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Studies on valyl-tRNA synthetase and tRNAVal from Escherichia coli,
J. Mol. Biol. (1969) 44, 1-15
Studies on Valyl-tRNA Syuthetase and tRNAvpl from Escherichia coli I. Purification and Properties of the Enzyme from Normal Escherichiu coli Strains M. Y~r.v
AND
F. GROS
Insti.tu.t de Bidogie Physico-Chirnipue Service de Phyeiologie ilficrobienne 13, rue Pierre et Marie Curie Paris 5e, France (Received 20 December 1968) A highly pursed valyl-tRNA synthetaae was prepared from Escherichia co& K12 strain. A sedimentation coe&ient (@G) of 6.6 s and a molecular weight of 110,000 were determined. One active site for valine and ATP was detected. Binding of these substrates to the active site changed the number of SH groups titrated. Some indications were found for a conformational change induced by an aminoacyl-AMP analog. Antiserum prepared against the pure enzyme inhibited all the
activity in extracts, indicating the existence of one activating enzyme for valine in E. co& No immunological cross-reaction was detected between valyl and isoleucyl activating enzymea in spite of similarities in structure and substrate
recognition.
1. Introduction The correct translation of a genetic message into a linear polypeptide sequence depends upon the attachment of the appropriate amino acid to a specific tRNA. The amino acid is first activated by an aminoacyl-tRNA synthetase in the presence of ATP and magnesium ions and subsequently transferred to the cognate tRNA (Norris & Berg, 1964). Activating enzymes therefore constitute one of the best models for studying the basic phenomenon of nucleic acid “recognition” by specific proteins. The amino acidtRNA-enzyme interaction can be studied with enzymes derived from normal or phage-infected cells (Neidhardt & Earhart, 1966) or from strains harboring a mutation in the enzyme or tRNA genes. We have undertaken a study of wild-type and genetically modified valyl-tRNA synthetase from Escher&&a coli. The present paper will report on the purification procedure and general properties of the wild-type enzyme. A highly purified VRSt has been obtained with a yield of 34%. Physical constants, certain chemical properties as well as the immunological behavior of the enzyme are described. Ammoacyl-AMPenzyme complexes were isolated with the pure enzyme indicating the presence of one active site per chain of 110,909. t Abbreviations used: DTNB, S,S’-dithiobio(2-nitrobenzoic mid); VRS, valyl-tRNA synthetaae; IRS, isoleucyl-tRNA synthetaee; pHMB, p-hydroxymercuribenzoate; BSA. bovine serum slbumin.
M. YANIV
2
AND
F. GROS
In the following papers (Yaniv & Gros, 1969a,b), we shall describe the isolation of tRNAVal-VRS complex and compare the characteristics of genetically modified enzyme with those of the normal one. a
2. Materials and Methods (a) Substrates,
reagents and chromdographic
absorbants
Unlabeled L- or m-amino acids, ATP, dithiothreitol and crystalline BSA were A grade products purchased from the California Corporation for Biochemical Research. DTNB was purchased from Aldrich end pHMB from Sigma. Sucrose and glycerol were analytical grade products from Merck. E. coli B-stripped tRNA ~8s purchased from General Biochemicals or Schwartz Bioresearoh. inorganic pyrophosphate and [a-32P]ATP were l*C-labeled amino acids, 32P-labeled products of C.E.A. Sephadex and DEAE-Sephadex A50 were from Pharmacia and Biogel HTP from Biorad. Calcium phosphate gel w&s prepared according to Keilin & Hartree (1938). (b) Valine-speci$c
tRNA
Valine-specific tRNA was purified from total E. coli B tRNA. Two-phase column chromatography, 8s described by Kelmers (1966), was run at 37°C and the tRNA was eluted by a sodium chloride gradient in 0.01 M-sodium acetate (pH 4.5). Vahne acceptor activity was separated into a minor tRNAV;’ peak (coded by GUU and GUC) and a major tRNAVfl peak (coded by GUA and GUG) eluted fkst and second, respectively. The tRNAVf peak fractions were pooled and rechromatographed twice on hydroxylapatite columns at pH 6.8 and 5.8 *cording to Muench t Berg (1966). The new peak fractions, having a constant 8cceptance activity of 1.6 mpmoles per o.D.~~~ unit, were desalted on a Seph8dex G25 column and concentrated by flash evaporation at 30°C under reduced pressure. (c) Vdyl-tRNA
synthetase
assay
Two assay methods were used to measure the activity of the enzyme: acylation of the tRNA by redioactive valine, or the 3aP-labeled pyrophosphate exchange in the presence of ATP and amino acid. (i) Acylation assay. A suitable amount of enzyme was added to 0.1 ml. reaction mixture containing, per ml., 40 rmoles Tris-HCl (pH 7.8), 5 pmoles magnesium acetate, 72 pmoles ammonium chloride, 6 pmoles j3-mercaptoeth8no1, 2 pmoles ATP, O-2 pmole of [14C]valine (specific activity, 20 &rmole), 250 pg of stripped tRNA capable of accepting about 0.3 mpmole of valine and 200 pg of cryst8lline BSA. Incubation w&s stopped after 10 min at 30°C unless stated otherwise, by addition of 0.7 ml. of 3.5% perchloric acid solution containing 1 mg of [‘sC]valine/ml. and 2.5 mg of lanthanum nitrate/ml. After 15 min, the precipitate was washed twice by centrifugation and resuspension in 0.5% perchloric acid solution containing 0.1 mg of valine/ml., filtered on Whatman GF/B glass filters, washed with cold water and alcohol. The dried filters were counted with an efficiency of 60% in a scintillation counter using a toluene PPO, POPOP scintillation liquid containing 0.5% hyamine base (modification of assay given by Martin, Yegian & Stent, 1963). (ii) Exchange assay. The reaction mixture (0.5 ml.) contained Tris-HCl, magnesium acetate, ammonium chloride, p-mercaptoethanol, ATP and BSA of the same concentrations as described above (section (i)) plus [‘zC]valine 2.0 /Imoles/ml. and 2 pmoles/sodium [3aP]pyrophosphate/m1. with a specific activity of IO4 to lo5 cts/min per pmole. Incubation ~8s stopped after 15 min at 30% and the [3aP]ATP adsorbed on the Norit was filtered on Whatman GF/C filters according to Calender & Berg (1966) and counted with a gas-flow counter. Enzyme dilutions were done in 0.01 &f-Tris buffer (pH 7.8) containing 0.064 M-ammonium chloride, 0.008 M-magnesium acetate, 0.006 M-meroaptoethanol, 1O-4 M-EDTA and 1 mg BSA/ml. Whenever pyrophosphate exchange activity was measured with crude extracts fractions, 0.05 M-sodium fluoride was added to the incubation mixture to inhibit the inorganic pyrophosphatase. The values obtained for VRS exchange activity in non-purified
VALYL-tRNA
SYNTHETASE
FROM
I#. COLl
3
fractions were corrected for the veline-dependent exchange with the isoleucyl-tRNA synthetaee. This latter aativity constitutes 62% of the isoleucine-dependent exchange of the s&me extract (Berg, Bergmann, Ofeng8nd & Dieckm8m, 1961). Another method consisted in precipitating the isoleucyl-tRNA synthetase with excess spectic antiserum and assaying the activity of the supernatant fraction. One unit of enzyme ~8s equivalent to the esteriflcation of 1 pmole of valine to tRNA under standard assay conditions. One unit of exchange activity was equal to 1 pmole of pyrophosphate incorporated into ATP under st8nd8rd assay conditions. Isoleucyl-tRNA synthetase wsa assayed under similar conditions in the presence of isoleucine. (iii) In some cases the VRS-dependent de8cyletion of [r4C]valyl-tRNA (1 mmole/ml.) in the presence of AMP (2 qoles/ml.) and inorganic pyrophosphate (2 qoles/ml.) w8s measured. The incubation mixture (0.1 ml.) contained the s8me concentrations of TrisHCl, ammonium chloride, megnesium acetate, mercaptoethanol snd BSA as used for the ecylation assay. Incubation ~8s st8rted by the addition of an 8ppropriate dilution of enzyme solution. At intervals, [14C]valyl-tRNA W&B precipiteted and counted by the procedure described for the acylation assay. One unit of descylation activity W&Bdefined 8s the diseppear8nce of 1 mole of [14C]valyl-tRNA in 10 mm 8t 30°C. (iv) Total valine-accepting cspecity of tRNA was measured under the conditions used for VRS ecylation assay. Total E. cc& tRNA was replaced by the sample to be assayed. Excess pure enzyme ~8s added (3.6 @/O-l ml.) and the solution ~8s incubated for 30 min at 30°C. Carrier yeast RNA (0.2 mg) ~8s added before acid precipitation. (d) Enzyme putifkation (i) Cell extraction: E. wZi K12 strain Hfr C w8s grown in M 63 mineral medium (Monod, Cohen-Bazire & Cohn, 1961) supplemented with 0.3% Cas8mino 8cids, O.1o/oyeast extract and 1*Oo/oglucose 8s carbon source; cells were harvested in 18te log phrase. Bacteria were washed twice with 0.01 ma-Tris-HCI (pH 73), 0.01 ~-magnesium chloride buffer rend kept frozen at -20°C. 1 kg (wet weight) of cells was suspended in 3 1. of 0.02 M-potassium phosphate buffer 8t pH 7.2, containing 0.16 M-KCl, 0.002 M-EDTA and 0.01 M-b-mercaptoethenol. Fr8ctions of 100 ml. were sonicated for 10 min with 8 M.E.L. 100-w sonicetor in the cold. The combined extracts were centrifuged for 30 min (811centrifugations were done with the GSA rotor of 8 refrigemted Sorv8ll centrifuge 86 8000 rev./min) and the supernat8nt fluid w&s collected (fraction I). (ii) Streptomycin precipitin. Streptomycin (320 ml. of 10% solution) W&Badded with stirring to each litre of fraction I. Stirring wan continued for 10 min in the cold. The precipitate that formed ~8s removed by centrifugation for 10 min and the supernetant freotion was collected (t&&ion II). Often the sonicetion and streptomycin precipitation steps were replaced by disruption with glaes beads in a Waring Blendor followed by autolysis 8t 37°C for 6 hr. The extrctction buffer in this c8se WEG+I 0.1 &r-potassium phosphate (pH 7.0) containing 0.01 na+mercaptoethanol. The precipitate formed during autolysis was removed by centrifirgation and the purification ~8s continued with the following step. (iii) A?n?ncn&m m.dfa?e. frah. Solid ammonium sulfate (242 g) ~8s added slowly to every litre of fraction II, and after 16 min the suspension WBB centrifuged for 30 min. The pellet ~8s discarded 8nd 188 g ammonium &f&e were added (66% saturation); after 16 min of stirring the suspension ~8s centrifuged for 30 min. The collected precipitate was dissolved in 700 ml. of 0.02 y-potassium phosphate (pH 7*0), containing 10m4 M-EDTA and 0.006 rd-jl-merc8ptoethanol (buffer 1). The enzyme was reprecipitated by bringing the solution to 66% saturation with solid ammonium sulfate and the final precipitate collected by centrifug8tion. A further purification w8s obtained by extraction of the pellet thus obtained with solutions of ammonium sulfate in buffer 1. The pellet ~8s resuspended in 376 ml. of 47% saturated ammonium sulfate by rapid stirring in 8 Waring Blendor and the suspension was centrifuged. The extraction wa8 repeated with the same volume of a 470, satur8ted eolution and twice with 376 ml. of 8 40% saturated solution. Fractions containing the highest specific activity were collected and the enzyme was precipitated by incre8sing the saturation to 66% ammonium sulfate. After centrifugation the precipit8te was dissolved in 200 ml. of buffer 1 (fraction III).
