Eur. J. Biochem. 54, 375- 184 (1975)

Two Enzymically Active Forms of Glycyl-tRNA Synthetase from Bacillus brevis Purification and Properties

Andrei P. SURGUCHOV and Trim G. SURGUCHOVA

Laboratory of Bio-Organic Chemistry. Moscow Stale University

(Received October. 8, 1974iJanuary 14, 1975)

Using sucrose density centrifugation and gel filtration of a 105000 x g supernatant of Bacillus hvevrs two enzymic activities of glycyl-tRNA synthetase were separated. Enzyme catalyzing the aminoacylation of tRNA (El) elutes in a high-molecular-weight region. Enzyme active in glycyl- hydroxamate formation (E,) elutes from a Sephadex gel column and sediments in sucrose density gradient in a region of relatively low molecular weight. The presence of two enzymic activities does not depend on the method of cell disruption; their proportion does not change when protease inhibitor (diisopropylphosphorofluoridate) is added to the extraction buffer.

Both El and E2 were purified to a nearly homogeneous state. Sedimentation coefficients ( s , , ~ ~ ) were found to be 8.6 S and 3.6 S and molecular weights 226000 and 66000 for E, and E,, respectively. During storage, El dissociates into two components, one of which has electrophoretic mobility identical to E,. The molecular weight of the other component is about 160000.

Electrophoresis of El in the presence of sodium dodecylsulfate reveals two bands corresponding to molecular weights of 81 000 and 30000. Under these conditions, E2 dissociates into a polypeptide with a molecular weight of 30000.

Valine was found to be the N-terminal amino acid for E2 and both valine and glutamic acid were N-terminal amino acids for El.

It is concluded that El is a tetrameric protein consisting of two large and two small subunits (a2&). E2 is a component of El with a structural formula a,.

Structural studies on aminoacyl-tRNh synthetases specific for different amino acids and isolated from various sources have shown that the majority of these enzymes may be divided into two groups [1,2]: syn- thetase consisting of one polypeptide chain and oligo- nieric enzymes consisting of identical subunits. Ex- ceptions to this rule are the enzymes with non-identical subunits : yeast and Escherichia coli phenylalanyl- tRNA synthetases [3,4] and glycyl-tRNA synthetase of E. coli [5] belong to the z2p2 type and glutamyl- tRNA synthetase of E. coli belongs to the ap type [6].

Aminoacyl-tRNA synthetases consisting of non- identical subunits are of special interest due to the bifunctional nature of these enzymes. In this paper it is demonstrated that glycyl-tRNA synthetase of Bacillus brevis consists of non-identical subunits (a2D2). Some structural and catalytic properties of glycyl- tRNA synthetase of B. brevis are described.

Etzzyme. Glycyl-tRNA synthetase (EC 6.1.1.14).

MATERIALS AND METHODS

P strain of B. brevis was grown at 40 ’C on the medium containing (gll): NaCI, 4; MgSO,, 0.16; K,HPO,, 7.9; ammonium oxalate, 6.5; glycerol, 1.6; lactic acid, 6.4; yeast extract, 0.3; casein hydrolysate, 0.3. The cells were harvested in the middle of the exponential phase and stored at -20 “C.

tRNA of B. hrevis was extracted according to Monier et al. [7] and purified on a DEAE-cellulose column as described elsewhere [8]. Endogenous ami- no acids were removed by NaHCO, treatment at pH 9.0. tRNA concentration was determined spectrophotometrically assuming that 24 ab- sorbance units correspond to a concentration of tRNA of 1 mg/ml. tRNA of E. cd i was purchased from Calbiochem. [3H]Glycine (2Ci/mmol) and [14C]glycine (114 Ci/mol) were purchased from Amersham. Tris and bovine serum albumin were products of Sigma. DEAE-cellulose (DE-52) and CM-cellulose (CM-82)

Eur. J. Biochetn. 54 (1Y75)

176 Glycyl-tRNA Synthetase of Hariilus hrrvis

were from Whatman. ATP (disodium salt), glutathione, 2-mercaptoethanol, dextran 2000, Coomassie blue R-250 and (NH,),SO, were purchased from Serva.

Sephadex G-100, G-200, DEAE-Sephadex A-50 werc from Pharmacia, hydroxylapatite from Bio Rad and acrylamide from Koch-Light. N,N,N',N'-Tetra- methylethylenediamine and N,N'-methylenebisacryl- amide were purchased from Schuchardt. Hydroxyl- amine base was prepared according to Beinert [9]. R N Ase-frcc sucrose and protamine sulfate were purchased from Calbiochem.

