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Affinity chromatography of rat liver aminoacyl-tRNA synthetase complex
vol. 80, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
February 28, 1978
Pages
AFFINITY
CHROMATOGRAPHY OF RAT LIVER
709-714
AMINOACYL-tRNA
SYNTHETASE COMPLEX Chi Van Dang and David Department
of Chemistry,
December
Yang
Georgetown
Washington, Received
C.H.
D.C.
University
20057
22,1977
Summary The affinity column lysyldiaminohexyl-Sepharose 4B has been synthesized for the purification of aminoacyl-tRNA synthetase, complexes. Lysyl-tRNA synthetase (EC 6.1.1.6) bound specifically to the Sepharose-bound lysine. The purified lysyl-tRNA synthetase was associated with arginyl-tRNA synthetase (EC 6.1.1.16) and sedimented at 18s and 12s. A 24s lysyl-tRNA synthetase bound specifically to the affinity column and also found associated with arginyl-tRNA synthetase. The results favor the model of a heterotypic multienzyme complex of mammalian aminoacyl-tRNA synthetases. Introduction The high acyl-tRNA
molecular
synthetase a brief
12s complexes
containing
the
from
rat
complexes
aminoacyl-tRNA graphy the
liver
and the
(10).
We report
interaction
structural complexes
models
least
enzyme
sults
o:E affinity complex
lysyl-
possible here
attempt
complex
with
structural a rapid
4B.
18S,
Taking
the
chromato-
advantage
of
and lysylthree
possible
complexes. cqmplex
carrier
of of the
column
lysine
multi-enzyme
and synthe-
purification
synthetase
favor
We
relationship
to differentiate
or without
chromatography
(l-9).
and arginyl-tRNA
by affinity
of aminoacyl-tRNA
amino-
of 24S,
Sepharose-bound
may be a heterotypic
typic
isolation
Sepharose of
we also
of the
complexes
on lysyldiaminohexyl
tRNA synthetase,
enzyme
at
of mammalian
have been documented
account
synthetase
specific
complexes
complexes
have reported
tases
weight
molecule. heterotypic
The
or a homoThe remulti-
model.
709
Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
vol. 80, NO.4, Materials
BIOCHEMICAL
1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and Methods
Radioactive IV- 14 CJ-L-arginine (290 mCi/mmol) and [U- 14 Cl-Llysine (282 mCi/mmol) were purchased from New England Nuclear, Inc. Rat liver transfer RNA was purchased from Biogenics Research COrpOrCatalase, B-galactosidase, orthoation (Chagrin Falls, Ohio). nitrophenylsufenyl chloride, lysine-HCl, and diaminohexyl-Sepharose 4B (AH Sepharose 4B, 6-10 umoles/ml capacity) were obtained from were analytical grade or Sigma Chemical Co. All other chemicals the purest form available. Buffer A contains 50 mM Tris-HC1 (pH 7.5), 5 mM Mg acetate, and 2 mM dithioerythreitol IDTE). Aminoacylation assays were perThe formed according to Som and Hardesty (5) with modifications. enzyme reaction was quenched after a lo-minute incubation at 37OC by spotting 50 ~1 aliquots from a total reaction volume of 125 ul on a 3 MM filter paper pad according to Yang and Sdll (11). All of the following procedures were performed at 0-4*C unless otherwise indicated. Fresh rat livers dissected from 6-P month cervitally dislocated WF male rats (Microbiological Associates) were homogenized, subjected to differential centrifugation, and subsequently chromatographed on a Sephadex G-200 column according to Vennegoor and Bloemendal (4). The excluded fractions from gel filtration were collected and concentrated with a PM30 membrane in an Amicon concentrator to the material designated as G2OOC (ca. 40 mg pzotein/ml). The G200C was stored in liquid nitrogen until