Eur. J. Biochem. 60, 363-369 (1975)

Purification and Properties of a Periplasmic Aminoendopeptidase from Escherichia coli Claude LAZDUNSKI, Jeanine BUSUTTIL, and Andrte LAZDUNSKI Centre de Biochimie et de Biologie Moleculaire and Laboratoire de Chimie Bacterienne, Centre National de la Recherche Scientifique, Marseille (Receivcd June 9, 1975)

A periplasmic aminoendopeptidase from Escherichia coli has been purified to homogeneity. It is a monomer of molecular weight 45000 and containing one - SH group that is necessary for catalytic activity. The study of its substrate specificity indicated that the enzyme has both aminopeptidase and endopeptidase activity. The pH optimum for L-alanine p-nitroanilide hydrolysis is between 7 and 7.5 and that for '251-labelledcasein proteolysis between 7.3 and 7.6. The activation energy for the hydrolysis of L-alanine p-nitroanilide was calculated to be 5.3 kcal x mol-' (22.2 kJ x mol-').

A number of investigators have reported one or more aminopeptidase activities in Escherichia coli [I -41. Most of the studies concerned with these enzymes were aimed at the characterization and isolation of the enzyme that functions in the removal of the NH,-terminal methionine from newly initiated proteins but all of those proteins are, of course, located in the cytoplasm. We have found a new aminopeptidase that has been localized near the cell surface, since it is directly assayable with intact cells and partially released by osmotic shock or by spheroplasts formation [5]. A four-fold increase in the differential rate of synthesis of this enzyme occurs when the inorganic phosphate (Pi) concentration is limiting [5]. Regulation is shared with alkaline phosphatase. The two enzymes are 'derepressed' at the same intracellular Pi concentration whether it is obtained by depletion of Pi from the medium or by mutation in the Pi transport system [6]. Mutations in the regulator gene phoR which controls the synthesis of alkaline phosphatase does not appear to affect the production of this periplasmic aminopeptidase [6]. In this study we report the purification and the properties of this enzyme. Since we will show that this enzyme displays both an aminopeptidase and endopeptidase activity we have called it aminoendopeptidase. MATERIALS AND METHODS Muter ials DEAE-cellulose (DE-32) was purchased from Whatman, QAE-Sephadex A-50 was obtained from

Pharmacia. L-Alanine p-nitroanilide and L-leucine p-nitroanilide were from Bachem Inc. ; L-alanyl-Lproline, L-alanyl P-naphthylamide, L-alanyl-L-valine, L-alanyl-L-serineand L-alanyl-glycyl-glycine were from Sigma ; L-leucine B-naphthylamide, L-proline P-naphthylamide, L-lysine P-naphthylamide and L-aspartyl b-naphthylamide were from Cyclo Chemical ; 5,5'dithio-bis(2-nitrobenzoic acid), L-phenylalanine 4-nitroanilide, N-ethylmaleimide and 1,4-dithioerythritol were from Merck. Enzyme Assays Routine measurements during enzyme purification were carried out at 37 "C, using 5 mM L-alanine p-nitroanilide as substrate, in 50 mM phosphate buffer (pH 7.4). The release of p-nitroaniline was spectrophotometrically followed at 410 nm as previously described [ 5 ] . The various L-amino acid b-naphthylamides were assayed according to Lee et al. [7]. Hydrolysis of dipeptides and tripeptides was followed by the ninhydrin method of Moore and Stein [8]. Endopeptidase Activity Casein, serum albumin and F, fragment from rabbit IgG were iodinated as previously described [9]. The '251-labelled casein and serum albumin obtained had a specific radioactivity of 8.66 x lo6 and 7.2 x lo6 counts min-' mg-' respectively. The release of acid-soluble radioactive products from the proteins was followed according to Reignier and Thang [3].

