Aminopeptidase a from Streptococcus Cremoris

Purification and Some Properties of a Membrane-Bound Aminopeptidase A from Streptococcus cremoris

FRED A. EXTERKATE* AND GERRIE J. C. M. DE VEER Netherlands Institute for Dairy Research, 6710 BA Ede, The Netherlands Received 25 August 1986/Accepted 24 November 1986

A membrane-bound L-a-glutamyl (aspartyl)-peptide hydrolase (aminopeptidase A) (EC 3.4.11.7) from Streptococcus cremoris HP has been purified to homogeneity. The free y-carboxyl group rather than the amino group of the N-terminal L-a-glutamyl (aspartyl) residue appeared to be essential for catalysis. No endopeptidase activity could be established with this enzyme. The native enzyme is a polymeric, most probably trimeric, metalloenzyme (relative molecular weight, approximately 130,000) which shows on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels apparent high relative molecular weight values due to (lipid?) material dissociable with butanol. The subunit (relative molecular weight, approximately 43,000) is catalytically inactive. The enzyme is inactivated completely by dithiothreitol, chelating agents, and the bivalent metal ions Cu2'and Hg2+. Of the sulfhydryl-blocking reagents tested, only p-hydroxymercuribenzoate appeared to

inhibit the enzyme. Activity lost by treatment with a chelating agent could be restored by Co2+ and Zn2+ . The importance of the occurrence of an aminopeptidase A in S. cremoris with respect to growth in milk is discussed.

Lactic streptococci possess proteinase activity associated successively, 25, 35, 45, and 55% (wt/vol) with solid AS. with the cell wall (7, 15, 22). In Streptococcus cremoris, After each addition, the solution was stirred for 15 min at 4°C peptidases have been detected which are located near the and the precipitate was collected by centrifugation (15 min at outside surface of and inside the membrane (8, 13). These 48,000 x g and 4°C). The precipitates were dissolved in peptidases are assumed to act in concert with the cell wall distilled water and either dialyzed overnight against an proteinase(s) to hydrolyze milk proteins to transportable excess of distilled water (4°C) and then freeze-dried or components (viz., amino acids and small peptides) (7), since concentrated by YM-10 (Amicon Corp., Lexington, Mass.) the size limit for transport through the membrane seems to filtration at 4°C. Usually >95% of the initial gluAP activity be approached with four to six residues in lactic streptococci was recovered and found entirely in the 25 to 35% (wt/vol) (14, 21). Subsequently, intracellular peptidases complete the AS fraction. This fraction was used for further purification. hydrolysis of transported peptides. In this way essential It contained 40 to 50% of the initial amount of protein in the amino acids are produced from milk proteins which other- cell extract. wise would limit growth of the organism. Butanol treatment. The AS fraction was dissolved in 0.05 Reports on the occurrence of membrane-bound peptidases M NaH2PO4-NaOH (pH 7.2), and n-butanol was added to are scarce, and knowledge of these peptidases is still scanty give a final concentration of 10% (vol/vol). The solution was (11, 13, 18). This is why our interest became focused stirred for 60 min at 25°C. After centrifugation (15 min, particularly on these enzymes in S. cremoris. Results (8) 48,000 x g), the supernatant was dialyzed twice against an have been obtained which indicate that a glutamate excess of distilled water at 4°C over a total period of 30 h and aminopeptidase (gluAP) is a membrane-bound enzyme. Ac- then centrifuged again to remove the precipitate obtained tivities detectable with the chymotrypsin substrate N- during dialysis. The supernatant was freeze-dried and dis- glutaryl-L-phenylalanine-4-nitroanilide and formerly desig- solved in 0.05 M NaH2PO4-NaOH buffer (pH 7.2). This nated as "endopeptidases" P37 and P50 have been found to known depend on a membrane-bound component (8) which might preparation exhibited gluAP activity, but all other be a peptidase responsible for the introductory release of the peptidase activities were inactivated by the butanol treat- glutaryl moiety. The gluAP activity could be due to a specific ment step (8). L-a-glutamyl (aspartyl) aminopeptidase (aminopeptidase A), Gel ifitration. For preparative purposes gel filtration was by the action of which the cell can provide itself with the performed at 4°C on a Sephacryl S-300 column (40 by 780 essential amino acid glutamic acid. The same enzyme could mm) equilibrated with 0.05 M NaH2PO4-NaOH buffer (pH be responsible for the release of a N-terminal glutaryl 7.2) with 350 mg of protein. Proteins were eluted with this moiety. To elucidate its function, the enzyme was purified buffer at a flow rate of approximately 0.6 ml min-', and 5-ml and its specificity was determined. fractions were collected. The pooled gluAP fraction was desalted and concentrated by filtration on an Amicon YM-10 MATERIALS AND METHODS filter. Analytical PAGE and IEF. Polyacrylamide gel electropho- Starting material for enzyme purification. A cell extract resis (PAGE) (8 or 8.5% acrylamide; 0.05 M imidazole buffer (500 ml) (7) obtained from milk-grown cells of S. cremoris system, pH 7.0) and analytical isoelectrofocusing (IEF) (pH HP (15-liter culture) suspended in 0.05 M NaH2PO4-NaOH 4 to 6) were performed essentially according to the instruc- buffer (pH 7.2) was subjected to fractionation by ammonium tions of LKB-Produkter AB, Bromma, Sweden (application sulfate (AS) precipitation. The extract (4°C) was brought to, notes 306 and 205, by respectively), by using the LKB 2117 Multiphor. The low-pl calibration kit (pH 2.5 to 6.5) was * Corresponding author. used for reference. 577 578 EXTERKATE AND DEVEER APPL. ENVIRON. MICROBIOL.

