The glutaminyl-transfer RNA synthetase of Escherichia coli. Purification, structure and function relationship

65

Biochimica et Biophysica Acta, 607 (1980) 65--80 © Elsevier/North-HollandBiomedicalPress

BBA 99617 THE GLUTAMINYL-TRANSFER RNA SYNTHETASE OF ESCHERICHIA COLI PURIFICATION, STRUCTURE AND FUNCTION RELATIONSHIP

D A N I E L K E R N a, S E R G E POTIER a, J A C Q U E S L A P O I N T E

b

and Y V E S B O U L A N G E R

a

a Institut de Biologie Moldculaire et Cellulaire du C.N.R.S., Laboratoire de Biochimie, 15, rue Rend Descartes, 67084 Strasbourg Cedex (France) and b Universitd Laval, Facultd des Sciences et de Gdnie, Ddpartement de Biochimie, Laval, Qudbec G I K T P 4 (Canada)

(Received June 5th, 1979)

Key words: Glutaminyl-tRNA synthetase; Aminoacyl-tRNA synthetase; tRNA; Aminoacylation; (E. coli)

Summary Glutaminyl-tRNA synthetase from Escherichia coli has been purified to homogeneity with a yield of about 50%. It is a monomer of about 69 000 daltons. Arginyl and glutamyl-tRNA synthetases are also monomeric synthetases of molecular weight significantly lower than 100 000. In addition it is well known that these three synthetases require their cognate tRNA to catalyze the [32P]PPi-ATP exchange. Like arginyl-tRNA synthetase, but unlike glutamyltRNA synthetase, glutaminyl-tRNA synthetase seems to contain some repeated sequences. Therefore no correlation can be established between the tRNA requirement of these synthetases for the catalysis of the isotope~xchange and the presence or the absence of sequence duplication. In the native enzyme four sulfhydryl groups react with dithiobisnitrobenzoic acid causing a loss of both the arninoacylation and the [32P]PPi-ATP exchange activities. The rate-limiting steps of the overall aminoacylation and its reverse reaction correspond, respectively, to the catalysis of the aminoacylation of tRNA Gin and of the deacylation of glutaminyl-tRNAGin. At acidic pH, glutaminyl-tRNA S u p p l e m e n t a r y data to this article are deposited with, and can be obt a i ne d from, Elsevier/North-Holland Biomedical Press B.V.. BBA Data Deposition. P.O. Box 1345, 1000 BM Amsterdam, The Netherlands. R e f e r e n c e should be made to No. B B A / D D / 1 2 9 / 9 9 6 1 7 / 6 0 7 (1980) 65--80. The s u p p l e m e n t a r y inform a t i o n includes: the tryptic map of reduced and [ 1 4 C ] c a r b o x y m e t h y l a t ed g i n t a m i n y l - t R N A synthetase. Abbreviations: Hepes, N-2ohydroxyethylpiPerazine-N'-2-ethanesulfonic acid; SDS, s odi um dodeeyl sulfate.

66 synthetase catalyzes the synthesis of the glutaminyl-tRNAGin and its deacylation at significantly lower rates than the [32P]PPi-ATP exchange, indicating that glutaminyl-tRNAmn cannot be an obligatory intermediate in this isotope exchange. These results suggest the existence of a two-step aminoacylation mechanism catalyzed by this enzyme.

Introduction

Up to now many aminoacyl-tRNA synthetases from various organisms have been purified to homogeneity and studied for their structural and enzymic properties (for a general review see Refs. 1--4). From a mechanistic view point they can be divided into two groups. The first one is constituted by arginyl-, glutamyl- and glutaminyl.tRNA synthetases which require their cognate tRNA to catalyze the [32P]PPi-ATP isotope-exchange reaction. The second one includes the 17 other aminoacyl-tRNA synthetases which are able to catalyze this reaction in the absence of the tRNA (see the general reviews Refs. 1--4). Apart from their mechanistic characteristics, arginyl-, glutaminyl- and glutamyl-tRNA synthetases differ structurally from the other synthetases: indeed all three are 'small' monomeric enzymes, respectively, 64 000 [ 5], 69 000 [6] and 56 000 [7,8] daltons in Escherichia coli, whereas in the second group large monomers of 100 000--120 000 daltons are found together with dimers ~2 (~ = 40 000--50 000 daltons), such as seryl- and tyrosyl-tRNA synthetases, dimers ~ ( a ' = 85000 daltons), such as methionyl-tRNA synthetase, and finally tetramers of the ~ 2 type (glycyl- and phenylalanyl-tRNA synthetases). Furthermore extensive sequence duplication has been found in large monomers [9--12] as well as in the subunits of large dimers and tetramers [9,13,14]. The occurrence of large repeated sequences looks like a general feature of those synthetases with subunits greater than 80 000 daltons. Recently repeated sequences have been found in both ~ and ~ subunits of yeast phenylalanyl-tRNA synthetase (73 000 and 62 000 daltons, respectively) [15] but not in E. coli glutamyl-tRNA synthetase (56 000 daltons) [7]. In order to find out whether or not this lack of sequence duplication is a general property of small monomeric synthetases (with molecular weight significantly lower than 100 000), and in an attempt to correlate the structural features with the kinetic properties of this group of synthetases, we have undertaken a study of E. coli glutaminyl-tRNA synthetase. This enzyme was first purified from E. coli K12 by Folk [6]. We describe here a simplified purification procedure of the glutaminyl-tRNA synthetase from E. coli MRE 600 which permits purification of the enzyme with a significantly higher yield. Kinetic and structural properties of the enzyme will be described, and evidence for a two-step catalytic aminoacylation mechanism reported. Materials and Methods Materials E. coil MRE 600 was grown in minimal medium [16] and harvested during