4
ill.
YANIV
AND
li’. C:ROS
(iv) Calcium phosphate gel fractionation. Fraction III was dialyzed overnight against 10 1. of 0.02 M-sodium acetate (pH 6.0), 0.006 M-fl-mercaptoethanol and 10e4 M-EDTA. The dialys8te ~8s diluted, before the addition of the gel, to a protein concentration of 10 mg/ml. Calcium phosphate gel (13.5 mg dry weight/ml.) was added in the proportion of 0.6 vol. to 1 vol. of diluted dialysate and the suspension ~8s centrifuged after an additional 10 min of stirring. The enzyme remaining in the supernatant fraction was adsorbed with another 0.6 vol. of gel. The 8mount of the gel used in the negative and positive absorption changed from one preparation to 8nother and was checked on a small portion every time. The enzyme WBB eluted from the gel by successive washings with potassium phosphrtte buffers of increased molarity and pH. The buffers used were: a, 0.02 M, pH 6.5; b, 0.02 M, pH 7.0; c, 0.05 M, pH 7.0; d, 0.05 M, pH 75; e, 0.2 M, pH 7.5; 811 contained 0.006 M-mercaptoethanol8nd 10e4 M-EDTA. The fractions containing the highest specific activity and most of the enzyme activity (usually fractions c to e) were pooled and the enzyme was precipitated by adding 472 g of solid 8mmonium sulfate/l. After 4 hr the suspension was centrifuged for 30 min, the pellet dissolved in 60 ml. of 0.01 M-Tris-HCl (pH 7.8), 0.1 M-NaCl, 0.006 M-&merc8ptoethanol, 10e4 M-EDTA (“dialysis buffer”) and dialysed twice against 3 1. of the same buffer (frrtction IV). (v) DEAE-Sephcdex fractionation. Dislyzed fraction IV w&s diluted to a protein concentration of 15 mg/ml. with the dialysis buffer before it was absorbed on 8 700-ml. A50 equilibrated with the same buffer. column (4.5 cm x 44 cm) of DEAE-Sephsdex After adsorption, the column w8s washed with 700 ml. of dialysis buffer and elution was effected with a linear gradient made by mixing 3 1. of dialysis buffer with 3 1. of 0.01 M-TrieHCl (pH 7.0), 0.4 ~-N&cl, 0.006 M-fi-mercaptoethanol and 10m4 M-EDTA. A flow rate of 1 ml./min was maintained and lo-ml. fractions were collected and assayed for the valine-dependent ATP-PPi exchange reaction. Since this reaction is also catalyzed by the IRS (Berg, Bergmann, Ofengand & Dieckmann, 1961), two peaks of activity were observed, one corresponding to the IRS (eluted at 0.25 M-salt buffer) and the other to the VRS (elution at O-3 M-s8lt buffer). The two enzyme fractions were pooled sep8rately (only fractions containing at least 60% of the peak activity were kept). The pooled VRS fractions are designated “fraction V”. The IRS was chromatographed separately on 8 hydroxylapatite column sccording to Baldwin & Berg (1966), yielding 30 mg of pure enzyme. The pure IRS moved as a single component in electrophoresis on polyacrylamide gel and exhibited 8 symmetrical sedimentation pattern in analytical ultracentrifugation. (vi) Hydroxykzpatite fractionation. Fraction V was applied directly to a 200-ml. (3 cm x 28 cm) hydroxylapatite column previously equilibrated with 0.001 m-potassium phosphate buffer (pH 6.75) containing 0*0005 M-dithiothreitol. The column was washed with 200 ml. of 0.02 M-potassium phosph8te buffer (pH 6.75) 8nd with the s8me volume of 0.05 IMpotassium phosphate buffer (pH 6.75). Enzyme w8s eluted with 8 linear gradient produced with 1000 ml. of 0.05 M-potassium phosphate (pH 6.75) to 1000 ml. of 0.2 M-potassium phosphate (pH 6.75). All the buffers contained 0.0005 M-dithiothreitol (Fig. 1). 7-ml. fractions were collected; those showing a constant specific activity were pooled and concentrated to about 10 mg/ml. by vacuum dialysis (the “Amicon diaflo” system with XM-50 membranes w&s used in recent purifications). The concentrated enzyme was dialyzed against 0.02 &r-potassium phosphate buffer (pH 7*5), contrtining 0.0005 M-dithiothreitol, and stored frozen in liquid nitrogen. In some c8ses it was adjusted to 50% glycerol and stored, either at -20°C or in liquid nitrogen. T8ble 1 summarizes the different purification steps. (e) Amino
acid analyses
A solution containing 0.65 mg of protein was dialyzed for 24 hr against 0.01 M-sodium acet8te (pH 6.0) and freeze dried. Performic oxidation was done at -8C according to Hirs (1956) followed by hydrolysis in wucuo at 105°C for 24, 48 and 72 hr in 6 N-HCl to which 8 crystal of phenol had been added. The hydrolysates were analyzed on a Beckman amino 8cid analyzer model 120C. Tryptophan was determined by spectral analysis in 0.1 M-N8OH with 8 Cary spectrophotometer (Beaven & Holiday, 1952). Serine 8nd threonine were estimated by 8 linear extrapolation to zero time of values obtained after 24, 46 and 72 hr of hydrolysis. Values for isoleucine, valine and leucine are based on 72-hr hydrolysis.
VALYL-tRNA I
I
SYNTHETASE I
I
0.30 -
FROM I
I
I
E. COLI
5
1000
I
I
PP / .-,‘\ \ ‘e,
3 0.20 8 2 _ 6 0: IO
k
-500 ‘1
/’
” .r $
‘Cc
P/’
‘hp.
+0-O
\ .’
, 50
-0-O
d
‘h
Pf 0’ 0’
al F 2 8 .k
‘-u I
I 70
I
I 90 Fraction
I
I 110
I 130
I
no.
FIQ. 1. Hydroxylapatite cbromatogmphy of valyl-tRNA synthetase fraction V. Samples (0.5 4.) were assayed for PPi exohange activity -- 0 ---- 0 -- and the optioel density was measured at 280 rnp -e-m--. TABLE
Puri$c&nz
Fraction
I II
Crude extract Streptomycin snpernatant
III
Aa elude
IV
Calcium phosphate eluate DEAFLSephadex Hydroxylepatite Concentration step
V VI
of valyl-tRNA
1
synthetme from E. coli K12
Total volnnle (ml.)
Protein hw*)
3300
26.2
83,000
17
o-7
66,000
3950
18
880
27.8
71,000 24,600
14.6 51.2
0.8 1.84
57,000 46,000
1600 200 262 4.5
1.9 1-l 0.21 9.4
Total protein units
2850 220 62 42.3
34.7 18.2 168 153 99 478 448 4226
62,006 33,600 24,000 19,000
Yield per oent
102 80 93 60 43.5 34
The purification prooedure is described in Materials and Methods. The activity is expressed in the ATP-PPi exchange units. (f) Titration of SH growp~ The number of the free SH groups in native and denatured enzyme was measured with DTNB according to Ellman (1969) and with pHMB aeeording to Bayer (1964). The enzyme was freed from protecting SH reagent by filtration on a G26 Sephadex column equilibrated with O-1 ar-Tris-HCl (pH 8-O). Enzyme concentration in the titration mixture was determined by ultraviolet absorption at 280 rnp. Velocity sedimentation studies were done with the Beckman model E ultraeentrifuge equipped with schlieren optios and temperature control. Epon-filled 12-mm, a = 25 cells, single or double sectored were used. Protein concentration was measured by ultraviolet absorption at 280 mp and by the refractive index of the enzyme solution. (h) S~eciJic antieerum ~eparo&a Rabbits were injected with 400 pg of purified IRS or 200 pg of VRS in Freund’s adjuvant. After 6 weeks, a second intravenous injection of the same amount of protein was given and
M. YANIV
6
blood was collected 7 days later. y-globulins were purified according
AND
F. GROS
The blood was centrifuged to Calendar Rr,Berg (I 966).
(i) Antiserum
after
coagulation
and the
iv,hibitiorL studies
Inhibition studies were done in 0.1 or 0.5 ml. of mixture containing, per ml. : 10 pmoles potassium phosphate (pH 7*0), 3 pmoles /I-mercaptoethanol, 200 pg BSA, 1 to 2 pg of pure enzyme and increasing amounts of antiserum. Tubes were incubated for 15 min at 4°C before assay, or otherwise centrifuged for 20 min at 20,000 g and the activity assayed in the supernatant fraction. (j) M&wJaneous Protein concentration was measured according Rosebrough, Farr & Randall, 1951) or the biuret with BSA used as standard.
