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

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mr. 1987, p /87/ $02.00/0 Copyright 1987, Americn Society for Microbiology Vol. 53, No. 3 Purifiction nd Some Properties of Membrne-Bound Aminopeptidse A from Streptococcus cremoris FRED A. EXTERKATE* AND GERRIE J. C. M. DE VEER Netherlnds Institute for Diry Reserch, 6710 BA Ede, The Netherlnds Received 25 August 1986/Accepted 24 November 1986 A membrne-bound L--glutmyl (sprtyl)-peptide hydrolse (minopeptidse A) (EC ) from Streptococcus cremoris HP hs been purified to homogeneity. The free y-crboxyl group rther thn the mino group of the N-terminl L--glutmyl (sprtyl) residue ppered to be essentil for ctlysis. No endopeptidse ctivity could be estblished with this enzyme. The ntive enzyme is polymeric, most probbly trimeric, metlloenzyme (reltive moleculr weight, pproximtely 130,000) which shows on sodium dodecyl sulfte-polycrylmide gel electrophoresis gels pprent high reltive moleculr weight vlues due to (lipid?) mteril dissocible with butnol. The subunit (reltive moleculr weight, pproximtely 43,000) is ctlyticlly inctive. The enzyme is inctivted completely by dithiothreitol, chelting gents, nd the bivlent metl ions Cu2'nd Hg2+. Of the sulfhydryl-blocking regents tested, only p-hydroxymercuribenzote ppered to inhibit the enzyme. Activity lost by tretment with chelting gent could be restored by Co2+ nd Zn2+. The importnce of the occurrence of n minopeptidse A in S. cremoris with respect to growth in milk is discussed. Lctic streptococci possess proteinse ctivity ssocited with the cell wll (7, 15, 22). In Streptococcus cremoris, peptidses hve been detected which re locted ner the outside surfce of nd inside the membrne (8, 13). These peptidses re ssumed to ct in concert with the cell wll proteinse(s) to hydrolyze milk proteins to trnsportble components (viz., mino cids nd smll peptides) (7), since the size limit for trnsport through the membrne seems to be pproched with four to six residues in lctic streptococci (14, 21). Subsequently, intrcellulr peptidses complete the hydrolysis of trnsported peptides. In this wy essentil mino cids re produced from milk proteins which otherwise would limit growth of the orgnism. Reports on the occurrence of membrne-bound peptidses re scrce, nd knowledge of these peptidses is still scnty (11, 13, 18). This is why our interest becme focused prticulrly on these enzymes in S. cremoris. Results (8) hve been obtined which indicte tht glutmte minopeptidse (gluap) is membrne-bound enzyme. Activities detectble with the chymotrypsin substrte N- glutryl-l-phenyllnine-4-nitronilide nd formerly designted s "endopeptidses" P37 nd P50 hve been found to depend on membrne-bound component (8) which might be peptidse responsible for the introductory relese of the glutryl moiety. The gluap ctivity could be due to specific L--glutmyl (sprtyl) minopeptidse (minopeptidse A), by the ction of which the cell cn provide itself with the essentil mino cid glutmic cid. The sme enzyme could be responsible for the relese of N-terminl glutryl moiety. To elucidte its function, the enzyme ws purified nd its specificity ws determined. MATERIALS AND METHODS Strting mteril for enzyme purifiction. A cell extrct (500 ml) (7) obtined from milk-grown cells of S. cremoris HP (15-liter culture) suspended in 0.05 M NH2PO4-NOH buffer (ph 7.2) ws subjected to frctiontion by mmonium sulfte (AS) precipittion. The extrct (4 C) ws brought to, * Corresponding uthor. successively, 25, 35, 45, nd 55% (wt/vol) with solid AS. After ech ddition, the