67

the exponential growth phase by centrifugation. The wet cells were frozen with liquid nitrogen and stored at --20°C. For some purifications, we started from the 100 000 × g supernatants of MRE 600 extracts, kindly provided by Dr. A.H. Wahba [17]. Unfractionated tRNA from E. coli B was purchased from Schwarz/Mann. It contains 4% tRNA 61n. tRNA GI~, pure at 65%, was obtained from unfractionated E. coli tRNA after two successive chromatographies on benzoylated DEAE-cellulose columns [6]. Phenylmethylsulfonylfluoride, 5,5'-dithiobis(2nitrobenzoic acid), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes), bovine serum albumin, ATP and spermidine were obtained from Sigma. Omnifluor, uniformly labeled L-[U-~4C, U-3H]glutamine and sodium [32P]pyrophosphate were from New England Nuclear. Microgranular DEAEcellulose (DE-52) and phosphocellulose (P~I) were bought from Whatman, the various chemicals for making polyacrylamide gels and 2-mercaptoethanol from Eastman Chemicals, poly(ethyleneglycol) 6000 from J.T. Baker and dextran T-500 from Pharmacia. Iodoacetamide was from the Commissariat ~ l'Energie Atomique (Saclay, France), trypsin (treated with L-l-tosylamino-2-phenylethyl ketone) from Worthington, and thin-layer cellulose plates were from Machery-Nagel (Polygram C400, 20 × 20 cm). Methods Protein concentrations were determined according to Lowry et al. [18] or for pure enzyme solutions, by their absorbance at 280 nm using the relation: E 2lmg/ml 8 0 n m = 0.57 (cf. Results). Aminoacylation reaction. The formation of Gln-tRNA G~n was followed in 0.1 ml reaction mixtures containing 50 mM sodium Hepes, pH 7.2, 2 mM ATP, 3 mg tRNA/ml, 0.1 mM L-[~4C]glutamine (specific activity: 25 Ci/mol) and the indicated amounts of enzyme. When necessary, the enzyme was diluted in 10 mM sodium Hepes, pH 7.2, 20 mM 2-mercaptoethanol and 1 mg bovine serum albumin/ml. After various incubation times at 37°C, an aliquot was transferred onto a disc of 3 MM Whatman filter paper, and the [14C]GlntRNA 61n synthesized determined as described previously [7]. For large-scale preparations of [14C]Gln-tRNA Gin, partially purified tRNA ~ln was aminoacylated under similar conditions and the [~4C]Gln-tRNAGln was then isolated on a short DEAE-cellulose column. Incorporation o f [32P]PPi into ATP. The reaction mixture contained, unless otherwise mentioned, 100 mM sodium Hepes, pH 7.2, 2 mM ATP, 16 mM MgC12, 6 mM L-glutamine, 3 mM [32P]PPi about 2000 cpm/nmol), 3 mg tRNA] ml and various amounts of enzyme. After various incubation times at 37°C, the amount of [32p]ATP present in the reaction mixture was determined as described previously [7]. Kinetic measurements. The aminoacylation reaction mixture corresponds to that described above. All substrates, except one were present at saturating concentrations: 2 mM ATP, 0.8 mM L-[14C]- or L-[3H]glutamine, 10 pM tRNA 61n in unfractionated E. coli tRNA. The [32p]PPi-ATP exchange reaction mixture corresponds to that described above. The reactions were started by adding the enzyme solution previously equilibrated at 37°C. The initial velocities of the reactions were determined from the amounts of [~4C]GIn-tRNAGIn

68 (for the aminoacylation reaction) or [32P]ATP (for the isotope-exchange reaction) synthesized during at least three different incubation times. For Km determinations, t h e kinetic data were analyzed according to Lineweaver and Burk [191. Concentration o f cellular fractions. Two techniques were used to concentrate various cellular fractions: dialysis against a buffer solution containing 30% (w/v) poly(ethyleneglycol) 6000, or dialysis under vacuum across a membrane (Schleicher and Schtill). Molecular weight determination. In the absence of denaturing agent, the molecular weight was measured either b y sedimentation on sucrose gradient under conditions described previously [20], or b y electrophoresis on polyacrylamide gels of various concentrations. The m e t h o d o l o g y described by Hedrick and Smith [21] was used to estimate the molecular weight of the native enzyme. In the presence of denaturing agent, we followed the protocol o f Weber and Osborn [22]. The experimental conditions and the molecular weight markers used for b o t h determinations were as described previously [7]. Sulfhydryl group titration. We used the m e t h o d described b y Ellman [23]. The enzyme was first dialyzed twice during 8 h under nitrogen against 5 1 of 10 mM Tris-HC1 (pH 7.4) to remove all traces of 2-mercaptoethanol. The titration was c o n d u c t e d at 25°C in the presence of 4.5 pM glutaminyl-tRNA synthetase and 250 #M dithiobisnitrobenzoic acid either in the absence of denaturing agent or in the presence of 8 M urea or 6 M guanidine-HC1. The titration was followed b y measuring the A412nm in a 1.0 cm pathlength quartz cell with a Zeiss s p e c t r o p h o t o m e t e r and using a molar extinction coefficient of dithiobisnitrobenzoic acid at 412 nm of 13 600. Measurements o f loss of the enzyme activities in the presence of dithiobisnitrobenzoic acid. The enzyme (final concentration: 20 p M ) w a s preincubated in the presence of 250 ~M dithiobisnitrobenzoic acid. After various times from 1 to 10 min, 10-/~1 aliquots were diluted in 3 ml of 0.01 M Tris-HC1, pH 7.4. The dilutions were then essayed for aminoacylation and isotope-exchange activities as described previously. Amino acids analysis. Samples were thoroughly dialyzed against diluted acetic acid {0.5%, v/v), freeze-dried and hydrolyzed under nitrogen in 6 N HC1 at l l 0 ° C for 25 h, with a crystal of phenol to protect tyrosine from oxidation. Cysteine and methionine were determined as cysteic acid and methionine sulfone, respectively, after hydrolysis of the performic-oxidized enzyme according to Moore [24]. Tryptophan was estimated b y the method of Liu and Chang [25]. Analyses were performed on a Durrum Ds00Analyzer. Carboxymethylation and tryptic digestion. Enzyme samples were carboxymethylated, using a technique derived from that of Crestfield et al. [26]. Samples (3.5 mg/ml) were reduced with 10 mM dithioerythritol for 1 h at r o o m temperature and thoroughly dialyzed under nitrogen against 0.2 M TrisHC1, pH 8.5, 0.1 mM EDTA and 0.1 mM diisopropylphosphorofluoridate. Solid recrystallized urea was added to yield a final concentration of 8 M. A 50-fold excess of iodo['4C]acetamide (0.5 Ci/mol) was added, and the labeling was carried o u t under nitrogen at r o o m temperature for a b o u t 30 min. A second addition of the same excess of non-radioactive reagent was made and the reaction was continued for another 30 min. During the whole incubation