to the modified Folin test (Gornall, Bardawill
reaction (Lowry, & David, 1949)
3. Results (a) Properties
of pure enzyme
The enzyme obtained after hydroxylapatite chromatography behaved mainly as a single physical constituent, although it still contained a small amount of contaminating material as revealed by polyacrylamide gel electrophoresis or by the asymmetry of the schlieren pattern apparent after 100 minutes of centrifugation at 59,780 rev./min (4’C). At this purification stage the degree of purity exceeded 95%, as deduced from the gel patterns. An enzyme preparation that showed lower contamination could be obtained by rechromatography of fraction VI on a hydroxylapatite column at pH 7.5. Polyacrylamide gel electrophoresis of this material showed only a minute band of contamination, and the schlieren pattern during sedimentation showed the presence of a single symmetric constituent. The specific activity of the enzyme obtained in four preparations varied between 400 to 450 exchange units/mg (the additional rechromatography step did not increase the specific activity significantly). Aoylation activity at the last step of purification was 32 units/mg. The ratio of exchange to acylation activity varied in the course of purification from 11, in the crude extract, to 14 in the iinal step. This difference may be attributed to a certain degree of inaccuracy of the exchange assay in crude extracts, since the valine-dependent activity of the IRS had to be subtracted from the activity measured (see Materials and Methods). Alternatively, it could be due to a slight inactivation of the acylation capacity during purification. The ratio of absorption at 280 rnp to that at 260 rnp was from 1.85 to 1.82 in different preparations, showing little contamination if any with nucleic acids. The absorption at 280 rnp of the protein solution (light path of 1 cm) was 1.13 mg/ml. (Folin) or 0.94 mg/ml. (biuret). Calculation of the total area in a synthetic boundary run in the ultracentrifuge gave an absorption at 280 rnp of 1.19 per mg/ml. In further calculations, a mean value of 1.10 was taken for the 280 rnp absorption of a solution of VRS containing 1 mg/ml. (b) Catalytic
indices of purified
valyl-tRNA
synthetase
Table 2 indicates the values of K,, V,,, and molecular activity for the exchange, acylation and deacylation reactions. Using the integral Michaelis-Menten equation (Dixon & Webb, 1964) the K, value for tRNA was determined either with total tRNA or with purified tRNAV,&‘. With the latter a value of 5.5 x 1O-7 M, not significantly different from that obtained with total tRNA, was calculated. When using
VALYL-tRNA
SYNTHETASE TABLE
FROM
V,.,
Acylation
6.7 X 1o-6 M 2.7 X lo-‘M 1.4 x 10-d M -
(pmoles per mg per min)
synthetase at 30°C KU7
Exchange
L-dine ATP PPi tRNA
7
2
V,,,,, and K, values of valyl-tRNA
Substrate
E. COLI
6.3 x 10-b ?d 13 x IO-4 M 6 x lo-’ (5.6 x lo-‘)t
29.8
Molecular aotivity (molecules of substrate 2470t reacting per min per molecule of enzyme)
Deayletion -
rd
3.18
l-02
368
118
t Determination with pure tRNAVal. $ In the presence of 2 x 10s4 ~-dine.
total tRNA, the relative concentration of specific tRNAVal was estimated after acylating total tRNA with excess purified enzyme. Confirming previous findings by Berg et a& (1961), we observed that the rate of ATP-PPi exchange was ten times higher than the rate of acylation of tRNA for the same amino acid concentration. L-Threonine and L-isoleucine gave low levels of exchange with high K, values, as did also the valine analog, DL-a-aminobutyric acid (Table 3). Magnesium was TAIZE 3 t!hbstrate e~eciIi$y of the amino acid-dependent p yrep?w8@katecxchawe
L-ThlVOllill~ DL-c&mino
L-Isoleucine L-valine
butyric
acid
7 x 10-s P 1.6 x10-3M 10-l M 6.7 x 1O-6 rd
21 6 22 100
necessary for the exchange and acylation reactions, but could be replaced by manganese in the acylation reaction with almost the same efiioiency (70% of V,,,). The precipitation of manganese pyrophosphate in the exchange assay mixture did not permit the study of this reaction with manganese ions. The inhibition of the acylation reaction by excess metal expressed as K’s (the metal concentration producing a 50% reduction of V ,,,) is equal to 2 x 10e3 M for manganese and 7 x 10q3 M for magnesium. Calcium or spermidine had no effect on enzyme activity. (c) Sedintentaticn and nbolccular weight measurement8 Figure 2 shows the sedimentation pro6le of pure VRS after zone sedimentation on a sucrose gradient. The total protein distribution coincides with the enzyme activity, which excludes an appreciable contamination of the enzyme with inactive protein of a different sedimentation coefficient. Calculation of the sedimentation coefficient and molecular weight of VRS, according to Martin k Ames (1961) gave values of 66 s
M. YANIV _~-
.- ..--
T-
AND ----T
F. GROS I-.--
--.--
Fraction no. FIQ. 2. Sedimentation of valyl-tRNA synthetase on sucrose gradient. Purified VRS (100 pg) and IRS (5 rg) were layered on a gradient of 5 to 20% sucrose in 0.02 &r-potassium phosphate (pH 6.75) buffer, containing 0.1 m-KC1 and 0.006 M-B-mercaptoethanol. Centrifugation was for 20 hr at 36,000 rev./min. Fractions were collected and assayed : valine-O--O--; protein (Folin), -~-~--; IRS activity, dependent exchange activity, y-J---o-.
and 106,000 respectively. Highly purified IRS was used as a marker, assuming a sedimentation coefficient of 5-9 s and a molecular weight of 112,000 (Baldwin & Berg, 1966). Sedimentation studies were also performed at different protein concentrations in the analytical ultracentrifuge at 4°C. Fignre 3 describes the dependence of S20,W on protein concentration. A value of 5.6 is obtained by extrapolation to zero protein
Protein (mg/ml.) Fro. 3. Variation of S value with concentration. Protein concentration was determined by absorption at 280 mp. The enzyme was diluted in a 0.02 M-potassium phosphate buffer (pH 7.0) containing 0.1 M-KCl. Temperature of runs, 4°C; speed 59,780 rev./min.
VALYL-tRNA
SYNTHETASE
FROM
E. COLI
9
Fraction no. FIG. 4. Isolation of valyl-AMP-enzyme complex by Sephadex gel fltration. VRS (195 H;) were mixed with 0.02 -010 ATP a-labeled with saP (16.7 ~c/~ole), 0.02 Fole [“C]valine (96 ~o/~ole) in presence of 0.02 M-Tris-HCl (pH 7.8). 0.036 M-NH&I, 0.0025 M-m&gneeium acetate and 0.006 M-/l-meroaptoethanol in a volume of 0.2 ml. After incubation for 6 min at 2O”C, the reaction mixture wae applied to a g-ml. (O-43 oma x 21 cm) Sephadex G76 column sucoineta (pH 6.0) buffer containing 0.06 X-KC& 0.006 M.y-merequilibrated with 0.05 ~-sodium captoethanol and 10m3 M-EDTA. Fraotiona of 0.16 ml. were oollected and 2&d. samples plated on GF/B glass filters and counted in e scintillation counter. Samples of 1 & were used for PPi exchange aotivity essay. Enzyme recovery was 69% (1.22 mFoles). The amount of valine and ATP found in the complex fraotiona wee 0.96 and 1.07 mwoles, respectively. [W]Valine, -a---a-; [a-3”P]AMP, -- X-- x --; [3aP]PPi exobange activity, -O-O-. In * separate experiment with the came amount of enzyme, the binding of [a-3aP]ATP in abeence of valine was followed: -n--n-.
concentration. Molecular weight determination by the approach to equilibrium method (Archibald, 1947) gave a value of 110,OOOf 8090. (d) Xubstrde binding Using the Sephadex gel filtration technique of Norris & Berg (1964), we could isolate a valyl-AMP-enzyme complex (Fig. 4). Valine was transferred to tRNA with an efbciency of 80%. Aa ah-eady observed for the activation and transfer of isoleucine by Norris & Berg (19&t), magnesium was necessary for valyl.AMP~nzyme formation, but not for the transfer to tRNA. The following paper will clearly show that tRNA can bind normally to the enzyme in the absence of bivalent cationa. The presence of magnesium in the complex isolated by Sephadex filtration is improbable, as the
M. YANIV
10
AND
1’. CR08
affinity of Mg 2+ to the enzyme is only lo4 I./mole as determined by fluorescence studies (Helene & Yaniv, manuscript in preparation). The fact that Allende, Mora, Gatica t Allende (1965) observed a bivalent cation requirement for the transfer of the activated threonine to tRNA from rat liver may be attributed to the stabilization of the tRNA structure by these cations, a possibility that was suggested by these authors. Calculation of the amount of valine and AMP bound indicates that 0.8 molecule of valine was bound per enzyme molecule. Thus the existence of one binding site can be inferred. In the following paper it is also shown, by another approach, that one molecule of tRNAVal can bind to each molecule of enzyme. This result is identical to the one obtained with the IRS (Norris & Berg, 1964). Both enzymes appear to have one active site per 110,000 molecular weight. (e) Amino acid composition and titration of SH groups The total amino acid composition of VRS obtained after performic acid oxidation of the protein is shown in Table 4. One outstanding feature is the low number of histidine residues. TABLET Amino acid composition of valyl-tRNA synthetase (in mole.3amino acid/mole enzyme) Tryptophan Lysine Histidine Arginine
11 47 5 37
Half cystine (as cyst&o acid) Aapartio acid Methionine (as methionine sulphone) Threonine Serine Glutamic acid Proline Glycine Alanine Vdine Isoleucine Leucine Tyrosine Phenylalanine
The method of protein Methods.
hydrolysis
and amino acid determination
11 140 17 32 35 150 28 93 145 78 58 67 39 22
is described
in Materials
and
It had been shown previously that for some of the activating enzymes studied the presence of substrates could modify the number of reactive SH groups. Accordingly, as already shown by George & Meister (1967), the number of reactive SH groups in VRS is changed after addition of valine and ATP. Figure 5(a) describes the titration of VRS SH groups with DTNB. After 30 minutes at 25”C, two groups react in the native enzyme and about 1.3 in the presence of magnesium, valine and ATP. When the enzyme was dissolved in 8 M-Urea, about five SH groups reacted with DTNB (a second enzyme preparation gave values of 2-5 and six SH groups for native and denaturated
VALYL-tRNA
SYNTHETASE
FROM
Minutes. at 25’C (a) SH groups/110,000 equivalents 0 2 4 6 8 IO I I 1 I ,
E. COLI
11
of enzyme
a 2
ro
-0-B
: u’q‘O -4 0~100 I ,” 0,200 pnvuuo
-
I
IO
20
30
$: of pHMB (IO’M) (b) FIQ. 5. (a) Titration of SH groups with 5,5’-dithiobio(2nitrobenzoic acid.) (0.14 mg) was titrated in a volume of 0.4 ml. adjusted to 0.1 r.r-Tri*HCl (pH 8-O), 2 x lo-’ M-EDTA. The increase of absorption at 412 mp was measured after the addition of DTNB to a final conoentration of 2 x lo-’ XI: no addition of substrata, -n-O--; in the and 10-s M-M&X,, -A----A-; in presence of lo-’ n%-vahne, presence of 3.6 x 10-s x-tRNAV*’ 4 x 10v6 M-ATP snd 10-s M-M&X, -O--O-; in presence of 8 M-ures -V-V -. Exp. 2: Titration of 0.17 mg enzyme from 8 second prsperstion, -a---a-; the number of SH groups titrated in experiment 2 after 160 min is equal to 3.3.