solution ws stirred for 15 min t 4 C nd the precipitte ws collected by centrifugtion (15 min t 48,000 x g nd 4 C). The precipittes were dissolved in distilled wter nd either dilyzed overnight ginst n excess of distilled wter (4 C) nd then freeze-dried or concentrted by YM-10 (Amicon Corp., Lexington, Mss.) filtrtion t 4 C. Usully >95% of the initil gluap ctivity ws recovered nd found entirely in the 25 to 35% (wt/vol) AS frction. This frction ws used for further purifiction. It contined 40 to 50% of the initil mount of protein in the cell extrct. Butnol tretment. The AS frction ws dissolved in 0.05 M NH2PO4-NOH (ph 7.2), nd n-butnol ws dded to give finl concentrtion of 10% (vol/vol). The solution ws stirred for 60 min t 25 C. After centrifugtion (15 min, 48,000 x g), the superntnt ws dilyzed twice ginst n excess of distilled wter t 4 C over totl period of 30 h nd then centrifuged gin to remove the precipitte obtined during dilysis. The superntnt ws freeze-dried nd dissolved in 0.05 M NH2PO4-NOH buffer (ph 7.2). This preprtion exhibited gluap ctivity, but ll other known peptidse ctivities were inctivted by the butnol tretment step (8). Gel ifitrtion. For preprtive purposes gel filtrtion ws performed t 4 C on Sephcryl S-300 column (40 by 780 mm) equilibrted with 0.05 M NH2PO4-NOH buffer (ph 7.2) with 350 mg of protein. Proteins were eluted with this buffer t flow rte of pproximtely 0.6 ml min-', nd 5-ml frctions were collected. The pooled gluap frction ws deslted nd concentrted by filtrtion on n Amicon YM-10 filter. Anlyticl PAGE nd IEF. Polycrylmide gel electrophoresis (PAGE) (8 or 8.5% crylmide; 0.05 M imidzole buffer system, ph 7.0) nd nlyticl isoelectrofocusing (IEF) (ph 4 to 6) were performed essentilly ccording to the instructions of LKB-Produkter AB, Bromm, Sweden (ppliction notes 306 nd 205, by respectively), by using the LKB 2117 Multiphor. The low-pl clibrtion kit (ph 2.5 to 6.5) ws used for reference. 577

2 578 EXTERKATE AND DEVEER Preprtive IEF. Preprtive fltbed IEF in grnulted gel ws performed with the LKB 2117 Multiphor essentilly ccording to the instructions described by Winters et l. in LKB ppliction note 198. A bed of Ultrodex contined Ampholine (LKB) crrier mpholytes of the ph rnge 4 to 6. The gluap preprtion (3 ml) supplemented with 5% (vol/vol) Ampholine ws pplied s zone fter prerun of 30 min, nd IEF ws performed overnight t 11 C. The seprte zones were collected by sectioning the gel bed nd trnsferred to smll columns. Elution of the gel frctions ws performed first with distilled wter (6 ml) (for ph determintion) nd then with 0.1 M NH2PO4-NOH buffer, ph 7.2 (6 ml). The pooled elutes were dilyzed ginst M NH2PO4-NOH buffer, ph 7.2. Preprtive PAGE. Preprtive PAGE ws performed by pplying the sme buffer system nd conditions s used for nlyticl PAGE. After completion of the electrophoretic run, the gels were first frozen t -80 C before the ctlyticlly ctive component ws cut out. Trnsfer of the protein from the gel into smll volume of buffer ws ccomplished by electroelution, with the ISCO model 1750 smple concentrtor (Instrumenttion Specilties Co., Lincoln, Nebr.) (3). The electrode comprtment buffer ws 0.05 M NH2PO4- NOH buffer, ph 7.2. Smll pieces of the cut gel were plced in the smple cups together with M NH2PO4-NOH buffer, ph 7.2. Electrophoresis ws crried out for 6 h t 4 C (100 V, 20 to 30 ma). The enzyme frction ws removed from the smll wells with plstic pipette. This frction ws dilyzed ginst distilled wter nd freeze-dried. Determintion of (subunit) moleculr weight. Reltive moleculr weights were estimted by electrophoresis