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OF BIOCHEMISTRY

Comparison

AND

BIOPHYSICS

172, 252-260 (1976)

of Free and Ribosome-Bound Phenylalanyl-tRNA Synthetase from Rabbit Reticulocytesl

WESLEY K. TANAKA,2 KAMALES SOM,3

AND

BOYD A. HARDESTY

Clayton Foundation Biochemical Institute, Department of Chemistry, The University of Texas, Austin, Texas 78712 Received June 26, 1975 Phenylalanyl-tRNA synthetase CL-phenylalanine:tRNA ligase [AMP], EC 6.1.1.b) from the ribosomal and the postribosomal cell supernatant fractions of rabbit reticulocytes were purified separately and characterized. Phenylalanyl-tRNA synthetase from the ribosomal fraction was purified 114-fold to a final specific activity of 1603 units/mg and is approximately 90% pure. Phenylalanyl-tRNA synthetase from the postribosomal supernatant fraction was purified 4186-fold to a final specific activity of 247 unitslmg. The enzymes from the two fractions appear to be identical based on their elution from various chromatographic media, sedimentation coefficient, pH, Mg2+, and K+ optima, and heat stability. Phenylalanyl-tRNA synthetase from rabbit reticulocytes has a molecular weight of approximately 245,000 with an a 1p 2 subunit structure. The molecular weights of the subunits are 57,000 and 67,200.

Increasing evidence in recent years has indicated that many aminoacyl-tRNA synthetases in eukaryotic cells are present in high molecular weight complexes or bound to ribosomes (l-8). These observations have led to the suggestion that synthetases in eukaryotic cells may be part of a structurally ordered system involving other components of protein synthesis (9, 10). The existence of ribosome-associated synthetases, in particular, suggests a greater complexity in the mechanism and regulation of protein synthesis in higher organisms than that appreciated to now. The significance of ribosome-associated synthetases has not yet been determined. It has been observed in some cases that synthetase activity for some amino acids can be found both bound to ribosomes and ’ This research was supported in part by Grant No. CA-16608 from the National Cancer Institute and by National Science Foundation Grant No. GB30902. 2 Recipient of USPHS Postdoctoral Fellowship No. lF22 CA00729-01 from the National Cancer Institute. 3 Present address: Department of Microbiology and Immunology, School of Medicine, UCLA, Los Angeles, Calif. 90025.

’ Abbreviations used: Phe-tRNA, tRNA; DEAE-, diethylaminoethyl. 252

Copyright All rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

free in the cell supernatant fluid. Preliminary evidence with Phe-tRNA4 synthetase in rat liver indicates that the supernatant enzyme form and that bound to the ribosomes are similar (9,ll). Small differences between the enzyme forms which could serve to point out the significance of ribosome-bound aminoacyl-tRNA synthetases, however, have not been ruled out. Phe-tRNA synthetase from the ribosomal and postribosomal supernatant fraction of rabbit reticulocytes was used in the present study to examine the differences between free and ribosome-bound synthetase. In the lysate of rabbit reticulocytes, approximately 90% of the Phe-tRNA synthetase activity is associated with the ribosomes (4). This observation is supported by similar but somewhat lower levels of binding of Phe-tRNA synthetase to ribosomes in Ehrlich ascites cells (31, Chinese hamster ovary cells (5), and rat liver (7). PhetRNA synthetase bound to reticulocyte ribosomes can be removed by treating the ribosomes with 0.5 M KC1 (4). The purification and partial characteriphenylalanyl-

RETICULOCYTE

PHENYLALANYL-tRNA

zation of Phe-tRNA synthetase from reticulocyte ribosomes and from reticulocyte ribosome-free cell supernatant fluid is presented in this paper. The enzyme isolated from the ribosomes appears to be the same as that from the postribosomal cell supernatant, on the basis of chromatography on various columns, molecular weight, sedimentation in sucrose and glycerol density gradients, sodium dodecyl sulfate-lo% polyacrylamide-gel electrophoresis, pH, Nit+, and K+ optima, and stability studies. MATERIALS