364

Gel Filtration Experiments

The estimation of the molecular weight was carried out according to Andrews [lo] in a Sephadex G-100 (1 cm x 100cm)column equilibrated and washed with 50 mM Tris-HCI (pH 8), 0.15 M NaCl at 4 "C. Proteins for calibration (2 mg) were bovine serum albumin dimer, ovalbumin, E. coli alkaline phosphatase, chymotrypsinogen and cytochrome c. Disc Gel Electrophoresis

Analytical disc gel electrophoresis experiments were carried out with 7.5 % acrylamide gels according to Davis [ l l ] except that a 7 mM imidazole/96 mM asparagine buffer was used and 0.1 mM 1,Cdithioerythritol was included in the gels and buffers. Determination of Terminal a-Amino Group

The dansylation of aminoendopeptidase to determine the terminal amino group was carried out according to Hartley [12]. Amino Acid Analysis

Amino acid composition was determined by the procedure of Moore and Stein [13] using norleucine as an internal standard. Hydrolyses were performed in vacuo at 105 f 1 "C for periods of 24,48 and 72 h. Hydrolysed samples were analyzed on a TSM (Technicon) automatic amino acid analyzer. Half-cystine was determined as cysteic acid following performic acid oxidation as described by Moore [13].Tryptophan was determined by spectrophotometry according to Spies and Chambers [14]. RESULTS Enzyme Ext ruc t ion

Strain C90, a derivative of E . coli K10 that carries a mutation in phoT and is affected in the Pi transport system [15], was used as a source of enzyme. The cells were grown at 37 "C in a 100 1fermenter (Chemap F.A.) to early stationary phase. In the Tris-bactopeptone medium previously described [6] we routinely obtained 150 g of cells (dry weight). Step 1. Extruction of Cells. The harvested bacteria were resuspended in 8 1 of iced 0.03 M Tris buffer pH 7.4 and successively 2.7 1 of 2 M sucrose, 500 ml of 1 % EDTA at pH 7 and 500ml of a solution of lysozyme at 2 mg/ml were successively added. Formation of spheroplasts was monitored by the decrease in absorbance at 600 nm of a 1/10 dilution. At room temperature, this process was completed within 10min ; 10 mM MgC1, (final concentration) was then added and the suspension was centrifuged at 16000 x g for

Purification and Properties of an Aminoendopeptidase from E. coli

20 min. The supernatant contained 35% of the total enzyme activity of the cells; 0.1 mM 2-mercaptoethanol was added to this solution before further use. Step 2, Ammonium Sulfate Fructionation. All the subsequent steps in the purification were carried out at 4 "C in the presence of 0.1 mM 2-mercaptoethanol. The supernatant of spheroplasts was dialyzed overnight against 0.02 M Tris pH 7.4,O.l mM 2-mercaptoethanol and this extract was brought to 40 saturation with solid ammonium sulfate. The precipitated proteins containing no aminoendopeptidase activity were discarded after centrifugation and the supernatant was brought to 60% saturation by further addition of (NH,), SO,. All the aminoendopeptidase activity was in the precipitated fraction. In this step almost 50 % of the activity was lost. However, this treatment was necessary for successful purification since part of the material eliminated cannot be removed in the next chromatographic steps. Step 3. DEAE-Cellulose Chromatography. The material from the preceding step was thoroughly dialyzed against 0.02 M Tris pH 7.4, 0.1 mM 2-mercaptoethanol. The dialyzed protein solution was then applied to a DEAE-cellulose column (3 x 30 cm) equilibrated in the above buffer. The column was then washed with 11 of this buffer. The adsorbed protein was eluted with a 2-1 linear gradient of 0.02 M to 0.3 M Tris-HC1 pH 7.4. The fractions containing the aminoendopeptidase activity (eluted at 0.26 M Tris-HCI) were pooled and dialyzed against 0.02 M Tris pH 7.4. Step 4 . QAE-Sephadex A-50 Chromatography. The dialyzate from step 3 was applied to a column of QAE-Sephadex (3 x 30 cm) equilibrated in 0.02 M Tris pH 7.4; the column was then washed with 1 1 of the same buffer and the protein then eluted with a 2-1 linear gradient of 0-0.4 M NaCl in this buffer. The fractions containing aminoendopeptidase activity were eluted with 0.25 M NaC1. They were pooled and dialyzed against 2 mM NH4H C 0 3 before lyophilization. Step 5 . Sephadex G-100 Chromatography. The lyophilized enzyme was solubilized in 5 ml of 0.02 M Tris pH 7.5, 0.15 M NaCl and applied to a Sephadex G-100 column (2 cm x 200 cm) equilibrated with the same buffer. In this step a 2.7-fold purification was obtained. The purification is summarised in Table 1. Evidencefor Homogeneity. The homogeneity of the enzyme prepared was checked by polyacrylamide gel electrophoresis. The results are shown in Fig. 1. 1,4-Dithioerythritol (0.1 mM) and imidazole buffer, as described under Materials and Methods, were found to be required to maintain the component as a single species during electrophoresis. The very thin band that appears in the middle of the gel (Fig.1) is a slow-moving form of bromophenol blue since it is also obtained when the dye is run alone under the same conditions. When unprotected by a reducing