in sodium dodecyl sulfte (SDS)-polycrylmide gels (8 or 8.5% crylmide) ccording to the method of Weber nd Osborn (26), by using the imidzole buffer system t ph 7.0 (LKB ppliction note 306). The smple buffer contined SDS nd dithiothreitol (DTT) or,3-mercptoethnol, but the smples were not heted unless mentioned otherwise. Low- nd high-moleculr-weight proteins (Bio-Rd Lbortories, Richmond, Clif.) were used s references: lysozyme (Mr 14,400), soyben trypsin inhibitor (Mr 21,500), crbonic nhydrse (Mr 31,000), ovlbumin (Mr 45,000), bovine serum lbumin (Mr 66,200), phosphorylse b (M' 92,500), - glctosidse (Mrll6,250), nd myosin (Mr 200,000). Fixing nd stining of gels. Gels were fixed nd stined with Coomssie brillint blue R-250 (Pierce Chemicl Co., Rockford, Ill.), s described in LKB ppliction note 306. Mesurement of gluap. Unless mentioned otherwise, gluap ctivity ws mesured t ph 7.2 nd 37 C with L-cx-glutmic cid-4-nitronilide (glu-pna) (2 mm) s described previously (8). Detection of ctlytic ctivity with different substrtes. The hydrolysis of peptides nd peptide derivtives ws detected by thin-lyer chromtogrphy s described previously (9). To 5 [li of solution of the pure enzyme in distilled wter ws dded 20,ul of 5 mm substrte solution in 0.05 M NH2PO4 buffer, ph 7.8. After vrious periods of incubtion t 37 C up to 24 h, smple ws withdrwn from the incubtion mixture nd exmined for hydrolysis of the substrte. Degrdtion of methyl-'4c-lbeled cseins or lbumin ws tested s described before (9). Stbility of the enzyme nd effect of bivlent ctions nd vrious regents. The enzyme preprtion (812,ug of protein ml-') used ws obtined by repeting the Sephcryl S-300 frctiontion fter completion of the first seprtion. This resulted in much higher ctivity yields, but the preprtion showed impurities s judged by SDS-PAGE. The enzyme (102 pug of protein) in 250,ul of HEPES (N-2-hydroxyethylpiperzine-N'-2-ethnesulfonic cid) buffer (25 mm), ph 7.5, ws preincubted for 15 min t tempertures rnging from 30 to 80 or t 37 C in the presence of bivlent ction (1 mm) or the indicted mount of regent. After preincubtion, enzyme ctivity ws mesured t 37 C nd ph 7.5 by the ddition of glu-pna solution (4 mm) in HEPES buffer (50 mm). Protein quntifiction. Proteins were estimted by the micromethod of Brdford (4), by using crystlline serum lbumin (frction V; BDH, Poole, Englnd) s the stndrd. Chemicls nd substrtes. All regents mentioned in this study were of gurnteed grde. The following substrtes were used: L-ot-Glu-L-Al (Sigm Chemicl Co., St. Louis, Mo.); Gly-L-o-Asp nd Gly-L-Phe (Fluk AG, Buchs, Switzerlnd); L-Al-L-Glu, L-0x-Glu-Gly, L-GluN-Gly, L-ot-Glu-L- Glu, L-oL-Glu-L-Al-L-Al, L-Al-L-Asp, L-o-Asp-L-Phe, L-- Asp-L-Leu, L-Phe-L-Asp, L-Lys-L-Glu-Gly, Gly-Gly-L-Glu- L-Al-methyl ester, L-y-Glu-L-Phe, nd L--y-Glu-pNA (pnitronilide) (Bchem AG, Bubendorf, Switzerlnd); pyroglu-pna (Serv, Heidelberg, Federl Republic of Germny); Z-L-Phe-L-Tyr nd pyroglu-l-al (Cyclo Chemicl, Los Angeles, Clif.); L--Glu-pN nd glutryl-l-phe-pn (Merck AG, Drmstdt, Federl Republic of Germny); Z-Gly-L-Phe, Z-L-(x-Glu-L-Phe, nd Z-L-o(-Glu-L-Tyr (Mnn Reserch Lbortories, New York, N.Y.); succinyl-l-phepna (Boehringer GmbH, Mnnheim, Federl Republic of Germny); N-cetyl-L-Ile-L-Glu-Gly-Arg-pNA (Kbi Dignostic, Stockholm, Sweden). L-Arg-L-Glu-L-Leu ws hydrolysis product obtined by the ction of chymosin on 1-csein (25). methyl-"4c-lbeled whole csein, 3-csein, 0.2 A280 ( 206) APPL. ENVIRON. MICROBIOL. glu AP ctivity ( units- ml1-1) I ~~~~~~~~~~~8 0.1I ~~~~~~~~ frction number FIG. 1. Sephcryl S-300 chromtogrphy of butnol-treted AS frction. A smple of 25 mg (dry weight) in 3 ml of 0.05 M NH2PO4-NOH (ph 7.2) ws subjected to chromtogrphy on column (2.5 by 35 cm) equilibrted nd eluted with the sme buffer. The flow rte ws 0.7 ml min-1; 5-ml frctions were collected. The A280 nd A206 were recorded, nd the gluap ctivities (0) were mesured ginst 1 mm glu-pna t 37 C nd expressed s rbitrry units ml of elute-1. For comprison, the ctive pek obtined by filtrtion of n untreted AS frction is lso shown (0). 10 2