Exp. 1: Enzyme
(b) p-Hydroxymemuribenzoste titration. Enzyme (0.14 me;) w&s titrated by serial additions of 2 4. pHMB 10-s M in a vol. of O-4 ml. in presence of 0.1 ar-Tris-HCl (pH 8-O). The inorease in absorbance at 260 rnp as oompamd to 8 blank sample was measured after eaoh addition of pHMB (no correction was made for dilution). No odd&ion, -O-O-; in presence of 10e4 M-Vslin0, 2 x 10-s M-ATP and lo-* M-M&&,, --n----o--.
12
M.
YANIV
ANI)
E’. UR08
enzyme after 30 min at 25°C). Addition of tRNAVsl in the presence of Mg2 + had no effect on the number of titrated SH groups in the native enzyme. Using pHMB, six SH groups could be titrated in the native enzyme and about 4.0 in the presence of substrates (Fig. 5(b)). The values obtained by the pHMB titration (and confirmed with the DTNB titration in presence of urea) are different from those obtained by George & Meister (1967) who detected with pHMB 16 sulfhydryl groups per 100,000 equivalent of enzyme. Moreover, the total number of cysteic acid residues which we found after performic oxidation of the enzyme is only 11 (it must nonetheless be recalled that the preparation used by George & Meister was isolated from E. coli B, whereas the one described here derives from a K12 strain). The discrepancy between the number of SH groups titrated and the number of cysteic acid residues detected in amino acid analysis is under study. A similar difference was also found with IRS by Baldwin & Berg (1966). (f) Antiserum
inhibition
Biochemical studies have unravelled a resemblance between the isoleucine- and valine-activating enzyme. IRS can form a complex efficiently with valine plus ATP (Norris & Berg, 1964), and an isoleucine-dependent ATP-PPi exchange is observed with VRS (Table 3). We have also just seen that both systems have similar molecular weights and that both contain one active site per molecule. It was of interest to prepare a specific antiserum against pure IRS and VRS and to look for possible cross-reactions. Figure 6 shows the specificity of each preparation against each homologous enzyme. No cross-inhibition was obtained. Each enzyme activity was
2000/rgA-VPS
1000 (a)
IO00
?OOOpgA-IRS
(b)
FIG. 6. Neutralization of isoleuoyl-tRNA synthetase (a) and valyl-tRNA synthctaae (b) by antiserum prepared against these enzymes. IRS (1.35 pg) and VRS (0.7 pg) were mixed with increasing amounts of antiserum. The exchange activity was assayed with 100 ~1. of supernatant obtained after oentrifugation as described in Materials and Methods. (a) IRS + anti-IRS, -()---()--; IRS t anti-VRS -n--u-: (b) VRS + anti-VRS, -O-O--; VRS + anti-IRS -n---n-.
VALYL-tRNA
SYNTHETASE
FROM
E. COLI
13
pgA-VRS FIQ. 7. Effect of antiserum on exchange and acylation activities of valyl-tRNA synthetaae. Enzyme (0.4 pg) W&B mixed with inoreasing amounts of 8n&ermn, end inoubated for 16 min at 4°C. Portions were assayed for exchange (100 4.) and eoylation (2 4.) sotivities respectively before and after centrifugcttion for 20 min at 20,000 g. Before centrifugation exchange, --O--O--; acylation, -@-a--; after centrifugation exchange, -- q -- q --; eoylation, -A---A-.
totally inhibited in crude extracts of E. coli by its corresponding antiserum, showing the existence of only one activating enzyme for each of these amino acids. Anti-WE3 serum also completely inhibited lysine and histidine incorporation in MS2 RNAdirected in vitro protein synthesis (Ghysen & Yaniv, unpublished observation). While the acylation of tRNA was inhibited instantaneously upon addition of the antiserum, the complex obtained between enzyme and y-globulin retained most of the PPi exchange activity as shown in Figure 7. Addition of substrate did not change the sensitivity to antiserum inhibition. The transfer of valine to tRNA from a pre-isolated valyl-AMP-enzyme complex was also inhibited completely by the antiserum. Another case in which the interaction of the enzyme with a protein leads to a preferential decrease in the aoylation activity is observed when the enzyme is converted into a dimer; when pure VRS was dialyzed against a buffer of low ionic strength, about 10% of the enzyme was converted into a dimer that sedimented at 7-8 s in sucrose gradients. The ratio of acylation to exchange activity was twofold lower in this dimer than in the native form. The decrease in acylation activity in these cases may be ascribed to several possible mechanisms: (i) binding of the protein to the RNA recognition site ; (ii) the steric hindrance towards a bulky substrate (tRNA) ; (iii) restriction of
14
M. YANIV
AND
F. GROS
the aminoacyl-AMP effect on the rate of enzyme-tRNA personal communication).
dissociation (Yams & Berg,
4. Discussion E. coli valyl-tRNA synthetase was first partially purified by Berg et al. (1961). Later, using a different preparation technique, George & Meister (1967) obtained in 5% yield a VRS preparation that behaved as a homogeneous protein on polyacrylamide gel electrophoresis. The present study describes a new procedure of purification, with a yield of 34%. The enzyme appears highly purified on the basis of chromatographic, electrophoretic and sedimentation characteristics. The specific activity of the enzyme as measured by the pyrophosphate exchange activity appears to be higher than that calculated from George & Meister’s data. Sedimentation studies under various conditions suggest that the protein has a molecular weight of 110,000 and a sedimentation coefficient of 5.6 at 20°C. The number of polypeptide chains present in the enzyme molecule is under investigation. Substrate binding experiments involving separation of the valyl-AMP-enzyme complex reveal the existence of one active site for the aminoacyl residue per molecule of enzyme. Addition of valine and ATP but not of tRNA Val affects the number of reactive SH groups of VRS, a result that may indicate a conformational change rather than a direct participation of cysteine in the active site. At least in the case of lysyl-tRNA synthetase, Stern, De Luca, Mehler t McElroy (1966) could exclude the presence of SH in the active site. Recent physicochemical studies of VRS confirm the existence of substrate-dependent changes in the conformation of the protein. Fluorescence intensity of the tryptophan residues of the enzyme was changed upon addition of Val-ol-AMP, an aminoalkyl adenylate competitive analog of aminoacyl AMP (Cassio, Lemoine, Weller, Sandrin & Boissonnas, 1967) or tRNAVal (Helene & Yaniv, manuscript in preparation). These results may indicate two distinct substrate-induced variations in the protein structure. Valine and isoleucine are synthesized by a common biosynthetic pathway in E. coli VRS and IRS have the ability to react with valine in the ATP-PPi exchange, isoleucine being recognized mainly by its specific enzyme and only with a low Vma, and high K, by the VRS. Both enzymes have almost equal molecular weights and an equal number of catalytic sites. In spite of these common features, VRS and IRS do not cross-react immunologically. Of interest is the finding that antiserum prepared against highly purified valine- and isoleucine-activating enzymes totally inhibits the capacity of E. coli extracts to acylate total valine or isoleucine acceptor tRNA’s with the specific amino acids. This result establishes that in E. coli there is only a single species of aminoacyl-tRNA synthetase for synonymous valine or isoleucine acceptor tRNA’s. The very skilful technic81 aesistance of Mrs A. Chestier is acknowledged. We thank J. P. Richardson and J. P. Wailer, for valuable criticisms of this mttnuscript. We are indebtetl to J. M. Dubert for help in the preparation of antiserum and to S. Filitti-Wurmser for the determin8tion of molecular weight. This work was supported by grants from the Fonds de Developpement de 18 Rechercho Scientifique et Technique, the Commissariat & 1’Energie Atomique, the Centre National de 18 Recherche Scientifique, the Ligue Nation& Frangaise contre le Center 8nd the Fondation pour 18 Recherche Medicale Franpaise.
VALYL-tRNA
SYNTHETASE
FROM
E. COLI
16
Note added irr proof. Reduction and polyacrylamide gel electrophoresis of the VRS in the presence of sodium dodecyl sulfate (Shapiro, A. L., Vinuela, E. & Maize& J. V. (1907). B&hem. Biophya. Rec. Comrn. 28, 815) revealed the presence of only one polypeptide chain with a molecular weight of 110,000. REFERENCES Allende, J. E., Mora, G., Gatica, M. & Allende, C. C. (1966). J. Biol. Chem. 240, PC 3229. Archibald, W. J. (1947). J. Phy8. Co&id. Chem. 51, 1204. Baldwin, A. N. 8z Berg, P. (1960). J. B&Z. Chem. 241, 831. Beaven, G. H. BEHoliday, E. R. (1952). Advanc. Protein Chem. 7, 319. Berg, P., Bergmann, F. H., Ofengand, E. J. & Dieckmann, M. (1961). J. Biol. Chem. 236, 1726. Boyer, P. D. (1964). J. Amer. Chem. Sot. 76, 4331. Calendar, R. & Berg, P. (1966). Biochemietry, 5, 1681. Cassio, D., Lemoine, F., Wailer, J. P., Sandrin, E. & Boissonnas, R. A. (1967). Biochemi&y, 6, 827. Dixon, M. & Webb, E. C. (1964). Enzymee, p. 114. London: Longmans. Ellman, G. L. (1969). Arch. Biochem. Biqvhy8. 82, 70. George, H. & Meister, A. (1967). Biochim. biophys. Acta, 182, 166. Gornall, A. G., Bardawill, C. J. & David, M. M. (1949). J. Biol. Chem. 177, 761. Him, C. H. W. (1966). J. Biol. Chem. 219, 611. Keilin, D. & Hartree, E. F. (1938). Proc. Roy. Sot. B, 124, 397. Kelmers, A. D. (1966). J. Biol. Chem. 241, 3540. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). J. BioZ. Chem. 198, 266. Martin, R. G. & Ames, B. N. (1961). J. BioZ. Chem. 236, 1372. Martin, E. M., Yegian, G. & Stent, G. (1963). 2. ~eer?&mg8Zehre, 94, 303. Monod, J., Cohen-Bazire, G. t Cohn, M. (1961). Biochim. biophye. Acta, 7,586. Muench, K. H. & Berg, P. (1966). Biochamietry, 5, 982. Neidhardt, F. C. BEEarhart, C. F. (1966). Cold Spr. Harb. Sm. Quant. BioZ. 31, 567. Norris, A. T. & Berg, P. (1964). Proc. Nat. Acad. Sci., Wuah. 52, 330. Stern, R., De Luca, M., Mehler, A. H. & McElroy, W. D. (1966). Biochemistry, 5, 126. Yaniv, M. & Gros, F. (1969a). J. Mol. BioZ. 44, 17. Yaniv, M. & Gras, F. (19698). J. Mol. BioZ. 44, 31.