AND

METHODS

Sephadex G-200 was from Pharmacia Fine Chemicals, Inc., Piscataway, NJ. ; [14Clphenylalanine was from New England Nuclear, Boston, Mass.; hydroxylapatite was from Clarkson Chemical Company, Williamsport, Pa.; DEAE-cellulose (DE 52) and carboxymethyl cellulose (CM 52) were from Whatman, Maidstone, England; ammonium sulfate (ultrapure y-globulin were from and porcine grade) SchwarzMann, Inc., Orangeburg, N.Y.;phosphocellulose was from Sigma Chemical Company, St. Louis, MO.; Diaflo ultrafiltration apparatus and type XM-50 membranes were from Amicon, Lexington, Mass. ; Escherichia coli /3-galactosidase (P-L Biochemicals, Inc., Milwaukee, Wis.) was assayed by hydrolysis of o-nitrophenyl-p-n-galacmpyranoside (Sigma) (12); beef heart lactate dehydrogenase (BHLDHC, Worthington Biochemical Corp., Freehold, N.J.) was assayed by the oxidation of NADH (Calbiochem, La Jolla, Calif.) (13); beef liver catalase (CTR, Worthington) was assayed according to the method of Beers and Sizer (14); rabbit liver tRNA was prepared as described previously (15). Protein concentration was determined by the method of Warburg and Christian (16). Solutions. Standard phosphate buffer contained potassium phosphate (pH 7.5), 5 mM 2-mercaptoethano1 and 1 mru dithioerythritol. The concentration of phosphate was as indicated in the text; e.g., 20 mM standard phosphate buffer. Standard Tris buffer contained 20 mru Tris-HCl (pH 7.5), 5 mM 2-mercaptoethanol and 1 mM dithioerythritol. KC1 was added as described in the text; e.g., 100 mM KC1 standard Tris buffer. Glycerol content of all designated buffers is indicated in volume per volume percentages. Cell fractionation. Reticulocytes were obtained from phenylhydrazine-injected rabbits and processed as previously described (15). Washed reticulocytes were lysed by hypotonic shock. The cell debris and unlysed cells were removed by centrifugation at 15,OOOg for 15 min. Ribosomes were pelleted from the lysate by centrifugation at 105,OOOgfor 90 min.

SYNTHETASE

253

Standard synthetase assay. Aminoacylation of tRNA was measured as incorporation of amino acid into cold trichloroacetic acid-insoluble material. The standard reaction mixture contained 100 mM TrisHCl (pH 7.51, 10 rntu MgCl*, 100 mM KCl, 20 mM 2mercaptoethanol, 20 pg of bovine serum albumin, 4 mM ATP, 0.1 mM [14Clphenylalanine (10 mCiimmo1) and 200 pg of rabbit liver tRNA in a total volume of 0.2 ml. A suitable aliquot, generally between 10 and 50 ~1 of enzyme solution, was added to the reaction mixture, mixed thoroughly and then incubated at 37°C for 5 min. The reaction was stopped by addition of 5 ml of cold 5% trichloroacetic acid (w/v). The resulting precipitate was filtered through Millipore Type HA membrane filters (Millipore Corp., Bedford, Mass.), washed three times with a total of 15 ml of 5% trichloroacetic acid (w/v), and then dried in an oven at 120°C. Dried filters were put into scintillation vials containing 10 ml of scintillation solution which contained 5 g of 2,5-diphenyloxazole (Sigma) in 1 liter of toluene. Vials were counted in a Beckman LS-150 scintillation system with an efficiency of 35%. Synthesis of aminoacyl-tRNA is proportional to added enzyme in the range from 50 to 200 pmol of product formed. A unit of enzyme activity is defined as the amount of enzyme required to form 1 nmol of aminoacyl-tRNA per minute. Sodium dodecyl sulfate-polyacrylamide-gel elecsulfate-polyacrylamide-gel trophoresis . Dodecyl electrophoresis and staining were carried out as described by Weber and Osborn (17) with minor modification. Prior to electrophoresis samples were heated at 90°C in a buffer containing 1% sodium dodecyl sulfate, 10 mM potassium phosphate (pH 7.0) and 1 mM 2-mercaptoethanol for 10 min. Gels were electrophoresed at a constant voltage of 50 V for 7 h. Gels were stained for 1 h with Coomassie blue (Sigma) and destained electrophoretically. Dialysis. Unless otherwise indicated, dialysis was carried out in the cold, with stirring, for about 12 h against two changes of 2 liters of the solution specified in the text. RESULTS