The extraction and purification of glycyl-tRNA synthetase was performed in 20 mM potassium phos- phate buffer (pH 7.8) containing 10 mM 2-mercapto- ethanol (buffer A). The buffer for cell disruption contained, in addition. 5 mM MgCI,.

G l ~ y 1 ~ 1 h ~ ~ d 1 - 0 uaniate Assaj!

The formation of labeled glycylhydroxamate was measured according to Parin e l al. [lo] with slight modifications. The incubation mixture contained (in a final volume of 250 pl, pH 7.5) 1 pmol ATP, 250 pmol NH,OH, 5 pmol MgCI,, 1 nmol [14C]glycine or [3H]- glycinc and rate-limiting amounts of enzyme. Incuba- tion was performed at 37 "C for 40 min. The reaction was stopped in a boiling water bath. The precipitated protein was removed by centrifugation. 50-p1 aliquots were poured on discs (22 mm diameter) of carboxy- nicthglcellulose paper (Whatman CM-30). The discs were washed five times with 1 1 of water, dried and counted in Mark-I scintillation counter in toluene scintillator.

/ 3 2 P ] P P , - 4 TP Isotope Exchange

The actibity of glycyl-tRNA synthetase in ATP-PP, isotopic exchange reaction was determined according to Lemoine (it ul. [I I ] .

1 R N A Ai12inoac~krtion

The initial rate of tRNA aminoacylation was determined using the incubation mixture containing, in a final volumc of 250 pl, 0.1 pmol ATP, 0.1 pmol glutathione, 100 pmol Tris, 5 pmol KCI, 5 pmol MgC12, 0.03 pmol [3H]glycine, 200 pg tRNA and rate- limiting amounts of enzyme. After incubation (for 10 min a t 37 C ) 3 ml o f cold 5 "/, trichloroacetic acid and, if necessary, 500 pg of bovine serum albumin were added to each sample. After 15 min in an ice bath, precipitates were collected on nitrocellulose filters (RIJFS. Czechoslovakia), washed with 5 ::.< cold trichloroacetic acid and 700! ethanol and counted as described above.

Protein Determination

Protein was determined as described by Lowry et al. [12] using bovine serum albumin as standard.

Sucrose Gradient Centrijuga tion

The general procedure of Martin and Ames [13] was followed. The centrifugation was carried out in an SW-39 rotor of the MSE-50 centrifuge using a 5 - 20 (w/v) gradient of sucrose prepared in 20 mM potassium phosphate buffer, pH 7.5 containing 1 niM EDTA and 10 mM 2-mercaptoethanol. 150 p1 of 105 000 x supernatant of E. coli or B. hrciiis, con- taining no more than 800 pg of protein was laqered on the sucrose gradient and centrifuged at 39000 rev.,' inin for 17 h. Fractions of 4 drops were collected and en7ymic activity was determined.

Analytical Cen trijiugat ion

Sw,zo were determined for purified E, and E, prep- arations in the analytical ultracentrifuge Spinco mod- el E equipped with a scanner. Experimcnts were performed at 40000 rev./min: absorbance at 280 nm was measured.

AT- Terminal Amino Acid Anuly.~is

The reaction of E, and E2 with 5-dimethylamino- 1 -naphthalenesulphonyl chloride (dansyl chloride) was carried out at pH 9.2 according to the method of Gray [14]. The products of the reaction were identified by thin-layer chromatography on silica gel plates or by paper electrophoresis in pyridineKH3COOH/I2O (2 : 4 : 944, v/v/v), pH 4.2. Prior to electrophoresis, the paper was soaked in a mixture of pyridine,' CH,COOH/H,O (50:2:448, viviv), pH 6.5. The time of electrophoresis was 2.5 h, at 14 Vkm. Identification of dansyl derivatives was carried out by their co- chromatography with the corresponding dansyl amino acids.

Amino Acid Anal-ysis

The purified enzymes E, or k 2 were dialysed against bidistilled water and hydrolysed in 3 times redistilled HC1 in evacuated tubes a t 105 'C, for 24, 48 and 72 h. Half-cystine residues were determined as cysteic acid after performic oxidation according to Schram [lj] . The hydrolysates were analysed in duplicate on a I-Iitachi amino acid automatic analyser. The values for threonine and serine were corrected for losses during hydrolysis bq extrapolation to 7cro time.