Preparative IEF. Preparative flatbed IEF in a granulated showed impurities as judged by SDS-PAGE. The enzyme gel was performed with the LKB 2117 Multiphor essentially (102 pug of protein) in 250 ,ul of HEPES (N-2-hydroxy- according to the instructions described by Winters et al. in ethylpiperazine-N'-2-ethanesulfonic acid) buffer (25 mM), LKB application note 198. A bed of Ultrodex contained pH 7.5, was preincubated for 15 min at temperatures ranging Ampholine (LKB) carrier ampholytes of the pH range 4 to 6. from 30 to 80 or at 37°C in the presence of bivalent cation (1 The gluAP preparation (3 ml) supplemented with 5% mM) or the indicated amount of reagent. After preincuba- (vol/vol) Ampholine was applied as a zone after a prerun of tion, enzyme activity was measured at 37°C and pH 7.5 by 30 min, and IEF was performed overnight at 11°C. The the addition of a glu-pNA solution (4 mM) in HEPES buffer separate zones were collected by sectioning the gel bed and (50 mM). transferred to small columns. Elution of the gel fractions was Protein quantification. Proteins were estimated by the performed first with distilled water (6 ml) (for pH determi- micromethod of Bradford (4), by using crystalline serum nation) and then with 0.1 M NaH2PO4-NaOH buffer, pH 7.2 albumin (fraction V; BDH, Poole, England) as the standard. (6 ml). The pooled eluates were dialyzed against 0.002 M Chemicals and substrates. All reagents mentioned in this NaH2PO4-NaOH buffer, pH 7.2. study were of guaranteed grade. The following substrates Preparative PAGE. Preparative PAGE was performed by were used: L-ot-Glu-L-Ala (Sigma Chemical Co., St. Louis, applying the same buffer system and conditions as used for Mo.); Gly-L-ao-Asp and Gly-L-Phe (Fluka AG, Buchs, Swit- analytical PAGE. After completion of the electrophoretic zerland); L-Ala-L-Glu, L-0x-Glu-Gly, L-GluN-Gly, L-ot-Glu-L- run, the gels were first frozen at -80°C before the catalyti- Glu, L-oL-Glu-L-Ala-L-Ala, L-Ala-L-Asp, L-o-Asp-L-Phe, L-a- cally active component was cut out. Transfer of the protein Asp-L-Leu, L-Phe-L-Asp, L-Lys-L-Glu-Gly, Gly-Gly-L-Glu- from the gel into a small volume of buffer was accomplished L-Ala-methyl ester, L-y-Glu-L-Phe, and L--y-Glu-pNA (p- by electroelution, with the ISCO model 1750 sample concen- nitroanilide) (Bachem AG, Bubendorf, Switzerland); trator (Instrumentation Specialties Co., Lincoln, Nebr.) (3). pyroglu-pNA (Serva, Heidelberg, Federal Republic of Ger- The electrode compartment buffer was 0.05 M NaH2PO4- many); Z-L-Phe-L-Tyr and pyroglu-L-Ala (Cyclo Chemical, NaOH buffer, pH 7.2. Small pieces of the cut gel were placed Los Angeles, Calif.); L-a-Glu-pNa and glutaryl-L-Phe-pNa in the sample cups together with 0.005 M NaH2PO4-NaOH (Merck AG, Darmstadt, Federal Republic of Germany); buffer, pH 7.2. Electrophoresis was carried out for 6 h at 4°C Z-Gly-L-Phe, Z-L-(x-Glu-L-Phe, and Z-L-o(-Glu-L-Tyr (Mann (100 V, 20 to 30 mA). Research Laboratories, New York, N.Y.); succinyl-L-Phe- The enzyme fraction was removed from the small wells pNA (Boehringer GmbH, Mannheim, Federal Republic of with a plastic pipette. This