2
Studies on Valyl-tRNA Syuthetase and tRNAvpl from Escherichia coli I. Purification and Properties of the Enzyme from Normal Escherichiu coli Strains M. Y~r.v
AND
F. GROS
Insti.tu.t de Bidogie Physico-Chirnipue Service de Phyeiologie ilficrobienne 13, rue Pierre et Marie Curie Paris 5e, France (Received 20 December 1968) A highly pursed valyl-tRNA synthetaae was prepared from Escherichia co& K12 strain. A sedimentation coe&ient (@G) of 6.6 s and a molecular weight of 110,000 were determined. One active site for valine and ATP was detected. Binding of these substrates to the active site changed the number of SH groups titrated. Some indications were found for a conformational change induced by an aminoacyl-AMP analog. Antiserum prepared against the pure enzyme inhibited all the
activity in extracts, indicating the existence of one activating enzyme for valine in E. co& No immunological cross-reaction was detected between valyl and isoleucyl activating enzymea in spite of similarities in structure and substrate
recognition.
1. Introduction The correct translation of a genetic message into a linear polypeptide sequence depends upon the attachment of the appropriate amino acid to a specific tRNA. The amino acid is first activated by an aminoacyl-tRNA synthetase in the presence of ATP and magnesium ions and subsequently transferred to the cognate tRNA (Norris & Berg, 1964). Activating enzymes therefore constitute one of the best models for studying the basic phenomenon of nucleic acid “recognition” by specific proteins. The amino acidtRNA-enzyme interaction can be studied with enzymes derived from normal or phage-infected cells (Neidhardt & Earhart, 1966) or from strains harboring a mutation in the enzyme or tRNA genes. We have undertaken a study of wild-type and genetically modified valyl-tRNA synthetase from Escher&&a coli. The present paper will report on the purification procedure and general properties of the wild-type enzyme. A highly purified VRSt has been obtained with a yield of 34%. Physical constants, certain chemical properties as well as the immunological behavior of the enzyme are described. Ammoacyl-AMPenzyme complexes were isolated with the pure enzyme indicating the presence of one active site per chain of 110,909. t Abbreviations used: DTNB, S,S’-dithiobio(2-nitrobenzoic mid); VRS, valyl-tRNA synthetaae; IRS, isoleucyl-tRNA synthetaee; pHMB, p-hydroxymercuribenzoate; BSA. bovine serum slbumin.
M. YANIV
2
AND
F. GROS
In the following papers (Yaniv & Gros, 1969a,b), we shall describe the isolation of tRNAVal-VRS complex and compare the characteristics of genetically modified enzyme with those of the normal one. a
2. Materials and Methods (a) Substrates,
reagents and chromdographic
absorbants
Unlabeled L- or m-amino acids, ATP, dithiothreitol and crystalline BSA were A grade products purchased from the California Corporation for Biochemical Research. DTNB was purchased from Aldrich end pHMB from Sigma. Sucrose and glycerol were analytical grade products from Merck. E. coli B-stripped tRNA ~8s purchased from General Biochemicals or Schwartz Bioresearoh. inorganic pyrophosphate and [a-32P]ATP were l*C-labeled amino acids, 32P-labeled products of C.E.A. Sephadex and DEAE-Sephadex A50 were from Pharmacia and Biogel HTP from Biorad. Calcium phosphate gel w&s prepared according to Keilin & Hartree (1938). (b) Valine-speci$c
tRNA
Valine-specific tRNA was purified from total E. coli B tRNA. Two-phase column chromatography, 8s described by Kelmers (1966), was run at 37°C and the tRNA was eluted by a sodium chloride gradient in 0.01 M-sodium acetate (pH 4.5). Vahne acceptor activity was separated into a minor tRNAV;’ peak (coded by GUU and GUC) and a major tRNAVfl peak (coded by GUA and GUG) eluted fkst and second, respectively. The tRNAVf peak fractions were pooled and rechromatographed twice on hydroxylapatite columns at pH 6.8 and 5.8 *cording to Muench t Berg (1966). The new peak fractions, having a constant 8cceptance activity of 1.6 mpmoles per o.D.~~~ unit, were desalted on a Seph8dex G25 column and concentrated by flash evaporation at 30°C under reduced pressure. (c) Vdyl-tRNA
synthetase
assay
Two assay methods were used to measure the activity of the enzyme: acylation of the tRNA by redioactive valine, or the 3aP-labeled pyrophosphate exchange in the presence of ATP and amino acid. (i) Acylation assay. A suitable amount of enzyme was added to 0.1 ml. reaction mixture containing, per ml., 40 rmoles Tris-HCl (pH 7.8), 5 pmoles magnesium acetate, 72 pmoles ammonium chloride, 6 pmoles j3-mercaptoeth8no1, 2 pmoles ATP, O-2 pmole of [14C]valine (specific activity, 20 &rmole), 250 pg of stripped tRNA capable of accepting about 0.3 mpmole of valine and 200 pg of cryst8lline BSA. Incubation w&s stopped after 10 min at 30°C unless stated otherwise, by addition of 0.7 ml. of 3.5% perchloric acid solution containing 1 mg of [‘sC]valine/ml. and 2.5 mg of lanthanum nitrate/ml. After 15 min, the precipitate was washed twice by centrifugation and resuspension in 0.5% perchloric acid solution containing 0.1 mg of valine/ml., filtered on Whatman GF/B glass filters, washed with cold water and alcohol. The dried filters were counted with an efficiency of 60% in a scintillation counter using a toluene PPO, POPOP scintillation liquid containing 0.5% hyamine base (modification of assay given by Martin, Yegian & Stent, 1963). (ii) Exchange assay. The reaction mixture (0.5 ml.) contained Tris-HCl, magnesium acetate, ammonium chloride, p-mercaptoethanol, ATP and BSA of the same concentrations as described above (section (i)) plus [‘zC]valine 2.0 /Imoles/ml. and 2 pmoles/sodium [3aP]pyrophosphate/m1. with a specific activity of IO4 to lo5 cts/min per pmole. Incubation ~8s stopped after 15 min at 30% and the [3aP]ATP adsorbed on the Norit was filtered on Whatman GF/C filters according to Calender & Berg (1966) and counted with a gas-flow counter. Enzyme dilutions were done in 0.01 &f-Tris buffer (pH 7.8) containing 0.064 M-ammonium chloride, 0.008 M-magnesium acetate, 0.006 M-meroaptoethanol, 1O-4 M-EDTA and 1 mg BSA/ml. Whenever pyrophosphate exchange activity was measured with crude extracts fractions, 0.05 M-sodium fluoride was added to the incubation mixture to inhibit the inorganic pyrophosphatase. The values obtained for VRS exchange activity in non-purified
VALYL-tRNA
SYNTHETASE
FROM
I#. COLl
3
fractions were corrected for the veline-dependent exchange with the isoleucyl-tRNA synthetaee. This latter aativity constitutes 62% of the isoleucine-dependent exchange of the s&me extract (Berg, Bergmann, Ofeng8nd & Dieckm8m, 1961). Another method consisted in precipitating the isoleucyl-tRNA synthetase with excess spectic antiserum and assaying the activity of the supernatant fraction. One unit of enzyme ~8s equivalent to the esteriflcation of 1 pmole of valine to tRNA under standard assay conditions. One unit of exchange activity was equal to 1 pmole of pyrophosphate incorporated into ATP under st8nd8rd assay conditions. Isoleucyl-tRNA synthetase wsa assayed under similar conditions in the presence of isoleucine. (iii) In some cases the VRS-dependent de8cyletion of [r4C]valyl-tRNA (1 mmole/ml.) in the presence of AMP (2 qoles/ml.) and inorganic pyrophosphate (2 qoles/ml.) w8s measured. The incubation mixture (0.1 ml.) contained the s8me concentrations of TrisHCl, ammonium chloride, megnesium acetate, mercaptoethanol snd BSA as used for the ecylation assay. Incubation ~8s st8rted by the addition of an 8ppropriate dilution of enzyme solution. At intervals, [14C]valyl-tRNA W&B precipiteted and counted by the procedure described for the acylation assay. One unit of descylation activity W&Bdefined 8s the diseppear8nce of 1 mole of [14C]valyl-tRNA in 10 mm 8t 30°C. (iv) Total valine-accepting cspecity of tRNA was measured under the conditions used for VRS ecylation assay. Total E. cc& tRNA was replaced by the sample to be assayed. Excess pure enzyme ~8s added (3.6 @/O-l ml.) and the solution ~8s incubated for 30 min at 30°C. Carrier yeast RNA (0.2 mg) ~8s added before acid precipitation. (d) Enzyme putifkation (i) Cell extraction: E. wZi K12 strain Hfr C w8s grown in M 63 mineral medium (Monod, Cohen-Bazire & Cohn, 1961) supplemented with 0.3% Cas8mino 8cids, O.1o/oyeast extract and 1*Oo/oglucose 8s carbon source; cells were harvested in 18te log phrase. Bacteria were washed twice with 0.01 ma-Tris-HCI (pH 73), 0.01 ~-magnesium chloride buffer rend kept frozen at -20°C. 1 kg (wet weight) of cells was suspended in 3 1. of 0.02 M-potassium phosphate buffer 8t pH 7.2, containing 0.16 M-KCl, 0.002 M-EDTA and 0.01 M-b-mercaptoethenol. Fr8ctions of 100 ml. were sonicated for 10 min with 8 M.E.L. 100-w sonicetor in the cold. The combined extracts were centrifuged for 30 min (811centrifugations were done with the GSA rotor of 8 refrigemted Sorv8ll centrifuge 86 8000 rev./min) and the supernat8nt fluid w&s collected (fraction I). (ii) Streptomycin precipitin. Streptomycin (320 ml. of 10% solution) W&Badded with stirring to each litre of fraction I. Stirring wan continued for 10 min in the cold. The precipitate that formed ~8s removed by centrifugation for 10 min and the supernetant freotion was collected (t&&ion II). Often the sonicetion and streptomycin precipitation steps were replaced by disruption with glaes beads in a Waring Blendor followed by autolysis 8t 37°C for 6 hr. The extrctction buffer in this c8se WEG+I 0.1 &r-potassium phosphate (pH 7.0) containing 0.01 na+mercaptoethanol. The precipitate formed during autolysis was removed by centrifirgation and the purification ~8s continued with the following step. (iii) A?n?ncn&m m.dfa?e. frah. Solid ammonium sulfate (242 g) ~8s added slowly to every litre of fraction II, and after 16 min the suspension WBB centrifuged for 30 min. The pellet ~8s discarded 8nd 188 g ammonium &f&e were added (66% saturation); after 16 min of stirring the suspension ~8s centrifuged for 30 min. The collected precipitate was dissolved in 700 ml. of 0.02 y-potassium phosphate (pH 7*0), containing 10m4 M-EDTA and 0.006 rd-jl-merc8ptoethanol (buffer 1). The enzyme was reprecipitated by bringing the solution to 66% saturation with solid ammonium sulfate and the final precipitate collected by centrifug8tion. A further purification w8s obtained by extraction of the pellet thus obtained with solutions of ammonium sulfate in buffer 1. The pellet ~8s resuspended in 376 ml. of 47% saturated ammonium sulfate by rapid stirring in 8 Waring Blendor and the suspension was centrifuged. The extraction wa8 repeated with the same volume of a 470, satur8ted eolution and twice with 376 ml. of 8 40% saturated solution. Fractions containing the highest specific activity were collected and the enzyme was precipitated by incre8sing the saturation to 66% ammonium sulfate. After centrifugation the precipit8te was dissolved in 200 ml. of buffer 1 (fraction III).
4
ill.
YANIV
AND
li’. C:ROS
(iv) Calcium phosphate gel fractionation. Fraction III was dialyzed overnight against 10 1. of 0.02 M-sodium acetate (pH 6.0), 0.006 M-fl-mercaptoethanol and 10e4 M-EDTA. The dialys8te ~8s diluted, before the addition of the gel, to a protein concentration of 10 mg/ml. Calcium phosphate gel (13.5 mg dry weight/ml.) was added in the proportion of 0.6 vol. to 1 vol. of diluted dialysate and the suspension ~8s centrifuged after an additional 10 min of stirring. The enzyme remaining in the supernatant fraction was adsorbed with another 0.6 vol. of gel. The 8mount of the gel used in the negative and positive absorption changed from one preparation to 8nother and was checked on a small portion every time. The enzyme WBB eluted from the gel by successive washings with potassium phosphrtte buffers of increased molarity and pH. The buffers used were: a, 0.02 M, pH 6.5; b, 0.02 M, pH 7.0; c, 0.05 M, pH 7.0; d, 0.05 M, pH 75; e, 0.2 M, pH 7.5; 811 contained 0.006 M-mercaptoethanol8nd 10e4 M-EDTA. The fractions containing the highest specific activity and most of the enzyme activity (usually fractions c to e) were pooled and the enzyme was precipitated by adding 472 g of solid 8mmonium sulfate/l. After 4 hr the suspension was centrifuged for 30 min, the pellet dissolved in 60 ml. of 0.01 M-Tris-HCl (pH 7.8), 0.1 M-NaCl, 0.006 M-&merc8ptoethanol, 10e4 M-EDTA (“dialysis buffer”) and dialysed twice against 3 1. of the same buffer (frrtction IV). (v) DEAE-Sephcdex fractionation. Dislyzed fraction IV w&s diluted to a protein concentration of 15 mg/ml. with the dialysis buffer before it was absorbed on 8 700-ml. A50 equilibrated with the same buffer. column (4.5 cm x 44 cm) of DEAE-Sephsdex After adsorption, the column w8s washed with 700 ml. of dialysis buffer and elution was effected with a linear gradient made by mixing 3 1. of dialysis buffer with 3 1. of 0.01 M-TrieHCl (pH 7.0), 0.4 ~-N&cl, 0.006 M-fi-mercaptoethanol and 10m4 M-EDTA. A flow rate of 1 ml./min was maintained and lo-ml. fractions were collected and assayed for the valine-dependent ATP-PPi exchange reaction. Since this reaction is also catalyzed by the IRS (Berg, Bergmann, Ofengand & Dieckmann, 1961), two peaks of activity were observed, one corresponding to the IRS (eluted at 0.25 M-salt buffer) and the other to the VRS (elution at O-3 M-s8lt buffer). The two enzyme fractions were pooled sep8rately (only fractions containing at least 60% of the peak activity were kept). The pooled VRS fractions are designated “fraction V”. The IRS was chromatographed separately on 8 hydroxylapatite column sccording to Baldwin & Berg (1966), yielding 30 mg of pure enzyme. The pure IRS moved as a single component in electrophoresis on polyacrylamide gel and exhibited 8 symmetrical sedimentation pattern in analytical ultracentrifugation. (vi) Hydroxykzpatite fractionation. Fraction V was applied directly to a 200-ml. (3 cm x 28 cm) hydroxylapatite column previously equilibrated with 0.001 m-potassium phosphate buffer (pH 6.75) containing 0*0005 M-dithiothreitol. The column was washed with 200 ml. of 0.02 M-potassium phosph8te buffer (pH 6.75) 8nd with the s8me volume of 0.05 IMpotassium phosphate buffer (pH 6.75). Enzyme w8s eluted with 8 linear gradient produced with 1000 ml. of 0.05 M-potassium phosphate (pH 6.75) to 1000 ml. of 0.2 M-potassium phosphate (pH 6.75). All the buffers contained 0.0005 M-dithiothreitol (Fig. 1). 7-ml. fractions were collected; those showing a constant specific activity were pooled and concentrated to about 10 mg/ml. by vacuum dialysis (the “Amicon diaflo” system with XM-50 membranes w&s used in recent purifications). The concentrated enzyme was dialyzed against 0.02 &r-potassium phosphate buffer (pH 7*5), contrtining 0.0005 M-dithiothreitol, and stored frozen in liquid nitrogen. In some c8ses it was adjusted to 50% glycerol and stored, either at -20°C or in liquid nitrogen. T8ble 1 summarizes the different purification steps. (e) Amino
acid analyses
A solution containing 0.65 mg of protein was dialyzed for 24 hr against 0.01 M-sodium acet8te (pH 6.0) and freeze dried. Performic oxidation was done at -8C according to Hirs (1956) followed by hydrolysis in wucuo at 105°C for 24, 48 and 72 hr in 6 N-HCl to which 8 crystal of phenol had been added. The hydrolysates were analyzed on a Beckman amino 8cid analyzer model 120C. Tryptophan was determined by spectral analysis in 0.1 M-N8OH with 8 Cary spectrophotometer (Beaven & Holiday, 1952). Serine 8nd threonine were estimated by 8 linear extrapolation to zero time of values obtained after 24, 46 and 72 hr of hydrolysis. Values for isoleucine, valine and leucine are based on 72-hr hydrolysis.
VALYL-tRNA I
I
SYNTHETASE I
I
0.30 -
FROM I
I
I
E. COLI
5
1000
I
I
PP / .-,‘\ \ ‘e,
3 0.20 8 2 _ 6 0: IO
k
-500 ‘1
/’
” .r $
‘Cc
P/’
‘hp.
+0-O
\ .’
, 50
-0-O
d
‘h
Pf 0’ 0’
al F 2 8 .k
‘-u I
I 70
I
I 90 Fraction
I
I 110
I 130
I
no.
FIQ. 1. Hydroxylapatite cbromatogmphy of valyl-tRNA synthetase fraction V. Samples (0.5 4.) were assayed for PPi exohange activity -- 0 ---- 0 -- and the optioel density was measured at 280 rnp -e-m--. TABLE
Puri$c&nz
Fraction
I II
Crude extract Streptomycin snpernatant
III
Aa elude
IV
Calcium phosphate eluate DEAFLSephadex Hydroxylepatite Concentration step
V VI
of valyl-tRNA
1
synthetme from E. coli K12
Total volnnle (ml.)
Protein hw*)
3300
26.2
83,000
17
o-7
66,000
3950
18
880
27.8
71,000 24,600
14.6 51.2
0.8 1.84
57,000 46,000
1600 200 262 4.5
1.9 1-l 0.21 9.4
Total protein units
2850 220 62 42.3
34.7 18.2 168 153 99 478 448 4226
62,006 33,600 24,000 19,000
Yield per oent
102 80 93 60 43.5 34
The purification prooedure is described in Materials and Methods. The activity is expressed in the ATP-PPi exchange units. (f) Titration of SH growp~ The number of the free SH groups in native and denatured enzyme was measured with DTNB according to Ellman (1969) and with pHMB aeeording to Bayer (1964). The enzyme was freed from protecting SH reagent by filtration on a G26 Sephadex column equilibrated with O-1 ar-Tris-HCl (pH 8-O). Enzyme concentration in the titration mixture was determined by ultraviolet absorption at 280 rnp. Velocity sedimentation studies were done with the Beckman model E ultraeentrifuge equipped with schlieren optios and temperature control. Epon-filled 12-mm, a = 25 cells, single or double sectored were used. Protein concentration was measured by ultraviolet absorption at 280 mp and by the refractive index of the enzyme solution. (h) S~eciJic antieerum ~eparo&a Rabbits were injected with 400 pg of purified IRS or 200 pg of VRS in Freund’s adjuvant. After 6 weeks, a second intravenous injection of the same amount of protein was given and
M. YANIV
6
blood was collected 7 days later. y-globulins were purified according
AND
F. GROS
The blood was centrifuged to Calendar Rr,Berg (I 966).
(i) Antiserum
after
coagulation
and the
iv,hibitiorL studies
Inhibition studies were done in 0.1 or 0.5 ml. of mixture containing, per ml. : 10 pmoles potassium phosphate (pH 7*0), 3 pmoles /I-mercaptoethanol, 200 pg BSA, 1 to 2 pg of pure enzyme and increasing amounts of antiserum. Tubes were incubated for 15 min at 4°C before assay, or otherwise centrifuged for 20 min at 20,000 g and the activity assayed in the supernatant fraction. (j) M&wJaneous Protein concentration was measured according Rosebrough, Farr & Randall, 1951) or the biuret with BSA used as standard.
to the modified Folin test (Gornall, Bardawill
reaction (Lowry, & David, 1949)
3. Results (a) Properties
of pure enzyme
The enzyme obtained after hydroxylapatite chromatography behaved mainly as a single physical constituent, although it still contained a small amount of contaminating material as revealed by polyacrylamide gel electrophoresis or by the asymmetry of the schlieren pattern apparent after 100 minutes of centrifugation at 59,780 rev./min (4’C). At this purification stage the degree of purity exceeded 95%, as deduced from the gel patterns. An enzyme preparation that showed lower contamination could be obtained by rechromatography of fraction VI on a hydroxylapatite column at pH 7.5. Polyacrylamide gel electrophoresis of this material showed only a minute band of contamination, and the schlieren pattern during sedimentation showed the presence of a single symmetric constituent. The specific activity of the enzyme obtained in four preparations varied between 400 to 450 exchange units/mg (the additional rechromatography step did not increase the specific activity significantly). Aoylation activity at the last step of purification was 32 units/mg. The ratio of exchange to acylation activity varied in the course of purification from 11, in the crude extract, to 14 in the iinal step. This difference may be attributed to a certain degree of inaccuracy of the exchange assay in crude extracts, since the valine-dependent activity of the IRS had to be subtracted from the activity measured (see Materials and Methods). Alternatively, it could be due to a slight inactivation of the acylation capacity during purification. The ratio of absorption at 280 rnp to that at 260 rnp was from 1.85 to 1.82 in different preparations, showing little contamination if any with nucleic acids. The absorption at 280 rnp of the protein solution (light path of 1 cm) was 1.13 mg/ml. (Folin) or 0.94 mg/ml. (biuret). Calculation of the total area in a synthetic boundary run in the ultracentrifuge gave an absorption at 280 rnp of 1.19 per mg/ml. In further calculations, a mean value of 1.10 was taken for the 280 rnp absorption of a solution of VRS containing 1 mg/ml. (b) Catalytic
indices of purified
valyl-tRNA
synthetase
Table 2 indicates the values of K,, V,,, and molecular activity for the exchange, acylation and deacylation reactions. Using the integral Michaelis-Menten equation (Dixon & Webb, 1964) the K, value for tRNA was determined either with total tRNA or with purified tRNAV,&‘. With the latter a value of 5.5 x 1O-7 M, not significantly different from that obtained with total tRNA, was calculated. When using
VALYL-tRNA
SYNTHETASE TABLE
FROM
V,.,
Acylation
6.7 X 1o-6 M 2.7 X lo-‘M 1.4 x 10-d M -
(pmoles per mg per min)
synthetase at 30°C KU7
Exchange
L-dine ATP PPi tRNA
7
2
V,,,,, and K, values of valyl-tRNA
Substrate
E. COLI
6.3 x 10-b ?d 13 x IO-4 M 6 x lo-’ (5.6 x lo-‘)t
29.8
Molecular aotivity (molecules of substrate 2470t reacting per min per molecule of enzyme)
Deayletion -
rd
3.18
l-02
368
118
t Determination with pure tRNAVal. $ In the presence of 2 x 10s4 ~-dine.
total tRNA, the relative concentration of specific tRNAVal was estimated after acylating total tRNA with excess purified enzyme. Confirming previous findings by Berg et a& (1961), we observed that the rate of ATP-PPi exchange was ten times higher than the rate of acylation of tRNA for the same amino acid concentration. L-Threonine and L-isoleucine gave low levels of exchange with high K, values, as did also the valine analog, DL-a-aminobutyric acid (Table 3). Magnesium was TAIZE 3 t!hbstrate e~eciIi$y of the amino acid-dependent p yrep?w8@katecxchawe
L-ThlVOllill~ DL-c&mino
L-Isoleucine L-valine
butyric
acid
7 x 10-s P 1.6 x10-3M 10-l M 6.7 x 1O-6 rd
21 6 22 100
necessary for the exchange and acylation reactions, but could be replaced by manganese in the acylation reaction with almost the same efiioiency (70% of V,,,). The precipitation of manganese pyrophosphate in the exchange assay mixture did not permit the study of this reaction with manganese ions. The inhibition of the acylation reaction by excess metal expressed as K’s (the metal concentration producing a 50% reduction of V ,,,) is equal to 2 x 10e3 M for manganese and 7 x 10q3 M for magnesium. Calcium or spermidine had no effect on enzyme activity. (c) Sedintentaticn and nbolccular weight measurement8 Figure 2 shows the sedimentation pro6le of pure VRS after zone sedimentation on a sucrose gradient. The total protein distribution coincides with the enzyme activity, which excludes an appreciable contamination of the enzyme with inactive protein of a different sedimentation coefficient. Calculation of the sedimentation coefficient and molecular weight of VRS, according to Martin k Ames (1961) gave values of 66 s
M. YANIV _~-
.- ..--
T-
AND ----T
F. GROS I-.--
--.--
Fraction no. FIQ. 2. Sedimentation of valyl-tRNA synthetase on sucrose gradient. Purified VRS (100 pg) and IRS (5 rg) were layered on a gradient of 5 to 20% sucrose in 0.02 &r-potassium phosphate (pH 6.75) buffer, containing 0.1 m-KC1 and 0.006 M-B-mercaptoethanol. Centrifugation was for 20 hr at 36,000 rev./min. Fractions were collected and assayed : valine-O--O--; protein (Folin), -~-~--; IRS activity, dependent exchange activity, y-J---o-.
and 106,000 respectively. Highly purified IRS was used as a marker, assuming a sedimentation coefficient of 5-9 s and a molecular weight of 112,000 (Baldwin & Berg, 1966). Sedimentation studies were also performed at different protein concentrations in the analytical ultracentrifuge at 4°C. Fignre 3 describes the dependence of S20,W on protein concentration. A value of 5.6 is obtained by extrapolation to zero protein
Protein (mg/ml.) Fro. 3. Variation of S value with concentration. Protein concentration was determined by absorption at 280 mp. The enzyme was diluted in a 0.02 M-potassium phosphate buffer (pH 7.0) containing 0.1 M-KCl. Temperature of runs, 4°C; speed 59,780 rev./min.
VALYL-tRNA
SYNTHETASE
FROM
E. COLI
9
Fraction no. FIG. 4. Isolation of valyl-AMP-enzyme complex by Sephadex gel fltration. VRS (195 H;) were mixed with 0.02 -010 ATP a-labeled with saP (16.7 ~c/~ole), 0.02 Fole [“C]valine (96 ~o/~ole) in presence of 0.02 M-Tris-HCl (pH 7.8). 0.036 M-NH&I, 0.0025 M-m&gneeium acetate and 0.006 M-/l-meroaptoethanol in a volume of 0.2 ml. After incubation for 6 min at 2O”C, the reaction mixture wae applied to a g-ml. (O-43 oma x 21 cm) Sephadex G76 column sucoineta (pH 6.0) buffer containing 0.06 X-KC& 0.006 M.y-merequilibrated with 0.05 ~-sodium captoethanol and 10m3 M-EDTA. Fraotiona of 0.16 ml. were oollected and 2&d. samples plated on GF/B glass filters and counted in e scintillation counter. Samples of 1 & were used for PPi exchange aotivity essay. Enzyme recovery was 69% (1.22 mFoles). The amount of valine and ATP found in the complex fraotiona wee 0.96 and 1.07 mwoles, respectively. [W]Valine, -a---a-; [a-3”P]AMP, -- X-- x --; [3aP]PPi exobange activity, -O-O-. In * separate experiment with the came amount of enzyme, the binding of [a-3aP]ATP in abeence of valine was followed: -n--n-.
concentration. Molecular weight determination by the approach to equilibrium method (Archibald, 1947) gave a value of 110,OOOf 8090. (d) Xubstrde binding Using the Sephadex gel filtration technique of Norris & Berg (1964), we could isolate a valyl-AMP-enzyme complex (Fig. 4). Valine was transferred to tRNA with an efbciency of 80%. Aa ah-eady observed for the activation and transfer of isoleucine by Norris & Berg (19&t), magnesium was necessary for valyl.AMP~nzyme formation, but not for the transfer to tRNA. The following paper will clearly show that tRNA can bind normally to the enzyme in the absence of bivalent cationa. The presence of magnesium in the complex isolated by Sephadex filtration is improbable, as the
M. YANIV
10
AND
1’. CR08
affinity of Mg 2+ to the enzyme is only lo4 I./mole as determined by fluorescence studies (Helene & Yaniv, manuscript in preparation). The fact that Allende, Mora, Gatica t Allende (1965) observed a bivalent cation requirement for the transfer of the activated threonine to tRNA from rat liver may be attributed to the stabilization of the tRNA structure by these cations, a possibility that was suggested by these authors. Calculation of the amount of valine and AMP bound indicates that 0.8 molecule of valine was bound per enzyme molecule. Thus the existence of one binding site can be inferred. In the following paper it is also shown, by another approach, that one molecule of tRNAVal can bind to each molecule of enzyme. This result is identical to the one obtained with the IRS (Norris & Berg, 1964). Both enzymes appear to have one active site per 110,000 molecular weight. (e) Amino acid composition and titration of SH groups The total amino acid composition of VRS obtained after performic acid oxidation of the protein is shown in Table 4. One outstanding feature is the low number of histidine residues. TABLET Amino acid composition of valyl-tRNA synthetase (in mole.3amino acid/mole enzyme) Tryptophan Lysine Histidine Arginine
11 47 5 37
Half cystine (as cyst&o acid) Aapartio acid Methionine (as methionine sulphone) Threonine Serine Glutamic acid Proline Glycine Alanine Vdine Isoleucine Leucine Tyrosine Phenylalanine
The method of protein Methods.
hydrolysis
and amino acid determination
11 140 17 32 35 150 28 93 145 78 58 67 39 22
is described
in Materials
and
It had been shown previously that for some of the activating enzymes studied the presence of substrates could modify the number of reactive SH groups. Accordingly, as already shown by George & Meister (1967), the number of reactive SH groups in VRS is changed after addition of valine and ATP. Figure 5(a) describes the titration of VRS SH groups with DTNB. After 30 minutes at 25”C, two groups react in the native enzyme and about 1.3 in the presence of magnesium, valine and ATP. When the enzyme was dissolved in 8 M-Urea, about five SH groups reacted with DTNB (a second enzyme preparation gave values of 2-5 and six SH groups for native and denaturated
VALYL-tRNA
SYNTHETASE
FROM
Minutes. at 25’C (a) SH groups/110,000 equivalents 0 2 4 6 8 IO I I 1 I ,
E. COLI
11
of enzyme
a 2
ro
-0-B
: u’q‘O -4 0~100 I ,” 0,200 pnvuuo
-
I
IO
20
30
$: of pHMB (IO’M) (b) FIQ. 5. (a) Titration of SH groups with 5,5’-dithiobio(2nitrobenzoic acid.) (0.14 mg) was titrated in a volume of 0.4 ml. adjusted to 0.1 r.r-Tri*HCl (pH 8-O), 2 x lo-’ M-EDTA. The increase of absorption at 412 mp was measured after the addition of DTNB to a final conoentration of 2 x lo-’ XI: no addition of substrata, -n-O--; in the and 10-s M-M&X,, -A----A-; in presence of lo-’ n%-vahne, presence of 3.6 x 10-s x-tRNAV*’ 4 x 10v6 M-ATP snd 10-s M-M&X, -O--O-; in presence of 8 M-ures -V-V -. Exp. 2: Titration of 0.17 mg enzyme from 8 second prsperstion, -a---a-; the number of SH groups titrated in experiment 2 after 160 min is equal to 3.3.
