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The purification of aminoacyl-tRNA synthetases by affinity chromatography
BIOCHIMICAET BIOPHYSICAACTA
137
BBA Report BBA 91353
The purification of aminoacyl-tRNA synthetases by affinity chromatography S. BARTKOWIAKand J. PAWEEKIEWICZ Institute o f Plant Genetics, Polish Academy of Sciences and Institute of Biochemistry, College o f Agriculture, Poznah (Poland)
(Received May 15th, 1972)
SUMMARY
The method of the purification of aminoacyl-tRNA synthetases by affinity chromatography is described on the basis of complex formation between the enzyme and aminoacyl-tRNA attached to bromoacetylamidobutyl-Sepharose. The purification of Escherichia coli isoleucyl-tRNA synthetase is given as an example.
The use of common enzymological techniques to prepare some of the aminoacyltRNA synthetases in a homogeneous state has sometimes failed. We have elaborated a method based on the technique of affinity chromatography that achieves the preparation of aminoacyl-tRNA synthetases of high purity in a simple manner. In this report we give as an example the purification of Escherichia coli isoleucyl-tRNA synthetase by this technique. The enzyme was already isolated in a homogeneous state by Baldwin and Berg 1, and by Yaniv and Gros 2, and its properties are well known 1' 3-6 Partially purified E. coli enzyme was chromatographed on modified Sepharose to which isoleucyl-tRNA molecules were attached. Sepharose was modified in the manner described by Cuatrecasas 7, 8. Sepharose 4B was activated with cyanogen bromide, coupled with tetramethylenediamine and then treated with bromoacetic ester of N-hydroxysuccinimide with the formation of bromoacetamidobutyl-Sepharose. To 20 ml of this derivative in 0.1 M potassium phosphate buffer, pH 7.5, 30 mg ofE. coli tRNA, aminoacylated previously with isoleucine and dissolved in 10 ml of phosphate buffer, was added, and the suspension was stirred for 3 days at 4°C. The aminoacylation reaction was carried out according to Yaniv.and Gros 2, and the isoleucyl-tRNA obtained was deproteinized carefully before use by phenol and then by chloroform-isoamyl alcohol (5:1, v/v) extraction. The suspension was filtered off and washed at 4°C with 500 ml of water, 100 Bioehim. Biophys. Acta, 272 (1972) 137 140
138
BBA REPORT
inl of 1 M NaC1 and 300 ml of water, successively. Next the preparation was stirred for 24 h in 0.1 M ethanolamine, adjusted to pH 7.5 with HCI in order to block all the unreacted active groups. After this reaction the suspension was washed with 500 ml of 0.3 M NaC1, and finally used for enzyme chromatography with an appropriate buffer. The amount of Sepharose-bound tRNA was calculated from the difference of its concentrations present in the supernatants before and after the binding reaction, or more directly after the hydrolysis of Sepharose-Ile-tRNA conjugate in 0.3 M KOH at 80°C for 50 rain. The hydrolysate of modified Sepharose without bound tRNA was taken in this case as the blank sample for the spectrophotometrical measurement. In the procedure given, about 25 #g of tRNA was bound per ml of Sepharose derivative. Attemps to bind isoleucyltRNA or unacylated tRNA directly to cyanogen bromide-activated Sepharose failed, in agreement with the data of Poonian et al. 9. Also bromoacetamidobutyl-Sepharose does not bind unacylated tRNA, which indicates that the a-amino group takes part in the coupling reaction. Thus the bromoacetamidobutyl "arm" attached to Sepharose plays two roles: it contains the reactive bromoacetyl group able to react with aminoacyl-tRNA, and, on the other hand, keeps the bound tRNA molecule away from the Sepharose matrix which facilitates the interaction between the enzyme and tRNA. Fractionation of partially purified isoleucyl-tRNA synthetase on a Sepharose -Ile-tRNA column was achieved in phosphate buffer at pH 5.5.