69

with iodoacetamide the vial was wrapped in aluminium foil to minimize iodine formation. The excess of reagent was destroyed by addition of 1% (v/v) 2-mercaptoethanol and the solution was exhaustively dialyzed against a 1% (w/v) solution of NH4HCOs. Tryptic digestion. The protein solution was freeze-dried, redissolved in 1% (w/v) NH4HCO3 to yield a final concentration of 5 mg/ml. Trypsin was added (1 : 100, w/w) and the mixture was incubated at 37°C for 2 h. Another sample of trypsin (1 : 100, w/w) was added and the incubation was continued for another 2 h. The mixture was then freeze-dried and dissolved in a minimum amount of 10% acetic acid. Fingerprinting techniques. The above solution was subjected to thin-layer electrophoresis and ascending chromatography on cellulose plates. Electrophoresis was run in the first dimension in a Camag apparatus, at pH 4.4 (pyridine/acetic acid/acetone/H20, 4 0 : 8 0 : 3 0 0 : 1 5 8 0 ) during 90 min at 400 V. The plates were dried and run in the second dimension in the following solvent: butan-l-ol/acetic acid/H20/pyridine (15 : 3 : 12 : 10). With these combined techniques about 100-#g peptides in 10 #1 could be fingerprinted and analyzed. Peptides were detected by ninhydrin staining (spray with 0.3% ninhydrin in C2HsOH, containing 3% collidine and 10% acetic acid). Radioactive peptides were detected by autoradiography of the map. Argininecontaining peptides were revealed by their fluorescence after spraying of the map with a solution of 0.01% phenanthrenequinone and 5% NaOH in 80% C2HsOH [27]. Tryptophan-containing peptides were specifically stained with the Ehrlich reagent (1% (w/v) p-dimethylaminobenzaldehyde in 10 ml of 12 N HC1, dissolved in 90 ml of acetone). Results

Purification of the glutaminyl-tRNA synthetase All the operations were performed between 0 and 4°C. All the buffers contained 10% (v/v) glycerol, 20 mM 2-mercaptoethanol and 0.1 mM phenylmethylsulfonylfluoride as a protective agent against proteases. In the buffer used for cell lysis, 10 mM phenylmethylsulfonylfluoride was present. The centrifugations were made in a GSA rotor in a Sorval RC2-B. I. Cell lysis. I kg of wet cells were suspended in 2 1 of 10 mM potassium phosphate, pH 8.0, and broken by sonication during 10 min in a Raytheon Sonic oscillator (model DF101), by fractions of 75 ml. The lysate was centrifuged at 8000 rev./min during 30 min to remove cell debris and intact cells, yielding 2350 ml of supernatant. IIa. Partition in a poly(ethyleneglycol).dextran two-phases system. Concentrated solutions of poly(ethyleneglycol) 6000 and dextran T-500 were added to reach the final concentrations of, respectively, 7% and 1.5% in the superuatant. As several aminoacyl-tRNA synthetases were found remaining in the poly(ethyleneglycol) phase in the presence of about 0.05 M potassium phosphate, pH 8.0 [8,28], 125 ml of I M potassium phosphate (pH 8.0) was added to the supematant. This suspension was mixed during 2 h, and the two phases were separated by centrifugation at 5000 rev./min during 20 min. The poly(ethyleneglycol)-rich top phase of about 3 1 contains most of the glutaminyl-tRNA synthetase activity.

70

IIb. 100 000 X g centrifugation: alternative second purification step. In some cases the two-phases partition step was replaced b y a centrifugation of the 8000 rev./min supernatant at 100 000 X g in a Spinco centrifuge for 3 h. In these experiments the following steps were then carried out on the 100 000 X g supernatant of a b o u t 1.8 1. III. Chromatography on DEAE-cellulose. The poly(ethyleneglycol) phase (when step IIa was used) was diluted by addition of 2 1 of 10% glycerol, 20 mM 2-mercaptoethanol, in order to reduce the ionic strength. In the purification procedures where step IIb was used, the 100 000 X g supernatant was adjusted to 2 1 with the same solution containing 10 mM potassium phosphate buffer, pH 7.2. Half of the solution (2.5 1 of the diluted poly(ethyleneglycol) phase, or 1 1 of the 100 000 X g supernatant) was then adsorbed on a 7 cm X 30 cm DEAE-cellulose (type DE-52) column, which was then washed with 1 1 of 10 mM potassium phosphate buffer, pH 7.2. The (macro)molecules left on the column were eluted at a b o u t 400 ml/h with a linear gradient from 20 mM potassium phosphate (pH 7.2) to 250 mM potassium phosphate buffer (pH 6.5) in a total volume of 6 1 (Fig. 1A). The glutaminyl-tRNA synthetase activity was eluted at the beginning of this gradient, and was already separated from most other aminoacyl-tRNA synthetases. The most active fractions were pooled and dialyzed against 10 mM potassium phosphate buffer, pH 7.0 (fraction DEAE). The same purification step was conducted on the other half of the poly(ethyleneglycol) phase or 100 000 X g supernatant. IV. Chromatography on phosphocellulose. Fraction DEAE-cellulose obtained from the chromatography of the 5-1 fraction of the diluted poly(ethyleneglycol) phase or the 2 1 one of the 1 0 0 0 0 0 X g supernatant was adsorbed onto a 3.5 cm X 35 cm column of phosphocellulose (Whatman Pll) equilibrated with a 10 mM potassium phosphate buffer, pH 7.0. After adsorption of the protein sample, the column was washed at a rate of 100 ml/h with 750 ml of this buffer and eluted with a linear gradient from 0.02 M to 0.3 M KC1 in 10 mM potassium phosphate buffer, pH 7.0 (total volume 4 I). The elution pattern (Fig. 1B) shows that only a few proteins were retained on the phosphoceUulose, and that the glutaminyl-tRNA synthetase was well separated from the other proteins. The fractions containing this enzymatic activity were pooled and concentrated (fraction Phospho-C). Purity o f the glutaminyl-tRNA synthetase obtained after step IV. Analysis of the Phospho-C fraction b y polyacrylamide gel electrophoresis in the absence or presence of denaturing agent revealed, respectively, the presence of one major protein band in b o t h cases (Fig. 1B, insert). The activity of the glutaminyl-tRNA synthetase was found to be associated to the major protein band when the electrophoresis was carried o u t under non-denaturing conditions (result n o t shown). The major band represented a b o u t 95% of the protein from the Phospho-C fraction. Yield o f purification. The results of a purification procedure using partition in a poly(ethyleneglycol)-dextran two-phases system as a second step is summarized in Table I. After the three purification steps the specific activity of the enzyme is enriched about 500-fold, and 54% of the initially present units are recovered. The extinction coefficient of the fraction Phospho-C at 280 nm ( b~280nm ~llTlg/mIJ was found equal to 0.57.

