Volume 4 Number 11 November 1977

Nucleic Acids Research

Purification and properties of tyrosyl tRNA synthetase of rat liver

Y. S. Prasada Rao and P. R. Srinivasan

Columbia University, College of Physicians and Surgeons, Department of Biochemistry*, 630 West 168th Street, New York, NY, 10032, USA Received 23 August 1977

ABSTRACT

Purification of rat liver tyrosine tRNA synthetase yields two protein fractions A and B and both fractions are required for charging of tyrosine to tRNAtyr. Fraction B catalyzes the activation of tyrosine. Fractions A and B have been purified to near homogeneity and they are composed of single polypeptide chains of 62,000 daltons each. Gel filtration studies suggest a molecular weight of 120,000 for the synthetase. INTRODUCTION

Tyrosyl tRNA synthetases have been isolated from various sources: E. coli, B. subtilis, Bakers yeast and B. Stearothermophilus (1-6). The molecular weights of the different tyrosyl tRNA synthetases vary between 43,000 and 100,000 and they are composed of identical subunits. The subunit structures vary between monomeric, dimeric and tetrameric. For studies on the nature and function of the isoaccepting species of tyrosyl tRNA from rat liver and Novikoff hepatoma (7), we were in need of a partially purified synthetase. The purification of this enzyme from rat liver resulted in its separation into two protein were necessary for the optimal charging of fractions and both tyrosine to tRNA. The two protein fractions were further purified to near homogeneity. Some structural and catalytic properties of this enzyme are described here. MATERIALS AND METHODS Transfer RNA from rat liver was prepared by phenol extractions, essentially as described by Wevers et al.(8). The tRNA was stripped of endogenous amino acids by incubation in 0.5 M Tris/Cl, pH 8.8, for one hr at 370. One-tenth volume of 4 M NaCl was then added and the tRNA was precipitated by the addition of two

C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England

3887

Nucleic Acids Research volumes of 95% ethanol and placed at -200. The precipitated RNAwas further purified by isopropanol fractiaiaticn (9). Hydroxylamine base was prepared by the method of Beinert et al. (10) as modified by Davie (11) and assayed by the rethod of Frear and Burrell (12). DEAE-cellulose, Type 20, '*as obtained fran Schleicher and Schuell; phosplocllulose, (Miatnan P-11) fran Wtatman Products and hydroxyapatite (Bio-Gel HTP) frcn Bio-Rad. The activity of the enzyme was Assay of tyr-tRN synttas measured by the formaticn of 3Htyr-tyrt in a reacticn mixture containing 100 mM Tris/Cl (pH 7.6), 4 mM magnesium acetate, 8 mrM ATCP, 50 mM KC1, 2 mM dithiothreitol, cne A260 unit of rat liver tRNA, 1 ,uCi of [3H]tyrosine (60 Ci/nmle) and enzyme in a total volume of 0.1 ml. After incubation at 370 for 5 min, 10% cold trichloroacetic acid was added to stop the reaction. The precipitate was collected on %tatman GF/C filters, washed with 5% cold trichloroacetic acid followed by 95% ethanol, dried and counted in a Beckman scintillation spectrameter in Cmnnifluor-toluene. After separaticn of the tyrosyl tRNA synthetase into factors A and B, each factor was assayed in the presence of the other factor. Tyrosylamate assay: The fonnation of labeled tyrosylhydrcnamte was measured by the method of Parin et al. (13) with the n-dification described by Surguchov and Surguchova (14). The details of the assay are given in the legend. Protein concentration: Protein concentration was determid by the method of lwry et al. (15) using bovine senrn albunin as standard. lDlecular weight by gel filtration chrtography: A column of Sephadex G-150 (1 x 76 an) was anployed with equilibration and elution carried out in Buffer B containing 0.1 M KC1. The flow rate was adjusted to 15 ml per haor and one ml fractions were collected. The void volume of the column was determined with blue dextran 2000 and a calibraticn curve was constructed with standard proteins: yeast aloohol dehydrogenase (151,000 M.W.), bacterial alkaline phosphatase 186, 000 M.W.), bovine serun albumin (68,000 M.W.) and ovalbumin (45,000 M.W.). The data are plotted according to the method of Andrews (16).