0.50I-. . . . . . . . . . . . . . . . . . . . . . --o.,40
i
m-- o.3o
~2
12500
i";
'~,
2000
I i
t~m<~
0.20
T
IO0O
0
-
o,o
Oi
i
I
tO
I
°--t':'--~--
20
~_
30
~[-~Izf~-z--~
40
50
--~
"r'60
• I
0
70
FRACTION NUHBER Fig. I. Chromatography ofE. coli isoleucyl-tRNA synthetase on Sephadex-Ile-tRNA column. The enzyme (10 nag of protein) partially purified to Stage IV as described by Yaniv and Gros 2 , was applied on the Sepharose- Ile-tRNA column (0.9 cm × 15 cm) equilibrated at 4°C with 0.1 M potassium phosphate buffer, pH 5.5, containing 1 mM MgC12 and 0.5 mM dithioerythriol, and washed with the same buffer until the absorbance at 280 nm decreased below 0.01. Elution was carried out with linear gradients from 0 to 0.3 M NaCI in the phosphate buffer. Fractions of 2 ml were collected at a flow rate of 20 ml/h. The absorbance at 280 n m ( - - - - ) and the enzyme activity (o . . . . o) were determined in each fraction. ~ A indicates the linear NaC1 gradient.
Biochim. Biophys. Acta, 272 (1972) 137
140
BBA REPORT
139
TABLE I PURIFICATION OF E. COLI ISOLEUCYL-tRNA SYNTHETASE ON SEPHAROSE lle-tRNA COLUMN The chromatographic procedure is described in the legend of Fig. 1. One enzyme unit was defined as the amount of enzyme catalysing the formation of 1 nmole of isoleucyl-tRNA during the initial 20 min at 30°C under the assay conditions given by Yaniv and Gros 2 for the valine-activating enzyme. Protein was measured by the turbidimetric tannin method ~3.
Fraction
VoL
Protein
Total activity
Specific activity
Recovery
{ml)
(mg)
{units)
(units~rag)
(%)
Starting solution
3
9.8
160
Column eluate
8
0.335
79.4
237
49.6
14.5
Peak fraction
2
0.075
33.7
450
21.0
27.5
16.3
Purification
100
L n m m
m
÷ A
B
Fig. 2. Polyacrylamide gel disc electrophoresis of isoleucyl-tRNA synthetase. A. Fraction of partially purified enzyme before affinity chromatography. B. Peak fraction of purified enzyme after chromatography on Sephadex-Ile-tRNA column.
Details o f the f r a c t i o n a t i o n are given in the legend o f Fig. 1, and the results summarized in Table I. The elution pattern o f i s o l e u c y l - t R N A synthetase indicates that the S e p h a r o s e - I l e - t R N A c o l u m n , besides the e n z y m e , retains considerable a m o u n t s o f o t h e r proteins eluted at a l o w e r c o n c e n t r a t i o n o f NaC1. In the p e a k fraction o f the purified e n z y m e insignificant activities o f leucine- and valine-activating e n z y m e s are s o m e t i m e s d e t e c t e d . The specific activity o f the i s o l e u c y l - t R N A synthetase
Biochim. Biophys. Acta, 272 (1972) 137-140
140
BBA REPORT
peak fraction was at least 28-fold higher than that applied on the column. Polyacrylamide disc gel electrophoresis revealed two near located bands (Fig. 2). The advantage of the described method is that it does not require the isolation of amino acid-specific tRNA. However, enzyme preparations applied on the column should be as free as possible from ribonuclease activity. In the experiment described we have used the same column 3 -4 times only because of its progressively decreasing capacity for enzyme retention. Simultaneously, in the fractions eluting laterObove the 50th fraction on Fig. 1), nacleotide material appeared, as demonstrated by measurements of the A26o/ A 28o ratio o f each fraction. In these experiments up to 50% of applied isoleucyl-tRNA synthetase activity was retained on