3 VOL. 53, 1987 o1-csein, nd lbumin were prepred s described previously (7). RESULTS Purifiction of the enzyme. The criterion for purity of the enzyme ws the number of bnds obtined fter nlyticl PAGE nd IEF. The individul steps re detiled below. After ech step in the purifiction, the gluap-positive frction ppered to coincide with the frction ble to relese, in combintion with the complementry fctor, p-nitroniline from glutryl-l-phe-pna (8). (i) Gel filtrtion. Figure 1 shows representtive elution pttern of butnol-treted AS frction on Sephcryl S-300. For comprison, the pek of gluap ctivity eluted under the sme conditions, by using n untreted AS frction, is lso shown. Proteolytic ctive mteril from both preprtions ws eluted from the column t the sme reltive elution volume. (ii) Preprtive IEF. The concentrted column frction obtined by preprtive gel filtrtion ws used to run preprtive fltbed IEF. The gluap ctivity ws recovered in zone with men ph vlue of 4.3. Anlyticl IEF of this pi -11~ b " FIG. 2. Anlyticl IEF of the gluap frction obtined fter preprtive fltbed IEF. Lnes: 1 nd 4, reference proteins; 2, Sephcryl S-300 column frction; 3, enzyme frction fter preprtive IEF. AMINOPEPTIDASE A FROM S. CREMORIS 579 ooo"".k.l - N FIG. 3. PAGE of the gluap frction obtined fter preprtive IEF () nd of the pure enzyme preprtion (b). Lnes: 1, Sephcryl S-300 column frction; 2, enzyme preprtion fter preprtive IEF; 3, the pure enzyme. Ctlyticlly ctive bnds re indicted (*). frction showed one min component (pl 4.35) nd wekly stined bnds close to it on both sides (Fig. 2). After PAGE one min component nd three fint bnds were detectble (Fig. 3). Incubtion of the IEF nd the polycrylmide gels, soked in 4 mm substrte solution t ph 7.8 nd 40 C, reveled tht in both cses only the min component ws ctlyticlly ctive. Figure 4 shows the frction fter SDS- PAGE. In this gel three components were detected with pproximte Mrs of 180,000 (III), 110,000 (A), nd 55,000 (B). (iii) Preprtive PAGE. The preprtion obtined fter preprtive IEF ws freeze-dried, dissolved in smll volume of smple buffer, nd used to run polycrylmide gels for preprtive purposes. The preprtion obtined fter electroelution of the gels showed only one bnd fter PAGE (Fig. 3b) nd IEF (not shown). A summry of these purifiction steps with mesures of their reltive efficiency is presented in Tble 1. Loss of ctivity, especilly during step 6, might be due to incresing instbility of the purified enzyme or loss of essentil ctions or both. In lter experiments it ppered tht prt of the lost ctivity could be restored by the ddition of Co2" ions (see below). Butnol tretment of the AS frction prior to gel filtrtion resulted in n increse in totl ctivity (2 nd 2.4 times in two seprte experiments), wheres protein ws reduced to 9 nd 11%, respectively, of the mount in the cell extrct. As result, the specific ctivity of the preprtion ws incresed by fctors of 8.2 nd 9.5, respectively. However, b