Polyacrylamide gel electrophoresis: Analytical electrophoresis on 7% polyacrylamide gels was performed by the methods described

by Gabriel (17). Sodium dodecylsulfate gel electrophoresis was carried out according to Shapiro et al.(18). Ten micrograms of tRNA synthetase factors A or B and the marker protein E. coli RNA 3888

Nucleic Acids Research polymerase precipitated with rabbit antibody were treated with 1% sodium dodecylsulfate and 1% 2-mercaptoethanol in 0.01 M sodium phosphate buffer, pH 7.0, for 10 min at 1000 and subjected to electrophoresis on 7.5% polyacrylamide gels. RESULTS

Purification of tyrosyl tRNA synthetase: The scheme for the purification and separation of the synthetase into two protein factions is illustrated in Fig. 1. All operations were performed RAT LIVER (500 g)

H0OMOGENATE I

10,000 x g

SUPERNATANT (S-10)

(NH4)2SO4 FRACTIONATION (50-75%) DEAE-CELLULOSE COLUMN

HYDROXYAPATITE COLUMN

FACTOR A DEAE-CELLULOSE COLUMN

FACTOR B PHOSPHOCELLULOSE COLUMN (0.1 mg PROTEIN)

BIOGEL P-300 COLUMN (0.9 mg PROTEIN) FIG. 1.

Scheme for the purification of tyrosyl tRNA synthetase.

The following buffers are used in the purification. Buffer A: 5 mM Tris/Cl buffer (pH 7.6), 0.25 M sucrose, 50 mM KC1, 5 mM MgC12, 5 mM 2-mercaptoethanol. Buffer B: 10 mM Tris/Cl buffer (pH 7.6), 1 mM magnesium acetate, 5 mM 2-mercaptoethanol, 10% glycerol. Buffer C: 25 mM Tris/Cl buffer (pH 7.6) 5 mM 2-mercaptoethanol, 10% glycerol. at 40C.

3889

Nucleic Acids Research Buffer D: 10 mM potassium phosphate buffer, (pH 7.2), 5 mM 2-mercaptoethanol, 10% glycerol. Buffer E: 10 mM potassium phosphate buffer, (pH 6.5), 5 mM 2-mercaptoethanol, 10% glycerol. Five hundred grams of rat liver were homogenized, in small portions, in a Waring blender for 2 min with 2 vol. of cold Buffer A. The homogenate was spun at 10,000 x g for 20 min. The post-mitochondrial supernatant (980 ml; 4,140 mg protein) was brought to 50% (NH4)2SO4. The solution was stirred for 30 min and the clear supernatant obtained by centrifugation at 10,000 x g for 30 min was brought to 70% (NH4)2SO4 saturation. The resulting precipitate was recovered by centrifugation and dissolved in Buffer B (85 ml, 1625 mg protein) and dialyzed against a 50-fold excess of the same buffer for 10 hrs. The protein solution (120 ml) was then applied to a DEAE-cellulose column (4.6 x 30 cm) that was previously equilibrated with Buffer B. The column was then eluted with a linear gradient from 0 to 0.4 M KC1 in Buffer B (volume in a mixer and reservoir chambers was 2 liters each) at a flow rate of 500 ml/hr. Fractions of 20 ml were collected and analyzed for enzyme activity and protein. The active fractions eluting between 0.12 to 0.14 M KC1 (Fig. 2) were pooled and precipitated by the addition of solid (NH4)2SO4 to 70% saturation. The resulting precipitate (290 mg) was dissolved in 25 ml of Buffer D and dialyzed against the same buffer. Separation of Factors A and B by hydroxyapatite chrcxnatography: A column of hydroxyapatite (2 x 30 cm) equilibrated in Buffer D was employed. After loading, the column was eluted with a linear gradient consisting of 400 ml of Buffer D containing 0.01 M potassium phosphate buffer, pH 7.2, in the mixing chamber and 400 ml of the same buffer containing 0.5 M potassium phosphate buffer, pH 7.2, in the reservoir. Seven ml fractions were collected at a flow rate of 20 ml/hr. Most of the protein was eluted in fractions 20 to 25 (Fig. 3). Only a small amount of enzymatic activity could be detected near Fraction 80. This finding indicated that either the protein was denatured on the column, or it was separated into two protein fracticns and both fractions are required for activity. A reassay of the individual fractions 3890

Nucleic Acids Research

DEAE- CELLULOSE-I

I E C

I

0

6

I0 w z

CD m 0

C.)

cn

CD

FRACTION NUMBER FIG. 2. DEAE-cellulose chromatography: tRNA synthetase is given under Methods.

The assay for tyrosyl For details, see Text.