The procedures used in the purification of Phe-tRNA synthetase from the ribosomes and from the supernatant fraction are summarized in Fig. 1. All steps were carried out on ice or at 4°C. Preparation of Phe-tRNA Synthetase from Ribosomal Salt Wash The ribosome pellet from the 105,OOOg centrifuge tion was resuspended and washed with 0.5 M KC1 as described by Miller and Schweet (18). The supernatant

254

TANAKA,

SOM AND

wash fraction was dialyzed for 12 h against three buffer changes, each containing 1 liter of 50 mM standard phosphate buffer/X% glycerol. Glycerol was added at this step to stabilize enzyme activity during phosphocellulose chromatography. The dialyzed enzyme containing about 300 mg of protein was applied to a 2.0 x 30-cm column of phosphocellulose equilibrated phosphate standard with 50 rnM buffer/E% glycerol. The column was eluted initially with one to two column volumes of starting buffer and then with a 350-ml linear gradient of 0.05-0.4 M standard phosphate buffer/l5% glycerol. Figure 2 shows a typical elution profile from phosphocellulose. Under these conditions two peaks of Phe-tRNA synthetase activity are eluted. The enzyme eluting in the initial wash fraction represents approximately 10% of the total enzyme activity. This does not appear to be due to overloading of the phosphocellulose since enlarging the column does not reduce appreciably the amount of activity in this initial fraction. In addition, this enzyme fraction will not bind to phosphocellulose upon rechromatography on a second column. Whether

FIG. 1. Purification wash and supernatsnt tase.

procedure for ribosomal salt phenylalanyl-tRNA synthe-

HARDESTY

FIG. 2. Phosphocellulose column chromatography of ribosomal salt wash Phe-tRNA synthetase. Dialyzed ribosomal salt wash was chromatographed as described in Results. Fractions (10 ml) were collected and IO-p1 aliquots were used in the standard synthetase assay.

this minor peak represents a different form of Phe-tRNA synthetase as compared to the bulk of the synthetase activity is uncertain. Preliminary observations suggest that this minor peak is related to the interaction of the enzyme with some other component in this crude fraction. PhetRNA synthetase in the 40-70% ammonium sulfate fraction will not bind to phosphocellulose equilibrated with 50 mM standard phosphate buffer/l5% glycerol. Partial purification of the ammonium sulfate fraction yields a preparation of PhetRNA synthetase that binds to phosphocellulose under the same conditions. In other experiments the 40-70% ammonium sulfate fraction was mixed with crude ribosomal salt wash. Phe-tRNA synthetase, which is abundant in this mixture, did not bind to phosphocellulose under these conditions. These results indicate that a component present in the 40-70% ammonium sulfate fraction from the ribosomal supernatant fluid, and to a lesser extent in the ribosomal wash fraction, interferes with the interaction of Phe-tRNA synthetase with phosphocellulose. The nature of this substance is not known. Approximately 90% of the Phe-tRNA synthetase activity is eluted from the phosphocellulose column by the phosphate gradient. This enzyme fraction was pooled and dialyzed against 50 mM standard phosphate buffer/l5% glycerol. The dialyzed enzyme fraction containing about 23 mg of protein was then chromatographed on a single column containing a 1.5 X 15-cm