4 580 EXTERKATE AND DEVEER b APPL. ENVIRON. MICROBIOL.,14400 N" A- B 0i t 8 FIG. 4. SDS-PAGE (8%) of the gluap frction obtined fter preprtive fltbed IEF () nd of the pure enzyme (b) performed with or without prior heting of the smple t 100 C. Lnes: 1, enzyme preprtion fter preprtive IEF (heted); 2, enzyme preprtion fter preprtive IEF (not heted); 3, Sephcryl S-300 column frction (not heted); 4, 5, nd 6, reference proteins; 7, pure enzyme (not heted); 8, pure enzyme (heted). loss of ctivity during the next steps ws not in proportion to this result. Properties of the purified enzyme. (i) Moleculr weight. SDS-PAGE of the purified enzyme without prior heting of the smple resulted in the detection of min component I (Mr 43,000) nd component III (Mr pproximtely 180,000) (Fig. 4b). Pretretment of the enzyme t 100 C (in the presence or bsence of DTT) resulted in the disppernce of III nd of n intensified component I (Fig. 4b). A similr result ws obtined with the frction obtined fter preprtive IEF (Fig. 4). Component B is more pronounced, pprently due to dissocition of component A. Other pure preprtions repetedly showed heterogeneous ppernce of component III (not shown). If the purifiction procedure ws strted with n AS frction which hd been treted with 10% (vol/vol) n-butnol, ctlyticlly ctive component (III') ws obtined with n Mr of pproximtely 130,000. Figure 5 shows Sephcryl S300 frction (pnel ) nd similr frction which hd been further purified by repeting the butnol extrction nd gel filtrtion (pnel b). The ltter preprtion contined component III' close to nother (inctive) component which, on heting in the sm- TABLE 1. Summry of purifiction procedure for L-ox-glutmyl minopeptidse Sp ct % of Purifiction step (nmol of pna mg (%) of totl initil min-' ml', 103) protein totl ctivity 1. Cell extrct ,525 (100) AS (25-35%, ,158 (48) 91 wt/vol) 3. YM10 filtrtion ,784 (39) Sephcryl S (6.3) YM10 filtrtion (2.25) Preprtive IEF (0.043) 0.97 NII ple buffer, generted subunits of 55,000 moleculr weight nd therefore is probbly identicl to component A. Component III' dissocites into component I. The second, less pure preprtion (Fig. S) showed, in ddition, component II (pproximte Mr 85,000) which pprently lso dissocited to give I. (ii) Substrte specificity. The substrte specificity of the purified peptidse ws investigted to understnd the role of this enzyme in the provision of the cell with nitrogen nd essentil mino cids. The hydrolytic ctivity towrds rnge of peptides nd peptide derivtives ws surveyed. A summry of the results is given in Tble 2. These results show tht the enzyme cleves peptide bonds in which the co-crboxyl group of glutmic cid or sprtic cid t the mino end of the chin is involved. An N-terminl glutryl or succinyl group is lso liberted but not the L-glutminyl or L--y-glutmyl group. No endopeptidse ctivity towrds methyl-'4c-lbeled cseins nd lbumin could be detected. (iii) Influence of bivlent ctions, chelting gents, nd group-specific inhibitors. Under the conditions pplied, Mn2+, Ni2 +, nd Cd2+ hd mrked inhibitory effect on the enzyme's ctivity, wheres with Cu2+ nd Hg2+full inctivtion ws estblished. The enzyme ws completely inctivted by 4-(hydroxymercuri)benzoic cid (1 mm) but not by other sulfhydryl group-blocking regents (viz., iodocetic cid, iodocetmide, nd N-ethylmleimide t concentrtions up to 10 mm). Complete inctivtion ws lso obtined with DTT (2 mm) nd with the chelting gent EDTA (1 mm) or 1,10-phennthroline (1 mm). In these cses SDS-PAGE did not revel dissocition into subunits (not shown). The ctivity lost by tretment with 1,10-phennthroline ws lmost completely restored by Co2 + nd Zn2 + dded t finl concentrtion of 5 mm. Mn2+ ws less effective, nd Cd2, Ni2+, Cu2+, Mg2+, Sr2+, C2+, nd Hg2+ hd no or only slight effect. (iv) Stbility. The optimum temperture for gluap ctivity in the cell extrct ws pproximtely 50 to 55 C (8). Heting