71

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Fraction number Fig. 1. E l u t i o n s o f g l u t a m i n y l - t R N A s y n t h e t a s e o n D E A E - c e l l u i o s e (A) a n d p h o s p h o c e l l u i o s e (B). Fract i o n s of 20 m] were c o l l e c t e d . F o r t h e e n z y m e assays, d i l u t i o n s ((A) 4 0 t i m e s ; (B) 500 t i m e s ) of t h e vario u s f r a c t i o n s were m a d e in t h e a m i n o a c y l a t i o n m i x t u r e . F o l l o w i n g a n i n c u b a t i o n of a b o u t 1 0 m i n a t 3 7 ° C , t h e a m o u n t o f [ 1 4 C ] G I n - t R N A G l n p r e s e n t in 50-/~1 s a m p l e s of t h e m i x t u r e s w e r e d e t e r m i n e d as d e s c r i b e d in Materials a n d M e t h o d s . o . . . . . . o, A 2 8 0 n m ; • -', e n z y m e a c t i v i t y ; - - - , Potassium p h o s p h a t e m o l a r l t y . I n s e t p h o t o g r a p h : a n a l y t i c a l p o l y a c r y l a m l d e gel e l e c t r o p h o r e s i s of a 10/~g s a m p l e o f t h e e n z y m e f r a c t i o n P h o s p h o - C i n t h e p r e s e n c e of r e d u c i n g a n d d e n a t u r i n g agents. TABLE I PURIFICATION OF THE GLUTAMYL-tRNA SYNTHETASE One u n i t of e n z y m e c a t a l y z e s t h e f o r m a t i o n 1 n m o l G l n - t R N A G i n / m i n a t 3 7 ° C . Step

I. II. III. IV.

Cell e x t r a c t Liquid polymer extract DEAE-csllulose Phosphoeellulose

Total protein (mg)

Total units

Specific a c t i v i t y (units/mg)

Recovery

33 150 19 4 6 0 1 130 35 *

82 900 80 3 0 0 68900 44 800

2.5 4.1 61 1280

100 97 83 54

(%)

0 n m value * The c o n c e n t r a t i o n o f t h e p u r e e n z y m e s o l u t i o n was c o r r e c t e d b y t a k i n g a c c o u n t o f t h e ~=12 8mgfl~al (see t h e t e x t ) .

72

Kinetic properties of the glutaminyl-tRNA synthetase Optimal aminoacylation conditions. The rate of Gln-tRNA Gin formation was f o u n d optimal at pH 8.5 (Fig. 2A) and at 45°C (result n o t shown). Concerning the ATP/magnesium requirement, the aminoacylation rate was found optimal at 2 mM ATP for a MgC12/ATP ratio equal to 2--5 (Fig. 2B). Mn 2÷ and C@ ÷ could replace partially magnesium in this reaction. In the presence of an optimal divalent cation/ATP ratio b o t h divalent cations stimulate the aminoacylation rate at half the value observed in the presence of magnesium (results n o t shown). Ca 2÷ and Zn 2÷ did n o t stimulate the aminoacylation reaction. Optimal [32P]PPi-ATP exchange conditions. Let us recall that the gluta-