used. Linear sucrose gradients (20-40%) were made in Buffer A with 25 mM KCl. Samples (0.2 ml) and standards (catalase, 11.3S, 8galactosidase, 16s) were loaded on identical gradients and centrifuged simultaneously for 14 hrs at 4'C and 40,000 rpm in an SW5OL rotor using a Spinco Model L ultracentrifuge. The 24s lysyl-tRNA synthetase was obtained by sedimenting GZOOC on a 5-20% sucrose gradient for 6 hrs as described previously (10). The diorthonitrophenylsulfenyl lysine (di-ONPS-Lys) derivative was prepared according to Zervas et rll(12). Coupling of 4 gm of AH Sepharose 4B (in 16 ml buffer) to 0.5 gm of di-ONPS-LYS and subsequent workup was done according to Robert-Gero and Waller (13). The affinity column was equilibrated with either Buffer B (Buffer A plus 200 mM KC1 and 1 mM ATP) or Buffer C (0.2 M potassium phosphate 25 mMKC1, 5 mM MgAc2, 2 mM DTE, and 1 mM ATP) and was used (pH 7.51, as the starting buffer; the high salt concentrations were used to eliminate nonspecific ionic interactions (13). After sufficient elution, a second buffer (pH 7.5) identical to the starting buffer plus 20 mM lysine was used to elute off the bound enzyme. Results A typical Lysyl-tRNA by affinity in
Buffer
synthetase
affinity
synthetase
column
was enriched
chromatography. B was used as the or arginyl-tRNA
chromatogram about
is 5000-fold
When 20 mM arginine second synthetase 710
buffer
shown in Figure with instead
no detectable
was eluted
1.
40% yield of
lysine
lysyl-tRNA
as shown in Table
I,
Vol. 80, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL
Fraction
Fig.
1
Chromatogram 1.2 mg G200C equilibrated Buffer B then fractions of
TABLE I. -
RESEARCH COMMUNICATIONS
Number
of G200C on lysyl-AH Sepharose 4B. Approximately was loaded to a 3 ml column (0.7 x 8 cm) prewith Buffer B. The column was first eluted with with 20 mM lysine in Buffer B at 10 ml/hr, and 1 ml were collected. 10 l.11 was assayed.
Recovery of Lysyl-tRNA Synthetase on Lysyl-AH Sepharose 4B
Experiment
Total Protein
Second Buffer
1.
G200C
1.2
Buffer
B + 20 mM Lysine
Fresh
85
2.
G200C
4.3
Buffer
B + 20 mM Lysine
Fresh
50
3.
G200C
4.3
Buffer
B + 20 mM Lysine
1st reuse
30
4.
G200C
4.3
Buffer
B + 20 mM Lysine
2nd reuse
0
5.
G200C
4.3
Buffer
B + 20 mM Arginine
Fresh
0
6.
24s
0.1
Buffer
C + 20 mM Lysine
Fresh
>90
(mg)a
loaded
was estimated
Affinity Gel
Synthetase
ratio
A
E
a.
Amount of protein A260 (14).
b.
Percentage of purified lysyl-tRNA synthetase activity was based on the total recovered lysyl-tRNA synthetase activities. Approximately 60% of the lysyl-tRNA synthetase activity loaded on the affinity column was recovered in all cases.
711
by the
% Lysyl-tR
of A280 and
BIOCHEMICAL
Vol. 80, No. 4, 1978
indicating covery vily
that
of
the elution
lysyl-tRNA
lysyl-AH
the gel cally
reduced
lysyl-tRNA
be used since
synthetase
of
repeated
I.
use of
the column drasti-
was associated
by aminoacylation. synthetase
at 18.5 and 12s in sucrose
two enzymes are physically We have previously at
isoleucyl-,
that
24s with
leucyl-
activities gradient
suggesting
the
synthetase
lysyl-AH
4B column as shown in Figure
tRNA synthetase
activity
specifically
eluted
with
lysyl-tRNA
synthetase
synthetase
activity.
co-
that
synthetase
the activities
and methionyl-tRNA
2,
complexes.
lysyl-tRNA
The 24s lysyl-tRNA Sepharose
tRNA
were found
in these
at least
(0.1
with
As shown in Figure
associated
reported
in G200C cosedimented arginyl-,
depends hea-
as shown in Table
and resolution
and arginyl-tRNA
sedimented
The re-
I).
determined
lysyl-
specific.