Eur. 1. Biochern. 54 (1975)

A. P. Surguchov and I . G. Surguchova 3 I7

10000c I

Fraction number

Fig. 1. Sucrose detisity centr(jiugutioiz ( 5 - 20 ?/;, iv /v J of’ 105000 x g supernutant of B. brevis and E. coli. (0) Activity of glycyl-tRNA synthetase of B. brevis in tRNA-acylation reaction; ( x ) activity of glycyl-tRNA synthetase of E. coli in tRNA-acylation reaction. The lo5000 x g supernatant of E. coli was obtaincd exactly as described for B. brevir (see Materials and Methods). For determination of

the activity of glycyl-IRNA synthetase of E. coli in tRNA acylation reaction, 200 pg of commercial preparation of tRNA (Calbiochem) was used per tube. (@j Activity of glycyl-tRNA synthetase of B. brevis in glycylhydroxamate formation; (A) bovine serum albumin (the absorbance at 280 nm was recorded). Other details in Materials and Methods

Gel Electrophoresis

Analytical polyacrylamide gel electrophoresis was carried out in Tris-EDTA-borate system (pH 8.3) according to Peacock and Dingman [16]. Electro- phoresis was performed at 4 “C and the gels were stained with 0.2% amino schwarz 10B. Gels were scanned in a densitometer “Chromoscan” equipped with filter 620. Determination of the molecular weights of native forms of glycyl-tRNA synthetase (El and E,) was done as described by Hedrick and Smith [17] using the buffer system mentioned above. The con- centration of acrylamide was 5, 6, 7 and 8 %, the con- centration of bisacrylamide accounting for 1/30 of the acrylamide concentration.

10- 50 pg of glycyl-tRNA synthetase (El or E,) or marker proteins were subjected to electrophoresis. Ovalbumin (Merck), bovine serum albumin (Koch- Light), yeast alcohol dehydrogenase (Calbiochem) and catalase from bovine liver (Calbiochem) were used as marker proteins.

Molecular Weight Determination in Dodecylsulphate Gels

The samples of enzymes (E, and E,) were denatured for 3 h at 37 “C in 0.1 M sodium phosphate buffer (pH 7.0) containing 1 sodium dodecylsulphate. Bovine serum albumin, ovalbumin, alcohol dehydro- genase, trypsin and cytochrome c were used as marker proteins. Electrophoresis was carried out as described [18] for 6- 7 h at 7 mA/tube. The gels were stained with 0.25 ‘x Coomassie brilliant blue and destained in 7 y/o acetic acid containing 5 methanol.

Determination of Molecular Weights of El and E, by Gel Filtration

Gel filtration was performed at 4 “C as described by Andrews [19] using a column of Sephadex G-200 (2 x 85 cm) equilibrated and developed in buffer A. 5 mg of crude enzyme preparation or marker protein were applied to the top of the column. Glycyl-tRNA synthetase of E. coli (crude preparation), bovine serum albumin, trypsin, ovalbumin and cytochrome c were used as standards. Fractions of 1 ml were collected at the rate of 5 ml/h.

RESULTS

Two Forms of Glycyl-tRNA Synthetase in Crude Extracts of’B. brevis

After ultracentrifugation of a crude extract in a sucrose density gradient, two enzymic activities of glycyl-tRNA synthetase were detected (Fig. 1). The first enzyme catalyses the aminoacylation of tRNA by glycine (El) and its sedimentation behaviour corre- sponds to that of glycyl-tRNA synthetase of E. coli which has a molecular weight 227 000 [20]. The second enzyme (E,) was revealed by the glycine hydroxamate formation test in the same fraction as bovine serum albumin (molecular weight 67 000). Similar values of molecular weights for El and E2 were found using gel filtration of the 105000 x g supernatant on a Cali- brated column of Sephadex G-200: 200000 for El and 70000 for E, (Fig. 2 and Fig. 3).

Both forms (E, and E,) were found irrespective of the methods of bacterial cell disruption (sonication,

Eur. J. Biochem. 54 (1975)

178 Glycyl-tRNA Synthetase of H t r c , i l h hrcv i s

0

Fractton number

Fig. 2. Gci fi/iiirriori of iO.jii00 x g ,sirpoitotarit of' B. brcvis r~irnugh S ~ p / 7 c c t / c . \ G-.?//O [ ~ ~ / i m u . ( ~ -) Absorbance at 280 nm; (0-4) activity ni' sly:) I-tR N A synthetase in tRNA-acylation reaction; (0 Q iictil it! ol'glycyl-t RNA synthetase in glycylhydroxamate foririation : I A -A) E , , inaclive species orglycyl-tRNA syiithetase loc;il17cd by coiiiplemcntalion with E L ;is described in thc tcxt and in Tahlc I

lysozyme treatment, disruption in press). Addition of diisopropylpliospliorofluoridate (50 pM) to the extraction buffer did not affect the position of El and Ez in gel filtration and ultraccntrifugation experiments.

