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Purification and characterisation of an aminotripeptidase from cytoplasm of Lactococcus lactis subsp. cremoris AM2
Int. Dairy Journal 3 (1993) 163-177
Purification and Characterisation of an Aminotripeptidase from Cytoplasm ofLactococcus lactis subsp, cremoris AM2
Christopher L. Bacon Department of Biochemistry, University College, Galway, Ireland
Martin Wilkinson National Dairy Product Research Centre, Moorepark. Fermoy, Co. Cork. Ireland
&
P. Vincent Jennings, Ide Ni Fhaolain & Gerard O'Cuinn* Department of Life Sciences, Regional Technical College, Galway, Ireland (Received 3 October, 1991; revised version accepted 13 April 1992)
ABSTRACT An aminopeptidase was purified 120-foM with a 78% recovery from the cytoplasm ofLactococcus lactis subsp, cremoris AM2. The purified enzyme exhibited no activity on dipeptides, dipeptideamides, tripeptideamides or tetrapeptides. As activity was observed only with tripeptides, from which it released the N-terminal amino acid, the enzyme was adjudged to be a strict aminotripeptidase. The enzyme had a Mr of 105 000 and showed one band, corresponding to an Mr of 55 000 on SDS-PAGE electrophoresis. Inhibition by EDTA. 8-hydroxyquinoline and 1,10 phenanthroline indicated that the peptidase was a metallo enzyme. Dithiothreitol, bestatin and amastatin caused total inhibition, whereas p-chloromercuribenzoate, bacitracin and phenyl methyl sulphonyl fluoride were without effect. K,, values for tripeptides were within the range 0.18 (Leu-Leu-Leu) to 0.38 m n (Trp-Gly-Gly). Substrate inhibition was noted with some tripeptides at concentrations above 1.5 mM. *To whom correspondence should be addressed. 163 Int. Dairy Journal 0958-6946/92/$05.00 © 1992 Elsevier Science Publishers Ltd, England. Printed in Ireland
164
C. L. Bacon, M. Wilkinson, P. l~ Jennings. I. N. Fhaolain, G. O'Cuinn
INTRODUCTION Because of the low concentration of free amino acids in milk, the proteolytic and peptidolytic systems of starter bacteria are vital for the release of free amino acids from casein to allow the growth of these starter bacteria to achieve high densities in milk (Thomas & Mills, 1981). The initial stages of casein breakdown are brought about by proteolytic enzymes located on the surface of starter bacteria (Bockelmann et al., 1989). The generation ofpeptides suitable for transport into the bacterial cell may involve further hydrolysis by, as yet unidentified, peptidases either on the surface of the bacteria or secreted by them. Peptides transported into bacteria are degraded to amino acids by a limited range of intracellular peptidases (Thomas & Pritchard, 1987). A definition of the range ofintracellular peptidases has been the subject of investigation by a number of groups. Kolstad & Law (1985) subjected cytoplasmic and particulate extracts from strains of starter bacteria to polyacrylamide gel electrophoresis and, using a variety of dipeptides and tripeptides in an activity stain, they identified a number of bands of activity. Their results suggested that a distinct band of activity was present which was responsible for the hydrolysis of tripeptides in cytoplasm. Kaminogawa etal. (1984) separated cytoplasm from a range of starter organisms by DEAE-cellulose chromatography. When the activity ofeluates from the column was scanned on a wide variety of dipeptides, tripeptides, tetrapeptides and aminoacyl naphthylamides, the results obtained showed that while two eluted activities could hydrolyse tripeptides, only one of these could hydrolyse tripeptides only. The suggestion that the cytoplasm of starter bacteria possesses an enzyme that is specific for tripeptides was confirmed by a recent publication on the purification and characterisation of such an activity from Lactococcus lactis subsp. cremoris Wg2 (Bosman et al., 1990). These authors purified this enzyme to homogeneity and showed that it hydrolysed tripeptides only, and that it was inhibited by EDTA and dithiothreitol. The native enzyme was found to have a molecular mass of between 103 000 and 105 000 daltons with two equal subunits of 52 000 daltons. The present study confirms the presence of a very similar aminotripeptidase in the cytoplasm of Lactococcus lactis subsp, cremoris AM2.
MATERIALS Lactococcus lactis subsp, cremoris AM2 and low heat skim milk were
provided by the National Dairy Product Research Centre, Moorepark,
Purification and characterisation of an aminotripeptidase
165
Fermoy, Ireland. EDTA, Tris, lysozyme, DNase (bovine pancreas, type IV), RNase (bovine pancreas, type Ill-A), fructose 1, 6 diphosphate, NADH (disodium salt), pyruvic acid, disodium ATP, p-hydroxyphenylacetic acid, L-amino acid oxidase (Type IV from Crotalus atrox), peroxidase (Type II horseradish), 1, 10-phenanthroline, 8-hydroxyquinoline, iodoacetamide, benzamidine, bacitracin, amastatin, bestatin and dithiothreitol were obtained from Sigma Chemical Company, Poole, Dorset, England. Butan-l-ol and formic acid were obtained from BDH, Poole. England. All peptides and peptide derivatives were obtained from Bachem Feinchemikalien, Bubendorf, Switzerland. Cellulose fine powder (CF 1) and DE-52 anionic exchanger were provided by Whatman, Maidstone, England. Sephacryl S-200 high resolution gel was obtained from Pharmacia Fine Chemicals AB (Uppsala, Sweden). Silica gel G plates were obtained from Merck.