by 20 LUMlysine
loaded
4B should
RESEARCH COMMUNICATIONS
was indeed
eluted
protein
the capacity
(see Table
synthetases
the
synthetase
Sepharose
The purified
the
by lysine
on the amount of total
Fresh
AND BIOPHYSICAL
of
synthetase
mg) was further
(10).
purified 3.
by a
The lysyl-
was retained
on the column and can be
20 mM lysine
in Buffer
was again
associated
C.
with
The purified
arginyl-tRNA
Discussion We report of
lysyl-tRNA
here
the purification
synthetase
and structural
complexes
by affinity
elucidation
column chromato-
graphy The multiple
forms of rat
have been documented working
hypothesis
aminoacyl-tRNA association synthetase
of
(3,4,8).
liver
We have previously
on the structural
synthetase
forms
lysyl-tRNA
synthetase
activities
was never
aminoacyl-tRNA
(10).
relationship However, with
other
demonstrated
712
beyond
synthetase proposed
a
of various the
physical
aminoacyl-tRNA CodeShenta-
BIOCHEMICAL
Vol. 80, No. 41, 1978
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
0.2 0
0
5
10 Fraction
15
TOP
Number
Fig.
2
Cos,edimentation of purified lysyl-tRNA synthetase (-VT) and. arginyl-tRNA synthetase (------o---o-) on a 20-40% sucrose gradient (see material and methods). 75 1.11was assayed.
Fig.
3
Chromatogram of 24s lysyl-tRNA Sepharose 4B. (cf. Table I.
tion
and coelution
to our knowledge
from various that
this
is
synthetase complex on lysyl-AH Experiment 6). 50 ~1 was assayed.
chromatographic the first
713
time
columns. that
It
is
mammalian amino-
Vol. 80, No. 4, 1978
acyl-tRNA
synthetases
BIOCHEMICAL
are observed
finity
column in contrast
(13).
Reconstitution
has been difficult. affinity ture
of
to coelute
to prokaryotic the large
The specific
column can provide of multienzyme
AND BJOPHYSICAL RESEARCH COMMUNICATIONS
aminoacyl-tRNA
complexes interaction
an alternative
on abiospecific
af-
synthetase
from smaller
complexes
of enzymes with to delineate
the
the struc-
complexes.
ACKNOWLEDGEMENTS Grant
This work was supported PCM 76-14955.
by National
Science
Foundation
References 1. 2. 3.
S&11, D. and Schimmel, P.R. (1974) in The Enzymes (Bayer, P .D., Vol 10, pp 489-538, Academic Press, New York. ea.). Neidhardt, F.C., Parker, J., and McKeever, W.G. (1975) Annu. Rev. Microbial. 29, 215-250. Bandyopadhyay, A.K. and Deutscher, M.P. (1971) J. Mol. Biof. 60,
4. 5. 6. 7. a. 9. 10. 11. 12. 13. 14.
113-122.
Vennegoor, C. and Bloemendal, H. (1972) Eur. J. Biochem. 26, 462-73. Som, K. and Hardesty, B. (1975) Arch. Biochem. Biophys. 166, 507-517. Berg, B.H. (1975) Biochim. Biophys. Acta 414, 93-98. Roberts, W.K. and Olsen, M.L. (1976) Biochim. Biophys. Acta. 454, 480-492. Goto, T. and Schweiger, A. (1973) Hoppe-Seyler's Z. Physiol. Chem. 354. 1027-1033. Hele, P. and Herbert, L. (1977) Biochim. Biophys. Acta 479, 311-321. (1978) in Cellular Functions and Dang , C.V. and Yang, D.C.H. Molecular Structure, (Agris, P.F., Leoppky, R., and Sykes, B., Academic Press (in press) ed.), Yang, C.H. and S&11, D. (1974) Proc. Natl. Acad. Sci., U.S.A., 71, 2838-2842. Zervas, L. and Hamalidis, C. (1965) J. Am. them. Sot. 87,99-104. Robert-Gero, M. and-Waller, J.P. (1974), Meth. in Enzymol., 24, 506-513. Warburg, 0. and Christian, W. (1942) Biochem. 2. 310, 384-421.
714
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
February 28, 1978
Pages
AFFINITY
CHROMATOGRAPHY OF RAT LIVER
709-714
AMINOACYL-tRNA
SYNTHETASE COMPLEX Chi Van Dang and David Department
of Chemistry,
December
Yang
Georgetown
Washington, Received
C.H.
D.C.
University
20057
22,1977
Summary The affinity column lysyldiaminohexyl-Sepharose 4B has been synthesized for the purification of aminoacyl-tRNA synthetase, complexes. Lysyl-tRNA synthetase (EC 6.1.1.6) bound specifically to the Sepharose-bound lysine. The purified lysyl-tRNA synthetase was associated with arginyl-tRNA synthetase (EC 6.1.1.16) and sedimented at 18s and 12s. A 24s lysyl-tRNA synthetase bound specifically to the affinity column and also found associated with arginyl-tRNA synthetase. The results favor the model of a heterotypic multienzyme complex of mammalian aminoacyl-tRNA synthetases. Introduction The high acyl-tRNA
molecular
synthetase a brief
12s complexes
containing
the
from
rat
complexes
aminoacyl-tRNA graphy the
liver
and the
(10).
We report
interaction
structural complexes
models
least
enzyme
sults
o:E affinity complex
lysyl-
possible here
attempt
complex
with
structural a rapid
4B.
18S,
Taking
the
chromato-
advantage
of
and lysylthree
possible
complexes. cqmplex
carrier
of of the
column
lysine
multi-enzyme
and synthe-
purification
synthetase
favor
We
relationship
to differentiate
or without
chromatography
(l-9).
and arginyl-tRNA
by affinity
of aminoacyl-tRNA
amino-
of 24S,
Sepharose-bound
may be a heterotypic
typic
isolation
Sepharose of
we also
of the
complexes
on lysyldiaminohexyl
tRNA synthetase,
enzyme
at
of mammalian
have been documented
account
synthetase
specific
complexes
complexes
have reported
tases
weight
molecule. heterotypic
The
or a homoThe remulti-
model.
709
Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
vol. 80, NO.4, Materials
BIOCHEMICAL
1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and Methods
Radioactive IV- 14 CJ-L-arginine (290 mCi/mmol) and [U- 14 Cl-Llysine (282 mCi/mmol) were purchased from New England Nuclear, Inc. Rat liver transfer RNA was purchased from Biogenics Research COrpOrCatalase, B-galactosidase, orthoation (Chagrin Falls, Ohio). nitrophenylsufenyl chloride, lysine-HCl, and diaminohexyl-Sepharose 4B (AH Sepharose 4B, 6-10 umoles/ml capacity) were obtained from were analytical grade or Sigma Chemical Co. All other chemicals the purest form available. Buffer A contains 50 mM Tris-HC1 (pH 7.5), 5 mM Mg acetate, and 2 mM dithioerythreitol IDTE). Aminoacylation assays were perThe formed according to Som and Hardesty (5) with modifications. enzyme reaction was quenched after a lo-minute incubation at 37OC by spotting 50 ~1 aliquots from a total reaction volume of 125 ul on a 3 MM filter paper pad according to Yang and Sdll (11). All of the following procedures were performed at 0-4*C unless otherwise indicated. Fresh rat livers dissected from 6-P month cervitally dislocated WF male rats (Microbiological Associates) were homogenized, subjected to differential centrifugation, and subsequently chromatographed on a Sephadex G-200 column according to Vennegoor and Bloemendal (4). The excluded fractions from gel filtration were collected and concentrated with a PM30 membrane in an Amicon concentrator to the material designated as G2OOC (ca. 40 mg pzotein/ml). The G200C was stored in liquid nitrogen until used. Linear sucrose gradients (20-40%) were made in Buffer A with 25 mM KCl. Samples (0.2 ml) and standards (catalase, 11.3S, 8galactosidase, 16s) were loaded on identical gradients and centrifuged simultaneously for 14 hrs at 4'C and 40,000 rpm in an SW5OL rotor using a Spinco Model L ultracentrifuge. The 24s lysyl-tRNA synthetase was obtained by sedimenting GZOOC on a 5-20% sucrose gradient for 6 hrs as described previously (10). The diorthonitrophenylsulfenyl lysine (di-ONPS-Lys) derivative was prepared according to Zervas et rll(12). Coupling of 4 gm of AH Sepharose 4B (in 16 ml buffer) to 0.5 gm of di-ONPS-LYS and subsequent workup was done according to Robert-Gero and Waller (13). The affinity column was equilibrated with either Buffer B (Buffer A plus 200 mM KC1 and 1 mM ATP) or Buffer C (0.2 M potassium phosphate 25 mMKC1, 5 mM MgAc2, 2 mM DTE, and 1 mM ATP) and was used (pH 7.51, as the starting buffer; the high salt concentrations were used to eliminate nonspecific ionic interactions (13). After sufficient elution, a second buffer (pH 7.5) identical to the starting buffer plus 20 mM lysine was used to elute off the bound enzyme. Results A typical Lysyl-tRNA by affinity in
Buffer
synthetase
affinity
synthetase
column
was enriched
chromatography. B was used as the or arginyl-tRNA
chromatogram about
is 5000-fold
When 20 mM arginine second synthetase 710
buffer
shown in Figure with instead
no detectable
was eluted
1.
40% yield of
lysine
lysyl-tRNA
as shown in Table
I,
Vol. 80, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL
Fraction
Fig.
1
Chromatogram 1.2 mg G200C equilibrated Buffer B then fractions of
TABLE I. -
RESEARCH COMMUNICATIONS
Number
of G200C on lysyl-AH Sepharose 4B. Approximately was loaded to a 3 ml column (0.7 x 8 cm) prewith Buffer B. The column was first eluted with with 20 mM lysine in Buffer B at 10 ml/hr, and 1 ml were collected. 10 l.11 was assayed.
Recovery of Lysyl-tRNA Synthetase on Lysyl-AH Sepharose 4B
Experiment
Total Protein
Second Buffer
1.
G200C
1.2
Buffer
B + 20 mM Lysine
Fresh
85
2.
G200C
4.3
Buffer
B + 20 mM Lysine
Fresh
50
3.
G200C
4.3
Buffer
B + 20 mM Lysine
1st reuse
30
4.
G200C
4.3
Buffer
B + 20 mM Lysine
2nd reuse
0
5.
G200C
4.3
Buffer
B + 20 mM Arginine
Fresh
0
6.
24s
0.1
Buffer
C + 20 mM Lysine
Fresh
>90
(mg)a
loaded
was estimated
Affinity Gel
Synthetase
ratio
A
E
a.
Amount of protein A260 (14).
b.
Percentage of purified lysyl-tRNA synthetase activity was based on the total recovered lysyl-tRNA synthetase activities. Approximately 60% of the lysyl-tRNA synthetase activity loaded on the affinity column was recovered in all cases.
711
by the
% Lysyl-tR
of A280 and
BIOCHEMICAL
Vol. 80, No. 4, 1978
indicating covery vily
that
of
the elution
lysyl-tRNA
lysyl-AH
the gel cally
reduced
lysyl-tRNA
be used since
synthetase
of
repeated
I.
use of
the column drasti-
was associated
by aminoacylation. synthetase
at 18.5 and 12s in sucrose
two enzymes are physically We have previously at
isoleucyl-,
that
24s with
leucyl-
activities gradient
suggesting
the
synthetase
lysyl-AH
4B column as shown in Figure
tRNA synthetase
activity
specifically
eluted
with
lysyl-tRNA
synthetase
synthetase
activity.
co-
that
synthetase
the activities
and methionyl-tRNA
2,
complexes.
lysyl-tRNA
The 24s lysyl-tRNA Sepharose
tRNA
were found
in these
at least
(0.1
with
As shown in Figure
associated
reported
in G200C cosedimented arginyl-,
depends hea-
as shown in Table
and resolution
and arginyl-tRNA
sedimented
The re-
I).
determined
lysyl-
specific.
by 20 LUMlysine
loaded
4B should
RESEARCH COMMUNICATIONS
was indeed
eluted
protein
the capacity
(see Table
synthetases
the
synthetase
Sepharose
The purified
the
by lysine
on the amount of total
Fresh
AND BIOPHYSICAL
of
synthetase
mg) was further
(10).
purified 3.
by a
The lysyl-
was retained
on the column and can be
20 mM lysine
in Buffer
was again
associated
C.
with
The purified
arginyl-tRNA
Discussion We report of
lysyl-tRNA
here
the purification
synthetase
and structural
complexes
by affinity
elucidation
column chromato-
graphy The multiple
forms of rat
have been documented working
hypothesis
aminoacyl-tRNA association synthetase
of
(3,4,8).
liver
We have previously
on the structural
synthetase
forms
lysyl-tRNA
synthetase
activities
was never
aminoacyl-tRNA
(10).
relationship However, with
other
demonstrated
712
beyond
synthetase proposed
a
of various the
physical
aminoacyl-tRNA CodeShenta-
BIOCHEMICAL
Vol. 80, No. 41, 1978
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
0.2 0
0
5
10 Fraction
15
TOP
Number
Fig.
2
Cos,edimentation of purified lysyl-tRNA synthetase (-VT) and. arginyl-tRNA synthetase (------o---o-) on a 20-40% sucrose gradient (see material and methods). 75 1.11was assayed.
Fig.
3
Chromatogram of 24s lysyl-tRNA Sepharose 4B. (cf. Table I.
tion
and coelution
to our knowledge
from various that
this
is
synthetase complex on lysyl-AH Experiment 6). 50 ~1 was assayed.
chromatographic the first
713
time
columns. that
It
is
mammalian amino-
Vol. 80, No. 4, 1978
acyl-tRNA
synthetases
BIOCHEMICAL
are observed
finity
column in contrast
(13).
Reconstitution
has been difficult. affinity ture
of
to coelute
to prokaryotic the large
The specific
column can provide of multienzyme
AND BJOPHYSICAL RESEARCH COMMUNICATIONS
aminoacyl-tRNA
complexes interaction
an alternative
on abiospecific
af-
synthetase
from smaller
complexes
of enzymes with to delineate
the
the struc-
complexes.
ACKNOWLEDGEMENTS Grant
This work was supported PCM 76-14955.
by National
Science
Foundation
References 1. 2. 3.
S&11, D. and Schimmel, P.R. (1974) in The Enzymes (Bayer, P .D., Vol 10, pp 489-538, Academic Press, New York. ea.). Neidhardt, F.C., Parker, J., and McKeever, W.G. (1975) Annu. Rev. Microbial. 29, 215-250. Bandyopadhyay, A.K. and Deutscher, M.P. (1971) J. Mol. Biof. 60,
4. 5. 6. 7. a. 9. 10. 11. 12. 13. 14.
113-122.
Vennegoor, C. and Bloemendal, H. (1972) Eur. J. Biochem. 26, 462-73. Som, K. and Hardesty, B. (1975) Arch. Biochem. Biophys. 166, 507-517. Berg, B.H. (1975) Biochim. Biophys. Acta 414, 93-98. Roberts, W.K. and Olsen, M.L. (1976) Biochim. Biophys. Acta. 454, 480-492. Goto, T. and Schweiger, A. (1973) Hoppe-Seyler's Z. Physiol. Chem. 354. 1027-1033. Hele, P. and Herbert, L. (1977) Biochim. Biophys. Acta 479, 311-321. (1978) in Cellular Functions and Dang , C.V. and Yang, D.C.H. Molecular Structure, (Agris, P.F., Leoppky, R., and Sykes, B., Academic Press (in press) ed.), Yang, C.H. and S&11, D. (1974) Proc. Natl. Acad. Sci., U.S.A., 71, 2838-2842. Zervas, L. and Hamalidis, C. (1965) J. Am. them. Sot. 87,99-104. Robert-Gero, M. and-Waller, J.P. (1974), Meth. in Enzymol., 24, 506-513. Warburg, 0. and Christian, W. (1942) Biochem. 2. 310, 384-421.
714