Blanqiiet eta/ . [31] found that potassium phosphate buffer inhibits the interaction of methionyl-tRNA synthetase with cognate tRNA. It was verified that the inability of form E, of the enzyme to amino- acylate t R K A was not due to the use of phosphate buffer. Identical results were obtained when the assay was pcrfcmned in Ihc presence or 20 mM imidazole- HC1 or t O O iiiM Tris-HC1 buffer both at pH 7.5.

Imzctivc Coniponeiit oJ'GlycyI-iRNA Synthetase

Results presented in Table 1 and Fig.2 show that in B. brc~vis extracts there is in addition to El and E,, an enzymically inactive component (Ei). Addition of this component (fraction 33. Fig. 2) to E,, (Traction 42, Fig.2) gives rise to the appearance of tRNA- acylating activity as shown in Table 1 ; neither E i nor E2 separately catalyse acylation of tRNA. E i elutes from thc Sephadex G-200 column in the fraction corresponding to a niolecular weight of about 160000 (Fig. 3). The appearance of tRNA-acylating activity is probably due to complementation of E, and E i forming an active complcx.

1 0 4 ~ 0 2 (2.4 Relative rnob l l i t y

Fig. 3. &fr~mimrion of m ~ l r ~ u i a r 11 r,i?hr of' U, . f.': toid E , h j gel f i lrrrrrion 0i7 Se.phades G-ZOO. 105000 x g wprrriatant of' 5. h r c ~ i s or E. coli containing 5 mp of protein was applied to the column. (1) Activity of glycyl-tRNA synthetase of 5. / i w i , i . s i n tRNA-acyla- lion reaction. (2) Activity o f glycyl-tRKA synthctase o f II. c d i in tKNA-acylation reaction. (4) Activity of $ycyl-tKNA synthetase of B. hi.evi.7 in glycylhydroxamate formation. (3. 5. 6) Marker proteins (the absorbance at 280 nm was recorded): ( 3 ) dehydrogenase. (5) bovine scrum albumin. (6) ovalbumin. 105000 x g supernatant of E. coli W ; I S obtained cxactl) a s described for B . b~ovis

Activity of' E, it7 C ; l ~ ~ ~ ~ l l ~ ~ ~ ~ l r r o s a ~ ~ 7 u t ~ ~ Fnrmrition

As shown in Fig. 1, El does not catalyse the reac- tion of glycylhydroxamate formation although it is active in the tRNA charging reaction. EIowever, glycylhydroxamate formation appears after addition of tRNA to the incubation mixture (Fig.4). In this respect, E, is similar to "tRNA-depcndent" form of glycyl-tRNA synthetase froin rat liver [22] . Both E, and E, catalyse the reaction of glycine-dependent ATP- PPi exchange detcrrnined as described in Materials and Methods (not shown). Thus the inability of El to catalyze glycylhydroxamate formation may be due to inaccessibility of the active site(s) of glycyl- tRNA synthetase to hydroxylamine molecules. It has been shown for rat liver glycyl-tRNA synthetase that glycylhydroxamate formation in the presence of tRNA proceeds via glycyl-tRNA formation and its sub- sequent hydroxylaminolysis [23] .

Both El and E, were purified from extracts of B. hrrvis to a nearly homogeneous state. All operations were carried out at 0-4 "C in buffer A.