HYDROXYAPATITE CHROMATOGRAPHY

E

C

0 co

N

w

L.

z

4

CD 0 C,) CD

60 40 FRACTION NUMBER FIG. 3. Separation of tyrosyl tRNA synthetase into Factors A and B on a hydroxyapatite column. For details, see Text.

a -A, tyrosyl tRNA synthetase activity. after admixture with Fraction 80 revealed an activity peak immediately after the elution of the bulk protein. This is referred to as "Factor A." With Factor A, the other fractions 3891

Nucleic Acids Research were again analyzed for charging activity. The total activity observed earlier near Fraction 80 was greatly enhanced. This protein fraction is designated "Factor B." Factors A and B were purified separately by the procedures described below. Purification of Factor B: The pooled fractions of Factor B was diluted to 0.1 M KC1 (210 ml; 0.5 mg protein) and applied to a phosphocellulose column (1.2 x 13 cm) previously equilibrated with Buffer C. After washing with two column volumes of Buffer C containing 0.1 M KC1, the column was developed with a linear gradient from 0.1 M to 0.5 M KC1 in Buffer C. The total volume of the gradient was 200 ml. Two ml fractions were collected at a flow rate of 20 ml/hr. The active fractions representing Factor B were pooled and concentrated to one ml in an Amicon Ultra Filtration Cell using PM-30 filters. The protein (0.1 mg) was stored in small aliquots at -700 after dialysis against Buffer B. Purification of Factor A: The fractions of Factor A (5 mg) from the hydroxyapatite column was diluted to 0.025 M salt concentration (200 ml) and loaded on to a DEAE-cellulose column (1.1 x 13 cm) previously equilibrated with Buffer D. The column was then washed with three column volumes of Buffer E and a linear gradient from 0.01 M to 0.3 M in potassium phosphate in Buffer E was applied. The total volume of the gradient was 200 ml. The active fractions were pooled (1.6 mg), dialyzed against Buffer C and concentrated by ultra filtration. Further purification was carried out by gel filtration on a Biogel P-300 column (1 x 76 cm; 200-400 mesh) in Buffer B containing 0.05 M KC1. The flow rate was 10 ml/hr and 2 ml fractions were collected. Fractions containing Factor A (0.9 mg) were pooled, concentrated in an Amicon Ultra Filtration Cell with a PM-30 filter, and after dialysis against Buffer B, it was stored at -70°. Reconstitution of tyrosyl tRNA synthetase: While Factor B shows some residual activity in the charging reaction of the order of 3%, Factor A is completely inactive. However, the two factors together does acylate tRNA with tyrosine (Table I). The addition of either bovine serum albumin or KC1 to the reaction mixture containing either A or B factor failed to promote 3892

Nucleic Acids Research TABLE I

RECONSTITUTION OF TYROSYL TRNA

SYNTHETASE ACTIVITY The formation of 3H-tyrosyl tRNA was assayed as described under Methods. Heat treatment was effected by placing the tube in a boiling water bath for 5 nin. Each assay was carried out in duplicate and the average value is given.

Tyrosyl-tRNA formed

Enzyme Factor

Experiment 1 A

(cpm) 0

B

500

A + B

15,860

A + preheated B

0

B + preheated A

520

A + BSA (10 lg)

0

B + BSA (10 iig)

640

A + KC1 ( 5imoles)

0

B + KC1 ( 5 pmoles)

520

*

Experiment 2

(pmol) 0

(cpm) 0

0.013

200

0.005

0.40

7290

0.182

0

0

200

0.013

0

0

220

0.006

0

0

190

0.005

0

0.013 0

0.016 0

0.013

(pmol) 0

BSA-Bovine Serum Albumin

the charging reaction. Neither the inclusion of preheated B Factor in reaction mixtures containing A Factor alone, nor the addition of preheated A Factor to reaction mixtures containing B Factor alone resulted in charging of tRNA. The requirement for both factors in the overall charging reaction is also illustrated in Fig. 4. In panel a, Factor A was kept at a concentration of 0.2 pg in the reaction mixture. In the absence of Factor B, the charging of tRNA with tyrosine was negligible. As the concentration of Factor B is increased, the incorporation of tyrosine is stimulated and a plateau is reached around 0.6 ig of Factor B. In panel b where Factor B was kept constant, similar results are obtained with increasing concentration of Factor A. Factors A and B were also checked for possible contamination of other aminoacyl synthetases. Aminoacylation activities for arginine, histidine, and phenylalanine were found to an extent of 1%, 1.2% and 0.8% respectively of the activity observed with 3893

Nucleic Acids Research FIG. 4. Saturation kinetics of Factors A and B. In panel (a), 0.2 vg of Factor A was assayed with various concentrations of Factor B, and in panel (b), 0.2 pg of Factor B was assayed with increasing concentrations of Factor A. Each value represents the average of two sets of experiments.

PI)

0 a. C)

FACTOR A (Mg

x

10)

tyrosine. No activity could be detected with the other 15 amino acids. Role of Factors A and B in the charging reaction: The possibility that one of the factors is a tRNA modifying enzyme, or tRNA repair enzyme like terminal nucleotidyl transferase, was also explored. For these studies, tRNA and the other components of the reaction mixture were first incubated with either A (or B), heat inactivated for 1 min in a boiling water bath to destroy the factor, and then incubated with B (or A). Pretreatment of tRNA with A (or B) before assaying with B (or A) failed to restore the charging activity observed when both factors are present together in the reaction mixture. 3894

Nucleic Acids Research TABLE II TYROSYLHYDROXAMATE FORMATION The assay mixture in 0.25 ml contained 1 imol ATP, 5 pmol MgCl2, 1 nmol 13H]tyrosine, 250 iimol of NH OH and appropriate amounts of Factors A or B. After incubatign at 370 for 40 min, the reaction was terminated by placing the tubes in a boiling water bath for 5 min. An aliquot (50 pl) of the assay mixture was placed on a Carboxymethylcellulose disc (Whatman CM-30), washed thoroughly with distilled water, dried and counted. The average values of duplicate sets of results are shown for each experiment. The concentration of tRNA when present was 10 A260 units per ml.

Enkyme Factor

A A B B A A

Tyrosylhydroxamate

+ tRNA

+ tRNA + B + B +

tRNA

Experiment I (cpm) (pmol) 0 0 0 0. 10,000 0.25 8,080 0.20 14,200 0.35 13,400 0.34

Experiment 2 (cpm) (pmol) 0 0 0 0 7,000 -

7,950 -

0.18 -

0.20 -

Evidence that one of the factors is involved in the activation of tyrosine is shown in Table II above. Factor A alcne or with tRNA fails to show any activation of tyrosine. In these studies, the 3H-tyrosyladenylate was analyzed as hydroxamate trapped on a Carboxymethylcellulose filter. However, B alone yields tyrosylhydroxamate and the degree of activation is of the same order of magnitude as when A and B are present together in the reaction mixture. Transfer RNA is not required for the formation of the hydroxamate. Molecular properties of tyrosyl tRNA synthetase: The molecular weights of A and B were determined by gel filtration on a Sephadex G-150 column at 40. A set of standard proteins of well-defined molecular weight served as reference. The elution volume of each protein species (Ve) was plotted against the logarithm of the molecular weight (Fig. 5). A and B each yielded a value of 62,000 daltons. The supernatant solution from 100,000 x g centrifugation of rat liver homogenate was also subjected to gel filtration to estimate the molecular weight of tyrosyl tRNA synthetase. The synthetase analyzed by its charging capacity eluted as a single peak corresponding to a 3895

Nucleic Acids Research molecular weight of 120,000 daltons (Figs. 5 & 6). Factors A and B were also examined by polyacrylamide gel electrophoresis on native and denaturing conditions. Factor B gave a single band on native gels and on sodium dodecylsulfate

SEPHADEX G-150 CHROMATOGRAPHY

FRACTION NUMBER FIG. 5. Gel filtration of 105,000 x g supernatant of rat liver homogenate on Sephadex G-150. TyRs, tyrosyl tRNA synthetase. The peak positions of Factors A and B are indicated by an arrow.

20 ADH (151 K)

2 10

TyRS (120 K)-

\

AP (86 K)

x

IAorB u

3:

5

BSA (68K)

(62K)-'

0

OA(45K)

I J

-J

0

_

._

30

Ve (ml) 3896

.__ 40

FIG. 6. Determination of molecular weights of

tyrosyl tRNA synthetase and Factors A and B by Sephadex G-150 column.

For details, see Text.

Nucleic Acids Research gels (Fig. 7). From the latter gel a molecular weight of 62,000 was calculated for this protein (Fig. 8). Factor A was found to be 85 to 90% pure on native gels and dodecylsulfate gel electrophoresis revealed a major protein band appearing around 62,000 daltons. These results lead to the conclusion that both Factors A and B are composed of single polypeptide chains of 62,000 daltons. Since the tyrosyl tRNA synthetase on gel filtration yields a molecular weight of 120,000, it is reasonable to suggest that the synthetase is a protein made up of two nonidentical subunits.

FIG. 7. Electorphoresis of Factor B on polyacrylamide gels under native conditions (bottom gel) and with sodium dodecylsulfate (top gel). About 10 ig of the protein was subjected to electrophoresis as described under Methods.

20 *

(160 K) 0 5O K)

x

10 1-J 3: CD)

FACTOR B

(62/

5

IgH(5OK)

(

Purification and properties of tyrosyl tRNA synthetase of rat liver.

Volume 4 Number 11 November 1977 Nucleic Acids Research Purification and properties of tyrosyl tRNA synthetase of rat liver Y. S. Prasada Rao and P...
1MB Sizes 0 Downloads 0 Views