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Fig. 2. E f f e c t s o f p H a n d b i v a l e n t c a t i o n s o n t h e i n i t i a l r a t e s o f t h e a m i n o a c y l a t i o n a n d t h e [ 3 2 p ] p P iATP isotope-exchange reactions catalyzed by the glutaminyl-tRNA synthetase. The aminoacylation mixt u r e s c o n t a i n e d : 1 0 0 m M s o d i u m H e p e s , p H 7.2, o r o t h e r w i s e i n d i c a t e d ; 0 . 8 m M L - [ 3 H ] g l u t a m i n e 4 3 2 0 0 0 c p m / n m o l ) , 2 m M A T P , 4 m M MgCI 2 u n l e s s o t h e r w i s e m e n t i o n e d , 5 0 m M KC1, 6 m g u n f r a c t i o n a t e d E. coli t R N A / m l ( a b o u t 1 0 /~M t R N A G i n ) a n d 0 . 6 /~g o f g l u t a m i n y l - t R N A s y n t h e t a s e / m l . T h e r e a c t i o n s w e r e c o n d u c t e d a t 3 7 ° C a n d t h e i n i t i a l r a t e s o f [ 3 H ] G I n - t R N A G i n f o r m a t i o n d e t e r m i n e d as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . In o r d e r t o m i n i m i z e t h e d e a c y l a t i o n o f t h e G I n - t R N A G l n a t h i g h p H , the incubation times did not exceed 1 rain at pH 7.2 and above. The [32P]PP;-ATP isotope-exchange mixtures contained: I00 mM sodium Hepes (pH 6.2) or otherwise indicated, I0 mM L-glutamine, 2 mM s o d i u m [ 3 2 P ] P P i ( a b o u t 2 0 0 0 e p m / n m o l ) , 2 m M A T P , 8 m M MgCI 2 u n l e s s o t h e r w i s e m e n t i o n e d , 1 0 / ~ M t R N A G l n in u n f r a c t i o n a t e d E. coli t R N A a n d 0 . 5 /~g o f g l u t a m i n y l - t R N A s y n t h e t a s e / m l . T h e r e a c t i o n s w e r e c o n d u c t e d a t 3 7 ° C a n d t h e i s o t o p e - e x c h a n g e r a t e s d e t e r m i n e d as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . ( A ) p H e f f e c t s o n t h e a m i n o a c y l a t i o n ( o ) a n d t h e [ 3 2 p ] P P i - A T P i s o t o p e - e x c h a n g e (o) r e a c t i o n s . (B) E f f e c t s o f t h e M g C 1 2 / A T P r a t i o o n t h e a m i n o a e y l a t i o n r e a c t i o n ( o ) a n d t h e MgCl 2 / A T P + PPi r a t i o o n t h e [ 3 2 p ] P P i - A T P i s o t o p e - e x c h a n g e r e a c t i o n (o). (C a n d D ) E f f e c t o f s p e r m i d i n e in t h e p r e s e n c e o f vario u s MgCI 2 c o n c e n t r a t i o n s o n t h e [ 3 2 p ] P P i - A T P i s o t o p e - e x c h a n g e (C) a n d o n t h e a m i n o a c y l a t i o n r e a c t i o n s (D): i n t h e a b s e n c e o f a d d e d MgCI 2 ( e ) , o r in t h e p r e s e n c e o f v a r i o u s M g C 1 2 / A T P + PPi (C) o r M g C 1 2 / A T P (D) r a t i o s : o, 0 . 1 2 5 ; o, 0 . 2 5 0 ; ~, 0 . 5 0 ; " , 1 . 0 ; o 2 . 0 .

73 minyl-tRNA synthetase catalyzes the incorporation of [32P]PP i into ATP only in the presence of tRNA G'n. The isotope exchange occurs faster at acidic than at neutral and alkaline pH: indeed in the range of pH tested (6.2--9.0) this rate was found the highest at pH 6.2 (Fig. 2A). In addition, the rate of isotope exchange is optimal in the presence of a MgC12/ATP + PPi ratio equal to 1--3 (Fig. 2B). In the absence of added MgCI:, CoCl2, partially stimulates this reaction (results not shown). Effect of spermidine on the aminoacylation and the [32P]PPi-ATP exchange reactions. In the absence of added MgC12, spermidine was found unable to promote the aminoacylation and the [32P]PPi-ATP isotope-exchange reactions. In the presence of limited MgC12 concentrations, spermidine significantly stimulated the two reactions (Fig. 2C and D); however, it can be shown, that spermidine is never able to totally replace magnesium (Fig. 2C and D). Kinetic constants of the glutaminyl-tRNA synthetase. The kinetic parameters of the glutaminylation reaction were estimated at pH 7.2 under the conditions defined in Materials and Methods. Lineweaver and Burk plots gave apparent Km values of 0.2 #M, 210 ~M and 150/IM, respectively, for tRNA G'n, ATP and glutamine. Turnover number of the glutaminyl-tRNA synthetase and rate-limiting steps o f the aminoacylation and its reverse reaction. At pH 7.2, 37°C, and in the presence of saturating concentrations of substrates one molecule of enzyme catalyzes the formation of 3.3 molecules of Gln-tRNAGln/s. When the aminoacylation reaction was effected under conditions where several successive catalytic cycles could be followed, no decrease of the aminoacylation rate was detected after the first catalytic cycle (Fig. 3A). Similarly, the rate of the AMP and PPi~iependent deacylation reaction catalyzed by this enzyme does not decrease after the first catalytic cycle (Fig. 3B). Furthermore under the same experimental conditions (0°C, pH 6.2, and in the presence of saturating substrates concentrations), the aminoacylation, as well as the AMP and PPi-dependent deacylation reactions occur at significantly slower rates than the [32P]PPi-ATP exchange: indeed the turnover numbers under these conditions are, respectively, 0.050 s-1 for the aminoacylation reaction (Fig. 3A), 0.0092 s-1 for the AMP and PPi-dependent deacylation reaction (Fig. 3B), and 2.15 s-1 for the [s2P]PPI-ATP exchange (result not shown).

Some structural properties of the glutaminyl-tRNA synthetase Molecular weight and monomeric structure. In the absence of denaturing agent, the molecular weight of the native enzyme was determined by sedimentation on sucrose gradients in the presence of either catalase (Mr 240 000), alcohol dehydrogenase (Mr 150 000) or hemoglobin (Mr 64 500). The values obtained were 65 400, 68 000 and 71 200, respectively. Electrophoresis of the native enzyme in polyacrylamide gels of various concentrations in the presence of molecular weight markers, gave a value of about 68 500 (not shown) whereas in the presence of SDS and reducing agent the enzyme gave rise to a unique band of 69 000 daltons (not shown). These results show that the native enzyme is a monomer. Amino acid composition and tryptic map. The amino acid composition of glutaminyl-tRNA synthetase is given in Table II. The tryptic maps of the [14C]-

74

/

/

c)

A

500 o

20( ¢1.