365

C. Lazdunski, J. Busuttil. and A. Lazdunski Table 1. Purification of E. coli atninoendopeptidase Step

Protein

Aminoendopeptidase ~

1 . Spheroplast supernatant 2. (NH& SO, precipitation 3 . DEAE-cellulose chromatography 4. QAE-Sephadex A-50 chromatography 5. Sephadex G-100 chromatography

total activity

Specific activity

mg

units

pmol x min-' x mg-'

10000 477

920000 450000

92 942

184

308389

1671

99

246711

2490

34

234475

6800

1

I 1

-

L

2.3

Fig. 2. Evidencefor slou autolysis. Dansylation and Edman degradation were carried out on freshly-prepared enzyme (A) and on the same enzyme after a 2-h incubation at 37 "C (B). NH,-terminal residues were then determined by bidimensional chromatography according to Hartley [12]

Fig. 1. Polyarryluniide gel electrophoresis of' the aminoendopeptidase. 50 pg of aminoendopeptidase purified as described was applied to a 7.5 yo polyacrylamide gel containing 0.1 mM 1,4-dithioerythritol. Electrophoresis was carried out at 5mA, and 4'C in a 7mM imidazolei96mM asparagine buffer containing 0.1 mM 1,4-dithioerythritol

reagent, aminoendopeptidase gives two additional bands that are inactive and are possibly oxidation products of the enzyme. A similar phenomenon has been recently reported for cytochrome 6-559 [16]. In order to reinforce the evidence for homogeneity the number and the nature of the N-terminal end of the protein which is composed of only one polypeptide chain (see further) was determined. The freshly purified protein had only one N-terminal end and that is a methionine residue (Fig. 2). Sequential degradation

by the Edman technique with identification of Nterminal amino acid residues after dansylation [12] indicates that the N-terminal end of the protein has the sequence Met-Ile-Thr. However, after storage at 4 "C, new bands appeared on polyacrylamide gel electrophoresis which correlated with the appearance of new N-terminal ends. This suggested a slow autolysis of the protein and is consistent with the endopeptidase activity (see later). To check this point we compared the N-terminal ends of the freshly prepared protein, and of the protein after a 2-h incubation at 37 "C. Results presented in Fig. 2 show the appearance of several new N-terminal ends. Glycine, valine and leucine are the main ones, phenylalanine, isoleucine and histidine are less intense, and traces of serine and aspartic acid are also found. Thus, two processes

Purification and Properties of an Aminoendopeptidase from E . coli 0.025

\

Cytchrome c

1.2

I

I

I

1.6

1.6

1 .a

I

2 .o

0

3.5

4 .0

4.5

0

vo Fig. 3. Estimation of the molecular weight of aminoendopeptidase. 1 ml of aminoendopeptidase (2 mg/ml) purified as described was applied to a Sephadex G-100 column (1 x 100 cm) equilibrated with 0.01 M Tris-HCI pH 7.4, 0.15 M NaCl buffer. As indicated, the column was calibrated using alkaline phosphatase of E. coli ( M , = 86000), bovine serum albumin ( M , = 61000), ovalbumin ( M , = 43000), chymotrypsinogen A ( M , = 25000), cytochrome c ( M , = 120001

Molecular weight Fig. 4. Theoretical analysis of' the umino acid composition. Curve ofy = .Z [n,/(Ni-1)l2[17]. The analysis of x mol of a protein sample

appear to contribute to the appearance of additional bands on polyacrylamide gels, the oxidation previously described and the slow autolysis.