5 VOL. 53, 1987 b AMINOPEPTIDASE A FROM S. CREMORIS 581 W. i,- _,14400% Z '2' I- e '- 1 2 I1 B ~92500 sii1 N> 16250/' i FIG. 5. SDS-PAGE (8.5%) of Sephcryl S-300 column frction from butnol-treted AS preprtion () nd similr frction subjected to second butnol tretment followed by gel filtrtion (b). The smples were either heted t 100 C or not heted. Lnes: 1 nd 8, high-moleculr-weight reference proteins; 2, first Sephcryl S-300 gluap frction (not heted); 3, first Sephcryl S-300 gluap frction (heted); 4 nd 5, low-moleculr-weight reference proteins; 6, second Sephcryl S-300 gluap frction (heted); 7, second Sephcryl S-300 gluap frction (not heted). of the enzyme solution between 50 nd 75 C t ph 7.5 for 15 min resulted in loss of ctivity up to mximum of 15 to 20%. Heting of the enzyme solution t these tempertures s function of time up to 60 min lso resulted in mximum loss of ctivity of pproximtely 20%. Above 75 C the ctivity loss ws considerble. DISCUSSION The present results suggest the occurrence in S. cremoris of peptide hydrolse specific for peptide bonds in which n mino-terminl L--glutmyl (sprtyl) residue is involved. The enzyme shows no strict requirement for the free mino group, since glutryl nd succinyl peptide derivtives re lso good substrtes. This my indicte tht the free -ycrboxyl group rther thn the mino group is essentil. If n mino group ws present next to the free crboxyl group (L--y-glutmyl peptide) or if the -mino group ws substituted, no hydrolysis could be detected, suggesting steric hindrnce. With respect to its nturl peptide substrtes, this enzyme my therefore be clssified s n L--glutmyl (sprtyl) minopeptidse (minopeptidse A) (EC ). Aminopeptidse A hs been found in vrious orgns nd ser of nimls (see reference 24). It hs been purified only prtilly from humn serum (17) nd from the brush border of rbbit smll intestine, since it ppered difficult to seprte the enzyme from other minopeptidses (1). Tobe et l. (24) hve succeeded in purifying the enzyme from pig kidney, using n ffinity column consisting of n grose mtrix linked to specific inhibitor, msttin, isolted from culture filtrtes of Streptomyces strin. A similr enzyme from hog intestinl brush border ws lso purified nd III' chrcterized (2). Both of these mmmlin minopeptidses A re more ctive towrds substrtes with N- terminl glutmic cid thn towrds those with sprtic cid. An enzyme from dog kidney removes sprtyl residues fster thn glutmyl residues (6). An minopeptidse with unique specificity for L--sprtyl peptides hs been prtilly purified from Slmonell typhimurium (5). A P- sprtyl peptidse from Escherichi coli hs been purified nd chrcterized nd shown to be specific for P-sprtyl dipeptides. In soil orgnisms of the genus Flvobcterium, L--glutmyl-L-glutmte ctivity hs been prtilly purified but not tested on other L--glutmyl peptides (20). We re wre of no cse of purifiction nd chrcteriztion of minopeptidse A from microorgnisms. Few dt re vilble concerning minopeptidse profiles of lctic streptococci which indicte the presence of L--glutmyl minopeptidse ctivity (12, 16). The existence of specific enzyme in S. cremoris ssocited with nd functioning t the outside surfce of the membrne hs been suggested (8), nd its specific ctlytic property hs been confirmed in this study. Our results indicte tht the minopeptidse A from S. cremoris is polymeric metlloenzyme consisting of identicl monomers (pproximte Mr 43,000). Tretment of the enzyme with chelting gents mintins n inctive conformtion of the polymer which llows the binding of metl ion ctivtors, Co2+ nd Zn2+ being exclusively effective. In the presence of DTT complete inctivtion ws obtined without dissocition of the polymer being observed. Apprently reduction of intrmoleculr rther thn intermoleculr disulfide bridges tkes plce. Heting t 100 C in the bsence of DTT nd SDS results in complete inctivtion nd dissocition into subunits. The reduction in