Exp. 1: Enzyme
(b) p-Hydroxymemuribenzoste titration. Enzyme (0.14 me;) w&s titrated by serial additions of 2 4. pHMB 10-s M in a vol. of O-4 ml. in presence of 0.1 ar-Tris-HCl (pH 8-O). The inorease in absorbance at 260 rnp as oompamd to 8 blank sample was measured after eaoh addition of pHMB (no correction was made for dilution). No odd&ion, -O-O-; in presence of 10e4 M-Vslin0, 2 x 10-s M-ATP and lo-* M-M&&,, --n----o--.
12
M.
YANIV
ANI)
E’. UR08
enzyme after 30 min at 25°C). Addition of tRNAVsl in the presence of Mg2 + had no effect on the number of titrated SH groups in the native enzyme. Using pHMB, six SH groups could be titrated in the native enzyme and about 4.0 in the presence of substrates (Fig. 5(b)). The values obtained by the pHMB titration (and confirmed with the DTNB titration in presence of urea) are different from those obtained by George & Meister (1967) who detected with pHMB 16 sulfhydryl groups per 100,000 equivalent of enzyme. Moreover, the total number of cysteic acid residues which we found after performic oxidation of the enzyme is only 11 (it must nonetheless be recalled that the preparation used by George & Meister was isolated from E. coli B, whereas the one described here derives from a K12 strain). The discrepancy between the number of SH groups titrated and the number of cysteic acid residues detected in amino acid analysis is under study. A similar difference was also found with IRS by Baldwin & Berg (1966). (f) Antiserum
inhibition
Biochemical studies have unravelled a resemblance between the isoleucine- and valine-activating enzyme. IRS can form a complex efficiently with valine plus ATP (Norris & Berg, 1964), and an isoleucine-dependent ATP-PPi exchange is observed with VRS (Table 3). We have also just seen that both systems have similar molecular weights and that both contain one active site per molecule. It was of interest to prepare a specific antiserum against pure IRS and VRS and to look for possible cross-reactions. Figure 6 shows the specificity of each preparation against each homologous enzyme. No cross-inhibition was obtained. Each enzyme activity was
2000/rgA-VPS
1000 (a)
IO00
?OOOpgA-IRS
(b)
FIG. 6. Neutralization of isoleuoyl-tRNA synthetase (a) and valyl-tRNA synthctaae (b) by antiserum prepared against these enzymes. IRS (1.35 pg) and VRS (0.7 pg) were mixed with increasing amounts of antiserum. The exchange activity was assayed with 100 ~1. of supernatant obtained after oentrifugation as described in Materials and Methods. (a) IRS + anti-IRS, -()---()--; IRS t anti-VRS -n--u-: (b) VRS + anti-VRS, -O-O--; VRS + anti-IRS -n---n-.
VALYL-tRNA
SYNTHETASE
FROM
E. COLI
13
pgA-VRS FIQ. 7. Effect of antiserum on exchange and acylation activities of valyl-tRNA synthetaae. Enzyme (0.4 pg) W&B mixed with inoreasing amounts of 8n&ermn, end inoubated for 16 min at 4°C. Portions were assayed for exchange (100 4.) and eoylation (2 4.) sotivities respectively before and after centrifugcttion for 20 min at 20,000 g. Before centrifugation exchange, --O--O--; acylation, -@-a--; after centrifugation exchange, -- q -- q --; eoylation, -A---A-.
totally inhibited in crude extracts of E. coli by its corresponding antiserum, showing the existence of only one activating enzyme for each of these amino acids. Anti-WE3 serum also completely inhibited lysine and histidine incorporation in MS2 RNAdirected in vitro protein synthesis (Ghysen & Yaniv, unpublished observation). While the acylation of tRNA was inhibited instantaneously upon addition of the antiserum, the complex obtained between enzyme and y-globulin retained most of the PPi exchange activity as shown in Figure 7. Addition of substrate did not change the sensitivity to antiserum inhibition. The transfer of valine to tRNA from a pre-isolated valyl-AMP-enzyme complex was also inhibited completely by the antiserum. Another case in which the interaction of the enzyme with a protein leads to a preferential decrease in the aoylation activity is observed when the enzyme is converted into a dimer; when pure VRS was dialyzed against a buffer of low ionic strength, about 10% of the enzyme was converted into a dimer that sedimented at 7-8 s in sucrose gradients. The ratio of acylation to exchange activity was twofold lower in this dimer than in the native form. The decrease in acylation activity in these cases may be ascribed to several possible mechanisms: (i) binding of the protein to the RNA recognition site ; (ii) the steric hindrance towards a bulky substrate (tRNA) ; (iii) restriction of
14
M. YANIV
AND
F. GROS
the aminoacyl-AMP effect on the rate of enzyme-tRNA personal communication).
dissociation (Yams & Berg,
4. Discussion E. coli valyl-tRNA synthetase was first partially purified by Berg et al. (1961). Later, using a different preparation technique, George & Meister (1967) obtained in 5% yield a VRS preparation that behaved as a homogeneous protein on polyacrylamide gel electrophoresis. The present study describes a new procedure of purification, with a yield of 34%. The enzyme appears highly purified on the basis of chromatographic, electrophoretic and sedimentation characteristics. The specific activity of the enzyme as measured by the pyrophosphate exchange activity appears to be higher than that calculated from George & Meister’s data. Sedimentation studies under various conditions suggest that the protein has a molecular weight of 110,000 and a sedimentation coefficient of 5.6 at 20°C. The number of polypeptide chains present in the enzyme molecule is under investigation. Substrate binding experiments involving separation of the valyl-AMP-enzyme complex reveal the existence of one active site for the aminoacyl residue per molecule of enzyme. Addition of valine and ATP but not of tRNA Val affects the number of reactive SH groups of VRS, a result that may indicate a conformational change rather than a direct participation of cysteine in the active site. At least in the case of lysyl-tRNA synthetase, Stern, De Luca, Mehler t McElroy (1966) could exclude the presence of SH in the active site. Recent physicochemical studies of VRS confirm the existence of substrate-dependent changes in the conformation of the protein. Fluorescence intensity of the tryptophan residues of the enzyme was changed upon addition of Val-ol-AMP, an aminoalkyl adenylate competitive analog of aminoacyl AMP (Cassio, Lemoine, Weller, Sandrin & Boissonnas, 1967) or tRNAVal (Helene & Yaniv, manuscript in preparation). These results may indicate two distinct substrate-induced variations in the protein structure. Valine and isoleucine are synthesized by a common biosynthetic pathway in E. coli VRS and IRS have the ability to react with valine in the ATP-PPi exchange, isoleucine being recognized mainly by its specific enzyme and only with a low Vma, and high K, by the VRS. Both enzymes have almost equal molecular weights and an equal number of catalytic sites. In spite of these common features, VRS and IRS do not cross-react immunologically. Of interest is the finding that antiserum prepared against highly purified valine- and isoleucine-activating enzymes totally inhibits the capacity of E. coli extracts to acylate total valine or isoleucine acceptor tRNA’s with the specific amino acids. This result establishes that in E. coli there is only a single species of aminoacyl-tRNA synthetase for synonymous valine or isoleucine acceptor tRNA’s. The very skilful technic81 aesistance of Mrs A. Chestier is acknowledged. We thank J. P. Richardson and J. P. Wailer, for valuable criticisms of this mttnuscript. We are indebtetl to J. M. Dubert for help in the preparation of antiserum and to S. Filitti-Wurmser for the determin8tion of molecular weight. This work was supported by grants from the Fonds de Developpement de 18 Rechercho Scientifique et Technique, the Commissariat & 1’Energie Atomique, the Centre National de 18 Recherche Scientifique, the Ligue Nation& Frangaise contre le Center 8nd the Fondation pour 18 Recherche Medicale Franpaise.
VALYL-tRNA
SYNTHETASE
FROM
E. COLI
16
Note added irr proof. Reduction and polyacrylamide gel electrophoresis of the VRS in the presence of sodium dodecyl sulfate (Shapiro, A. L., Vinuela, E. & Maize& J. V. (1907). B&hem. Biophya. Rec. Comrn. 28, 815) revealed the presence of only one polypeptide chain with a molecular weight of 110,000. REFERENCES Allende, J. E., Mora, G., Gatica, M. & Allende, C. C. (1966). J. Biol. Chem. 240, PC 3229. Archibald, W. J. (1947). J. Phy8. Co&id. Chem. 51, 1204. Baldwin, A. N. 8z Berg, P. (1960). J. B&Z. Chem. 241, 831. Beaven, G. H. BEHoliday, E. R. (1952). Advanc. Protein Chem. 7, 319. Berg, P., Bergmann, F. H., Ofengand, E. J. & Dieckmann, M. (1961). J. Biol. Chem. 236, 1726. Boyer, P. D. (1964). J. Amer. Chem. Sot. 76, 4331. Calendar, R. & Berg, P. (1966). Biochemietry, 5, 1681. Cassio, D., Lemoine, F., Wailer, J. P., Sandrin, E. & Boissonnas, R. A. (1967). Biochemi&y, 6, 827. Dixon, M. & Webb, E. C. (1964). Enzymee, p. 114. London: Longmans. Ellman, G. L. (1969). Arch. Biochem. Biqvhy8. 82, 70. George, H. & Meister, A. (1967). Biochim. biophys. Acta, 182, 166. Gornall, A. G., Bardawill, C. J. & David, M. M. (1949). J. Biol. Chem. 177, 761. Him, C. H. W. (1966). J. Biol. Chem. 219, 611. Keilin, D. & Hartree, E. F. (1938). Proc. Roy. Sot. B, 124, 397. Kelmers, A. D. (1966). J. Biol. Chem. 241, 3540. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). J. BioZ. Chem. 198, 266. Martin, R. G. & Ames, B. N. (1961). J. BioZ. Chem. 236, 1372. Martin, E. M., Yegian, G. & Stent, G. (1963). 2. ~eer?&mg8Zehre, 94, 303. Monod, J., Cohen-Bazire, G. t Cohn, M. (1961). Biochim. biophye. Acta, 7,586. Muench, K. H. & Berg, P. (1966). Biochamietry, 5, 982. Neidhardt, F. C. BEEarhart, C. F. (1966). Cold Spr. Harb. Sm. Quant. BioZ. 31, 567. Norris, A. T. & Berg, P. (1964). Proc. Nat. Acad. Sci., Wuah. 52, 330. Stern, R., De Luca, M., Mehler, A. H. & McElroy, W. D. (1966). Biochemistry, 5, 126. Yaniv, M. & Gros, F. (1969a). J. Mol. BioZ. 44, 17. Yaniv, M. & Gras, F. (19698). J. Mol. BioZ. 44, 31.
2