Table 2. Amino acid composition ofthe aminoendopeptidase Results were measured as mol per 44000g amino acids, to the nearest integer

ve

gave the number of mol x i of amino acid i; ni = x i / x N i represents the number of amino acid residues of type i in 1 mol of protein. The correct value of x , that is of ni, is that which gives a minimum for y . Therefore, the minimum in the curve represents the most probable value of the molecular weight

Amino acid

Numbers of residues

Half-cystine a Aspartic acid Threonine Serine Glutamic acid Proline G1y cin e Akanine Valine Methionine Isoleucine Leucine Tyrosine Phen ylalanine Lysine Histidine Arginine Tryptophan

5 38 23 20 54 17 33 35 28 5 21 36 8 16 21 8 17 9

Enzyme Properties Stability. When it is stored in the absence of reducing reagent the homogenous enzyme is rather unstable. In the presence of 0.1 mM 2-mercaptoethanol, solutions of aminoendopeptidase in 50 mM phosphate buffer pH 7.4 can be kept frozen at -20 "C for one month without detectable loss of activity. Molecular Weight Determination. The molecular weight of aminoendopeptidase was estimated by molecular sieve chromatography on a Sephadex G-100 column as indicated in Fig.3. The molecular weight of native aminoendopeptidase was 45000 f 1000. Electrophoresis in dodecylsulfate-polyacrylamidegel confirmed this molecular weight and showed that the enzyme has only one polypeptide chain. Amino Acid Composition. The amino acid composition of the aminoendopeptidase is presented in Table2. Assay with the Ellman reagent shows the presence of one free sulfhydryl group in the denatured enzyme ;the content of half-cystines (Table 2) indicates therefore that there are two disulfide bridges per mole of protein. The theoretical analysis of the amino acid composition according to Delaage [17] is presented in Fig.4; it indicates a molecular weight of 44000 in good agreement with gel filtration experiments. Metal Ion Requirement. Incubation of enzyme preparations in the presence of 10 mM EDTA did not cause any loss of activity. Thus, divalent cations do not appear to be necessary for the catalytic activity.

Determined as cysteic acid. Determined according to Spies and Chambers [14].

Suljhydryl Requirement. One - SH group per mole of enzyme can be titrated with the Ellman reagent, 5,5'-dithio-bis(2-nitrobenzoicacid). The - SH group reacts with 100 mM N-ethylmaleimide with concomitant loss of activity. 50% inactivation is observed within 1 h in the absence of urea or within 30 min in the presence of 1 M urea. Effect of an Inhibitor of Serine Protease. The effect of diisopropylphosphorylfluoridate, which covalently binds the serine of the active site of many proteases [18], was investigated. We did not find any inhibition

367

C. Lazdunski, J. Busuttil, and A. Lazdunski

Table 3. Suhstrate specificit), of arninopeptidasc Substrate

Relative rates of hydrolysis

L-Alaninep-nitroanilide L-Leucine p-nitroanilide L-Phen ylalanine p-nitroanilide ~-Alanine11-naphthylamide L-Leucine P-naphthylamide t.-Proline P-naphthylamide L-Lysine 8-naphthylamide L-Aspartyl P-naphthylamide L-Aspartyl 8-naphthylamide A 1a - G 1y - G 1y Ala-Val Ala-Pro Ala-Ser

100 32 0 16

3 2 5 0.8 0.8 0 0 0 0

U

10

5

0

1 500

0

4"

or any binding when 10 mM dii~opropyl[~~P]phosphorylfluoridate was used.

500

n

Substrate Specijicity Aminopeptiduse Activity. The relative rates of hydrolysis of various substrates catalyzed by aminoendopeptidase are summarized in Table 3. L-Alanine p-nitroanilide was the best substrate for the enzyme. However, it appears that the hydrophobicity of the non-amino-acid part of this substrate plays a central role since other dipeptides or tripeptides containing L-alanine were not hydrolyzed. L-Alanine P-naphthylamide was hydrolyzed but at only about one-fifth the rate of hydrolysis of L-alanine p-nitroanilide. If L-alanine is replaced by leucine in thep-nitroanilide, there is a three-fold decrease in the rate of hydrolysis, and replacement of L-alanine by L-phenylalanine prevents the breakdown of thep-nitroanilide. Similarly, when various amino acids were substituted for Lalanine in the P-naphthylamide, the rate of breakdown was much lower. We did not find any esterase activity with benzoylarginine ethyl ester, acetyl-tyrosine ethyl ester or p-nitrophenyl acetate. Endopeptiduse Activity. The endopeptidase activity was determined by the hydrolysis of I-labelled proteins. Various proteins were tested as substrates. Casein and serum albumin were good ones whereas the F, fragment from purified rabbit IgG was poorly hydrolyzed [9]. Casein and serum albumin hydrolyses decrease as a function of time. The rates shown in Fig. 5 A are initial rates (first 5 min of incubation). Under these conditions the amount of acid-soluble casein degradation products released is proportional to the amount of enzyme present (Fig.5B). As a control, a highly purified preparation of intestinal brush border aminopeptidase (generous gift from Dr Maroux) was used. This enzyme did not produce

