463 Biochem. J. (1973) 135, 463-468 Printed in Great Britain Molecular Weight and Amino Acid Composition of the Exoceliular DD-Carboxypeptidase-Transpeptidase of Streptomyces R61 By JEAN-MARIE FRtRE and JEAN-MARIE GHUYSEN* Service de Microbiologie, Faculte de Medecine, Institut de Botanique, Universite de Liege, Sart-Tilman, 4000 Liege, Belgium and HAROLD R. PERKINS and MANUEL NIETO National Institute for Medical Research, Mill Hill, London NW7 1AA, U.K. (Received 28 March 1973) A procedure allowing the purification of milligram amounts of the exocellular DD-carboxypeptidase-transpeptidase from Streptomyces R61 to protein homogeneity (95% purity) is described. The isolated protein has a molecular weight of about 38000 and consists of one polypeptide chain. Its amino acid composition is presented. Previous papers have described the isolation, substrate specificity and other enzymic properties of the exocellular DD-carboxypeptidase-transpep- tidase produced by Streptomyces strain R61 (Leyh- Bouille et al., 1971; Pollock et al., 1972; Nieto et al., 1973a; Perkins et al., 1973). This enzyme is believed to be a soluble form of the membrane-bound transpeptidase which catalyses the peptide cross- linking of the nascent cell-wall peptidoglycan (Dusart et al., 1973). In the course of these studies, only small amounts of the enzyme were isolated. The present paper describes the purification of milligram amounts of enzyme, and some physico- chemical properties of the isolated protein. Materials and Methods Enzyme activity One unit of enzyme catalyses the hydrolysis of 1 equiv. of n-alanyl-D-alanine linkage/min at 37°C when the enzyme is exposed to Ac2f-L-Lys-D-Ala-D- Ala at concentrations of 10 times the K. value (12mM) in 5mM-sodium phosphate buffer, pH 7.5 (Leyh-Bouille et al., 1971). In previous papers, one unit referred to the conversion of lnmol of substrate/h. Physicochemical methods Diffusion coefficient (D20,W). The enzyme solution (3mg of protein/ml) was dialysed against 0.01M- Tris-HCI buffer, pH8.0, containing 0.09M-NaCl * To whom correspondence should be sent. t Abbreviations: Ac, acetyl; A2pm, diaminopimelic acid. Vol. 135 and analysed at 20°C in a Spinco model E ultracentri- fuge, equipped with a 'Rotor internal temperature control unit' and a schlieren optical system. Experi- ments were carried out in the epon-aluminium double-sector capillary-type synthetic-boundary cell at a speed of 13410 rev./min. The rate of diffusion was measured by plotting A2/(H2F2) versus time (A, area; H, maximum height of the peak; F, total enlargement used, i.e. 15). Equilibrium sedimentation and molecular-weight de- termination. Theequilibriumsedimentations were per- formed in 0.01M-Tris - HCl buffer, pH8.0, containing 0.09M-NaCl at 12590rev./min for 22h at an initial protein concentration of 3mg/ml, by using cells with a 12mm filled-epon double-sector centrepiece and sapphire windows. The solution columns were about 2mm long. Initial solute concentrations were determined by a complementary run with the double- sector capillary-type synthetic-boundary cell. The apparent molecular weight (M,,pp.) RT 1 dC (I - ( p)w2 Cx dx' at any point x of the column, where v is the partial specific volume of the protein, p is the density of the solvent and C the concentration at point x was calculated according to O'Donnell & Woods (1962). The v value was not experimentally measured and a value of 0.75cm3. g-I was used in the calculations. The molecular weight was determined by plotting I/Mapp. versus concentration at 0.1 mm intervals of the column. Polyacrylamide-gel electrophoresis. This was done at pH8.4 as previously described (Leyh-Bouille et al., 1972).