10C

Minutes

Minutes Fig. 3. D e t e r m i n a t i o n s o f t h e r a t e - l i m i t i n g s t e p s o f t h e a m i n o a c y l a t i o n r e a c t i o n ( A ) a n d o f t h e A M P a n d P P i - d e p e n d e n t d e a c y l a t i o n r e a c t i o n (B) c a t a l y z e d b y t h e g l u t a r n i n y l - t R N A s y n t h e t a s e , ( A ) T h e i n c u b a t i o n m i x t u r e s c o n t a i n e d 1 0 0 m M s o d i u m H e p e s , p H 6 . 2 , 2 m M A T P , 4 m M MgCI 2, 5 0 m M KC1, 5 0 I.~M E. coli t R N A Gln, 0 . 8 m M L - [ 3 H ] g l u t a m i n e ( 5 5 0 0 0 c p m / n m o l ) , a n d v a r i o u s c o n c e n t r a t i o n s o f g l u t a m i n y l t R N A s y n t h e t a s e : m 0 . 5 #M~ ©, 1 . 0 ~M; e, 2.0 p M . (B) T h e i n c u b a t i o n m i x t u r e s c o n t a i n e d 1 0 0 m M s o d i u m H e p e s , p H 6 . 2 , 1 0 m M A M P , 2 m M PPi, 4 m M MgC12. 50 m M KCI, 1 2 . 6 / ~ M [ 1 4 C ] G I n - t l ~ N A G l n ( 6 5 3 0 0 c p m / n m o l ) a n d v a r i o u s c o n c e n t r a t i o n s o f g l u t a m i n y l - t R N A s y n t h e t a s e : m, 1 . 0 ~M~ o, 2 . 0 /~M; e , 3 . 0 $zM. T h e a m o u n t s o f [ 3 H ] - o r [ 1 4 C ] G I n - t R N A G I n p r e s e n t a f t e r v a r i o u s i n c u b a t i o n t i m e s a t 0 ° C w e r e d e t e r m i n e d i n 50-/~1 a l i q u o t s as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . In t h e c o n t r o l e x p e r i m e n t (~) t h e h i g h e s t e n z y m e c o n c e n t r a t i o n t e s t e d ( 3 . 0 # M ) w a s i n c u b a t e d w i t h o u t A M P a n d PPi u n d e r t h e s a m e conditions. No significant enzymatic and chemical Oin-tRNA Gln deacylations could be detected. The i n s e r t f i g u r e r e p r e s e n t s t h e d e p e n d a n c e o f l o g ( G l n - t R N A G i n d e a c y l a t e d ) v e r s u s t i m e . I n ( A ) a n d (B), t h e s u c c e s s i v e c a t a l y t i c c y c l e s are i n d i c a t e d b y a r r o w s .

carboxymethylated enzyme showed after autoradiography 11 radioactive spots, among which four major ones; phenantrene quinone and Ehrlich stainings revealed 37 and two peptides, respectively, whereas after ninhydrin spraying about 55 peptides could be visualized. Reproductions of these tryptic maps are deposited in the Biochim. Biophys. Acta Data Bank. Given the fact that one

75 T A B L E II A M I N O A C I D C O M P O S I T I O N O F T H E G L U T A M I N Y L - t R N A S Y N T H E T A S E O F E. C O L I A m i n o acid

N u m b e r o f r e s i d u e s p e r 69 3 0 0

Alanine Arginine A s p a r t i c acid + a s p a r a g i n e Cysteine * Glutamic acid + glutamine Glycine Histidine Isoleucine Leucine Lysine Methionine * Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine

55 40 73 10 59 48 16 32 48 39 12 24 32 32 35 2 22 41 Total:

620

* D e t e r m i n e d . r e s p e c t i v e l y , as c y s t e i c acid a n d m e t h i o n i n e s u l f o n e .

enzyme molecule contains 40 arg~nines, 39 lysines, two tryptophans and ten cysteines, and taking into account of the very high sensitivities of the staining methods, these results leave room to a limited but significant amount of duplicated sequences. Titration o f the sulfhydryl groups o f the glutaminyl-tRNA synthetase with dithiobisnitrobenzoic acid and p-hydroxymercuribenzoate and their influence on the enzymatic activities. A strong decrease of the aminoacylation and the [a2P]PPi-ATP isotope-exchange activities was observed after several months storage of enzyme solution at --20°C in the presence of 10 mM potassium phosphate, pH 7.5, 50% glycerol and 5 mM 2-mercaptoethanol. However, both activities could be completely recovered after exhaustive dialysis of the enzyme against a buffer containing 20 mM 2-mercaptoethanol. Titration of the suifhydryl groups was performed in the absence and in the presence of denaturing agent. In the absence of denaturing agent, the reaction of dithiobisnitrobenzoic acid with the native enzyme (followed by measuring the change of absorbance at 412 nm) is complete after 30 s, when 3.8 sulfhydryl groups per enzyme molecule have reacted with dithiobisnitrobenzoic acid. In the presence of 8 M urea, the reaction of the glutaminyl-tRNA synthetase with dithiobisnitrobenzoic acid is over after 2 min, when 5.7 sulfhydryl groups per enzyme have reacted. In the presence of 6 M guanidin~HC1, 6.5 sulfhydryl groups per enzyme react with dithiobisnitrobenzoic acid (for further experimental details see Materials and Methods). Dithiobisnitrobenzoic acid was found to inhibit the aminoacylation and the [32P]PPi-ATP isotope-exchange reactions catalyzed by the glutaminyl-tRNA synthetase. Indeed a 10-fold excess of this reagent causes the loss of 100% of

76

lo4r-c, A

5C

\

>

100

o

B .._.

50

j•a L

•

II

1 2;o MINUTES Fig. 4. E f f e c t o f p - h y d r o x y m e r c u r i b e n z o a t e o n t h e a m i n o a c y l a t i o n ( A ) a n d o n t h e [ 32 p ] p p i . A T P i s o t o p e exchange (B) reactions catalyzed by the glutamlnyl-tRNA synthetase. (A) The enzyme (1.2/~M) was incub a t e d i n t h e a b s e n c e ( o ) or in t h e p r e s e n c e o f 1 . 2 5 # M ( e ) , 2 . 5 p M ( g ) o r 6 ~ M (4) p - h y d r o x y m e r c u r i b e n z o a t e . A f t e r various i n c u b a t i o n t i m e s , 1 0 - # l s a m p l e s w e r e m i x e d w i t h 1 9 0 #1 0 . 0 1 M Tris-HC1, p H 7 . 5 , a t 0 ° C . T h e residual a m i n o a e y l a t i o n a c t i v i t y w a s m e a s u r e d b y i n c u b a t i n g 10/~1 o f this d i l u t i o n w i t h 9 0 ~l o f t h e s t a n d a r d a m t n o a e y l a t i o n assay m i x t u r e a t 3 7 ° C d u r i n g 3 r a i n , a n d d e t e r m i n i n g t h e a m o u n t o f [ 1 4 C ] G I n - t R N A G l n in 50-~tl s a m p l e s . We verified t h a t u n d e r t h e s e c o n d i t i o n s , the initial v e l o c i t y o f the a m i n o a c y l a t i o n r e a c t i o n w a s m e a s u r e d . (B) T h e c o n d i t i o n s o f r e a c t i o n w i t h p - h y d r o x y m e r c u r i b e n z o a t e a n d d i l u t i o n w e r e i d e n t i c a l w i t h t h o s e d e s c r i b e d a b o v e . T h e rate o f [ 3 2 p ] P P i - A T P i s o t o p e - e x c h a n g e react i o n w a s m e a s u r e d a t p H 6 . 2 as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s .

both activities within 2 min (not shown). p-Hydroxymercuribenzoate strongly inhibits the catalytic activities of this enzyme (Fig. 4). In the presence of a stoichiometric concentration of this reagent 35% of the aminoacylation (Fig. 4A) and 25% of the [32P]PPi-ATP exchange activities (Fig. 4B) are lost after 2 min. A 5-fold excess o f p - h y d r o x y mercuribenzoate causes the loss of 100% of both activities within 2 min. Discussion

New purification procedure of the glutaminyl-tRNA synthetase The approach described here for the purification of this enzyme has three major differences with that described by Folk [6]. First, we used as second step either partition in a poly(ethyleneglycol)-dextran two-phases system or a 1 0 0 0 0 0 X g centrifugation instead of streptomycin sulfate, followed by an (NH4)2SO4 precipitations. This last step is well known to be denaturing for enzymes. Secondly, we used a different linear salt and pH gradient to elute the enzyme from the DEAE-cellulose column. Finally, we replaced the

77 hydroxyapatite chromatography and the Sephadex G-200 gel filtration by a phosphocellulose chromatographic step. The glutaminyl-tRNA synthetase is eluted from this column with a purity of about 95% and its activity enriched about 20-fold as compared to the preceding step. This chromatographic step was also found to be very selective for glutamyl-, threonyl-, tryptophanyl-, lysyl- and histidyl-tRNA synthetases (Kern, D. and Lapointe, J., unpublished results). The enzyme obtained after these three purification steps exhibits a specific activity about 500 times higher than that in the crude extract. Structural features o f the enzyme Glutaminyl-tRNA synthetase consists of a single polypeptide chain of 69000 daltons. In the absence of denaturing agent, it migrates on a gel or sediments on a sucrose gradient as a globular protein of Mr 65 000--70 000 indicating a monomeric structure. These molecular weight determinations agree with those reported by Folk [6]. There are, however, significant discrepancies between the amino acid composition published by this author and our determination. The results listed in Table II give a total of 620 residues instead of 530 reported by this author. However, it must be pointed out that calculations made with Folk's data gave a value of 60 000 which thus falls 9000 short of the molecular weight measured in both laboratories. This monomeric enzyme may contain some repeated sequences as seen above: indeed there are discrepancies between the actual numbers of various tryptic peptides and the theoretical ones as expected from the amino acid composition. About 25 tryptic peptides are missing out of the expected 80. Besides only four major radioactive spots could be visualized after autoradiography of the map and the seven minor ones could very well correspond to overlapping peptides arising from incomplete cleavage of some bonds. A very similar situation was found in the a subunit of yeast phenylalanyl-tRNA synthetase (Robbe-Saul, S., Potier, S. a n d Boulanger, Y., unpublished results) which contains a significant amount of sequence duplication [ 15 ]. Therefore such repeat, look very likely in E. coli glutaminyl-tRNA synthetase in the light of these preliminary results. But of course further structural studies are necessary in order to fully establish their existence. Involvement o f SH groups in the enzymatic reaction Our results indicate that at least one SH group is involved in the overall catalytic activity of the enzyme: dithiobisnitrobenzoic acid and p-hydroxymercuribenzoate inhibit both the [32P]PPi-ATP isotope exchange and aminoacylation reactions. Fig. 4 shows that both activities are equally inhibited by p-hydroxymercuribenzoate. In this respect glutaminyl-tRNA synthetase resembles glutamyl-tRNA synthetase [7] which belongs to the same group of synthetases as defined from a mechanistic view point (see Introduction), and differs from other synthetases such as yeast phenylalanyl-tRNA synthetase for which cysteines were shown to be essential for tRNA aminoacylation but not for amino acid activation [29,30]. In spite of their requirement of the cognate tRNA for the [32P]PPi-ATP

isotope exchange, arginyl-, glutamyl- and glutaminyl-tRNA synthetases can

78 function with a two-step catalytic aminoacylation mechanism as supported by various results (see discussion further on). In this two-step mechanism sulfhydryl reagents equally inhibit both the isotope-exchange and the aminoacylation reactions, whereas in systems where tRNA is not required for the amino acid activation step sulfhydryl reagents inhibit the aminoacylation step to a much higher extent than the isotope-exchange reaction, thus suggesting an essential role of some thiol groups in the productive interaction of the tRNA with the enzyme.

The catalytic properties o f the glutaminyl-tRNA synthetase The pH affects the aminoacylation and the [32P]PPi-ATP isotope-exchange reactions in an opposite manner. The increase in the stimulation of the aminoacylation rate from acidic to alkaline pH seems to be a general characteristic of the synthetases [1--4]. Concerning the [32P]PPt-ATP isotope exchange various pH effects have been reported [1--4]. The glutaminyl-tRNA synthetase exhibits a much faster isotope-exchange rate at acidic than at alkaline pH. A t pH 6.2 the rate of [14C]Gln-tRNAGI" formation is the same during the first catalytic cycle than at the steady-state (Fig. 3A). As a consequence, no end-product dissociation is rate-limiting of the overall aminoacylation reaction. Thus the steady-state rate of aminoacylation is a measure of that of the catalytic step of Gin-tRNA ~ln synthesis. Similar results were obtained for the E. coli glutamyl-tRNA synthetase [31]. However, it was shown that the dissociation of the aminoacylated tRNA constitutes the rate-limiting step of the E. coli isoleucylation [32] and the yeast valylation [33] and arginylation [34] systems. At pH 6.2 the [32P]PPi-ATP exchange occurs about 40 times faster than the synthesis of Gln-tRNA ~ln either at 37°C (Fig. 2A) or at 0°C (see Results). As Gln-tRNA GIn cannot be consumed in the [32P]PPi-ATP isotope exchange faster than it is synthesized, it cannot be an obligatory intermediate in the isotope exchange. The analysis of the kinetics of the AMP and PPi-dependent deacylation reaction leads to the same conclusion. Indeed, the rate-limiting step of this reaction is the catalysis of the deacylation of Gln-tRNA Gin (Fig. 3B) which occurs at a significantly slower rate than that of incorporation of the [32P]PP i into ATP (Fig. 2A). Thus the deacylation step does not obligatory take part in the isotope exchange. These results strongly suggest that glutaminyl-tRNA synthetase catalyses at least at acidic pH the aminoacylation of tRNA GI~ via a two-step mechanism: first, activation of the glutamine resulting in the synthesis of the enzyme adenylate intermediate, followed secondly by the transfer of the activated glutamine to tRNAGI": E + tRNA

Gin + A T P

+ Gin.

MgCI 2 " E • AMP

~ G i n + PPi + t R N A G I n

E • AMP ~ Gin + tRNA ~ln -* E + AMP + Gln-tRNA ~ln

(1)

(2)

In this pathway the [32P]PPi-ATP exchange occurs at the equilibrium of the amino acid activation step, and does not involve the transfer step and its reversal. As a consequence the requirement of tRNA GI~ for the catalysis of the

79 isotope exchange indicates that the enzyme must be activated by the t R N A GIn in order to catalyze the activation of glutamine. Arguments favoring a two-step aminoacylation mechanism have been reported for the E. coli glutamyl- [31] and arginyl-tRNA synthetases [35] and for the yeast and Neurospora crassa arginyl-tRNA synthetases [34,36 ]. The opposite kinetic behaviors of the isotope-exchange and the aminoacylation reactions when p H is varied, probably result from the competition between t R N A GIn and PPi for the enzyme adenylate intermediate. Increasing p H values decrease the reactivity of PP~ and increase that of t R N A 61n for this intermediate. So, at acidic pH, the adenylate intermediate reacts faster with PPi than with t R N A °I", whereas at alkaline p H it reacts faster with t R N A °In than with PPi. The glutarninyl-tRNA synthetase does not require a large excess of MgCI2 with respect to A T P or A T P + PPi present to catalyze the aminoacylation and the [32P]PPi-ATP isotope-exchange reactions at the optimal rates. Furthermore no sharp MgCl2 dependence of these reactions is observed. Different MgCl2 dependences were found for the various aminoacylation systems studied, some being even strongly inhibited in the presence of a slight excess of this bivalent cation with respect to A T P [12,37]. The stimulation of the aminoacylation and the [32P]PPi-ATP isotopeexchange reactions by other bivalent cations can be interpreted in two ways. First, some of these cations are able to replace magnesium efficiently in the formation of certain reactive substrates: ATP-magnesium, PPi-magnesium, tRNA-magnesium. Second, added bivalent cations can displace magnesium bound either to t R N A or to protein thus rendering M g 2+ available to magnesium-requiring substrates. The synergistic effect observed between magnesium and spermidine probably arises from this phenomenon. It was shown that in many cases spermidine could replace MgCI2 in the structuration of the t R N A [38], but cannot replace it in the combination with A T P and PPi [39]. The glutaminyl-tRNA synthetase and the group o f small monomeric aminoacyl.tRNA synthetases The glutaminyl-tRNA synthetase is one the smallest monomeric aminoacyltRNA synthetases. It is noticable that arginyl- and glutamyl-tRNA synthetases which share the exceptional catalytic property of requiring their cognate tRNA to catalyze the incorporation of [32P]PPi into ATP are monomeric enzymes too with molecular weights in the same range [5,7]. Unlike E. coli glutamyltRNA synthetase [7], E. coIi glutaminyl-tRNA synthetase seems to contain some repeated sequences. Our latest results show that this is also the case for yeast arginyl-tRNA synthetase: in particular most cysteine and tryptophancontaining peptides are repeated in this monomeric enzyme of 74 000 daltons (Potier, S. and Boulanger, Y., unpublished results). Thus the requirement of the cognate tRNA for the amino acid activation step, a common property of these three monomeric synthetases, cannot be correlated with an absence of sequence duplication. On the contrary it looks as though all aminoacyl-tRNA synthetases (monomeric or oligomeric) with subunits greater than 56000 daltons contain repeated sequences. At the present time the structural or func-

80 tional role of these repeated units has not been elucidated and obviously more facts must be brought to help us to understand their true significance.

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