"

6

7

8

9

10

PH

Fig. 5. Endopeptidasc activity. (A) As a function of time 5 pg of the purified aminoendopeptidase was incubated at 37 "C in a reaction mixture (500 pl) containing: 10 mM Tris-HCI pH 7.5, 1200 pg '2S-I-kdbelledcasein. Aliquots (100 pl) were taken at intcrvals and the radioactivity not precipitated by 10 "/, trichloracetic acid was measured after 1 h at 0 T. (B) Reaction mixtures (100 pl) as above but containing various amount of enzyme were incubated for 5 min at 37 "C before acid addition, and the unprecipitated radioactivity was again measured. (C) As a function of pH. Reaction mixtures (100 pl) buffered respectively at pH 6 (10 mM morpholinoethane sulfonate), pH 7 (10 mM Na,H PO,/NaH, PO,), pH 8 (10 mM Tris/ HCI) and pH 9 (10 mM ethanolamine) and containing 5 pg of enzymc were incubated for 5 min at 37 'C. Acid-soluble radioactivity was determined in each case

any acid-soluble casein peptides under the conditions used. Ejfect of p H on the Aminopeptiduse and Endopeptiduse Activities. The maximum ielocity ( V ) of the hydrolysis of L-alanine p-nitroanilide was measured as a function of pH (Fig.6). The decrease of V at alkaline pH might reflect the necessity for a protonated amino acid residue of the active site of the enzymesubstrate complex or it might be a consequence of the deprotonation of the a-amino group of the substrate. In fact both effects might be involved. The K, under standard conditions (37 'C, 50 mM NazH PO4 buffer pH 7.4) is 0.18 mM. The endopeptidase activity also

Purification and Properties of an Aminoendopeptidase from E . coli

368

0.5 3.2

PH

I

I

I

3.3

3.4

3.5

lo3/T

'

(K-1)

Fig. 6. pH dependencefor the hydrolysis of' L-ulanine p-nitroanilide. The buffers used are the same as in Fig. 5

Fig. I . Temperature dependence fbr the hydrolysis of' L-alanine pnitroanilide. The release of p-nitroaniline was followed spectrophotometrically at 410 nm in 50 mM phosphate buffer (pH 7.4) at various temperatures ; the Arrhenius plot is presented

varies with pH (Fig.5C). The maximum activity is obtained at pH 7.5. Temperature Dependence of ~-Alaninep-Nitroanilide Hydrolysis. This study was carried out in 50mM Na,H PO, buffer 7.4. Results are presented as an Arrhenius plot (Fig. 7) from which an activation energy of 5.3 kcal x mol-' (22.2 kJ x mol-') was calculated. This plot is not linear above 45 "C which suggests thermal denaturation of the enzyme.

a more restricted specificity. When the cleavage of an N-terminal amino acid is involved, L-alanine peptides are very much favored provided that adjacent amino acid is a bulky hydrophobic one. Endopeptidic cleavage might also depend upon the nature of the amino acid residues at the carboxyl and amino side of the splitting point. Hydrophobic residues might be preferentially recognized on the amino side since slow autolysis causes the appearance of mainly hydrophobic N-terminal ends (Fig. 2). Since the enzyme purified is not inhibited by diisopropylphosphorofluoridateor by EDTA but is sensitive to N-ethylmaleimide it might be classified as a thiol proteinase. Other microbial proteinases already described include serine proteinases and metalchelator-sensitive proteinases [21]. Blocking of only one -SH group per molecule results in a complete loss of activity. The optimum pH for aminopeptidase activity is about 7; it differs very significantly from the optimum pH for endopeptidase activity which is about 7.5. Most microbial proteinases found are predominantly extracellular. They have been tentatively classified into four groups according to the system of Hartley based on mechanism of action [22]. There are (a) serine proteinases, (b) thiol proteinases, (c) metal-chelator-sensitive proteinases, (d) acid proteinases. However, the enzyme described in this study has a. peculiar behavior in that it shows both amino peptidase and endopeptidase activity. At present, we are carrying out a more thorough characterization of the enzyme specificity and studying the molecular mechanisms of catalysis of peptide bond hydrolysis.

DISCUSSION A procedure for the isolation of a periplasmic aminoendopeptidase is reported. In 'derepressed' C90 cells, this enzyme accounts for about 1 of the total cell protein. It might be identical with a periplasmic protease identified by Regnier and Thang [3] after osmotic shock. However, since the latter protease has not been purified nor studied in detail the likelihood of its being the same enzyme is hard to assess. Like approximately 40% of E. coli proteins, the aminoendopeptidase has an N-terminal methionine, which suggests that after deformylation the initiating methionine has not been removed. This is supported by the fact that the amino acid in second position is isoleucine since it has been shown that methionine is not cleaved efficiently in this case [4]. The aminoendopeptidase hydrolyzes L-alanine p nitroanilide, a typical substrate of true aminopeptidases such as that from pig kidney or intestinal brush-border. However, its specificity towards other substrates is quite different [19,20]. By comparison with most microbial proteinases, the enzyme that we have purified from E. coli shows

369

C. Lazdunski, J. Busuttil, and A. Lazdunski

The authors are grateful to Dr M. Rovery for her advice on the amino-terminus identification and to Dr D. Grossman for careful reading of the manuscript. This work was partly supported by the DPlPgation Generule a la Recherche Scientijique et Technique.

REFERENCES 1. Matheson, A T. & Marayama, T. (1966) Can. J . Biochem. 44,

1407-1415. 2. Vogt, V. M. (1970) J . Biol. Chem. 245, 4760-4769. 3. Reignier, P. & Thang, M. N. (1972) Biochimie (Paris) 54, 1227-1236. 4. Brown, J. L. (1973) J . B i d . Chem. 248, 409-416. 5. Lazdunski, A,, Murgier. M. & Lazdunski, C. (1975) Eur. J . Biochem. 60, 349- 355. 6. Lazdunski, A,, Pelissier, C. & Lazdunski, C. (1975) Eur. J . Biochem. 60, 357- 362. 7. Lee, H. J., La Rue, J. W. &Wilson, I. P. (1971) Anal. Biochem. 41,307-401. 8. Moore, S. & Stein, W. H. (1954) J . Biol. Chem. 211, 907-912.

9. Lazdunski, C., Pages, J. M. & Louvard, D. (1975) J . Mul. Biol. 97, 309- 335. 10. Andrews, P. (1970) Methods Biochem. Anal. 18, 1 - 53. 11. Davis, B. J. (1964) Ann. N . Y . Acad. Sci. 121, 404-427. 12. Hartley, B. S. (1963) Biochem. J . 89, 379-380. 13. Moore, S. & Stein, W. H. (1963) Methods Enzymol. 6 , 819831. 14. Spies, J. R. & Chambers, D. C. (1967) Anal. Chem. 39, 14121419. 15. Willsky, G. R., Bennett, R. L. & Malamy, M. (1973) J. Bacteriol. 113, 529- 539. 16. Garewal, H. S. & Wasserman, A. R. (1974) Biochemistry, 13, 4063 - 4071. 17. Delaage, M. (1968) Biochim. Biophys. Acta, 168, 573-575. 18. Jansen, E. F., Nutting, M. D., Jung, R. & Balls, A. K. (1949) J . Biol. Chem. 179, 201 -206. 19. Smith, E. L. & Spackman,D. H. (1955) J . Biol. Chem. 212, 271 -283. 20. Maroux, S., Louvard, D. & Baratti, J. (1973) Biochim. Biophys. Acta, 321, 282 - 295. 21. Morihara, K. (1974) Adv. Enzymol. 41, 179-243. 22. Hartley, B. S. (1960) Annu. Rev. Biochem. 29, 45-70.

C. Lazdunski and J. Busuttil, Centre de Biochimie et de Biologie Moltculaire du C.N.R.S., 31 Chemin Joseph-Aiguier, F-I 3274 Marseille-Cedex-2, France A. Lazdunski, Laboratoire de Chimie Bacttrienne du C.N.R.S., 31 Chemin Joseph-Aiguier, F-13274 Marseille-Cedex-2, France

Purification and properties of a periplasmic aminoendopeptidase from Escherichia coli.

Eur. J. Biochem. 60, 363-369 (1975) Purification and Properties of a Periplasmic Aminoendopeptidase from Escherichia coli Claude LAZDUNSKI, Jeanine B...
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