6 582 EXTERKATE AND DEVEER TABLE 2. Substrte specificity of the purified gluap Substrte Hydrolysis' L-ot-Glu-L-Al L-Al-L-Glu L-ct-Glu-Gly L-GluN-Gly L--Glu-L-Glu + L--GlU-L-Al-L-Al L-Al-L-Asp L-Gly-L-Asp L--ASp-L-Phe L---ASp-L-Leu L-Phe-L-Asp - L-Lys-L-Glu-Gly - Gly-Gly-L-Glu-L-Al-methyl ester L-Arg-L-GlU-L-Leu - Z-L--Glu-L-Tyr Z-L--Glu-L-Phe N-Acetyl-L-Ile-L-Glu-Gly-L-Arg-pNA L--Glu-pNA + + Glutryl-L-Phe-pNA + + Succinyl-L-Phe-pNA + + Pyroglu-L-Al Pyroglu-pNA L-y-Glu-pNA - L-y-GIU-L-Phe - Gly-L-Phe Z-L-Phe-L-Tyr - Z-Gly-L-Phe 4CH3-lbeled whole csein 4CH3-lbeled,-csein "CH3-lbeled o,1-csein '4CH3-lbeled lbumin Hydrolysis of the vrious substrtes is described s follows: +++ = hydrolysis (nerly) completed within 60 min; + + = good substrte, but hydrolysis is not completed within 60 min; + = slight hydrolysis detectble; - = no detectble hydrolysis fter 24 h. ctivity t reltively low tempertures my be due to either conformtionl chnge or the presence of more unstble frction. Pretretment of crude enzyme preprtion with butnol results in still ctlyticlly ctive component of moleculr weight 130,000, suggesting trimer. Component II (moleculr weight, 85,000) might be the intermediry dimer. The higher-moleculr-weight (heterogeneous) component III might represent this trimer still ssocited with mteril (lipid?) which interferes with the ction of SDS under the present conditions nd which otherwise hs been removed by butnol. Tht both components III nd III' re eluted during gel filtrtion t exctly the sme reltive elution volume sustins this view. Alterntively, the ntive enzyme might be tetrmer, nd trimeric complex is induced by butnol tretment. Compred with the well-chrcterized enzyme from pig kidney (24), both enzymes hve sensitivity to chelting gents, optimum ph nd optimum temperture, nd insensitivity to sulfhydryl-blocking gents (except p-hydroxymercuribenzote) in common. In contrst, the pig enzyme (Mr 300,000) ws not inctivted by S-S dissociting gents, ws completely inhibited by Zn+ nd ws (re)ctivted by lkline ctions, especilly C2. Glutmic cid, the product of the ction of the present enzyme, is n essentil nimo cid nd in ddition hs the highest minimum concentrtion required for protein synthesis nd mximum growth of S. cremoris (23). The occurrence of n minopeptidse A in this orgnism my ensure tht, by its specific ction subsequent to the ction of the cell wll APPL. ENVIRON. MICROBIOL. proteinse(s) nd in concert with other minopeptidses or not, the cell will be efficiently provided with this essentil mino cid. Severl results re indictive of membrnebound stte of this enzyme (8). These include results which show tht the enzyme in situ is ffected by chnges ttended by the energiztion nd de-energiztion of the membrne (unpublished results). An involvement of the enzyme in specific nionic permese system such s tht detected in Streptococcus feclis might be postulted (19). Such trnsport system, restricted to glutmyl (sprtyl) peptides, could be device to stisfy most efficiently the need of this orgnism for glutmic cid. LITERATURE CITED 1. Andri, G., A. Mrzi, nd S. Auricchio Glutmyl-pnphthyl mide hydrolse of rbbit smll intestine. Locliztion in the brush border nd seprtion from other brush border peptidses. Biochim. Biophys. Act 419: Benjib, A., nd S. Mroux Purifiction nd chrcteriztion of n minopeptidse A from hog intestinl brush-border membrne. Eur. J. Biochem. 