Transcript
463Biochem. J. (1973) 135, 463-468Printed in Great Britain
Molecular Weight and Amino Acid Composition of the ExoceliularDD-Carboxypeptidase-Transpeptidase of Streptomyces R61
By JEAN-MARIE FRtRE and JEAN-MARIE GHUYSEN*Service de Microbiologie, Faculte de Medecine, Institut de Botanique,
Universite de Liege, Sart-Tilman, 4000 Liege, Belgiumand
HAROLD R. PERKINS and MANUEL NIETONational Institute for Medical Research, Mill Hill,
London NW7 1AA, U.K.(Received 28 March 1973)
A procedure allowing the purification of milligram amounts of the exocellularDD-carboxypeptidase-transpeptidase from Streptomyces R61 to protein homogeneity(95% purity) is described. The isolated protein has a molecular weight of about 38000and consists of one polypeptide chain. Its amino acid composition is presented.
Previous papers have described the isolation,substrate specificity and other enzymic propertiesof the exocellular DD-carboxypeptidase-transpep-tidase produced by Streptomyces strain R61 (Leyh-Bouille et al., 1971; Pollock et al., 1972; Nieto et al.,1973a; Perkins et al., 1973). This enzyme is believedto be a soluble form of the membrane-boundtranspeptidase which catalyses the peptide cross-linking of the nascent cell-wall peptidoglycan(Dusart et al., 1973). In the course of these studies,only small amounts of the enzyme were isolated.The present paper describes the purification ofmilligram amounts of enzyme, and some physico-chemical properties of the isolated protein.
Materials and MethodsEnzyme activityOne unit of enzyme catalyses the hydrolysis of
1 equiv. of n-alanyl-D-alanine linkage/min at 37° Cwhen the enzyme is exposed to Ac2f-L-Lys-D-Ala-D-Ala at concentrations of 10 times the K. value(12mM) in 5mM-sodium phosphate buffer, pH 7.5(Leyh-Bouille et al., 1971). In previous papers, oneunit referred to the conversion of lnmol ofsubstrate/h.
Physicochemical methodsDiffusion coefficient (D20,W). The enzyme solution
(3mg of protein/ml) was dialysed against 0.01M-Tris-HCI buffer, pH8.0, containing 0.09M-NaCl
* To whom correspondence should be sent.t Abbreviations: Ac, acetyl; A2pm, diaminopimelic
acid.
Vol. 135
and analysed at 20° C in a Spinco model E ultracentri-fuge, equipped with a 'Rotor internal temperaturecontrol unit' and a schlieren optical system. Experi-ments were carried out in the epon-aluminiumdouble-sector capillary-type synthetic-boundarycell at a speed of 13410 rev./min. The rate ofdiffusionwas measured by plotting A2/(H2F2) versus time(A, area; H, maximum height of the peak; F, totalenlargement used, i.e. 15).
Equilibrium sedimentation and molecular-weight de-termination. Theequilibriumsedimentations were per-formed in 0.01M-Tris -HCl buffer, pH8.0, containing0.09M-NaCl at 12590rev./min for 22h at an initialprotein concentration of 3mg/ml, by using cellswith a 12mm filled-epon double-sector centrepieceand sapphire windows. The solution columns wereabout 2mm long. Initial solute concentrations weredetermined by a complementary run with the double-sector capillary-type synthetic-boundary cell. Theapparent molecular weight (M,,pp.)
RT 1 dC(I -( p)w2 Cx dx'
at any point x of the column, where v is the partialspecific volume of the protein, p is the density of thesolvent and C the concentration at point x wascalculated according to O'Donnell & Woods (1962).The v value was not experimentally measured and avalue of 0.75cm3. g-I was used in the calculations.The molecular weight was determined by plottingI/Mapp. versus concentration at 0.1mm intervals ofthe column.
Polyacrylamide-gel electrophoresis. This was doneat pH8.4 as previously described (Leyh-Bouilleet al., 1972).
J.-M. FRiRE, J.-M. GHUYSEN, H. R. PERKINS AND M. NIETO
Polyacrylamide-gel electrophoreses were alsocarried out at pH7 in the presence of sodiumdodecyl sulphate, according to the technique ofWeber & Osbom (1969) and after pretreatment with1% (final vol.) of mercaptoethanol. Gels (85mm x6mm) contained 10.5 % acrylamide, 0.27 % NN'-methylenebisacrylamide and 0.1 % sodium dodecylsulphate. They were pre-run overnight at 2mA/tubebefore application of the samples.
Gel electrofocusing. This was performed as des-cribed by Wrigley (1969). Gels (6cmxO.5cm) con-tained 2.5 % carrier ampholytes (pH3-6), 7 %acrylamide, 0.18% NN'-methylenebisacrylamideand 0.08 % ammonium persulphate. The pH gradientwas formed by passing the current (2mA/tube,up to a maximum of 240V) for 30min. The sample,in 10% sucrose, was layered on the top of the geland protected from direct contact with the cathodesolution by a layer of 2.5% ampholytes in 5%sucrose. Current was passed for 75min, the gelswere removed from the tubes, the ampholyteseliminated by diffusion in 2.5 % trichloroacetic acidfor several days and the precipitated proteins werestained with Coomassie Brilliant Blue G 250, 0.25 %in methanol -acetic acid -water (10:1:9, by vol.)for 2h at 0° C. Destaining was carried out at roomtemp. for 2 days by diffusion in methanol-acetic acid-water (1: 1:8, by vol.).
Performic acid oxidation. This was carried outaccording to the technique of Hirs (1956). Samples ofthe R61 enzyme (5-10l,g of protein) were evaporatedto dryness and redissolved in 30,u1 of formic acid -methanol (5: 1, v/v) previously cooled at -5° C.Performic acid (50ul) was then added and the mix-ture kept at -5° C. After 150min, 0.75ml ofwater wasadded and the mixture was evaporated to dryness.
Separation of radioactive peptides by high-voltageelectrophoresis. Peptides were separated from eachother by electrophoresis on Whatman 3MM paperat pH6.5 (collidine-acetic acid-water; 7:2.5:1000,by vol.) for 210min, a Gilson High Voltage (IOOOOV)Electrophorator model DW (60V/cm) being used.The radioactive peptides were located on the stripsby using a Packard Radiochromatogram Scanner.The radioactivity was determined by cutting stripsinto sections of 10mm, which were counted in aPackard Tri-Carb liquid-scintillation spectrometer.Amino acid composition of radioactive peptides.
The radioactive peptides, purified by paper electro-phoresis, were hydrolysed in6M-HCl at 100° C for 16h.After being freeze-dried, the residues were dissolved in1.5ml of 0.2M-sodium citrate buffer, pH2.2. Thesolutions were injected in a Bio-Cal BC200 aminoacid analyser programmed for single-column runs.When diaminopimelic acid was not present in thehydrolysates, the temperature was kept constant at50° C and the following buffers were used: (1)0.2M-sodium citrate, pH3.25, in 20% ethanol (for
elution of glycine and alanine); (2) 1.2M-sodiumcitrate, pH6.5 (for elution of lysine). When diamino-pimelic acid was present in the hydrolysates, theconditions used were as follows: (1) 0.2M-sodiumcitrate, pH3.25, in 20% ethanol at 30° C (for elutionof glycine and alanine); (2) 0.2M-sodium citrate,pH3.50, at 60° C (for elution of diaminopimelicacid; (3) 1.2 M-sodium citrate, pH6.5, at 60° C (forelution of lysine).Amino acid composition of the R61 protein.
This was determined on a sample hydrolysed for24h at 110° C in constant-boiling HCl (made fromAnalaR HC1 by addition of water and redistillation)after removal of air in a vacuum. The instrument wasa Bio-Cal automatic analyser.
Chemicals and reagentsCarrier ampholytes were obtained from LKB
(ampholine 8142). Myoglobin, chymotrypsinogen,carbonic anhydrase, aldolase and ovalbumin werepurchased from Sigma Chemical Co., St. Louis, Mo.,U.S.A. Sodium dodecyl sulphate was recrystallizedtwice from ethanol before use.
ResultsPurification of the enzyme
Step 1. Streptomyces R61 (150 litres) was grown,the enzyme adsorbed from the culture super-natant on Amberlite CG 50 at pH4.0 anddesorbed from the resin by 0.1 M-K2HPO4 at pH8.0,as previously described by Leyh-Bouille et al. (1971).The adsorption of the enzyme on the resin and itselution as well as all subsequent steps were performedat 4° C. Solid (NH4)2S04 was added to the elutedenzyme solution and the precipitate obtained at40% saturation was discarded. Protein precipitatedwhen the (NH4)2S04 concentration was raised to.65% saturation was redissolved in a minimum ofO.OlM-Tris-HCl buffer, pH8.0 (1 litre), and dialysedagainst 50 litres of the same buffer. The dialysedsolution was divided into four 250ml portions, whichwere separately adsorbed on four 800ml columns ofDEAE-cellulose previously equilibrated against0.O1M-Tris-HCI buffer, pH8.0. The enzyme waseluted from the DEAE-cellulose with an increasingNaCl gradient as described by Leyh-Bouille et al.(1971). The active fractions from the four columnswere pooled, concentrated by ultrafiltration througha UM-10 membrane in an Amicon cell, dialysed andrechromatographed on one 200ml column ofDEAE-cellulose under the same conditions as above. Theactive fractions were pooled, concentrated to 25mlby ultrafiltration and dialysed against water.
Step 2. After step 1, the enzyme preparation wasstill heavily contaminated by a dark-brown pigment.It was divided into three lots, which were separately
transpeptidase (step 2)Fractions (7.5ml) were collected with a flow rate of1 ml/min. DD-Carboxypeptidase activity was esti-mated by incubating 2,ul ofeach fraction with 50nmolof Ac2-L-Lys-D-Ala-D-Ala for 15min at 37° C in atotal volume of30,u. x, Enzyme activity; e, E280.
(b)
- 2,69 M = 37150 _- /
D 0.01 0.02Cx
0.03 0.04
Fig. 2. Reciprocal of the apparent molecular weightofR61 DD-carboxypeptidase-transpeptidase in 0.01 M-Tris-HCI buffer (pH8.0, ionic strength adjusted to0.1 with NaCl) at initial concentration of 3mg ofprotein/ml, as a function of the concentration in the
column (C.) during sedimentation equilibriumFor conditions, see the text. (a) Enzyme preparationafter step 3; (b) enzyme preparation after step 4.
submitted to preparative polyacrylamide disc-electro-phoresis in a Shandon apparatus (height of the gel:5cm; current intensity: 40mA), by using the same gel/buffers as those described for the analytical procedure(Leyh-Bouille et al., 1972). The brown pigment waseluted from the gel with the tracking dye (Bromo-phenol Blue) and the enzyme was eluted 60minlater. The active fractions were pooled, concentratedto 8ml by ultrafiltration, dialysed against water andsubmitted to an additional electrophoresis under thesame conditions as above. Fractions 11-22 (Fig. 1)were pooled and concentrated to 20ml by ultra-filtration.
Step 3. After step 2, the concentrated solution wasfaintly yellow. It was filtered through a 400mlcolumn of Sephadex G-75 in 0.01 M-Tris - HCI buffer,pH8.0. The active fractions were pooled and appliedto a 100ml column of DEAE-cellulose equilibratedagainst 0.01 M-Tris - HCI buffer, pH8.0. The enzymeand all the material absorbing at 280nm was fixedon DEAE-cellulose. The resin was treated with anincreasing linear gradient of NaCI (500ml of Trisbuffer+500ml of 0.2M-NaCl in Tris buffer). Theelution profile of the enzyme activity closely followedthat of the absorbancy at 280nm. The active fractionswere concentrated by ultrafiltration. Althoughthe preparation was colourless, analysis by equili-brium sedimentation (Fig. 2a) and polyacrylamide-gelelectrophoresis at pH8.4 indicated the presence ofcontaminating materials of low molecular weight.Vol. 135
Step 4. The enzyme solution was dialysed against0.015M-HCI-piperazine buffer, pH6.0, and appliedto a short column (10cm xl cm) of DEAE-Sephadex A-50 previously equilibrated against thesame HCI - piperazine buffer. The enzyme was elutedfrom the column with an increasing linear gradient ofNaCl (200ml of HCl-piperazine buffer+200ml of0.15M-NaCl in the HCl-piperazine buffer). The en-zyme activities of the fractions were exactly propor-tional to their protein contents. The active fractionswere pooled and concentrated to 0.5ml by ultrafil-tration. Analysis by equilibrium sedimentation(Fig. 2b), polyacrylamide-gel electrophoresis atpH8.4 and electrofocusing (see below) indicatedthat not more than 5% of contaminating materialwas present in the final preparation. The specificactivity was equal to 86 units/mg of protein, i.e. avalue five times higher than that of the preparationpreviously obtained (Leyh-Bouille et al., 1971).Table 1 gives the total recoveries and enrichments inspecific activity after each step of the purificationprocedure.
Transpeptidase activity of the purified enzyme.During its purification, the enzyme was monitored byfollowing its DD-carboxypeptidase activity uponAC2-L-Lys-D-Ala-D-Ala. The enzyme preparations oflower specific activity, which had been obtainedearlier (17 units/mg of protein; Leyh-Bouille et al.,1972), catalysed transpeptidation reactions when a
465
0
J.-M. FRERE, J.-M. GHUYSEN, H. R. PERKINS AND M. NIETO
* The protein concentration was determined either by measuring extinction at 280 and 260nm and using the formulaC (mg/ml) = 1.54E280-0.76E260, or by measuring the amount of total amino groups available to fluorodinitrobenzeneafter 6M-HCI hydrolysis (100° C, 20h; standard: bovine serum albumin).
Table 2. Amino acid composition ofR61 DD-carboxypeptidase-transpeptidaseThe amount of enzyme hydrolysed was 31.5.ug
350* Calculated from the u.v. spectrum in alkali (Nieto et al., 1973b).t Cystine alone was found; no cysteine occurred. Evidently the protein must contain one, or at most two, disulphide
bridges.
suitable acceptor was added to the incubation mixture(Pollock et al., 1972; Perkins et al., 1973; Nieto et al.,1973a). The demonstration that the highly purifiedpreparation was also able to catalyse such trans-peptidation reactions was made by using 'IC-labelledAc2-L-Lys-D-Ala-D-Ala as donor and either meso-diaminopimelic acid or glycyl-L-alanine as accep-tors. A detailed study ofthe kinetics of these reactionsis presented elsewhere (Fr6re et al., 1973). Afterreaction, the excess of radioactive tripeptide donor,
the hydrolysis product "4C-labelled AC2-L-LyS-D-Alaand the transpeptidation products [either "4C-labelledAc2-L-Lys-D-Ala4D)-meso-A2pm or "4C-labelled Ac2-L-Lys-D-Ala-Gly-L-Ala] were separated from eachother by paper electrophoresis at pH6.5 (see theMaterials and Methods section). The electrophoreticmigrations were: 53cm for the tripeptide donor, 60cmfor the hydrolysis product, 46cm for the trans-peptidation product with meso-diaminopimelic acidand 48cm for the transpeptidation product with
From left to right: A, electrophoresis at pH8.4 of 20,g of R61 DD-carboxypeptidase-transpeptidase;B, electrophoresis of 7,ug of R61 DD-carboxypeptidase-transpeptidase at pH7.1 in the presence of 0.1 % sodiumdodecyl sulphate (the fast moving band is myoglobin, used as an internal standard); C, electrophoresis of 7,ug ofperformic acid-oxidized R61 DD-carboxypeptidase-transpeptidase at pH7.1 and in the presence of 0.1 %sodium dodecyl sulphate (the fast-moving band is myoglobin); D, electrofocusing of 4jtg of R61 DD-carboxy-peptidase-transpeptidase. For conditions see the text.
J.-M. FRERE. J.-M. GHUYSEN, H. L. PERKINS AND M. NIETO
Plate 1
(Facing p. 466)
CARBOXYPEPTIDASE-TRANSPEPTIDASE COMPOSITION
glycyl-L-alanine. The radioactive compounds wereeluted from the paper strips, purified by filtration onSephadex G-15 in water and characterized bydetermining their amino acid composition afterHCI hydrolysis (see the Materials and Methodssection).
Molecular weight and diffusion coefficient of thepurifiedR61 DD-carboxypeptidase-transpeptitdase. Fig.2(a) and Fig. 2(b) compare the plots of 1/Mapp. versusconcentration for the preparation obtained afterstep 3 (Fig. 2a) and the final enzyme preparation afterstep 4 (Fig. 2b). From this lastexperiment, a molecularweight of 37150 was obtained by extrapolation.The R61 enzyme exhibited a D20,,, value (see theMaterialsand Methods section) of8.45 x 10-7cm2_S-.Since a spherical, non-hydrated protein of molecularweight 37150 has a diffusion constant Do of9.50x10 cm2 .S-1, it follows that the purifiedenzyme has a frictional ratio (f/fo = DO/D20,,)of 1.12.Electrophoresis
Polyacrylamide-gel electrophoresis of the purifiedR61 DD-carboxypeptidase-transpeptidase. After elec-trophoresis of 20p,g of enzyme at pH8.4 and colora-tion with Coomassie Blue (Plate IA), two very faintbands were visible in addition to the main proteinband. As previously observed (Leyh-Bouille et al.,1971), the R61 enzyme is a very anionic protein atpH8.4. Scanning of the stained gel indicated thataltogether the two contaminating compounds repre-sented less than 5 % of the total protein content of theenzyme preparation.
Gel electrophoresis in the presence of sodiumdodecyl sulphate. Chymotrypsinogen, carbonicanhydrase, aldolase and ovalbumin were used asmolecular-weight standards. The mobility of theR61 enzyme and that of each protein wereexpressed as the ratio of their migration to themigration of myoglobin used as an internalstandard. The mercaptoethanol-treated R61 enzyme(7,ug) migrated as a single band. Its mobility, whencompared with that of the likewise-treated proteinstandards, indicated a molecular weight of 39000±1600 (4 determinations) (Plate 1B).
Gel electrophoresis in the presence of sodiumdodecyl sulphate after performic acid oxidation ofthe purified DD-carboxypeptidase-transpeptidase. Ifthe enzyme were composed of two or severalpolypeptide chains held together through disulphidebridges, oxidation with performic acid (see theMaterials and Methods section) would give rise tocompounds of lower molecular weight. The dryresidue obtained after performic oxidation of 7,gofenzyme was dissolved in 0.12ml of0.1 M-phosphatebuffer, pH7.1, containing 1% mercaptoethanol andI % sodium dodecyl sulphate, and submitted topolyacrylamide-gel electrophoresis under the sameVol. 135
conditions as described above. A single band wasdetected, which had the same mobility as thatexhibited by the non-oxidized enzyme (Plate 1Q,thus demonstrating that the protein was composed ofa single polypeptide chain.
Isoelectric point of the purified R61 DD-carboxy-peptidase-transpeptidase
The R61 enzyme (4,g) was submitted to gel iso-electrofocusing (see the Materials and Methodssection). Another gel, to which no protein had beenapplied, was also submitted to isoelectrofocusingunder the same conditions but was not stained byCoomassie Blue. This second gel was sliced intosections, 3mm thick, which were extracted separatelywith 1 ml ofwater. After 2h, the pH ofeach eluate wasmeasured. The isoelectric point of the R61 enzymewas determined from the position of the protein bandon the stained gel. It was found to be 4.8±0.14(4 determinations). As shown in Plate 1ID, a very faintbatid was seen about 1-2mm below the main band.Scanning of the stained gel indicated that this con-taminating compound represented less than 5%ofthe total protein content ofthe enzyme preparation.
Amino acid composition ofR61 enzymeAmino acids, except tryptophan, were estimated
after HCI hydrolysis. Tryptophan content wasestimated from the u.v. spectrum given by the enzymein alkali (Nieto et al., 1973b). Results are given inTable 2. The total mass of amino acid residues,29.58,ug, compared fairly well with the nominalamount of enzyme analysed, namely 31.5,ug.
tidase excreted by Streptomyces R61 has been purifiedto near homogeneity (at least 95% purity), and thepurified protein has been shown to perform bothenzymic activities (see also Frere et al., 1973). Amolecular weight of 38000±1000 was assigned tothe protein either by equilibrium sedimentationor by polyacrylamide-gel electrophoresis in thepresence ofsodium dodecyl sulphate ofthe mercapto-ethanol-treated protein. The turnover number was3300mol of D-alanine liberated from Ac2-L-Lys-D-Ala-D-Ala per mol of enzyme per min. Treatment ofthe protein with performic acid did not affect itsmolecular weight. Hence at ionic strength 0.1, theenzyme molecule is one single polypeptide chain.The enzyme molecule contains about three half-cystine residues. The acidic residues (20%) largelyoutnumber the basic ones (8%.). This propertyexplains the negatively charged character of theprotein and its low isoelectric point (4.8). About50% of the residues are non-polar ones, suggesting
467
468 J.-M. FRtRE, J.-M. GHUYSEN, H. R. PERKINS AND M. NIETO
the existence of two areas within the molecule.One of-them might be strongly polar and the other onehighly hydrophobic. From the experiments reportedhere and by Nieto et al. (1973b), the fluorophorictryptophan and at least some of the tyrosine residuesmight be somehow buried in the latter hydrophobicarea.
This research has been supported in part by the FondsNational de la Recherche Scientifique, the Fonds de laRecherche Fondamentale Collective, Brussels, Belgium(contracts no. 515 and no. 1000) and by the Institut pourl'Encouragement de la Recherche Scientifique dansl'Industrie et l'Agriculture, Brussels, Belgium (contractno. 1699). J.-M. F. is Charg6 de Recherches du FondsNational de la Recherche Scientifique, Brussels, Belgium.
References
Dusart, J., Marquet, A., Ghuysen, J.-M., Frere, J.-M.,Moreno, R., Leyh-Bouille, M., Johnson, K., Lucchi,C., Perkins, H. R. & Nieto, M. (1973) Antimicrob.Agents Chemother. 3, 181-187
Frere, J.-M., Ghuysen, J.-M., Perkins, H. R. & Nieto, M.(1973) Biochem. J. 135,483492
Hirs, C. H. W. (1956) J. BioL Chem. 219, 611-621Leyh-Bouille, M., Coyette, J., Ghuysen, J.-M., Idczak,,J.,
Perkins, H. R. & Nieto, M. (1971) Biochemistry 10,2163-2170
Leyh-Bouille, M., Nakel, M., Frere, J.-M., Johnson, K.,Ghuysen, J.-M., Nieto, M. & Perkins, H. R. (1972)Biochemistry 11, 1290-1298
Nieto, M., Perkins, H. R., Leyh-Bouille, M., Frere, J.-M.& Ghuysen, J.-M. (1973a) Biochem. J. 131, 163-171
Nieto, M., Perkins, H. R., Frere, J.-M. & Ghuysen,J.-M. (1973b) Biochem. J. 135, 493-505
O'Donnell, I. J. & Woods, E. F. (1962) in Modern Methodsof Plant Analysis (Linskens, H. F. & Tracey, M. V.,eds.), pp. 250-324, Springer, Berlin
Perkins, H. R., Nieto, M., Frere, J.-M., Leyh-Bouille, M.& Ghuysen, J.-M. (1973) Biochem. J. 131, 707-718
Pollock, J. J., Ghuysen, J.-M., Linder, R., Salton, M. R. J.,Perkins, H. R., Nieto, M., Leyh-Bouille, M., Frere,J.-M. & Johnson, K. (1972) Proc. Nat. Acad. Sci. U.S.69, 662-666
Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244,4406-4412
Wrigley, C. W. (1969) Shandon Instrument ApplicationBulletin 29
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