the column. The isolation conditions for different synthetases may differ and, therefore, they should be established individually for each system investigated. For example, isoleucyl-tRNA synthetase is not retained on the Sepharose -Ile-tRNA column at pH 7.5 as it is at pH 5.5. The effect can be attributed to differences in affinity of aminoacyl-tRNA synthetase to tRNA Ile at both pH values because it is generally assumed that the binding of tRNAs to their specific aminoacyltRNA synthetases is enhanced under acidic conditions (see, e.g. refs 10 and I 1). There are, of course, the same relations on the column where the enzyme interacts with immobilized tRNA ne. The method elaborated was successfully employed for the purification o f isoleucyl-tRNA synthetase of plant origin ~2. This work was supported by the Polish Academy o f Sciences within Project 09.3.1. 1 2 3 4 5 6 7
8 9 10 11 12 13
A.N. Baldwin and P. Berg, J. Biol. Chem., 241 (1966) 831. M. Yaniv and F. Gros, J. Mol. Biol., 44 (1969) 1. M. Yarus and P. Berg,,/. Mol. Biol., 42 (1969) 171. D.J. Arndt and P. Berg, J. Biol. Chem., 245 (1970) 665. F. Berthelot and M. Yaniv, Eur. J. Biochem., 16 (1970) 123. G.R. Penzer, E.L. Bennett and M. Calvin, Eur. J. Biochem., 20 (1971) i. P. Cuatracasas, Nature, 228 (1970) 132. P. Cuatracasas, J. Biol. Chem., 245 (1970) 3059. M.P. Poonian, A.J. Schlabach and A. Weissbach, Biochemistry, 10 (1971)425. C. Helene, F. Brun and M. Yaniv, J. Mol. BioL, 58 (1971) 349. H.M. Kosakowski and A. B6ck, Eur. J. Biochem., 24 (1971) 190. S. Bartkowiak, to be published. W. Mejbaum-Katzenellenbogen,Acta Biochem. Polon., 2 (1955) 279.
Biochim. Biophys. Acta, 272 (1972) 137 140
137
BBA Report BBA 91353
The purification of aminoacyl-tRNA synthetases by affinity chromatography S. BARTKOWIAKand J. PAWEEKIEWICZ Institute o f Plant Genetics, Polish Academy of Sciences and Institute of Biochemistry, College o f Agriculture, Poznah (Poland)
(Received May 15th, 1972)
SUMMARY
The method of the purification of aminoacyl-tRNA synthetases by affinity chromatography is described on the basis of complex formation between the enzyme and aminoacyl-tRNA attached to bromoacetylamidobutyl-Sepharose. The purification of Escherichia coli isoleucyl-tRNA synthetase is given as an example.
The use of common enzymological techniques to prepare some of the aminoacyltRNA synthetases in a homogeneous state has sometimes failed. We have elaborated a method based on the technique of affinity chromatography that achieves the preparation of aminoacyl-tRNA synthetases of high purity in a simple manner. In this report we give as an example the purification of Escherichia coli isoleucyl-tRNA synthetase by this technique. The enzyme was already isolated in a homogeneous state by Baldwin and Berg 1, and by Yaniv and Gros 2, and its properties are well known 1' 3-6 Partially purified E. coli enzyme was chromatographed on modified Sepharose to which isoleucyl-tRNA molecules were attached. Sepharose was modified in the manner described by Cuatrecasas 7, 8. Sepharose 4B was activated with cyanogen bromide, coupled with tetramethylenediamine and then treated with bromoacetic ester of N-hydroxysuccinimide with the formation of bromoacetamidobutyl-Sepharose. To 20 ml of this derivative in 0.1 M potassium phosphate buffer, pH 7.5, 30 mg ofE. coli tRNA, aminoacylated previously with isoleucine and dissolved in 10 ml of phosphate buffer, was added, and the suspension was stirred for 3 days at 4°C. The aminoacylation reaction was carried out according to Yaniv.and Gros 2, and the isoleucyl-tRNA obtained was deproteinized carefully before use by phenol and then by chloroform-isoamyl alcohol (5:1, v/v) extraction. The suspension was filtered off and washed at 4°C with 500 ml of water, 100 Bioehim. Biophys. Acta, 272 (1972) 137 140
138
BBA REPORT
inl of 1 M NaC1 and 300 ml of water, successively. Next the preparation was stirred for 24 h in 0.1 M ethanolamine, adjusted to pH 7.5 with HCI in order to block all the unreacted active groups. After this reaction the suspension was washed with 500 ml of 0.3 M NaC1, and finally used for enzyme chromatography with an appropriate buffer. The amount of Sepharose-bound tRNA was calculated from the difference of its concentrations present in the supernatants before and after the binding reaction, or more directly after the hydrolysis of Sepharose-Ile-tRNA conjugate in 0.3 M KOH at 80°C for 50 rain. The hydrolysate of modified Sepharose without bound tRNA was taken in this case as the blank sample for the spectrophotometrical measurement. In the procedure given, about 25 #g of tRNA was bound per ml of Sepharose derivative. Attemps to bind isoleucyltRNA or unacylated tRNA directly to cyanogen bromide-activated Sepharose failed, in agreement with the data of Poonian et al. 9. Also bromoacetamidobutyl-Sepharose does not bind unacylated tRNA, which indicates that the a-amino group takes part in the coupling reaction. Thus the bromoacetamidobutyl "arm" attached to Sepharose plays two roles: it contains the reactive bromoacetyl group able to react with aminoacyl-tRNA, and, on the other hand, keeps the bound tRNA molecule away from the Sepharose matrix which facilitates the interaction between the enzyme and tRNA. Fractionation of partially purified isoleucyl-tRNA synthetase on a Sepharose -Ile-tRNA column was achieved in phosphate buffer at pH 5.5.
0.50I-. . . . . . . . . . . . . . . . . . . . . . --o.,40
i
m-- o.3o
~2
12500
i";
'~,
2000
I i
t~m<~
0.20
T
IO0O
0
-
o,o
Oi
i
I
tO
I
°--t':'--~--
20
~_
30
~[-~Izf~-z--~
40
50
--~
"r'60
• I
0
70
FRACTION NUHBER Fig. I. Chromatography ofE. coli isoleucyl-tRNA synthetase on Sephadex-Ile-tRNA column. The enzyme (10 nag of protein) partially purified to Stage IV as described by Yaniv and Gros 2 , was applied on the Sepharose- Ile-tRNA column (0.9 cm × 15 cm) equilibrated at 4°C with 0.1 M potassium phosphate buffer, pH 5.5, containing 1 mM MgC12 and 0.5 mM dithioerythriol, and washed with the same buffer until the absorbance at 280 nm decreased below 0.01. Elution was carried out with linear gradients from 0 to 0.3 M NaCI in the phosphate buffer. Fractions of 2 ml were collected at a flow rate of 20 ml/h. The absorbance at 280 n m ( - - - - ) and the enzyme activity (o . . . . o) were determined in each fraction. ~ A indicates the linear NaC1 gradient.
Biochim. Biophys. Acta, 272 (1972) 137
140
BBA REPORT
139
TABLE I PURIFICATION OF E. COLI ISOLEUCYL-tRNA SYNTHETASE ON SEPHAROSE lle-tRNA COLUMN The chromatographic procedure is described in the legend of Fig. 1. One enzyme unit was defined as the amount of enzyme catalysing the formation of 1 nmole of isoleucyl-tRNA during the initial 20 min at 30°C under the assay conditions given by Yaniv and Gros 2 for the valine-activating enzyme. Protein was measured by the turbidimetric tannin method ~3.
Fraction
VoL
Protein
Total activity
Specific activity
Recovery
{ml)
(mg)
{units)
(units~rag)
(%)
Starting solution
3
9.8
160
Column eluate
8
0.335
79.4
237
49.6
14.5
Peak fraction
2
0.075
33.7
450
21.0
27.5
16.3
Purification
100
L n m m
m
÷ A
B
Fig. 2. Polyacrylamide gel disc electrophoresis of isoleucyl-tRNA synthetase. A. Fraction of partially purified enzyme before affinity chromatography. B. Peak fraction of purified enzyme after chromatography on Sephadex-Ile-tRNA column.
Details o f the f r a c t i o n a t i o n are given in the legend o f Fig. 1, and the results summarized in Table I. The elution pattern o f i s o l e u c y l - t R N A synthetase indicates that the S e p h a r o s e - I l e - t R N A c o l u m n , besides the e n z y m e , retains considerable a m o u n t s o f o t h e r proteins eluted at a l o w e r c o n c e n t r a t i o n o f NaC1. In the p e a k fraction o f the purified e n z y m e insignificant activities o f leucine- and valine-activating e n z y m e s are s o m e t i m e s d e t e c t e d . The specific activity o f the i s o l e u c y l - t R N A synthetase
Biochim. Biophys. Acta, 272 (1972) 137-140
140
BBA REPORT
peak fraction was at least 28-fold higher than that applied on the column. Polyacrylamide disc gel electrophoresis revealed two near located bands (Fig. 2). The advantage of the described method is that it does not require the isolation of amino acid-specific tRNA. However, enzyme preparations applied on the column should be as free as possible from ribonuclease activity. In the experiment described we have used the same column 3 -4 times only because of its progressively decreasing capacity for enzyme retention. Simultaneously, in the fractions eluting laterObove the 50th fraction on Fig. 1), nacleotide material appeared, as demonstrated by measurements of the A26o/ A 28o ratio o f each fraction. In these experiments up to 50% of applied isoleucyl-tRNA synthetase activity was retained on the column. The isolation conditions for different synthetases may differ and, therefore, they should be established individually for each system investigated. For example, isoleucyl-tRNA synthetase is not retained on the Sepharose -Ile-tRNA column at pH 7.5 as it is at pH 5.5. The effect can be attributed to differences in affinity of aminoacyl-tRNA synthetase to tRNA Ile at both pH values because it is generally assumed that the binding of tRNAs to their specific aminoacyltRNA synthetases is enhanced under acidic conditions (see, e.g. refs 10 and I 1). There are, of course, the same relations on the column where the enzyme interacts with immobilized tRNA ne. The method elaborated was successfully employed for the purification o f isoleucyl-tRNA synthetase of plant origin ~2. This work was supported by the Polish Academy o f Sciences within Project 09.3.1. 1 2 3 4 5 6 7
8 9 10 11 12 13
A.N. Baldwin and P. Berg, J. Biol. Chem., 241 (1966) 831. M. Yaniv and F. Gros, J. Mol. Biol., 44 (1969) 1. M. Yarus and P. Berg,,/. Mol. Biol., 42 (1969) 171. D.J. Arndt and P. Berg, J. Biol. Chem., 245 (1970) 665. F. Berthelot and M. Yaniv, Eur. J. Biochem., 16 (1970) 123. G.R. Penzer, E.L. Bennett and M. Calvin, Eur. J. Biochem., 20 (1971) i. P. Cuatracasas, Nature, 228 (1970) 132. P. Cuatracasas, J. Biol. Chem., 245 (1970) 3059. M.P. Poonian, A.J. Schlabach and A. Weissbach, Biochemistry, 10 (1971)425. C. Helene, F. Brun and M. Yaniv, J. Mol. BioL, 58 (1971) 349. H.M. Kosakowski and A. B6ck, Eur. J. Biochem., 24 (1971) 190. S. Bartkowiak, to be published. W. Mejbaum-Katzenellenbogen,Acta Biochem. Polon., 2 (1955) 279.
Biochim. Biophys. Acta, 272 (1972) 137 140