107: Bhown, A. S., J. E. Mole, F. Hunter, nd J. C. Bennett High-sensitivity sequence determintion of proteins quntittively recovered from sodiumdodecylsulphte gels using n improved electrodilysis procedure. Anl. Biochem. 103: Brdford, M. M A rpid nd sensitive method for the quntittion of microgrm quntities of protein utilizing the principle of protein dye binding. Anl. Biochem. 72: Crter, T. H., nd C. G. Miller Asprtte-specific peptidses in Slmonell typhimurium: mutnts deficient in peptidse E. J. Bcteriol. 159: Cheung, H. S., nd D. W. Cushmn A soluble sprtte minopeptidse from dog kidney. Biochim. Biophys. Act 242: Exterkte, F. A An introductory study of the proteolytic system of Streptococcus cremoris strin HP. Neth. Milk Diry J. 29: Exterkte, F. A Loction of peptidses outside nd inside the membrne of Streptococcus cremoris. Appl. Environ. Microbiol. 47: Exterkte, F. A., nd G. J. C. M. de Veer Prtil isoltion of nd degrdtion of cseins by cell wll proteinse(s) of Streptococcus cremoris HP. Appl. Environ. Microbiol. 49: Hley, E. E Purifiction nd properties of P-sprtyl peptidse from Escherichi coli. J. Biol. Chem. 243: Hermsdorf, C. L., nd S. Simmonds Role of peptidses in utiliztion nd trnsport of peptides by bcteri, p In J. W. Pyne (ed.), Micro-orgnisms nd nitrogen sources. John Wiley & Sons Ltd., Chichester, Englnd. 12. Kminogw, S., T. Ninomiy, nd K. Ymuchi Aminopeptidse profiles of lctic streptococci. J. Diry Sci. 67: Kolstd, J., nd B. A. Lw Comprtive peptide specificity of cell wll, membrne nd intrcellulr peptidses of group N streptococci. J. Appl. Bcteriol. 58: Lw, B. A., E. Sezgin, nd M. E. Shrpe Amino cid nutrition of some commercil cheese strters in reltion to their growth in peptone-supplemented whey medi. J. Diry Res. 43: Mills, 0. E., nd T. D. Thoms Relese of cell-wll ssocited proteinse(s) from lctic streptococci. N. Z. J. Diry Sci. Technol. 13: Mou, L., J. J. Sullivn, nd G. R. Jgo Peptidse ctivities in group N streptococci. J. Diry Res. 42: Ngtsu, I., T. Ngtsu, T. Ymmoto, G. G. Glenner, nd J. W. Mehl Purifiction of minopeptidse A in humn serum nd degrdtion of ngiotensin II by the purified enzyme. Biochim. Biophys. Act 198: Pyne, J. W Micro-orgnisms nd nitrogen sources, p John Wiley & Sons Ltd., Chichester, Englnd. 19. Pyne, J. W., G. M. Pyne, nd T. M. Nisbet An nionic

7 VOL. 53, 1987 peptide trnsport system in Streptococcus feclis. FEMS Microbiol. Lett. 14: Prtt, A. G., E. J. Crwford, nd M. Friedkin The hydrolysis of mono-, di- nd triglutmte derivtives of folic cid with bcteril enzymes. J. Biol. Chem. 243: Rice, G. H., F. H. C. Stewrt, A. J. Hillier, nd G. R. Jgo The uptke of mino cids nd peptides by Streptococcus lctis. J. Diry Res. 45: Thoms, T. D., B. D. W. Jrvis, nd N. A. Skipper Locliztion of proteinse(s) ner the cell surfce of Streptococcus lctis. J. Bcteriol. 118: AMINOPEPTIDASE A FROM S. CREMORIS Thoms, T. D., nd 0. E. Mills Proteolytic enzymes of strter bcteri. Neth. Milk Diry J. 35: Tobe, H., F. Kojim, T. Aoygi, nd H. Umezw Purifiction by ffinity chromtogrphy using msttin nd properties of minopeptidse A from pig kidney. Biochim. Biophys. Act 613: Visser, S Proteolytic enzymes nd their ction on milk proteins. A review. Neth. Milk Diry J. 35: Weber, K., nd M. Osborn The relibility of moleculr weight determintions by dodecylsulphte polycrylmide gel electrophoresis. J. Biol. Chem. 244: