the 24s complex (3,7,10 and fig. 4).
ACKNOWLEDGMENTS C. Bianchi is supported by a fellowship from the Associazione We are grateful to G. Martinotti for typing the manuscript, to technical assistance. These studies are supported by Consiglio grants from Minister0 dell’Universit8 e della Ricerca Scientifica
Italiana per la Ricerca sul Cancro. E. Gavazzeni and M. Urban0 for Nazionale delle Ricerche and by e Tecnologica (40% and 60%).
REFERENCES ::
Dang, C.V.and Dang, C.V. (1986) Biochem. J. 239,249-255. Mirande, M. (1991) Progress Nucl. Acid Res. Mol. Biol.40,951075
142.
Vol.
187,
3. 54: 6. 7. ; 10: 11. 12. 13. 14. E: 17. 18. ii: 21. 2;: 24.
No.
2, 1992
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AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Cirakoglu, B. and Waller, J.P. (1985) Biochem. Biophys. Acta 829, 173-179. Mirande, M., Le Corre, D. and Wailer, J.P. (1985) Eur. J. Biochem. 147, 281-289. Del Monte, U., Capaccioli, S., Neri Cini, G., Perego, R., Caldini, R. and Chevanne, M. (1986) Biochem. J. 236, 163-169. Perego, R. and Del Monte, U. (1986) CeII Biol. Int. Rep. 10,477. Godar, D.E., Godar, D.E., Garcia, V., Jacabo, A., Aebi, U. and Yang, D.C.H. (1988) Biochemistry 27,692 l-6928. Norcum, M.T. (1989) J. Biol. Chem.264, 15043-1505 1. Kerjan, P., Triconnet, M. and Waller, J.P. (1992) Biochimie 74, 195205. Cerini, G., Kerjan, P., Astier, M., Grotecas, D., Mirande, M. and Semeriva, M.(1991) EMBO J. 10,4267-4277. Robert Gero, M. and Wailer, J.P. (1974) Methods Enzymol. 24,506-5 13. Bianchi, C., Perego, R. and Del Monte, U. (1990) Cell Biol. Int. Rep. 14 abstracts suppl., 14. Laemmli, U.K. (1970) Nature 227, 680-685. Fairbanks, G., Steck, T.L., Wallech, D.F.H. (1971) Biochemistry 10, 2606-2617. Bradfor, M.M. (1976) Anal. Biochem. 72, 248-258. Perego, R., Riccio, D. and Del Monte, U. (1982) IRCS Med. Sci. 10, 536-537. Venegoor, C. and Bloemendal, H. (1976) Eur. J. B&hem. 15, 161-170. Dang, C.V. and Yang D.C.H.(1979) J. Biol. Chem.254, 5350-5356. Whelly, S.M. and Barker, K.M. (1985) Eur. J. Biochem. 146, 245-253. Price, C.A. (1982) Centrifugation in Density Gradients, ~~323-326. Academic Press, New York. Eriani,G., Delarue, M., Poch, O., Gangloff, J. and Moras, D.(1990) Nature 347, 203206. Huang, S. and Deutscher, M.P. (1991) Biochem. Biophys. Res. Comm. 180,702-708. Bianchi, C., Costa, R., Perego, R. and Del Monte, U. (1989) Eur. J. Cell. Biol. 49,suppl. 28, 7. Bianchi, C. (1991) Thesis, research doctorate in experimental pathology, University of Firenze.
1076
187,
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No.
2, 1992
BIOCHEMICAL
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COMMUNICATIONS
Pages 1071-1076
POLYPEPTIDE COMPOSITION OF THE 8s FORM OF PROLYL-tRNA SYNTHETASE FROM RAT LIVER Cristina Bianchi, Roberto Peregoand Ugo Del Monte Institute of General Pathology, University of Milan0 and C.N.R. Center for Researchin Cellular Pathology, Via L. Mangiagalli 31,20133 Milano, Italy Received
August
3, 1992
SUMMARY - Rat liver Fraction X containing the 24s complex of nine aminoacyl-tRNA synthetases,including prolyl-tRNA synthetase, was centrifuged on a 1535% sucrosedensity gradient to obtain the 8s form of prolyl-tRNA synthetase. The enzyme was purified on a prolyldiaminohexyl-Sepharose4B affinity column, specifically binding prolyl-tRNA synthetaseto Sepharose-boundproline. After SDS-polyacrylamide gel electrophoresis,two peptidesof 58 and 61 kDa were detected in the peak of prolyl-tRNA synthetaseactivity eluted from the affinity column. The 58 and 61 kDa peptides were also present in the 24s complex containing prolyltRNA synthetaseactivity isolatedon the sucrosedensity gradient. z 199~~~~~~~~~ ~~~~~~ inc.
The aminoacyl-tRNA synthetases(EC 6.1.1), a ubiquitous class of enzymes essential to aminoacidactivation andtRNA aminoacylationduring protein synthesis,may be obtainedfrom the higher eukaryotic cell cytosol either asmultiple high molecular weight complexesor asfree forms or both. Different-sized complexescontaining severalaminoacyl-tRNA synthetaseactivities were isolated from different tissues(1,2). The largest stablecomplex contains nine synthetases(the specific ones for Lys, Arg, Ile, Leu, Met, Gln, Glu, Asp, Pro) and sedimentsat 24s (3-5). Prolyl-tRNA synthetaseis bounded to the 24s complex in a labile way and a stable complex containing prolyl-tRNA synthetaseactivity wasonly isolatedin a few cases(3,4,6-g). Under mild conditions of homogenization, we constantly isolated a stable 24s complex of nine aminoacyltRNA synthetasesfrom both Yoshida hepatomacells (6) and rat liver. Furthermore, we observed that the prolyl-tRNA
synthetasepartition between the noncomplex-bound form (KS) and the
complex-bound form (24s) shifts towards the latter in growing tissues(5). In the literature, different methodshave beenusedto identify the peptide composition of the 24s complex (2). It hasrecently been publishedthat the largestpolypeptide in sheepliver complex and in Drosophila complex is a bifunctional protein encoding both glutamyl-tRNA synthetase and prolyl-tRNA synthetaseactivity (9,lO). We report here a rapid and simpleprocedureto identify and isolate the peptidesassociatedwith the 8s foml of rat liver prolyl-tRNA synthetaseactivity. The procedure, based upon density gradient centrifugation followed by affinity chromatography on prolyldiaminohexyl-Sepharose4B, takes advantageof the specific interaction betweenSepharose-
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2, 1992
BIOCHEMICAL
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RESEARCH
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bound proline and the functional forms of prolyl-tRNA synthetase in the presence of ATP (11). A preliminary account of this paper has already been published as an abstract (12).
MATERIALS
AND METHODS
[U-t4C]-L-proline (264 Ci/mol) was from Amersham, Bucks, U.K. Diorthonitrophenylsulfenyl proline (di-ONPS-Pro) was from SIGMA, St. Louis, MO, USA. Diaminohexyl-Sepharose 4B (AH-Sepharose 4B; 6-10 l.rmoles/ml capacity) was from Pharmacia, Uppsala, Sweden. DEAEcellulose (DE-52; 1 mequiv/g capacity) was from Whatman, Maidstone, U.K. Molecular weight protein standards were from BioRad, Richmond, CA, USA. Gradient calibration markers were as described (5). All other chemicals were analytical grade, in the purest form available. tRNA was prepared from rat liver as reported (5). Aminoacylation assays described in ref. 5 (Method B) were performed at 37” C with a reaction time of 2 or 10 minutes in a volume of 100 ~1 containing 5.5 mM (U-t4C)-L-proline (0.15 PCi) and 9.5 mM L-proline. One unit of enzyme activity is the amount of enzyme required to form 1 nmol of aminoacyl-tRNA/min. Direct coupling of di-ONPS-proline to AH-Sepharose 4B was carried out according to Robert-Gero and Waller (11) and a 3 ml affinity column was prepared and equilibrated with buffer A (0.2 M potassium phosphate, pH 7.5,25 mM KCl, 5 mM Mg Acetate, 2 mM dithiothreitol and 1 mM ATP). SDSpolyacrylamide gel electrophoresis was performed either according to Laemmli (13) (T= 10%; C=2.6%) or Fairbanks et al. (14) (T=5.6%; C=3.6%; SDS=l%). Densitometric tracings of gel patterns were obtained with an LKB 2202 Ultroscan Laser Densitometer. Protein was assayed according to Bradford et al. (15). All the following procedures were performed at 0-4°C unless otherwise mentioned. Male Wistar rats (2-3 months old), housed and fed as reported (5), were killed by decapitation after 12-h starvation, livers were dissected and homogenized (16). The homogenization medium was 0.35 M sucrose containing 50 mM Tris/HCl, pH 7.5, 25 mM KCl, 5 mM magnesium acetate, 2 mM dithiothreitol, 0.44 mM phenylmethylsulfonylfluoride. Fraction X (17) (20 mg) obtained from cell sap was layered on the top of a 25 ml exponential density gradient (15-35% w/v sucrose containing all other components of the homogenization medium) (16). Centrifugation was at 22500 rpm for 18 h at 2°C in a Beckman-Spinco rotor SW27. 2 ml fractions were collected and assayed for aminoacyl-tRNA synthetase activities. The gradient was calibrated with several markers covering the 4-288 range (5). The prolyl-tRNA synthetase fraction (0.8 mg) from 8s sucrose gradient region (fraction 3) was Ioaded on the affinity coIumn. Elution was carried out at 10°C with Buffer A (20 ml) at first and later with 20 r&l L-proline in Buffer A (30 ml) to elute the purified prolyl-tRNA synthetase. 1 ml fractions were collected and assayed for aminoacyl-tRNA synthetase activities. 2 ug of affinity-column purified prolyl-tRNA synthetase (fraction 23) was analyzed according to Laemmli (13) and 14 ug of 24s aminoacyl-tRNA synthetase complex from the sucrose density gradient (fraction 9) according to Fairbanks et al. (14).
RESULTS
AND
DISCUSSION
A rapid and simple method was used to identify on an analytical scale the peptides associated with prolyl-tRNA synthetase activity. Rat livers were homogenized under very mild procedures (16) in order to obtain the 24s complex containing nine aminoacyl-tRNA synthetases. A “Fraction X” (17) enriched with 24s complex, prepared from cell sap by ultracentrifugation, was sedimented on a 15-35% sucrose density gradient (Fig. l), to obtain roughly-purified synthetase. The sedimentation pattern of prolyl-tRNA synthetase activity on
8s prolyl-tRNA the density gradient
shows two major peaks. The first one, sedimenting around 8S, contains both part of the complex bound form of prolyl-tRNA synthetase detached from 24s complex during the gradient sedimentation (18) and the free form, present in the cytosol and pelleted in Fraction X during its preparation. The second peak contains the prolyl-tRNA synthetase bound to the 24s complex. Prolyl-tRNA synthetase sedimented in fraction 3 of the sucrose gradient was purified on the 1072
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Fig. Sucrose density gradient sedimentation of prolyl-tRNA synthetase activity. 20 mg of Fraction X was layered on a 1535% w/v sucrose density gradient. 2 ml fractions were collected and 16 pl assayed for 2 minutes at 37O C. Arrows indicate sedimentation markers. 0-a ProlyltRNA synthetase activity; gsz Protein concentration.
affinity column (Fig. 2). This steptakesadvantageof the specificity of the aminoacylationreaction and of the ability of this affinity chromatography to separatefunctional from non-functiona enzyme (11). The column eluate assayedfor prolyl-tRNA synthetaseactivity showsa main peak
5
10
15
20
25
30
35
40
45
50
FRACTION NUMBER
Fig. 2. Purification of prolyl-tRNA synthetase by affinity chromatography on prolyldiaminohexylSepharose column. Protein (0.8 mg) from fraction 3 of the density gradient (Fig. 1) was loaded on affinity column (3.8 x 1 cm). After washing the column with buffer A (20 ml), the enzyme was eluted with 20 mh4 L-proline in Buffer A (30 ml, 10 ml/In). 1 ml fractions were collected and 16 pl assayed for 10 min. at 37’ C. 0-O Prolyl-tRNA synthetase activity.
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Table 1 - Prolyl-tRNA synthetase activity from 2.9 g rat liver during purification Steps
Cellsap FractionX
Protein
Activity
Specific
Recovery (mg)
Recovery (units)
(units/mg)
180
56
20
Activity
0.31
9.88
0.49 0.24 2.65
DensityGradient region8s (fraction3) region24s(fraction9)
3.8 0.2
0.92 0.53
Affinity Chromatography (fraction23)
0.0033
8.10
2454
containingnearly all its activity, without other aminoacyl-tRNA synthetaseactivities. AS shownin Table 1, the specific activity of prolyl-tRNA synthetaseisolatedby this method is very high. The presencein the cytosol of someinhibitors that affect the enzymatic activity and are lost during the purification procedures, might explain these data. Inhibitors severely affecting prolyl-tRNA synthetaseactivity have recently beendescribed(19). Moreover, we shouldstressthat the reported activity values reflect the non-Iinear kinetic propertiesof the free form of prolyl-tRNA synthetase that interfere with accuratepurification measurement,asalsonoted by others(9). The SDS-polyacrylamide gel electrophoresisanalysisof the fraction containing the prolyl-tRNA synthetaseactivity peak eluted from the affinity column showedtwo peptidesof 58 and 61 kDa, with a molar ratio of about 1 (Fig. 3). In literature, it has beenreported as unpublisheddata that the same-sizedpeptides were presentin the purified prolyl-tRNA synthetasefrom rat and rabbit liver (7). In our preparation of the 24s complex, obtained by centrifugation on a 15-35% sucrose density gradient, the 58 and 61 kDa peptidesare also clearly represented.Our 24s complex also contains the bandscorrespondingto the other eight aminoacyl-tRNA synthetasesand somestill unidentified bandsdescribedin the literature (Fig. 4) (2). To characterize the 8s form of prolyltRNA synthetasebetter, the main peak eluted from the affinity column wascentrifuged on a linear 7-20% sucrosedensity gradient. The prolyl-tRNA synthetaseactivity sedimentedaround 8S, the sedimentationvalue typical of proteinsof about 150 kDa (20). In addition to this, the gel filtrate of rat liver cell sap showed an heterogeneousprofile of prolyl-tRNA synthetase activity whose smallest molecular weight peak was 120 kDa (data not shown). The sedimentation and gel filtration data suggestthat the 8s prolyl-tRNA synthetaseexists in a dimeric form. Our Iater preparationsof prolyl-tRNA synthetasefrom rat liver by a different method specifically aimed at purifying the free form only,revealed no heterodimeric structure (unpublisheddata), like E. cob. prolyl-tRNA synthetase(21). Perhapsthe 58 and 61 kDa peptides are analogousto the recently describedtwo different forms of arginyl-tRNA synthetase(22), one a complex-bound form (72 kDa> with a hydrophobic NH, terminal extension and the other a free form (60 l&a). Even in our purified preparation of prolyl-tRNA synthetase,the two peptidesmight representthe complex1074
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kDa kDa
109 98
iW9.S GlnPS
= -ZZ
“3; * 24
*
m SDS-polyacrylamide gel electrophoresis, according to Laemmli (13), of purified prolyltRNA synthetase. Lane 1: Protein standards. Lane 2: fraction 23 of Fig. 2 (2 pg loaded). The corresponding densitomemc pattern is shown. Fig,. 4. SDS-polyacrylamide gel electrophoresis, according to Fairbanks et al (14), of 24s aminoacyl-tRNA synthetase complex. Lane 1: protein standards. Lane 2: fraction 9 of Fig. 1 (14 pg loaded). The protein bands are assigned to synthetases according to the literature (2). The corresponding densitomemc pattern is shown. * Unidentified peptides.
bound form of the synthetase detached from the 24s complex during sucrose density gradient sedimentation (18) and the native free form of prolyl-tRNA
synthetase pelleted in the Fraction X
during its preparation. Recent reports suggest that during the preparation of the 24s complex from sheep liver, the complex-bound form of prolyl-tRNA synthetase is excised from a larger bifunctional
peptide encoding glutamyl-
and prolyl-tRNA
synthetase activity, by a proteolytic
reaction. As a matter of fact, in this peptide, the region joining the two catalytic domains can be a proteolytic target (9). If this should be the case in rat liver, too, the two different-sized could be the result of proteolytic
peptides
cleavage in different sites of the joining region, which might
bring about different functional forms of prolyl-tRNA
synthetase. The preliminary evidence that
the 24s complex is coeluted from an affinity column and cosediments on a density gradient with an ATP-independent protease activity (23, 24) is of particular interest. This tight association between the 24s complex and the proteases could also account for the low molecular weight peptides always present in the various preparations of the 24s complex (3,7,10 and fig. 4).
ACKNOWLEDGMENTS C. Bianchi is supported by a fellowship from the Associazione We are grateful to G. Martinotti for typing the manuscript, to technical assistance. These studies are supported by Consiglio grants from Minister0 dell’Universit8 e della Ricerca Scientifica
Italiana per la Ricerca sul Cancro. E. Gavazzeni and M. Urban0 for Nazionale delle Ricerche and by e Tecnologica (40% and 60%).
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