RETICULOCYTE

PHENYLALANYL-tRNA

layer of carboxymethyl cellulose (CM 52) over a 1.5 x 3.5~cmlayer of DEAE-cellulose (DE 52) equilibrated with 50 mM standard phosphate buffer/l5% glycerol. Enzyme was applied to the column and washed with starting buffer. Nonadsorbed protein (11 mg> was collected, pooled and applied directly to a 1.5 x 15-cm hydroxylapatite column equilibrated with 50 mM standard phosphate buffer/l5% glycerol. The column was washed with two column volumes of starting buffer. Phe-tRNA synthetase activity was eluted by a 400-ml linear gradient of 0.05-0.4 M standard phosphate buffer/l5% glycerol. A typical elution profile from the hydroxylapatite column is given in Fig. 3. Fractions containing the peak of the synthetase activity were pooled and concentrated by ultrafiltration with an XM 50 filter at an operating pressure of 40 psi. The concentrated enzyme from hydroxylapatite chromatography containing 2.2 mg of protein was applied to a 2.0 x 55-cm column of Sephadex G-200 that had been equilibrated with 20 mM standard phosphate buffer containing 250 mM KC1 and 15% glycerol. The column was developed at a flow rate of approximately 8 ml/h. Figure 4 shows an elution profile from the G-200 column. Fractions containing the peak of the Phe-tRNA synthetase activity were pooled, concentrated by ultrtiltration to 3-4 ml and then dialyzed against 20 mM standard phosphate buffer/l5% glycerol. The purified enzyme preparation

i a~~~~~‘~~~ 75 90 15 10 15 FRICTION NUMBER FIG. 3. Hydroxylapatite column chromatography of ribosomal salt wash Phe-tRNA synthetase. Eluate from the CM 52 plus DE 52 column was chromatographed as described in Results. Fractions (6.5 ml) were collected at a column flow rate of 30 ml/h and aliquots of 1-5 ~1 were used in the stand_ _ ard synthetase assay.

255

SYNTHETASE

I \

I i

FRACTION NUMBik

FIG. 4. Sephadex G-200 chromatography

of ribosomal salt wash Phe-tRNA synthetase. The concentrated enzyme fraction from hydroxylapatite chromatography was applied to Sephadex G-200 as described in Results and eluted at a flow rate of approximately 8 ml/h. Fractions of 2.6 ml were collected and l-k1 aliquots were used in the standard synthetase assay.

shows less than 10% loss of enzymatic activity when stored at -90°C for up to 6 months. Preparation of Phe-tRNA Synthetase from Postribosomul Supernatant Fraction

The supernatant solution from the 105,OOOg centrifugation was adjusted to pH 6.5 with 1 M acetic acid. Solid ammonium sulfate was added to give 40% saturation (22.6 g/100 ml of supernatant). The solution was stirred for 30 min and the resulting precipitate was removed by centrifugation at 15,000g for 15 min. Phe-tRNA synthetase was precipitated by addition of 18.2 g of ammonium sulfate for each 100 ml of initial solution to give 70% saturation. The resulting precipitate was collected by centrifugation and resuspended in standard Tris buffer equal to about 1% of the original volume. The resuspended ammonium sulfate precipitate was dialyzed against standard Tris buffer for approximately 24 h. This 40-70% ammonium sulfate fraction has been stored at -90°C for more than 6 months with negligible loss of Phe-tRNA synthetase activity. About 4.4 g of the 40-70% ammonium sulfate fraction was applied to a 5.0 x lo-cm column of CM 52 equilibrated with standard Tris buffer. Phe-tRNA synthetase is not retained on the column under these conditions. Fractions near the void volume containing the enzymatic activity were pooled and made 50 mM in KC1 with the addition of a suita-

256

TANAKA,

SOM AND

ble volume of 1 M KC1 standard Tris buffer. About 60% of the total protein applied to the column is contained in the pooled enzyme fraction. The enzyme fraction from the CM 52 column containing approximately 2.7 g of protein was then applied to a 2.0 x 35-cm column of DE 52 equilibrated with 50 mM KC1 standard Tris buffer and eluted with the same buffer. Fractions at the void volume containing the enzyme were pooled and dialyzed against 0.1 M standard phosphate buffer/l5% glycerol. Glycerol was added at this stage to stabilize enzyme activity during and after hydroxylapatite chromatography. The dialyzed enzyme fraction containing about 1 g of protein was chromatographed on hydroxylapatite by a procedure similiar to that described for Phe-tRNA synthetase from the ribosomal salt wash. The only modifications were that the hydroxylapatite column (2.0 x 30 cm) was equilibrated and washed with 0.1 M standard phosphate buffer/l5% glycerol, and the enzyme was eluted in a 500-ml linear gradient of O.l0.4 M standard phosphate buffer. Under the conditions used, supernatant PhetRNA synthetase elutes from hydroxylapatite at a phosphate concentration of 0.25 M. The enzyme peak was pooled and dialyzed against 50 mM standard phosphate buffer/l5% glycerol. This enzyme fraction containing 14 mg of protein was then chromatographed on a 1.5 x lo-cm phosphocellulose column by a procedure similar to that used for ribosomal salt wash PhetRNA synthetase. After an initial wash

step

Salt wash fraction Phosphocellulose Carboxymethyl plus DEAE-cellulose Hydroxylapatite Sephadex G-200 a Unit milligram

the column was eluted with a loo-ml linear gradient of 0.05-0.4 M standard phosphate buffer/l5% glycerol. Under these conditions only one peak of enzyme activity is eluted from the column. This peak elutes in the phosphate gradient at a phosphate concentration of 0.125 M. The peak tubes of activity were pooled and concentrated by ultrafiltration. The concentrated enzyme was then chromatographed on Sephadex G-200 as previously described. The final enzyme, representing 2% of the starting material, was stored at -90°C in 20 mM standard phosphate buffer/l5% glycerol and shows storage stabilities similar to those of ribosomal salt wash PhetRNA synthetase. Tables I and II summarize the purification data at each step for the two enzyme fractions. Differences in the overall yields were due primarily to differences in the order of the columns used and the amount of contaminating protein present in the starting material. Purification of the ribosomal salt wash fraction by the same procedure used for the postribosomal supernatant fraction reduced overall yield from 22 to about 5%.

Characterization Results of gel electrophoresis of the purified Phe-tRNA synthetase preparations are shown in Fig. 5. The purified PhetRNA synthetase from the ribosomal salt wash yields two bands of equal staining intensity along with three minor bands that show up on gels overloaded with pro-

TABLE I SYNTHETASE FROM RIBOSOMAL SALT WASH .FRACTION

Pns-tRNA Fractionation

HARDESTY

Volume (ml)

43 116 110

of enzyme is defined of protein.

8 1.22

Total protein (mg)

Specific activity” (units/mg)

Total units

Yield (%)

Purilication

305 23.2 11.0

14 111 234

4270 2575 2574

100 60 60

-

2.2 0.6

756 1603

1663 962

39 22

54 114

activity

is units per

as 1 nmol of Phe-tRNA

formed per minute.

Specific

7.8 16.7

RETICULOCYTE

PHENYLALANYL-tRNA TABLE

Pnx-tRNA

II

SYNTHETASE FROM POSTRIBOSOMAL SUPERNATANT FRACTION OF THE RABBIT RETICULQCXTE LYSATE

Fractionation step Postribosomal supernatant 40-70% WHMO, Carboxymethyl cellulose DEAE-cellulose Hydroxylapatite Phosphocellulose Sephadex G200 a Unit milligram

257

SYNTHETASE

Volume (ml) 2510

Total protein (mg)

Specific activity” (units/mg)

Total units

Yield (%)

4.57 x 1oL

0.059

2696

115

4393

0.465

2043

75.8

132

2660

0.76

2026

75.1

12.9

121

1042

1.7

1771

65.7

28.8

34.0

490

18.2

576

180

14.4

100

Purifkation 7.88

3.8

1.18

160

189

7.0

2712

1.4

0.21

247

52

1.9

4186

of enzyme is defined of protein.

as 1 nmol of Phe-tRNA

FIG. 5. Sodium dodecyl sulfate-polyacrylamide gels of purified Phe-tRNA synthetase from ribosomal salt wash and high speed supematant fraction. Samples, consisting of (A) 20 pg of purified ribosomal salt wash Phe-tRNA synthetase and (B) 40 pg of purified supernatant Phe-tRNA synthetase, were electrophoresed as described in Materials and Methods.

formed per minute.

Specific

activity

is units

per

tein and have molecular weights of 57,000 and 67,200, as determined by dodecyl sulfate-gel electrophoresis with known standards (Fig. 6). Purified Phe-tRNA synthetase from the supernatant fraction yields at least 13 bands on gel electrophoresis. Two of these bands corn&rate with the two major bands from purified ribosomal salt wash Phe-tRNA synthetase. Molecular weight determinations were carried out on a calibrated Sepharose 4B column under conditions in which the PhetRNA synthetase retains full activity. A 2.0 x 70-cm column of Sepharose 4B was equilibrated with 20 mM standard phosphate buffer containing 250 mM KC1 and 15% glycerol. Samples containing 60 units of Phe-tRNA synthetase from ribosomal salt wash (756 units/mg) or postribosomal supernatant fluid (160 units/mg) in combination with various standards were chromatographed at a flow rate of 12 ml/h. Fractions of approximately 1 ml were collected and assayed for enzymatic activity. Figure 7 summarizes results obtained with the Sepharose 4 B column. Phe-tRNA synthetase activity from both the supernatant fraction and ribosomal salt wash elute identically, slightly after the activity for beef heart catalase W, 248,000) (24). The

258

TANAKA,

SOM AND

FIG. 6. Molecular weight determination of ribosomal salt wash Phe-tRNA synthetase by sodium dodecyl sulfate-gel electrophoresis. A sample containing 10 pg of ribosomal salt wash Phe-tRNA synthetase; (1) 10 pg of bovine serum albumin (M, 68,000) (17) and 10 pg of porcine y-globulin; (2) H Chain (M, 50,000); and (3) L Chain (M, 23,500) (17) was run as described in Materials and Methods. Relative mobility of the various protein species was determined as described by Weber and Osborn (17) using bromophenol blue as a tracking dye.

I

I

I

HARDESTY

Both synthetases sediment identically on 7.5-20% sucrose gradients. As shown in Fig. 8, Phe-tRNA synthetase sediments slightly slower than the lactate dehydrogenase marker (M, 140,000; s2,,,,,, = 7.4s (13)). This observation indicates a sedimentation coefficient of 6.8 and suggests that the enzyme is capable of dissociating into enzymatically active dimers with a molecular weight of approximately 120,000. Since glycerol stabilizes both enzyme forms during purification and storage we also examined the sedimentation of both purified synthetases in lo-30% glycerol gradients. The results obtained with glycerol gradient centrifugation were identical to those with sucrose gradients. Both purified synthetase preparations show identical magnesium optimum (10 mM), KC1 optimum (100 mM) and pH optimum (pH 8.5). Figure 9 also shows that both purified enzyme fractions display a similar stability to heat inactivation. Phe-tRNA synthetase purified from the ribosomal salt wash fraction is virtually free of contaminating synthetase activities. The two major contaminating species are asparaginyl- and glutamyl-tRNA synthetases that exhibit specific activities of 17.5 and 8.3 units/mg of protein, respec-

170

160

ELUTION

VOLUME

(ml1

FIG. 7. Molecular weight determination by Sepharose 4B chromatography. Purified Phe-tRNA synthetase from ribosomal salt wash or supernatant fluid was chromatographed on a calibrated Sepharose 4B column. Arrows indicate the volume at which different enzymatic activities are eluted from the column: (1) E. coli p-galactosidase (M, 540,000) (12); (2) beef liver catalase (M, 248,000) (24); and (3) beef heart lactate dehydrogenase (IV, 140,000) (13).

molecular weight for Phe-tRNA synthetase obtained by this method is approximately 245,000. This molecular weight is slightly lower than the molecular weight of 270,000 reported by Igarashi and Zmean (19). In conjunction with the electrophoretie data, the molecular weight suggests an cr& 2 quaternary structure. This is consistent with similar observations in rat liver (20) and yeast (21).

‘“k

J

‘b~o,,,,,,.-,e-e

J

10 IS 20 25 FRACTION NUMBER FIG. 8. Sucrose gradient centrifugation. A 0.4-ml sample containing Phe-tRNA synthetase from either the ribosomal salt wash or postribosomal supernatant fluid (10 units), lactate dehydrogenase (82 pg), catalase (7 pg), and p-galactosidase (100 pg) was layered over an 11.2-ml linear gradient of 7.520% sucrose containing 20 mM standard phosphate buffer with 250 mM KCl. Centrifugation was carried out at 40,000 rpm in an SW 41 rotor at 4°C for 12 h. Fractions of 0.5 ml were collected and assayed as described in Materials and Methods for the various activities. Sedimentation was from left to right. Arrows indicate the peak tubes of activity for: (1) ,&galactosidase (16s) (12); (2) catalase (11.69 (24); and (3) lactate dehydrogenase (7.453) (13). 5

RETICULOCYTE

PHENYLALANYL-tRNA

FIG. 9. Time course of heat inactivation of ribosomal salt wash (A) and supernatant fraction (Cl) Phe-tRNA synthetase at 45°C. Purified Phe-tRNA synthetase from ribosomal salt wash (0.2 pg) or postribosomal supernatant fluid (1.2 gg) was heated to 45°C in 0.1 ml of 20 mM standard phosphate buffer. At the times indicated, lo-n1 aliquots were taken and assayed in the standard synthetase assay. Activity of 100% represents approximately 1500 cpm.

tively. All other synthetases are present in lesser amounts or below detectable levels. DISCUSSION

Phe-tRNA synthetase has been purified from the ribosomes and from the supernatant fluid of rabbit reticulocytes. Both enzyme fractions elute identically from various chromatographic media including hydroxylapatite, phosphocellulose and Sephadex G-200. The purified Phe-tRNA synthetase derived from ribosomes has an eightfold higher specific activity than the corresponding preparation from the supernatant solution. This difference is due to contaminating protein in the purified supernatant Phe-tRNA synthetase preparation. The molecular weight of both purified synthetase fractions, as determined by Sepharose 4B chromatography, is approximately 245,000. Sedimentation in sucrose or glycerol density gradients yields identical sedimentation values (6.88 for the two enzyme fractions. Both enzyme fractions show identical Mg2+, K+, and pH optima. All available data are consistent with the conclusion that the Phe-tRNA synthetase bound to the ribosomes is the same as that found free in the cell supernatant fluid. This conclusion is in agreement with the preliminary observations of Tscherne et al. (9) and Nelson and Haschemeyer (11) on Phe-tRNA synthetase from rat liver.

SYNTHETASE

259

The present studies do not rule out possible differences in K, values for substrates, although preliminary studies indicate that both purified enzyme fractions have a similar K, value for phenylalanine. Alternatively, the two enzyme fractions may be composed of a single enzyme species and may differ with respect to their activities due to interaction with the ribosome. Graf (22) has shown that the activity for PhetRNA synthetase is stimulated in the presence of reticulocyte ribosomes. In addition, the synthetase activity for methionine, lysine, arginine, and valine, all synthetases that can be found in a ribosome-bound form in reticulocytes, are stimulated in the presence of reticulocyte ribosomes. The significance of ribosome-bound synthetases may ultimately lie in the ordered arrangement produced. The proximity of the ribosome bound aminoacyl-tRNA synthetase to the site of amino acid incorporation implies a spatial significance for efficient incorporation of these amino acids into protein. The observations of Airhardt et al. (23) suggest that a general intracellular amino acid pool is not the sole precursor of aminoacyl-tRNA. Rather, both extracellular and intracellular amino acids contribute to a restricted compartment, possibly associated with the membrane system, from which amino acids are drawn for protein synthesis. Association of aminoacyltRNA synthetase and tRNA (25) with membrane-bound ribosomes may provide the necessary spatial arrangement for the efficient in viuo incorporation of amino acids into protein. ACKNOWLEDGMENTS We thank D. Konecki for excellent technical assistance, Dr. J. Miguel Cimadevilla for many helpful suggestions during the course of these studies, Dr. Gisela Kramer for her advice during the preparation of this manuscript, and B. Anderson for her patience in the preparation of the typescript. REFERENCES 1. BANDYOPADHYAY, A. K., AND DEUTSCHER, M. P. (1971) J. Mol. Biol. 60, 113-122. 2. VENNEGOOR, C. J. G. M., STOB, A. L. H., AND BLOEMENDAL, H. (1972) J. Mol. Bill. 65,375 378. 3. ROBERTS, W. K., AND COLEMAN, W. H. (1972) B&hem. Biophys. Res. Commun. 46, 206-

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214. 4. IRVIN, J. D., ANDHARDESTY, B. (1972)Bicchemistry 11,1915-1920. 5. HAMPEL, A., AND ENGER, M. D. (1973) J. Mol.

Biol. 79, 285-293. 6. GOTO, T., AND SCHWEIGER, A. (1973) Hoppe-Sey-

ler’s Z. Physiol. Chem. 354,1027-1033. 7. MOLINE, G., HAMPEL, A., AND ENGER, M. D. (1974) Biochem. J. 143, 191-195. 8. SOM, K., AND HARDESTY, B. (1975) Arch. Biothem. Biophys. 166,507~517. 9. TSCHERNE, J. S., WEINSTEIN, I. B., LANKS, K. W., GERSTEN, N. B., AND CANTOR, C. R. (1973) Biochemistry 12, 3859-3865. 10. HARDESTY, B., SOM, K., CIMADEVILLA, M., AND IRVIN, J. (1974) in Modern Trends in Human Leukemia (R. Neth, ed.), pp. 314-326, J. F. Lehmanns, Verlag, Munich. 11. NIEISEN, J. B., AND HASCHEMEYER, A. E. V. (1973) Fed. Proc. 32, 459. 12. CRAVEN, G. R., STEERS, E., JR., AND ANFINSEN, C. B. (1965) J. Biol. Chem. 240, 2468-2476. 13. PESCE, A., MCKAY, R. H., STOLZENBACH, F., CAHN, R. D., AND KAPLAN, N. 0. (1964) J. Biol. Chem. 239, 1753-1761. 14. BEERS, R. F., JR., AND SIZER, I. W. (1952) J. Biol. Chem. 195, 133-140.

HARDESTY

15. HAFUIESTY, B., MCKEEHAN, W., AND CULP, W. (1971) in Methods in Enzymology (Moldave, K., and Grossman, L., eds.), Vol. 20, Part C, pp. 316-330, Academic Press, New York. 16. WARBURG, O., AND CHRISTIAN, W. (1942) Biothem. Z. 310, 384-421. 17. WEBER, K., AND OSBORN, M. (1969) J. Biol. Chem. 244, 4406-4412. 18. MILLER, R. L., AND SCHWEET, R. (1968) Arch. Biochem. Biophys. 125, 632-646. 19. IGARASHI, S. J., ANDZMEAN, J. A. (1975)Can. J. Biochem. 53, 120-123. 20. TSCHERNE, J. S., LANKS, K. W., SALIM, P. D., GRUNBERGER, D., CANTOR, C. R., AND WEINSTEIN, I. B. (1973) J. Biol. Chem. 248, 4052-

4059. 21. FASIOLO, F., BEFORT, N., BOULANGER, Y., AND EBEL, J. P. (1970) Biochim. Biophys. Actu 217, 305-318. 22. GRAF, H. (1973) Fed. Proc. 32,459. 23. AIRHART, J., VIDRICH, A., AND KHAIRALLAH, E. A. (1974) Biochem. J. 140,539-548. 24. SAMEJIMA, T., KAMATA, M., AND SHIBATA, K. (1962) J. B&hem. (Tokyo) 51, 181-187. 25. SMITH, D. W. E., AND MCNAMARA, A. L. (1974) J. Biol. Chem. 249, 1330-1334.

Comparison of free and ribosome-bound phenylalanyl-tRNA synthetase from rabbit reticulocytes.

ARCHIVES OF BIOCHEMISTRY Comparison AND BIOPHYSICS 172, 252-260 (1976) of Free and Ribosome-Bound Phenylalanyl-tRNA Synthetase from Rabbit Retic...
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