fraction was dialyzed against Germany); N-acetyl-L-Ile-L-Glu-Gly-Arg-pNA (Kabi distilled water and freeze-dried. Diagnostica, Stockholm, Sweden). L-Arg-L-Glu-L-Leu was a Determination of (subunit) molecular weight. Relative mo- hydrolysis product obtained by the action of chymosin on lecular weights were estimated by electrophoresis in sodium 1-casein (25). methyl-"4C-labeled whole casein, 3-casein, dodecyl sulfate (SDS)-polyacrylamide gels (8 or 8.5% acrylamide) according to the method of Weber and Osborn glu AP activity (26), by using the imidazole buffer system at pH 7.0 (LKB A280 ( 206) ( units- ml1-1) application note 306). The sample buffer contained SDS and 0.2 dithiothreitol (DTT) or ,3-mercaptoethanol, but the samples were not heated unless mentioned otherwise. Low- and high-molecular-weight proteins (Bio-Rad Laboratories, 10 Richmond, Calif.) were used as references: lysozyme (Mr 14,400), soybean trypsin inhibitor (Mr 21,500), carbonic anhydrase (Mr 31,000), ovalbumin (Mr 45,000), bovine serum albumin (Mr 66,200), phosphorylase b (M' 92,500), - I ~~~~~~~~~~~8 galactosidase (Mrll6,250), and myosin (Mr 200,000). Fixing and staining of gels. Gels were fixed and stained with Coomassie brilliant blue R-250 (Pierce Chemical Co., Rockford, Ill.), as described in LKB application note 306. 0.1I4 6 Measurement of gluAP. Unless mentioned otherwise, gluAP activity was measured at pH 7.2 and 37°C with 280 ~~~~~~~~4 L-cx-glutamic acid-4-nitroanilide (glu-pNA) (2 mM) as described previously (8). Detection of catalytic activity with different substrates. The hydrolysis of peptides and peptide derivatives was detected 2 by thin-layer chromatography as described previously (9). To 5 [lI of a solution of the pure enzyme in distilled water was added 20 ,ul of a 5 mM substrate solution in 0.05 M NaH2PO4 buffer, pH 7.8. After various periods of incubation 0 10 20 30 40 50 60 at 37°C up to 24 h, a sample was withdrawn from the fraction number incubation mixture and examined for hydrolysis of the FIG. 1. Sephacryl S-300 chromatography of a butanol-treated AS substrate. Degradation of methyl-'4C-labeled caseins or al- fraction. A sample of 25 mg (dry weight) in 3 ml of 0.05 M bumin was tested as described before (9). NaH2PO4-NaOH (pH 7.2) was subjected to chromatography on a of the column (2.5 by 35 cm) equilibrated and eluted with the same buffer. Stability enzyme and effect of bivalent cations and The flow rate was 0.7 ml min-1; 5-ml fractions were collected. The various reagents. The enzyme preparation (812 ,ug of protein A280 and A206 were recorded, and the gluAP activities (0) were ml-') used was obtained by repeating the Sephacryl S-300 measured against 1 mM glu-pNA at 37°C and expressed as arbitrary fractionation after completion of the first separation. This units ml of eluate-1. For comparison, the active peak obtained by resulted in much higher activity yields, but the preparation filtration of an untreated AS fraction is also shown (0). VOL. 53, 1987 AMINOPEPTIDASE A FROM S. CREMORIS 579 ao1-casein, and albumin were prepared as described previously (7). a b RESULTS

Purification of the enzyme. The criterion for purity of the enzyme was the number of bands obtained after analytical PAGE and IEF. The individual steps are detailed below. After each step in the purification, the gluAP-positive fraction appeared to coincide with the fraction able to release, in combination with the complementary factor, p-nitroaniline from glutaryl-L-Phe-pNA (8). (i) Gel filtration. Figure 1 shows a representative elution pattern of a butanol-treated AS fraction on Sephacryl S-300. For comparison, the peak of gluAP activity eluted under the same conditions, by using an untreated AS fraction, is also shown. Proteolytic active material from both preparations was eluted from the column at the same relative elution volume. (ii) Preparative IEF. The concentrated column fraction obtained by preparative gel filtration was used to run preparative flatbed IEF. The gluAP activity was recovered in a - zone with a mean pH value of 4.3. Analytical IEF of this N.... pi ooo"".k .l

-11~3.5 1 2 3 3.75 FIG. 3. PAGE of the gluAP fraction obtained after preparative IEF (a) and of the pure enzyme preparation (b). Lanes: 1, Sephacryl S-300 column fraction; 2, enzyme preparation after preparative IEF; - 4.15 3, the pure enzyme. Catalytically active bands are indicated (*). b4.55 fraction showed one main component (pl 4.35) and weakly stained bands close to it on both sides (Fig. 2). After PAGE one main component and three faint bands were detectable (Fig. 3a). Incubation of the IEF and the polyacrylamide gels, - 5.2 soaked in a 4 mM substrate solution at pH 7.8 and 40°C, revealed that in both cases only the main component was catalytically active. Figure 4a shows the fraction after SDS- PAGE. In this gel three components were detected with approximate Mrs of 180,000 (III), 110,000 (A), and 55,000 (B). (iii) Preparative PAGE. The preparation obtained after preparative IEF was freeze-dried, dissolved in a small volume of sample buffer, and used to run polyacrylamide gels for preparative purposes. The preparation obtained after electroelution of the gels showed only one band after PAGE (Fig. 3b) and IEF (not shown). 5.85 A summary of these purification steps with measures of their relative efficiency is presented in Table 1. Loss of 4"6.55 activity, especially during step 6, might be due to increasing instability of the purified enzyme or loss of essential cations or both. In later experiments it appeared that part of the lost activity could be restored by the addition of Co2" ions (see below). Butanol treatment of the AS fraction prior to gel filtration 1 2 3 4 resulted in an increase in total activity (2 and 2.4 times in two separate experiments), whereas protein was reduced to 9 FIG. 2. Analytical IEF of the gluAP fraction obtained after of the amount in the cell extract. As preparative flatbed IEF. Lanes: 1 and 4, reference proteins; 2, and 11%, respectively, of the was in- Sephacryl S-300 column fraction; 3, enzyme fraction after prepara- a result, the specific activity preparation tive IEF. creased by factors of 8.2 and 9.5, respectively. However, 580 EXTERKATE AND DEVEER APPL. ENVIRON. MICROBIOL. a b

,14400 N".21 500

B 0i45000 -66 20 A- - -20000 NII

1 2 3 4 5 6 t 8 FIG. 4. SDS-PAGE (8%) of the gluAP fraction obtained after preparative flatbed IEF (a) and of the pure enzyme (b) performed with or without prior heating of the sample at 100°C. Lanes: 1, enzyme preparation after preparative IEF (heated); 2, enzyme preparation after preparative IEF (not heated); 3, Sephacryl S-300 column fraction (not heated); 4, 5, and 6, reference proteins; 7, pure enzyme (not heated); 8, pure enzyme (heated). loss of activity during the next steps was not in proportion to ple buffer, generated subunits of 55,000 molecular weight this result. and therefore is probably identical to component A. Com- Properties of the purified enzyme. (i) Molecular weight. ponent III' dissociates into component I. The second, less SDS-PAGE of the purified enzyme without prior heating of pure preparation (Fig. Sa) showed, in addition, a component the sample resulted in the detection of a main component I II (approximate Mr 85,000) which apparently also dissoci- (Mr 43,000) and component III (Mr approximately 180,000) ated to give I. (Fig. 4b). Pretreatment of the enzyme at 100°C (in the (ii) Substrate specificity. The substrate specificity of the presence or absence of DTT) resulted in the disappearance purified peptidase was investigated to understand the role of of III and of an intensified component I (Fig. 4b). A similar this enzyme in the provision of the cell with nitrogen and result was obtained with the fraction obtained after prepar- essential amino acids. The hydrolytic activity towards a ative IEF (Fig. 4a). Component B is more pronounced, range of peptides and peptide derivatives was surveyed. A apparently due to dissociation of component A. Other pure summary of the results is given in Table 2. These results preparations repeatedly showed a heterogeneous appear- show that the enzyme cleaves peptide bonds in which the ance of component III (not shown). If the purification co-carboxyl group of a glutamic acid or aspartic acid at the procedure was started with an AS fraction which had been amino end of the chain is involved. An N-terminal glutaryl or treated with 10% (vol/vol) n-butanol, a catalytically active succinyl group is also liberated but not the L-glutaminyl or component (III') was obtained with an Mr of approximately L--y-glutamyl group. No endopeptidase activity towards 130,000. Figure 5 shows a Sephacryl S300 fraction (panel a) methyl-'4C-labeled caseins and albumin could be detected. and a similar fraction which had been further purified by (iii) Influence of bivalent cations, chelating agents, and repeating the butanol extraction and gel filtration (panel b). group-specific inhibitors. Under the conditions applied, The latter preparation contained component III' close to Mn2+, Ni2 +, and Cd2+ had a marked inhibitory effect on the another (inactive) component which, on heating in the sam- enzyme's activity, whereas with Cu2+ and Hg2+full inactivation was established. The enzyme was completely inactivated by 4-(hydroxymercuri)benzoic acid (1 mM) but not by TABLE 1. Summary of purification procedure for other sulfhydryl group-blocking reagents (viz., iodoacetic L-ox-glutamyl aminopeptidase acid, iodoacetamide, and N-ethylmaleimide at concentra- % of tions up to 10 mM). Complete inactivation was also obtained Sp act agent Purification step (nmol of pNA mg (%) of total initial with DTT (2 mM) and with the chelating EDTA (1 mM) min-' ml', 103) protein total or 1,10-phenanthroline (1 mM). In these cases SDS-PAGE activity did not reveal dissociation into subunits (not shown). The 1. Cell extract 27.8 4,525 (100) 100 activity lost by treatment with 1,10-phenanthroline was 2. AS (25-35%, 54.0 2,158 (48) 91 almost completely restored by Co2 + and Zn2 + added at a wt/vol) final concentration of 5 mM. Mn2+ was less effective, and 3. YM10 filtration 70.5 1,784 (39) 99 Cd2 , Ni2+, Cu2+, Mg2+, Sr2+, Ca2+, and Hg2+ had no or 4. Sephacryl S-300 394.3 286 (6.3) 88 only a slight effect. 5. YM10 filtration 855.1 101.8 (2.25) 68 The optimum temperature for gluAP activity 6. Preparative IEF 634.1 1.958 (0.043) 0.97 (iv) Stability. in the cell extract was approximately 50 to 55°C (8). Heating VOL. 53, 1987 AMINOPEPTIDASE A FROM S. CREMORIS 581 a b

W i,-. _,14400% Z -21 500 - 31 000 '2' I- e -0 45000- - --- 1141'- B- - 66200- I1 __~92500 sI1I N> 16250/' i --200 000 III'

1 2 3 4 5 6 7 8 FIG. 5. SDS-PAGE (8.5%) of a Sephacryl S-300 column fraction from a butanol-treated AS preparation (a) and a similar fraction subjected to a second butanol treatment followed by gel filtration (b). The samples were either heated at 100°C or not heated. Lanes: 1 and 8, high-molecular-weight reference proteins; 2, first Sephacryl S-300 gluAP fraction (not heated); 3, first Sephacryl S-300 gluAP fraction (heated); 4 and 5, low-molecular-weight reference proteins; 6, second Sephacryl S-300 gluAP fraction (heated); 7, second Sephacryl S-300 gluAP fraction (not heated). of the enzyme solution between 50 and 75°C at pH 7.5 for 15 characterized (2). Both of these mammalian aminopep- min resulted in a loss of activity up to a maximum of 15 to tidases A are more active towards substrates with N- 20%. Heating of the enzyme solution at these temperatures terminal glutamic acid than towards those with aspartic acid. as a function oftime up to 60 min also resulted in a maximum An enzyme from dog kidney removes aspartyl residues loss of activity of approximately 20%. Above 75°C the faster than glutamyl residues (6). An aminopeptidase with activity loss was considerable. unique specificity for L-a-aspartyl peptides has been partially purified from Salmonella typhimurium (5). A P- DISCUSSION aspartyl peptidase from Escherichia coli has been purified and characterized and shown to be specific for P-aspartyl The present results suggest the occurrence in S. cremoris dipeptides. In soil organisms of the genus Flavobacterium, of a peptide hydrolase specific for peptide bonds in which an L-a-glutamyl-L-glutamate activity has been partially purified amino-terminal L-a-glutamyl (aspartyl) residue is involved. but not tested on other L-a-glutamyl peptides (20). The enzyme shows no strict requirement for the free amino We are aware of no case of purification and characteriza- group, since glutaryl and succinyl peptide derivatives are tion of aminopeptidase A from microorganisms. Few data also good substrates. This may indicate that the free -y- are available concerning aminopeptidase profiles of lactic carboxyl group rather than the amino group is essential. If an streptococci which indicate the presence of L-a-glutamyl amino group was present next to the free carboxyl group aminopeptidase activity (12, 16). The existence of a specific (L--y-glutamyl peptide) or if the a-amino group was substi- enzyme in S. cremoris associated with and functioning at the tuted, no hydrolysis could be detected, suggesting steric outside surface of the membrane has been suggested (8), and hindrance. With respect to its natural peptide substrates, this its specific catalytic property has been confirmed in this enzyme may therefore be classified as an L-a-glutamyl study. Our results indicate that the aminopeptidase A from (aspartyl) aminopeptidase (aminopeptidase A) (EC 3.4.11.7). S. cremoris is a polymeric metalloenzyme consisting of Aminopeptidase A has been found in various organs and identical monomers (approximate Mr 43,000). Treatment of sera of animals (see reference 24). It has been purified only the enzyme with chelating agents maintains an inactive partially from human serum (17) and from the brush border conformation of the polymer which allows the binding of of rabbit small intestine, since it appeared difficult to sepa- metal ion activators, Co2+ and Zn2+ being exclusively rate the enzyme from other aminopeptidases (1). Tobe et al. effective. In the presence of DTT complete inactivation was (24) have succeeded in purifying the enzyme from pig obtained without dissociation of the polymer being ob- kidney, using an affinity column consisting of an agarose served. Apparently reduction of intramolecular rather than matrix linked to a specific inhibitor, amastatin, isolated from intermolecular disulfide bridges takes place. Heating at culture filtrates of a Streptomyces strain. A similar enzyme 100°C in the absence of DTT and SDS results in complete from hog intestinal brush border was also purified and inactivation and dissociation into subunits. The reduction in 582 EXTERKATE AND DEVEER APPL. ENVIRON. MICROBIOL.

TABLE 2. Substrate specificity of the purified gluAP proteinase(s) and in concert with other aminopeptidases or Substrate Hydrolysis' not, the cell will be efficiently provided with this essential amino acid. Several results are indicative of a membrane- L-ot-Glu-L-Ala + + + bound state of this enzyme (8). These include results which L-Ala-L-Glu show that the enzyme in situ is affected by changes attended L-ct-Glu-Gly + + + by the energization and de-energization of the membrane L-GluN-Gly An involvement of the in a L-a-Glu-L-Glu + (unpublished results). enzyme L-a-GlU-L-Ala-L-Ala + + + specific anionic permease system such as that detected in L-Ala-L-Asp Streptococcus faecalis might be postulated (19). Such a L-Gly-L-Asp transport system, restricted to glutamyl (aspartyl) peptides, L-a-ASp-L-Phe + + + could be a device to satisfy most efficiently the need of this L--a-ASp-L-Leu + + + organism for glutamic acid. L-Phe-L-Asp - L-Lys-L-Glu-Gly - LITERATURE CITED Gly-Gly-L-Glu-L-Ala-methyl ester 1. Andria, G., A. Marzi, and S. Auricchio. 1976. a-Glutamyl-p- L-Arg-L-GlU-L-Leu - naphthyl amide hydrolase of rabbit small intestine. Localization Z-L-a-Glu-L-Tyr in the brush border and separation from other brush border Z-L-a-Glu-L-Phe peptidases. Biochim. Biophys. Acta 419:42-50. N-Acetyl-L-Ile-L-Glu-Gly-L-Arg-pNA 2. Benajiba, A., and S. Maroux. 1980. Purification and character- L-a-Glu-pNA + + ization of an aminopeptidase A from hog intestinal brush-border Glutaryl-L-Phe-pNA + + membrane. Eur. J. Biochem. 107:381-388. Succinyl-L-Phe-pNA + + 3. Bhown, A. S., J. E. Mole, F. Hunter, and J. C. Bennett. 1980. Pyroglu-L-Ala High-sensitivity sequence determination of proteins quantita- Pyroglu-pNA tively recovered from sodiumdodecylsulphate gels using an im- L-y-Glu-pNA - proved electrodialysis procedure. Anal. Biochem. 103:184-190. L-y-GIU-L-Phe - 4. Bradford, M. M. 1976. A rapid and sensitive method for the Gly-L-Phe quantitation of microgram quantities of protein utilizing the Z-L-Phe-L-Tyr - principle of protein dye binding. Anal. Biochem. 72:248-254. Z-Gly-L-Phe 5. Carter, T. H., and C. G. Miller. 1984. Aspartate-specific 4CH3-labeled whole casein peptidases in Salmonella typhimurium: mutants deficient in 4CH3-labeled ,-casein peptidase E. J. Bacteriol. 159:453-459. "CH3-labeled oa,1-casein 6. Cheung, H. S., and D. W. Cushman. 1971. A soluble aspartate '4CH3-labeled albumin aminopeptidase from dog kidney. Biochim. Biophys. Acta 242:190-193. a Hydrolysis of the various substrates is described as follows: +++ = 7. Exterkate, F. A. 1975. An introductory study of the hydrolysis (nearly) completed within 60 min; + + = good substrate, but proteolytic hydrolysis is not completed within 60 min; + = slight hydrolysis detectable; system of Streptococcus cremoris strain HP. Neth. Milk Dairy - = no detectable hydrolysis after 24 h. J. 29:303-318. 8. Exterkate, F. A. 1984. Location of peptidases outside and inside the membrane of Streptococcus cremoris. Appl. Environ. Mi- activity at relatively low temperatures may be due to either crobiol. 47:177-183. conformational change or the presence of a more unstable 9. Exterkate, F. A., and G. J. C. M. de Veer. 1985. Partial isolation fraction. of and degradation of caseins by cell wall proteinase(s) of Pretreatment of a crude enzyme preparation with butanol Streptococcus cremoris HP. Appl. Environ. Microbiol. results in a still catalytically active component of molecular 49:328-332. weight 130,000, suggesting a trimer. Component II (molecu- 10. Haley, E. E. 1968. Purification and properties of a P-aspartyl lar weight, 85,000) might be the dimer. The peptidase from Escherichia coli. J. Biol. Chem. 243:5748-5752. intermediary 11. Hermsdorf, C. L., and S. Simmonds. 1980. Role of peptidases in higher-molecular-weight (heterogeneous) component III utilization and transport of peptides by bacteria, p. 301-334. In might represent this trimer still associated with material J. W. Payne (ed.), Micro-organisms and nitrogen sources. John (lipid?) which interferes with the action of SDS under the Wiley & Sons Ltd., Chichester, England. present conditions and which otherwise has been removed 12. Kaminogawa, S., T. Ninomiya, and K. Yamauchi. 1984. by butanol. That both components III and III' are eluted Aminopeptidase profiles of lactic streptococci. J. Dairy Sci. during gel filtration at exactly the same relative elution 67:2483-2492. volume sustains this view. Alternatively, the native enzyme 13. Kolstad, J., and B. A. Law. 1985. Comparative peptide speci- might be a tetramer, and a trimeric complex is induced by ficity of cell wall, membrane and intracellular peptidases of butanol treatment. group N streptococci. J. Appl. Bacteriol. 58:449-456. 14. Law, B. A., E. Sezgin, and M. E. Sharpe. 1976. Amino acid Compared with the well-characterized enzyme from pig nutrition of some commercial cheese starters in relation to their kidney (24), both enzymes have sensitivity to chelating growth in peptone-supplemented whey media. J. Dairy Res. agents, optimum pH and optimum temperature, and insen- 43:291-300. sitivity to sulfhydryl-blocking agents (except p-hydroxy- 15. Mills, 0. E., and T. D. Thomas. 1978. Release of cell-wall mercuribenzoate) in common. In contrast, the pig enzyme associated proteinase(s) from lactic streptococci. N. Z. J. Dairy (Mr 300,000) was not inactivated by S-S dissociating agents, Sci. Technol. 13:209-215. was completely inhibited by Zn+ and was (re)activated by 16. Mou, L., J. J. Sullivan, and G. R. Jago. 1975. Peptidase alkaline cations, especially Ca2 . activities in group N streptococci. J. Dairy Res. 42:147-155. Glutamic acid, the product of the action of the present 17. Nagatsu, I., T. Nagatsu, T. Yamamoto, G. G. Glenner, and J. W. enzyme, is an Mehl. 1970. Purification of aminopeptidase A in human serum essential animo acid and in addition has the and degradation of angiotensin II by the purified enzyme. highest minimum concentration required for protein synthe- Biochim. Biophys. Acta 198:255-270. sis and maximum growth of S. cremoris (23). The occurrence 18. Payne, J. W. 1980. Micro-organisms and nitrogen sources, p. of an aminopeptidase A in this organism may ensure that, by 211-256. John Wiley & Sons Ltd., Chichester, England. its specific action subsequent to the action of the cell wall 19. Payne, J. W., G. M. Payne, and T. M. Nisbet. 1982. An anionic VOL. 53, 1987 AMINOPEPTIDASE A FROM S. CREMORIS 583

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