Purification of El

Table 2. The steps of purification are summariLed in

Eur. J. Biochem. 54 (1975)

A. P. Surguchov and 1. G. Surguchova 179

Table 1. Discovevj. of enzymaticniiy inactive f rom (Ei) of g/j.cy/- f R N A Jyrztiwfase of B. brevis Enzyme was obtained from aliquots of 100 pl withdrawn from each fraction obrained after gel filtration on Sephadex G-200 (Fig. 2). Activity in tRNA aininoacylation was determined as described in Materials and Methods

Source of cnzynic Formation of glycyl-tRNA

Fraction 42 (k2)

Fraction 33 (E i )

Fraction 42 + fraction 31

Fraction 42 + fraction 33

Fraction 42 + fraction 35

Fraction 42 + fraction 33"

Fraction 33 + fraction 42"

Fraction 42 + albumin"

Fraction 33 + alhumin''

pmol

0

0.02

1 .0

1.6

0.6

0

0.02

0

0.02

a Added after incubation. 100 pg bovine serum albumin

Crude Extract. 600- 800 g cells (wet weight) were suspended in 2-fold volume of buffer A, containing 5 mM MgC12. The cells were disrupted in an ultrasonic desintegrator (MSE) at 20 kHz for 3.5 min in portions of 50ml. The suspension was centrifugated at 23 000 x g for 40 min and the supernatant fluid at 105000xg for 2 h.

Protarnine Sulphate Treatment. To the supernatant fluid after high-speed centrifugation a 5 solution of protamine sulphate was added dropwise under constant stirring to give 0.5 % final concentration. The suspension was stirred for 30min and then centrifuged at 23000 x g for 20 min.

Ammonium Sulphate Fractionation. To the prot- amine sulphate supernatant, solid ammonium sulphate was added slowly under stirring to give 309.1, satura- tion. The mixture was allowed to stand for 30 min, with stirring, and then centrifuged for 40 min at 23 000 x g. The supernatant fluid was brought to 50 :A saturation by the addition of solid ammonium sul- phate, the suspension was stirred for 1 h, and centri- fuged at 23000 x g for 1 h. The precipitate was taken up in buffer A and adjusted to pH 5.0 by the dropwise addition of cold 1 N acetic acid. The suspension was allowed to stand for 20 min, with stirring, and then centrifuged at 23 000 x g for 1 h. The precipitate was dissolved in buffer A and dialysed overnight against a 100-fold excess of the same buffer. The precipitate which appeared during dialysis was discarded after centrifugation at 23000 x g for 15 min.

"0 50 100 tRNA (Fg)

Fig. 4. Acrivilj of E, in gl.i?~~~ll~,i?dro,urrn2utr fomzntinn iir theyvr.wncr of iRNA. 350 pg of partially purified preparation of E, obtaincd after gel filtration on Sephadex G-200 column (see Fig.2) were placed in each test tube for determination of glycylhydroxaniatc activity. (0) Crude tRNA of B. hwvis was addcd; (0) crude tRNA of E. coli was added

DEAE- Cellulo.se Chromatography. The supernatant was appliedon a column of DEAE-cellulose ( 3 x 21 cm), equilibrated with buffer A. The column was washed with buffer A until disappearance of absorbance at 280 nm. The column was then eluted with a linear gradient from 0.02 M to 0.4 M phosphate buffer pH 7.8 containing 0.01 M 2-mercaptoethanol, the total volume of the eluant being 2400 ml. The flow rate was adjusted to 100 ml:h and fractions of 50 ml were collected and analysed for enzymic activity. The pool of active fractions was precipitated by addition of solid ammonium sulphate to 707, satu- ration.

Gel-Filtrat ion through Sephadex (3-200. The pre- cipitate was collected by centrifugation 40000 x g for 40 min, dissolved in 8 ml buffer A, and applied on a column of Sephadex G-200 (5 x 80 cm) equilibrated with buffer A. The flow rate was adjusted to 20 inljh and fractions of 8 nil were collected.

Hydroxyapatite Fractionation. The pool of active fractions (about 40 ml) was applied directly on a column of hydroxyapatite (1.5 x 15 cm) equilibrated with buffer A. The column was washed with the same buffer and eluted with a linear gradient from 0.02 M to 0.4 M phosphate buffer pH 7.8 with 10 mM 2-mer- captoethanol, the total volume of the eluant being 800 ml. The flow rate was 25 ml/h and fractions of 6 ml were collected. Active fractions were pooled and concentrated to 10 mg/ml in an Amicon ultrafiltra- tion cell.

Purfication of E2

The steps of purification are summarized in Table 3.

The first steps of the purification procedure of E2 (protamine sulphate treatment and ammonium sul- phate fractionation) were carried out exactly as de-

Eur. J. Biochem. 54 (1975)

1x0 Glycyl-tKKA Synthetasc of Badlus b r e w

able 2. Yurrf imtion n.f glycyl-tR!VA s.vntheruse ,lrom B. brevis (iRNA-ucylufing enzyme El)

Fraction Volume Protein A,,O:A260 Specific activity Purification

105000 x supcrnatant Protamrne sulphate Ammoiii urn s u Ipha tc pH 5.0 DEAF-cellulose (DE-52) Sepliadex G-200 Hydroxyapatite

ml

1010 1100 400 120 500 60 32

mk? 40 500 39000 18 450

1620 293 28 14

units 'mg

0.53 0.0073

0.88 0.030 1.34 1.56 0.646 1.74 1.28 1.76 2.93

- -

-

-fold

1

4.2 -

-

89 177 402

Table 3. Pirrific aiiori ( ~ f g l y c j ~ l - r R N A synthetu.ue ,fr.om B. brevis (Xlwine-uctivating oiz jme El)

Fraction Volume Protein Al*OIA,," Specific activitj Purification

ml mg units,mg -fold

105000 xi: supernatant 1000 43 200 0.53 0.0025 1 Protamine sull7hate 975 41 700 - - Ammonium sulphate 350 19700 0.88 0.0052 2.1

DCAE-Srphadex A-50 (11) 160 16 1.70 0.855 342

-

DEAC-cellulose (DE-52) 1400 1180 1.40 0.0775 31 DEAE-Sephadex A-50 (I) 800 76 1.58 0.450 180

Sephadex Cr-100 20 5.2 1.78 1.810 720

scribed for E, (see above). The precipitate after addi- tion of 50 ",, ammonium sulphate was dissolved in buffer A, dialysed overnight against the same buffer and subjected to DEAE-cellulose chromatography.

11 EA E- Ce Nulose Chroma tograpliy . The dialysed enzyme was loaded onto a column of DEAE-cellulose (6 x 21 cm) equilibrated with buffer A. The column was washed with the same buffer until the disap- pearance of absorbance at 280nm in the effluent, and then eluted with a linear gradient from 0.02 M to 0.25 M phosphate buffer pH 7.8 containing 0.01 M 2-mercaptoethanol. The total volume of the eluant was 12 1. The flow rate was adjusted to 200 ml/h and fractions of 50 ml were collected. The active fractions were combined and dialysed overnight against buffer A.

DEAE-Sephadex Chromatography (1). The dia- lysed solution was loaded onto a column of DEAE- Sephadex A-50 ( 5 x 16 cm) equilibrated with buffer A. The column was washed with the same buffer until the disappearance of absorbance at 280 nm and the enzyme was eluted with a linear gradient of phosphate bufl'er 0.06 M--0.2 M, pH 7.8, the total volume of the eluant being 3600 ml. The flow rate was 90 ml/h and fractions of 35 ml were collected. The fractions containing entyme activity were dialysed overnight against buffer A.

DEA E-Seplzadex Chromatography ( 2 ) . The dialysed enzyme was applied on a second column of DEAE- SephadexA-50 (2.2 x 10 m) equilibrated with bufferA.

The column was washed with buffer A and eluted with a linear gradient formed from 800 ml of 0.1 M potassium phosphate buffer (pH 7.8) and 800 ml of 0.25 M potassium phosphate (pH 5.8). The flow rate was 60 mlih and fractions of 25 ml were collected. To the combined active fractions, solid ammonium sulphate was added slowly under stirring to give 70 :4 saturation. After centrifugation at 23000 x g for 40 min, the precipitate was taken up in 6 ml buffer A.

Gel-Filtrution through Seplzades G-100. The solu- tion was applied on a column of Sephadex G-100 (4x 95 cm> at a flow rate of 20 ml/h. Fractions of 6 ml were collected. Both equilibration and elution of the column were performed with buffer A. Active fractions were pooled and concentrated in an Aniicon ultrafiltration cell.

Properties of the Purified Enzyines El and E2

Analytical gel electrophoresis of the purified El revealed a single band (Fig.5). Electrophoresis of E2 showed one main band comprising more than 90'j;, of the total protein which, after elution of the protein from the gel, contained all of the enzymic activity.

The ultraviolet absorption spectrum of both E, and E2 showed a maximum at 279 nm. The ratio of the absorbance at 280 and 260 nni was 1.78- 1.80.

Only one N-terminal amino acid, valine, was found in the E2 preparation whereas El contained

Eur. J. Biochem. 54 ( I 975)

A. P. Surguchov and J. G. Surguchova 181

II

a b C

Fig. 5. Electrophoresis ofglycyI-~RNA synthetase oj'B. brevis in paly- ucry/untide ,ye/, (a) 10 pg of El in 5 , 6, 7 and 8 "4 gels (from left lo right). (b) 10 pg of El in 5% gel. (c) 30 pg of El in 77:) gcl

both valine and glutamic acid in about equimolar ratio. The discovery of two N-terminal amino acids in E, is in good agreement with the observation that Ei consists of polypeptides of two different types (see below).

Amino acid composition of El and E, is shown in Table 4. For comparison, the amino acid composi- tion of glycyl-tRNA synthetase from E. coli is pre- sented [20]. As shown in the Table 4; El contains more methione, isoleucine and cysteine and less alanine and leucine than glycyl-tRNA synthetase from E. coli. In general, the amino acid composition is rather similar with the exception of cysteine which is 3 times more abundant in El. Titration of El with p-chlormercuri- benzoate [24] reveals the same amount of cysteine residues as was determined after acid hydrolysis of performic-acid-treated protein. It is interesting to note that tyrosyl-tRNA synthetase from E. coli and from B. subtilis have a similar amino acid composition but vary significantly in cysteine content [25].

Analytical ultracentrifugation of El reveals one component having s,,,~ = 8.6 S (in some prepara- tions a heavier component with s , , ~ ~ = 14- 15 S was detected; this component is probably the dimer of El).

Table 4. Amino ucid composition of glycq'l t R N A .s~jxthetasr of' B. brevis and E. coli Calculations are based on molecular weights of 226000. 66000 and 227000 rcspectivcly for E,. E, and glycyl-tRNA synthetase of t'. coli [ZO]. n. d. = not determined

Amino acid Number of residues per molecule

El E2 Glycyl-tRNA synthetase of E. coli

Alanine Arginine Aspartic acid Half-cystine Glutamic acid Glycine Hialidinc lsolcucinc Leucine Lysine Methionine P henylalaninc Prolinc Serine Threonine Tyrosine Valinc

167 144 193 25

214 177 42

117 152 152 74 93 64

120 91 55

165

60 26 49 n. d 76 45

5 40 44 50 4

21 17 34 33 18 22

220 123 220

8 259 138 31 79

222 112 36 92

108 75

105 57

134

E, gives rise to one protein component with s , , , ~ = 3.6 S.

Storage of E, at 2-4 "C results in the appearance of two protein bands with higher mobility in poly- acrylamide gel (Fig. 6). Molecular weights of these components estimated by the method of Hedric and Smith [17] correspond to 160000 and 66000, respec- tively (Fig.7). By the same technique the molecular weight of El has been found to be 228000 and molecular weight of E,, 66000. The molar ratio of the dissociation products according to the scanning of the gels is about 1 : 1, assuming that the amount of dye is proportional to the amount of protein in the band. Electrophoretic mobility of the light com- ponent formed from E, during storage coincides exactly with the electrophoretic mobility of E, at all gel concentrations.

When El is treated with 1 % sodium dodecyl- sulphate and 1 2-mercaptoethanol and run in the presence of 0.1 % sodium dodecylsulphate, two pro- tein bands are revealed. The molecular weights of these bands were determined from their relative mobilities as compared to several marker proteins and were found to be 81 000 and 30000 (Fig.8). The two bands were found to be in about equimolar ratio. E2, in these conditions, gives rise to one band with molecular weight about 30000. Assuming the molec- ular weight of native E, to be 226000 the subunit

Eur. .I. Biochem. 54 (1975)

1x2 Glycyl-tKNA Synthetasc of Hmiilirs hi.e~i.7

Vobi I i ty'

Fig. 6 S w t i ! ? f i i z o fp i , /w iwj krff i i ikgc'/s . (A) Fresh prcparation of E , ; (13) dissociatiuii of F., during storage for 10 days in buffer A. Con- ccntmtioii oi'protein = 300 pg:ml

structure of z2/j2 is highly probable. E, is apparently a component of Ll with a structural formula CI: and I, consists o f two large subunits (p2). All the data are summarized in Table 5 .

Rela t ive mcb i . , t

Fig. 8. E/Wlrop/?or.e,sis of El i i n d E2 it1 p / ) m r i ~ i a n ~ i d i ~ X L ' I ill lhr

pre.wtire of dodecjisuiphate. The coIicentriitm!i of the gel was 7",,. (1 ) Cytochrorne c : (2) Lrypsin: ( 3 ) tz and E l ; (4) alcohol dehydro- genase; ( 5 ) ovalbumin: (6) hovinc serum albumin: (7) E , . El gives rise to two bands (points 7 and 3). whereab II. to band 3 on1y

DISCUSSION

Ostrom and Berg [20] using both an ATP-PPi isotope exchange method and the tRNA acylation reaction described onc form of glycyl-tRNA syn- thetase in E. coli: a tetramer with a molccular weight of 228000 and structural formula x2J2. From the inaterial presented here it is evident that glycyl-tRNA synthetase El from B. bwis closely resembles the enzyme from E. cali. The discovery that B. bret1i.q besides the tRNA-acylating form (El ) , contains two other forms of glycyl-tRNA synthetase. one of which (Ei) is enzymatically inactive and thc other (E2) catalyses the reactions of ATP- PPi isotope exchange and glycylhydroxamate formation but not the tRNA- charging reaction, is of special interest.

Special controls demonstrate that appearance of E2 and E i is probably not a consequence of the pro- cedure of cell disruption. The addilion of the protease inhibitor (diisopropylphosphorofluoridate) in the ex- traction buffer changes neither the ratio of El to E2 nor their position in gel filtration and sucrose density centrifugation.

The possibility should not be excluded at thc mo- ment that both E i and E2 do exist in viw in B. brrvis cells and the conversion El E i + E, plays some regulatory role.

From the catalytic properties of the three forms of glycyl-tRNA synthetase, one can suggest that sinall-molecular-weight subunits (E2) bind low-molec- ular-weight substrates (glycine and ATP) and catalyse

Eur .I Biochetn 54 11975)

A. P. Surguchov and I . G. Surguchova 183

Table 5. Striiciure and propt~riit~s ofg(rql- tRNA sjmlhetuse of B. brevis n. d . = not determined

Property Form E, Form EL Form F,,

Sedimentation coefficient Molecular weight Aminoacylation of tRNA Formation of glycylhydroxamate - in the absence of tRNA - in the presence of tRNA

ATP =g PP, isotope exchange Subunit structure Molecular weight of aubunits

8.6 S 3.6 S n . d. 226 000 66000 160 000

+ -

+ + + n. d.

the activation of glycine; El alone or together with E2 interacts with tRNAGly. The complete oligomer catalyses the physiologically significant reaction, aminoacylation of tRNAGlS.

Parfait [26] demonstrated that the binding of tRNAArN to the monomeric form of synthetase provokes the dimerisation The dimeric form of arginyl-tRNA formed is active in the aminoacylation other substrates, ATP and arginine,

argin yl-tRN A of this form. synthetase so reaction. Two also influence -

the monomer to dimer transition. One may suppose that the binding of tRNAClY to the p2 and/or ATP and glycine to the a2 components of glycyl-tRNA synthetase can push the equilibrium towards the formation of El. In this case, the activity of glycyl- tRNA synthetase can be regulated by substrate con- centrations.

N-terminal analysis of glycyl-tRNA synthetase of B. brevis revealed that valine is the N-terminal amino acid of the a chain and glutamic acid that of the f l chain. The amino acid composition of glycyl- tRNA synthetase from E. coli and B. brevis is rather similar; only the content of cysteine significantly differs for these enzymes, being three times higher for glycyl-tRNA synthetase of B. hrevis.

Besides resemblance with E. coli glycyl-tRNA synthetase [20], the B. brevis enzyme has some common features with rat liver glycyl-tRNA synthetase 1221 ; the presence of two enzymically active forms in crude extracts, the dependence of one of the enzymic activities (glycylhydroxamate formation) on the pres- ence of tRNA, the existence of the second, “tRNA- independent” enzyme form. This similarity allows us to predict that rat liver glycyl-tRNA synthetase has the same type of quaternary structure, a2&, and that the existence of the two enzyme forms described [21] are connected with the oligomeric state of enzyme.

In the light of our data it is of interest to try to dissociate the E. coli glycyl-tRNA synthetase with

consequent dissociation of the functional activities. We have described here three molecular species of glycyl-tRNA synthetase of B. brevis but only one of them (El) catalyses the physiologically significant reaction, aminoacylation of tRNA. This observation is in accord with the absence of multiplicity of amino- acyl-tRNA syiithetases in procaryotic cells.

We thank D r L. Kisselev for his interest in the course of this work and many helpful discussions and D r J.-P. Waller for critical reading of manuscript.

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A . S~irguchoi. Lahoratoriya Metaboliima Miocarda, Institut Cardiologii, Petroverigsky pereuloh 10. Moskva. U S.S.R. 301837

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Fur .I. Hiochem. 54 (1075)