METHODS Assay for tripeptidase Tripeptidase activity was measured by incubating 50/~1 of sample with 450/~1 of 1-11 mM L-Phe-Gly-Gly or L-Leu-L-Leu-L-Leu in 75 mM borate, pH 7.5, for 10 min at 30°C. The reaction was terminated by heating to 90°C for 5 min and the phenylalanine or leucine released measured, after cooling, by the coupled L-amino acid oxidase-peroxidase system (Nicholson & Peters, 1978) in which oxidised p-hydroxyphenylacetic acid was determined fluorimetrically using excitation and emission wavelengths of 317 and 414nm, respectively. The unit of aminotripeptidase activity is defined as the amount of enzyme which releases 1 nmol of N-terminal Phe from Phe-Gly-Gly min -I in 75 mM borate, pH 7.5, at 30°C. Protein determination Protein concentration was determined by the method of Lowry et ai. (1951), using bovine serum albumin as standard. Production of subcellular fractions from Lactococcus lactis subsp, cremoris AM2 Lactococcus lactis subsp, cremoris AM2 cells were grown, harvested,
washed and subjected to subcellular fractionation as described previously
166
C. L. Bacon, M. Wilkinson, P. V. Jennings, I. N. Fhaolain, G. O'Cuinn
by Booth et al. (1989, 1990). The presence of cell m e m b r a n e material in recovered fractions was monitored by measurement of ATP-ase activity (Abrams, 1965) in which liberated phosphate was measured by the method of King (1932). The presence of cell wall material was monitored by measuring glucosamine using a modification (Aidoo et al., 1981) of the method of Rondle & Morgan (1955), while lactate dehydrogenase activity (Wittenberger et al., 1970) was indicative of cytoplasm.
Preparation of calcium phosphate-cellulose Calcium phosphate gel was prepared as described by Barranger et al. (1972). Batches of cellulose powder were prepared as described by Donlon & Kaufman (1980). Prior to column packing, the washed cellulose powder was made into a 10% (w/v) suspension in 20 mM potassium phosphate buffer, pH 7-5, containing 0.15M KC1. Five volumes of this suspension were then added to one volume of the calcium phosphate gel. The resultant mixture was mixed well at 4°C until a homogeneous suspension was obtained.
Purification of aminotripeptidase The cytoplasmic fraction was dialysed against 50 mM Tri-HC1, pH 7.5, containing 0.1 M NaCI and applied to a Sephacryl S-200 column. Activity was eluted from the column in 50 mM Tris-HC1, pH 7-5, containing 0.1 M NaC1 at a flow rate of 16 ml h-~; 3 ml fractions were collected. The tubes containing aminotripeptidase activity were poole& dialysed against 50 mM Tris-HC1, pH 7.5~ and applied to a W h a t m a n DE-52 anion exchange column (3.4 cm X 1.5 cm). After washing with two column volumes of 50 mM Tris-HCl, pH 7-5, the activity was eluted in a gradient established between 50 mM Tris-HC1, pH 7.5, and 50 mM Tris-HC1, pH 7.5, containing 0.75 M NaC1. The eluates were collected in 2 ml fractions at a flow rate of 10 ml h -~. The active fractions were pooled, dialysed against 20 mM potassium phosphate, pH 7.2, containing 0.15 M KC1 and loaded onto a calcium phosphate-cellulose column ( 5 c m X 1 cm) pre-equilibrated with 20mM potassium phosphate, pH 7.2, containing 0.15 M KC1. The column was washed with two volumes of the equilibration buffer and activity was eluted using a linear gradient established between 20 mM potassium phosphate, pH 7-2, containing 0.15 M KCI and 250mM potassium phosphate, pH 7.2, containing 0.15 M KC1. The eluates were collected in 1 ml fractions and active fractions were pooled. Prior to assaying, with a view to
Purification and characterisation of an aminotripeptidase
167
determining specific activity and yield, aliquots of each fraction were dialysed against water. Effect of inhibitors on the activity of purified tripeptidase Aliquots (50/.tl) of a solution of purified enzyme (9 activity units) were preincubated at 30°C for 15 min with 50/A of inhibitor solution in water. (For final concentrations see Table 3). Tripeptide hydrolysis was initiated by adding 400:11 of 1.25 mM P h e - G l y - G l y in 75 mM borate, pH 7.5, and the reaction mixture incubated for 10 min. The reaction was terminated by heating to 90°C for 5 min and the phenylalanine released was estimated as described before. The activity observed with each inhibitor was related to the activity observed when water (50/~1) was used instead of inhibitor solution. Some of the inhibitors employed, notably 1,10-phenanthroline, dithiothreitol, 8-hydroxyquinoline and captopril, inhibited the L-amino acid oxidase-peroxidase system. In experiments where these inhibitors were used, standard curves relating phenylalanine concentration to fluorescence in the presence of the appropriate concentration of each inhibitor were constructed. Determination of Mr A solution of tripeptidase activity (540 activity units) was loaded onto a Sephacryl S-200 column (88 cm X 2-5 cm diam.), previously equilibrated with 50 mM Tris-HCl, pH 7.5, containing 0.1 M NaC1 and calibrated using ferritin (440000), aldolase (158 000), bovine serum albumin (67 000), ovalbumin (43 000) and chymotrypsin (26 000). The tripeptidase activity was eluted from the column with 50 mM Tris-HCl, pH 7.5, containing 0.1 M NaC1 and collected in 3 ml fractions at a flow rate of 16 ml h -~. The Mr of the tripeptidase was computed by the relationship of its elution volume to elution volumes of standards of known Mr. Subunit molecular weight determination Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) was performed as described by Laemmli (1970) using 12% acrylamide gels. Coomassie brilliant blue G250 was used to visualize the proteins after electrophoresis and the molecular weight of the protein band was computed by reference to the migration of low molecular weight SDS-PAGE standards (Bio-Rad labs, Richmond, California).
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C L. Bacon, M. Wilkinson, P. V. Jennings, 1. N. Fhaolain, G. O'Cuinn
Stability of purified tripeptidase at 10°C and at pH 5.2 A solution of the purified tripeptidase (180 activity units) was dialysed against 100 mM sodium acetate, pH 5.2, containing 5%, w/v, NaCI. The dialysed solution was then incubated at 10°C and samples were withdrawn and assayed for tripeptidase activity using P h e - G l y - G l y as substrate on days 0, 8, 17, 30 and 67 after the c o m m e n c e m e n t of incubation. The release of phenylalanine was measured as described above.
Stability of purified tripeptidase at different temperatures A solution of the purified tripeptidase in 100 mM potassium phosphate, pH 7.2, containing 0.15 M KC1 was incubated at 25°C, 37°C or 50°C. After 0, 10~ 20, 30 and 60 min, samples were taken from each incubation mixture and assayed for tripeptidase activity at 30°C using P h e - G l y - G l y as substrate.
Substrate specificity of the purified tripeptidase Aliquots (50pl) of a solution of purified tripeptidase (9 activity units) were incubated with 450 pl of 1.11 mM solutions of each peptide to be tested in 75 mM borate, pH 7-5, at 30°C. After 10 min, the reaction was terminated by heating to 90°C for 5 min and the amino acids released quantified as described above using appropriate standard curves, relating the amount of each amino acid to the intensity of fluorescence. Alternatively, where release of the amino terminal amino acid could not be quantified by the coupled L-amino acid oxidase-peroxidase system, incubation of 50 pl of tripeptidase solution with 50pl of 4 mM peptide in 75 mM borate, pH 7.5, was allowed to proceed for 16 h. Portions of each reaction mixture (50pl) were chromatographed on silica gel G plates using butanol:formic acid:water (20:6:5, v:v:v) as solvent system. Appropriate standards were co-chromatographed.
Determination of kinetic parameters Tripeptidase solutions (9 activity units) were incubated with various concentrations oftripeptides in 450 pl of 75 mM borate, pH 7.5, yielding a range of final concentrations oftripeptide from 0-065 to 1-5 raM. After 10 min, the reaction was terminated and the released amino acids measured as described above. Km values were calculated from Lineweaver-Burk plots.
Purification and characterisation of an aminotripeptidase
169
RESULTS The data presented in Table 1 indicate that while the majority of the tripeptidase activity was located in the cytoplasm, a significant level of activity was associated with the cell m e m b r a n e fraction. No tripeptidase activity was detected in cell wall or wash fractions or in the extracellular fluid. The subcellular fractionation procedure employed in this study was monitored for its efficiency in yielding fractions enriched in cell walls, cell m e m b r a n e s and cytoplasm by the use of markers. The results obtained were very similar to those reported in a previous study (Booth etal., 1989) and showed enrichment of Mg2+ATP-ase in the cell m e m b r a n e fraction, of glucosamine in the cell wall fraction and of lactate dehydrogenase in the cytoplasm. Chromatography of the cytoplasmic fractions on Sephacryl S-200 resolved the activity against P h e - G l y - G l y into three peaks (Fig. 1). Activity against Lys-pNA was associated with the third peak, as was activity against P r o - L e u - G l y - G l y from which both Pro and Leu were released. The third peak of activity was also able to remove Leu and Trp from Leu-Trp-Met-Arg and Leu, Trp and Met from L e u - T r p - M e t - A r g Phe. The second peak of activity was pooled and chromatographed on DE-52, from which it eluted as a single peak at 0.55 M NaC1 in a linear salt gradient. The pooled peak of activity was then chromatographed on a calcium phosphate-cellulose column from which it also eluted as a single peak at 45 mM potassium phosphate. The data in Table 2 show that the three step purification resulted in a 120-fold purification of one of the P h e - G l y - G l y hydrolysing activities of the cytoplasm with a recovery of 78%. TABLE I S u b c e l l u l a r D i s t r i b u t i o n o f H y d r o l a s e Activity a g a i n s t L e u L e u - L e u a n d P h e - G l y - G l y in S u b c e l l u l a r F r a c t i o n s D e r i v e d f r o m Lactococcus lactis subsp, cremoris A M 2
nmoles of N-terminal aminoacid re~eased (min -I tota/ fraction -1)
E x t r a c e l l u l a r fluid Wash Cell wall Cell m e m b r a n e Cytoplasm
Phe-Gly-Gly
Leu-Leu-Leu
---1 540 2 800
---2 520 3 850
170
C. L. Bacon, M. Wilkinson, P V. Jennings, I. N. Fhaolain, G. O'Cuinn 1
2
3
12
-6 1-0
10 -4
~E
2 2 <
0.50 -2
A~
...Z~,~
-5
I~
tt tXl~ A
~. 0 100
I
i
2OO
30O
. . . .
Elution Volume (ml) Fig. 1. The clution of cytoplasm (predialysed into 50 mM Tris-HCl, pH 75) from Laetococcus lactis subsp, cremoris AM2 off Sephacryl S-200 in 50 mM Tris-HCL pH 7-5. Tripeptidase activity measured using P h e - G l y - G l y as substrate ( l - - - - - l ) . Aminopeptidase activity using Lys-pNA as substrate ( O - I - O ) . Protein measured by absorbance at 280 mM (A . . . . A). Fractions that released Pro and Leu from Pro-LeuGly-Gly, Leu and Trp from Leu-Trp-Met-Arg and Leu, Trp and Met from Leu-TrpMet-Arg-Phe are indicated by < . . . . >. Void volume was calculated using ferritin (mol wt. 440 000) and by measuring eluates at 280 nm: 3-ml fractions were collected at a flow rate of 16 ml h t.
TABLE 2 Purification of Aminotripeptidase from Cytoplasm of Lactococcus lactis subsp, cremoris AM2. (Aminotripeptidase Activity Assayed Using P h e - G l y - G l y as Substrate)
Total protein (rag)
Cytoplasm Post Sephacryl S-200 Post DE-52 Post calcium phosphatecellulose
Total enzyme activity (nmol Phe min -1)
Specific activity (nmol Phe min -1 mg -/)
Purification
Yield (%)
9'0 1"38
I 150 5 250
127-7 3 804
1"0 29"78
100 456
0'507 0'0585
2 340 900
4 615 15 384
36' 14 120'47
203 78
Purification and characterisation of an aminotripeptidase
171
Incubation of the purified tripeptidase with a range of possible inhibitors (Table 3) showed that no inhibition occurred with p-chloromercuribenzoate, N-ethylmaleimide, bacitracin or phenyl methyl sulphonyl fluoride. Modest inhibition was noted with benzamidine and iodoacetamide while more significant inhibition was caused by EDTA and puromycin. Bestatin and amastatin caused total inhibition. After allowing for the effect of captopril, 1,10-phenanthroline, dithiotreitol and 8-hydroxyquinoline on the L-amino acid oxidase-peroxidase system, 1,10-phenanthroline and captopril were found to significantly inhibit the activity of the tripeptidase while total inhibition was caused by dithiotreitol and 8-hydroxyquinoline. These observations were confirmed by chromatography of incubates containing each inhibitor on silica gel G plates where total inhibition of the tripeptidase was observed with dithiothreitol and 8-hydroxyquinoline while only partial inhibition occurred with 1,10-phenanthroline and captopril. The enzyme was active over a wide pH range (6.8-9.1), with m a x i m u m activity at pH 8.6. The relative molecular mass of the purified tripeptidase was computed to be 105 000 and S D S - P A G E showed only one b a n d with a relative molecular mass of 55 000. The ability of the purified enzyme to hydrolyse a range of dipeptides, tripeptides and tetrapeptides was tested. Only tripeptides were hydrolysed and in each case, only the amino terminal amino acid of the tripeptide TABLE 3
The Effect of Inhibitors on the Activity of an Aminotripeptidase Purified from Cytoplasm of Lactococcus lactis subsp, cremoris AM2
Inhibitor used
1~ 10 Phenanthroline 8-Hydroxyquinoline EDTA lodoacetamide N-ethylmaleimide p-Chloromercuriben zoate Captopril Bestatin Dithiothreitol Amastatin Puromycin Benzamidine Bacitracin Phenylmethylsulphonylfluoride
Concentration of inhib#or (mM)
Remaining activiW (% of control)
0"05 1-0 1.0 5"0 1-0 0" 1 1-0 1.0 2"0 1-0 5"0 5"0 0" 1 1"0
12-5 0 32"0 72"9 100'0 100-0 46.0 0-0 0'0 3"0 62"5 83"3 100.0 100.0
172
C L. Bacon. M. Wilkinson, P. V. Jennings, I. N. Fhaolain, G. O'Cuinn
was released (Table 4). Dipeptide amides (Leu-Leu-NH2, Leu-GlyNH2) or tripeptide amides (Gly-GIy-GlyNH2, Leu-Leu-LeuNH2) were not hydrolysed. The tripeptidase released a range of amino terminal amino acids from tripeptides presented, including proline from Pro-Gly-Gly and aspartic acid from Asp-Tyr-Met. No hydrolysis was observed, however, when proline was present in the central position in a tripeptide. Km values of the purified enzyme with eight substrates (Table 5) revealed little variation in affinity for these substrates. Trp-Gly-Gly, with a Km value of 0.38 mM appeared to have least affinity for the enzyme, gca t values also did not vary notably with Met-Gly-Gly (201-6s -t) showing the highest value and Trp-Gly-Gly (117.6s -I) showing the lowest value. The purified aminotripeptidase showed no loss of activity at 25°C or 37°C over a 60 min incubation at these temperatures. Incubation at 50°C, however, resulted in a 50% loss in activity after 13 min. When the purified aminotripeptidase was
TABLE 4 Substrate Specificity of an Aminotripeptidase Purified from Cytoplasm of Lactococcus lactis subsp, cremoris A M 2 Arg-Asp Ala-Gln
-"
lie-lie-lie
+h
-
Phe-Gly-Gly
+
Ala-Ala
-
Thr-Gly-Gly
+
Ala-Gly
-
Tyr-Gly-Gly
+
Ala-Gly-Gly
+
Met-Gly-Gly
+
Ala-Gly-Gly-Gly
-
Trp-Gly-Gly
+
Ala-Gly-Gly-Gly-Gly
-
Lys-Gly-Gly
+
Tyr-Pro
-
Arg-Gly-Gly
+
Gly-Pro
-
Pro-Gly-Gly
+
Gly-Gly
-
Glu-Gly-Phe
+
-
Glu-Asp
-
+
Asp-Lys
-
Gly-GlyNH
2
Gly-Gly-Gly GIy-GIy-GIyNH
2
Gly-Gly-Gly-Gly Leu-Gly
-
Asp-Tyr-Met
+
-
Ala-Pro-Arg
-
-
Ala-Pro-Gly
-
-
Arg-Pro-Pro
-
Leu-Gly-Gly
+
Ile-Pro-lle
-
Leu-Leu
+
Pro-Pro-Pro Pro-Pro
-
-
Pro-Phe
-
Leu-GlyNH
2
Leu-Leu-Leu Leu-Leu-LeuNH Leu-Ala-Pro
2
+
" - indicates no hydrolysis. t'+ indicates cleavage of N-terminal amino acid.
-
Purification and characterisation of an aminotripeptidase
173
TABLE 5
Km, Vmaxand Kcat Values Derived from the Interaction of the Purified Aminotripeptidase with Tripeptide Substrates
Substrate
Met-Gly-Gly Trp-Gly-Gly Phe-Gly-Gly Leu-Gly-Gly Leu-Ala-Pro Leu-Leu-Leu Leu-Gly-Pro Tyr-Gly-Giy
K,,,
V.no.,-
K~.a,
(raM)
(nmol min -l m1-1)
(s -t)
0"23 0"38 0'20 0.19 0' 24 0' 18 0'33 0"22
571 540 434 400 392 370 344 333
201"6 190.7 153"2 141-2 132 '4 i 30"6 12 !'5 117-6
incubated in sodium acetate, pH 5.2, containing 5% NaC1 (w/v) at 10°C, 88% of starting activity was recovered after 30 days incubation. After 67 days only 25% of the initial activity remained.
DISCUSSION The results presented in Table 1 indicate that the majority of peptidase activity against either Phe-Gly-Gly or Leu-Leu-Leu in Lactococcus lactis subsp, cremoris AM2 resided in the cytoplasmic fraction. No activity against either tripeptide was observed in the extracellular fraction, in the wash or in the cell wall fraction. Significant activity against these tripeptides was found in the cell membrane fraction. The relationship between the cytoplasmic activity and that of the cell membrane remains to be determined. When the cytoplasmic activity against Phe-Gly-Gly was chromatographed on Sephacryl S-200 and when the eluted fractions were monitored for activity against Phe-GlyGly, Lys-pNA, Pro-Leu-Gly-Gly, Leu-Trp-Met-Arg and Leu-TrpMet-Arg-Phe, two adjacent peaks of activity against Phe-Gly-Gly were observed, preceded by a minor peak of activity (Fig. 1). Peak 3 was found to contain activity against Lys-pNA, Pro-Leu-Gly-Gly, Leu-Trp-MetArg and Leu-Trp-Met-Arg-Phe, suggesting the presence of a general aminopeptidase similar to that previously described for Lactococcus lactis subsp, cremoris Wg2 (Tan & Konings, 1990). The second peak, active only against tripeptides, was selected for further purification as a possible aminotripeptidase similar to those previously described in
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C. L. Bacon, M. Wilkinson, P. V. Jennings, I. N. Fhaolain, G. O'Cuinn
Lactococcus lactis subsp, cremoris Wg2 (Bosman etal., 1990) and in Streptococcus diacetylactis 267 (Desmazeud & Zevaco, 1979).
The purification data (Table 2) show that while chromatography on Sephacryl S-200 separated three enzymes in cytoplasm capable of hydrolysing Phe-Gly-Gly (Fig. 1), a dramatic increase was observed in Phe-Gly-Gly hydrolysing activity of the partially purified aminotripeptidase relative to the level observed in the cytoplasm. Precautions had been taken to dilute the cytoplasm such that the Phe-Gly-Gly hydrolysing activity was linear with respect to the dilution of the cytoplasm. In spite of this precaution, the increase in activity was observed consistently during repeated purifications. When the enzyme activity recovered from Sephacryl S-200 gel filtration column was incubated with aliquots of other fractions from the column no significant reduction in activity was noted. Studies on the apparent cytoplasmic inhibition of this Phe-Gly-Gly hydrolysing activity are continuing. In addition, this step separated the aminotripeptidase not only from the broad specificity peptidase which hydrolysed Phe-Gly-Gly, Lys-pNA, Pro-Leu-Gly-Gly, Leu-Trp-Met-Arg and Leu-Trp-Met-Arg-Phe but also from the minor peak of activity. The purified aminotripeptidase removed the amino terminal amino acid from all the tripeptides presented with the exception of those which contained proline in the central position, indicating the inability of the enzyme to hydrolyse the imido linkage. The inability to hydrolyse dipeptides, dipeptideamides, tripeptideamides or tetrapeptides indicates that the enzyme is a strict aminotripeptidase. In a previous study, no proline aminopeptidase activity could be detected in Lactococcus lactis subsp, cremoris AM2 using Pro-AMC as substrate (Booth et al:, 1990). The observation that the broad specificity aminopeptidase, separated on Sephacryl S-200 chromatography from the aminotripeptidase (Fig. 1), could release N-terminal proline from Pro-Leu-Gly-Gly and that the purified aminotripeptidase could release proline from Pro-Gly-Gly (Table 4) may indicate that both these enzymes supplement any apparent deficiency in classical proline aminopeptidase in this culture strain. The purified aminotripeptidase was also found to release aspartic acid from Asp-Tyr-Met and glutamic acid from Glu-Gly-Phe. In this respect, the substrate specificity overlaps with that of aminopeptidase A, an enzyme purified and assayed using Asp-pNA as substrate (Bacon, C. L., unpublished data). The inhibition of the purified enzyme by 1,10-phenanthroline, EDTA and to a lesser extent by 8-hydroxyquinoline, suggests that it is a metalloenzyme. This finding is in agreement with Bosman et al. (1990).
Purification and characterisation of an aminotripeptidase
175
Failure to observe significant inhibition with N-ethylmaleimide, iodoacetamide or PCMB indicates that sulphydryl groups are not involved in the active site. The possibility that the enzyme is a serine peptidase can be eliminated since phenyl methyl sulphonyl fluoride caused no inhibition. The total inhibition of activity by dithiothreitol is in agreement with results obtained with the tripeptidase from Lactococcus lactis subsp, cremoris Wg2 (Bosman et al., 1990). Amastatin, previously noted as an inhibitor of mammalian alanine aminopeptidase and glutamine aminopeptidase (Tobe et al., 1980), was found to totally inhibit the activity of the tripeptidase. Captopril, an inhibitor of mammalian peptidyl dipeptidase I, (Cushman et al., 1977), also caused significant inhibition. These unusual inhibition features may prove helpful in characterising the tripeptidase activity. A relative molecular mass of 105 000 was obtained for the present enzyme which was very similar to the values reported for Lactococcus lactis subsp, cremoris Wg2 (Bosman et al., 1990) but larger than the 75 000 reported for Streptococcus diacetylactis 267 (Desmazeud & Zevaco, 1979). A subunit relative molecular mass of 55 000 for the present enzyme was similar to the value 52 000 for the aminotripeptides, obtained from Lactococcus lactis subsp, cremoris Wg2 (Bosman et al., 1990) and suggests that the enzyme consists of two equally sized monomers. This study confirms the findings of Bosman etal. (1990) of a strict aminotripeptidase in Lactococcus lactis subsp, cremoris. It also indicates that at least one further enzyme is present in the cytoplasm which may hydrolyse Phe-Gly-Gly and that a significant proportion of the cell's capacity to hydrolyse this tripeptide is present in the cell membrane. It also shows that the tripeptidase is unable to release the amino terminal amino acid from dipeptide amides or tripeptide amides. The study demonstrated that the g m of the enzyme for tripeptide substrates (Table 5) is not greatly affected by the nature of the amino terminal amino acid. Finally, the observed inhibition with amastatin and bestatin may prove useful in distinguishing this activity from other tripeptide hydrolysingactivities in the cell.
ACKNOWLEDGEMENTS The authors wish to acknowledge financial support from Eolas, the Irish Science and Technology Agency, and the National Dairy Product Research Centre, Moorepark, Fermoy, Co. Cork.
176
C L. Bacon. M. Wilkinson. P V. Jennings. 1. N. Fhaolain. G. O'Cuinn
REFERENCES Abrams, A. (1965). The release of bound adenosine triphosphate from isolated bacterial membranes and the properties of the solubilised enzymes. J. Biol. Chem., 240, 3657-81. Aidoo, K. E., Hendry, R. & Wood, B. J. B. (1981). Estimation of fungal growth in a solid state fermentation system. Eur. J. App. Microbiol. Biotechnol.. 12, 6-9. Barranger, J. A., Geiger, P. J., Nuzino, A. & Bossman, S. (1972). lsozymes of rat phenylalanine hydroxylase. Science, 175, 903-5. Bockelmann, W., Monnet, V., Geis, A., Teuber, M. & Gripon, J. C. (1989). Comparison of cell wall proteinases from Lactococcus lactis subsp, cremoris AC1 and Lactococcus lactis subsp, lactis NCDO763. Specificity towards casein. App. Microbiol. Biotechnol., 31, 112-8. Booth, M., Donnelly, W. J., Ni Fhaolain, I., Jennings, P. V. & O'Cuinn, G. (1989). Preparation of subcellular fractions from strains of Streptococcus crernoris AM2. Biochem. Soc. Trans., 17, 385-6. Booth, M., Donnelly, W. J., Ni Fhaolain, I., Jennings, P. V. & O'Cuinn, G. (1990). Proline specific peptidases of Streptococcus cremoris AM2. J. Dairy Res., 57, 79-88. Bosman, B. W., Tan, P. S. T. & Konings, W. N. (1990). Purification and characterisation of a tripeptidase from Lactococcus lactis subsp, cremoris Wg2. App. Environ. Microbiol., 56, 1839-43. Cushman, D. W., Cheung, H. S., Sabo, E. F. & Ondetti, M. A. (1977). Design of a potent competitive inhibitor of angiotensin converting enzyme. Carboxy alkanoyl and mercapto alkanoyl amino acids. Biochem., 16, 5484-91. Desmazeaud, M. J. & Zevaco, C. (1979). Isolation and general properties of two intracellular aminopeptidases of Streptococcus diacetylactis. Milchwissenschaft, 34, 606-10. Donlon, J. & Kaufman, S. (1980). Relationship between the multiple forms of rat hepatic phenylalanine hydroxylase and degree of phosphorylation. J. Biol. Chem., 255, 2146-52. Kaminogawa, S., Ninomiya, T. & Yamauchi, K. (1984). Aminopeptidase profiles of lactic streptococci. J. Dairy Sci., 67, 2483-92. King, E. J. (1932). The coiorimetric determination of phosphorus. Biochem. J., 26, 292-7. Kolstad, J. & Law, B. A. (1985). Comparative peptide specificity of cell wall, membrane and intracellular peptidases of group N streptococci. J. App. Bacteriol.. 58, 449-56. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227, 680-5. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). Protein measurement with the Folin reagent. J. Biol. Chem., 193, 265-75. Nicholson, J. A. & Peters, T. J. (1978). Fluorimetric assay for intestinal peptidases. Anal, Biochem.. 87, 418-24. Rondle, C. J. M. & Morgan, W. T. J. (1955). The determination of glucosamine and galactosamine. Biochem. J., 61,586-9. Tan, P. S. T. & Konings, W. N. (1990). Purification and characterisation of an
Purification and characterisation of an aminotripeptidase
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aminopeptidase from Lactococcus lactis subsp, cremoris Wg2. App. Environ. Microbiol., 56, 526-32. Thomas, T. D. & Mills, O. E. (1981). Proteolytic enzymes of starter bacteria. Netherlands Milk and Dairy J., 35, 255-73. Thomas, T. D. & Pritchard, G. G. (1987). Proteolytic enzymes of dairy starter cultures. FEMS Microbiology Reviews, 46, 245-68. Tobe, H., Kojima, F., Aoyagi, T. & Umezawa, H. (1980). Purification by affinity chromatography using amastatin and properties of aminopeptidase A from pig kidney. Biochimica et Biophysica Acta, 613, 459-68. Wittenberger, C. L. & Angelo, N. L. (1970). Purification and properties of a fructose 1,6 diphosphate activated lactate dehydrogenase from Streptococcus faecalis. J. Bacteriol., 26, 717-24.
Purification and Characterisation of an Aminotripeptidase from Cytoplasm ofLactococcus lactis subsp, cremoris AM2
Christopher L. Bacon Department of Biochemistry, University College, Galway, Ireland
Martin Wilkinson National Dairy Product Research Centre, Moorepark. Fermoy, Co. Cork. Ireland
&
P. Vincent Jennings, Ide Ni Fhaolain & Gerard O'Cuinn* Department of Life Sciences, Regional Technical College, Galway, Ireland (Received 3 October, 1991; revised version accepted 13 April 1992)
ABSTRACT An aminopeptidase was purified 120-foM with a 78% recovery from the cytoplasm ofLactococcus lactis subsp, cremoris AM2. The purified enzyme exhibited no activity on dipeptides, dipeptideamides, tripeptideamides or tetrapeptides. As activity was observed only with tripeptides, from which it released the N-terminal amino acid, the enzyme was adjudged to be a strict aminotripeptidase. The enzyme had a Mr of 105 000 and showed one band, corresponding to an Mr of 55 000 on SDS-PAGE electrophoresis. Inhibition by EDTA. 8-hydroxyquinoline and 1,10 phenanthroline indicated that the peptidase was a metallo enzyme. Dithiothreitol, bestatin and amastatin caused total inhibition, whereas p-chloromercuribenzoate, bacitracin and phenyl methyl sulphonyl fluoride were without effect. K,, values for tripeptides were within the range 0.18 (Leu-Leu-Leu) to 0.38 m n (Trp-Gly-Gly). Substrate inhibition was noted with some tripeptides at concentrations above 1.5 mM. *To whom correspondence should be addressed. 163 Int. Dairy Journal 0958-6946/92/$05.00 © 1992 Elsevier Science Publishers Ltd, England. Printed in Ireland
164
C. L. Bacon, M. Wilkinson, P. l~ Jennings. I. N. Fhaolain, G. O'Cuinn
INTRODUCTION Because of the low concentration of free amino acids in milk, the proteolytic and peptidolytic systems of starter bacteria are vital for the release of free amino acids from casein to allow the growth of these starter bacteria to achieve high densities in milk (Thomas & Mills, 1981). The initial stages of casein breakdown are brought about by proteolytic enzymes located on the surface of starter bacteria (Bockelmann et al., 1989). The generation ofpeptides suitable for transport into the bacterial cell may involve further hydrolysis by, as yet unidentified, peptidases either on the surface of the bacteria or secreted by them. Peptides transported into bacteria are degraded to amino acids by a limited range of intracellular peptidases (Thomas & Pritchard, 1987). A definition of the range ofintracellular peptidases has been the subject of investigation by a number of groups. Kolstad & Law (1985) subjected cytoplasmic and particulate extracts from strains of starter bacteria to polyacrylamide gel electrophoresis and, using a variety of dipeptides and tripeptides in an activity stain, they identified a number of bands of activity. Their results suggested that a distinct band of activity was present which was responsible for the hydrolysis of tripeptides in cytoplasm. Kaminogawa etal. (1984) separated cytoplasm from a range of starter organisms by DEAE-cellulose chromatography. When the activity ofeluates from the column was scanned on a wide variety of dipeptides, tripeptides, tetrapeptides and aminoacyl naphthylamides, the results obtained showed that while two eluted activities could hydrolyse tripeptides, only one of these could hydrolyse tripeptides only. The suggestion that the cytoplasm of starter bacteria possesses an enzyme that is specific for tripeptides was confirmed by a recent publication on the purification and characterisation of such an activity from Lactococcus lactis subsp. cremoris Wg2 (Bosman et al., 1990). These authors purified this enzyme to homogeneity and showed that it hydrolysed tripeptides only, and that it was inhibited by EDTA and dithiothreitol. The native enzyme was found to have a molecular mass of between 103 000 and 105 000 daltons with two equal subunits of 52 000 daltons. The present study confirms the presence of a very similar aminotripeptidase in the cytoplasm of Lactococcus lactis subsp, cremoris AM2.
MATERIALS Lactococcus lactis subsp, cremoris AM2 and low heat skim milk were
provided by the National Dairy Product Research Centre, Moorepark,
Purification and characterisation of an aminotripeptidase
165
Fermoy, Ireland. EDTA, Tris, lysozyme, DNase (bovine pancreas, type IV), RNase (bovine pancreas, type Ill-A), fructose 1, 6 diphosphate, NADH (disodium salt), pyruvic acid, disodium ATP, p-hydroxyphenylacetic acid, L-amino acid oxidase (Type IV from Crotalus atrox), peroxidase (Type II horseradish), 1, 10-phenanthroline, 8-hydroxyquinoline, iodoacetamide, benzamidine, bacitracin, amastatin, bestatin and dithiothreitol were obtained from Sigma Chemical Company, Poole, Dorset, England. Butan-l-ol and formic acid were obtained from BDH, Poole. England. All peptides and peptide derivatives were obtained from Bachem Feinchemikalien, Bubendorf, Switzerland. Cellulose fine powder (CF 1) and DE-52 anionic exchanger were provided by Whatman, Maidstone, England. Sephacryl S-200 high resolution gel was obtained from Pharmacia Fine Chemicals AB (Uppsala, Sweden). Silica gel G plates were obtained from Merck.
METHODS Assay for tripeptidase Tripeptidase activity was measured by incubating 50/~1 of sample with 450/~1 of 1-11 mM L-Phe-Gly-Gly or L-Leu-L-Leu-L-Leu in 75 mM borate, pH 7.5, for 10 min at 30°C. The reaction was terminated by heating to 90°C for 5 min and the phenylalanine or leucine released measured, after cooling, by the coupled L-amino acid oxidase-peroxidase system (Nicholson & Peters, 1978) in which oxidised p-hydroxyphenylacetic acid was determined fluorimetrically using excitation and emission wavelengths of 317 and 414nm, respectively. The unit of aminotripeptidase activity is defined as the amount of enzyme which releases 1 nmol of N-terminal Phe from Phe-Gly-Gly min -I in 75 mM borate, pH 7.5, at 30°C. Protein determination Protein concentration was determined by the method of Lowry et ai. (1951), using bovine serum albumin as standard. Production of subcellular fractions from Lactococcus lactis subsp, cremoris AM2 Lactococcus lactis subsp, cremoris AM2 cells were grown, harvested,
washed and subjected to subcellular fractionation as described previously
166
C. L. Bacon, M. Wilkinson, P. V. Jennings, I. N. Fhaolain, G. O'Cuinn
by Booth et al. (1989, 1990). The presence of cell m e m b r a n e material in recovered fractions was monitored by measurement of ATP-ase activity (Abrams, 1965) in which liberated phosphate was measured by the method of King (1932). The presence of cell wall material was monitored by measuring glucosamine using a modification (Aidoo et al., 1981) of the method of Rondle & Morgan (1955), while lactate dehydrogenase activity (Wittenberger et al., 1970) was indicative of cytoplasm.
Preparation of calcium phosphate-cellulose Calcium phosphate gel was prepared as described by Barranger et al. (1972). Batches of cellulose powder were prepared as described by Donlon & Kaufman (1980). Prior to column packing, the washed cellulose powder was made into a 10% (w/v) suspension in 20 mM potassium phosphate buffer, pH 7-5, containing 0.15M KC1. Five volumes of this suspension were then added to one volume of the calcium phosphate gel. The resultant mixture was mixed well at 4°C until a homogeneous suspension was obtained.
Purification of aminotripeptidase The cytoplasmic fraction was dialysed against 50 mM Tri-HC1, pH 7.5, containing 0.1 M NaCI and applied to a Sephacryl S-200 column. Activity was eluted from the column in 50 mM Tris-HC1, pH 7-5, containing 0.1 M NaC1 at a flow rate of 16 ml h-~; 3 ml fractions were collected. The tubes containing aminotripeptidase activity were poole& dialysed against 50 mM Tris-HC1, pH 7.5~ and applied to a W h a t m a n DE-52 anion exchange column (3.4 cm X 1.5 cm). After washing with two column volumes of 50 mM Tris-HCl, pH 7-5, the activity was eluted in a gradient established between 50 mM Tris-HC1, pH 7.5, and 50 mM Tris-HC1, pH 7.5, containing 0.75 M NaC1. The eluates were collected in 2 ml fractions at a flow rate of 10 ml h -~. The active fractions were pooled, dialysed against 20 mM potassium phosphate, pH 7.2, containing 0.15 M KC1 and loaded onto a calcium phosphate-cellulose column ( 5 c m X 1 cm) pre-equilibrated with 20mM potassium phosphate, pH 7.2, containing 0.15 M KC1. The column was washed with two volumes of the equilibration buffer and activity was eluted using a linear gradient established between 20 mM potassium phosphate, pH 7-2, containing 0.15 M KCI and 250mM potassium phosphate, pH 7.2, containing 0.15 M KC1. The eluates were collected in 1 ml fractions and active fractions were pooled. Prior to assaying, with a view to
Purification and characterisation of an aminotripeptidase
167
determining specific activity and yield, aliquots of each fraction were dialysed against water. Effect of inhibitors on the activity of purified tripeptidase Aliquots (50/.tl) of a solution of purified enzyme (9 activity units) were preincubated at 30°C for 15 min with 50/A of inhibitor solution in water. (For final concentrations see Table 3). Tripeptide hydrolysis was initiated by adding 400:11 of 1.25 mM P h e - G l y - G l y in 75 mM borate, pH 7.5, and the reaction mixture incubated for 10 min. The reaction was terminated by heating to 90°C for 5 min and the phenylalanine released was estimated as described before. The activity observed with each inhibitor was related to the activity observed when water (50/~1) was used instead of inhibitor solution. Some of the inhibitors employed, notably 1,10-phenanthroline, dithiothreitol, 8-hydroxyquinoline and captopril, inhibited the L-amino acid oxidase-peroxidase system. In experiments where these inhibitors were used, standard curves relating phenylalanine concentration to fluorescence in the presence of the appropriate concentration of each inhibitor were constructed. Determination of Mr A solution of tripeptidase activity (540 activity units) was loaded onto a Sephacryl S-200 column (88 cm X 2-5 cm diam.), previously equilibrated with 50 mM Tris-HCl, pH 7.5, containing 0.1 M NaC1 and calibrated using ferritin (440000), aldolase (158 000), bovine serum albumin (67 000), ovalbumin (43 000) and chymotrypsin (26 000). The tripeptidase activity was eluted from the column with 50 mM Tris-HCl, pH 7.5, containing 0.1 M NaC1 and collected in 3 ml fractions at a flow rate of 16 ml h -~. The Mr of the tripeptidase was computed by the relationship of its elution volume to elution volumes of standards of known Mr. Subunit molecular weight determination Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) was performed as described by Laemmli (1970) using 12% acrylamide gels. Coomassie brilliant blue G250 was used to visualize the proteins after electrophoresis and the molecular weight of the protein band was computed by reference to the migration of low molecular weight SDS-PAGE standards (Bio-Rad labs, Richmond, California).
168
C L. Bacon, M. Wilkinson, P. V. Jennings, 1. N. Fhaolain, G. O'Cuinn
Stability of purified tripeptidase at 10°C and at pH 5.2 A solution of the purified tripeptidase (180 activity units) was dialysed against 100 mM sodium acetate, pH 5.2, containing 5%, w/v, NaCI. The dialysed solution was then incubated at 10°C and samples were withdrawn and assayed for tripeptidase activity using P h e - G l y - G l y as substrate on days 0, 8, 17, 30 and 67 after the c o m m e n c e m e n t of incubation. The release of phenylalanine was measured as described above.
Stability of purified tripeptidase at different temperatures A solution of the purified tripeptidase in 100 mM potassium phosphate, pH 7.2, containing 0.15 M KC1 was incubated at 25°C, 37°C or 50°C. After 0, 10~ 20, 30 and 60 min, samples were taken from each incubation mixture and assayed for tripeptidase activity at 30°C using P h e - G l y - G l y as substrate.
Substrate specificity of the purified tripeptidase Aliquots (50pl) of a solution of purified tripeptidase (9 activity units) were incubated with 450 pl of 1.11 mM solutions of each peptide to be tested in 75 mM borate, pH 7-5, at 30°C. After 10 min, the reaction was terminated by heating to 90°C for 5 min and the amino acids released quantified as described above using appropriate standard curves, relating the amount of each amino acid to the intensity of fluorescence. Alternatively, where release of the amino terminal amino acid could not be quantified by the coupled L-amino acid oxidase-peroxidase system, incubation of 50 pl of tripeptidase solution with 50pl of 4 mM peptide in 75 mM borate, pH 7.5, was allowed to proceed for 16 h. Portions of each reaction mixture (50pl) were chromatographed on silica gel G plates using butanol:formic acid:water (20:6:5, v:v:v) as solvent system. Appropriate standards were co-chromatographed.
Determination of kinetic parameters Tripeptidase solutions (9 activity units) were incubated with various concentrations oftripeptides in 450 pl of 75 mM borate, pH 7.5, yielding a range of final concentrations oftripeptide from 0-065 to 1-5 raM. After 10 min, the reaction was terminated and the released amino acids measured as described above. Km values were calculated from Lineweaver-Burk plots.
Purification and characterisation of an aminotripeptidase
169
RESULTS The data presented in Table 1 indicate that while the majority of the tripeptidase activity was located in the cytoplasm, a significant level of activity was associated with the cell m e m b r a n e fraction. No tripeptidase activity was detected in cell wall or wash fractions or in the extracellular fluid. The subcellular fractionation procedure employed in this study was monitored for its efficiency in yielding fractions enriched in cell walls, cell m e m b r a n e s and cytoplasm by the use of markers. The results obtained were very similar to those reported in a previous study (Booth etal., 1989) and showed enrichment of Mg2+ATP-ase in the cell m e m b r a n e fraction, of glucosamine in the cell wall fraction and of lactate dehydrogenase in the cytoplasm. Chromatography of the cytoplasmic fractions on Sephacryl S-200 resolved the activity against P h e - G l y - G l y into three peaks (Fig. 1). Activity against Lys-pNA was associated with the third peak, as was activity against P r o - L e u - G l y - G l y from which both Pro and Leu were released. The third peak of activity was also able to remove Leu and Trp from Leu-Trp-Met-Arg and Leu, Trp and Met from L e u - T r p - M e t - A r g Phe. The second peak of activity was pooled and chromatographed on DE-52, from which it eluted as a single peak at 0.55 M NaC1 in a linear salt gradient. The pooled peak of activity was then chromatographed on a calcium phosphate-cellulose column from which it also eluted as a single peak at 45 mM potassium phosphate. The data in Table 2 show that the three step purification resulted in a 120-fold purification of one of the P h e - G l y - G l y hydrolysing activities of the cytoplasm with a recovery of 78%. TABLE I S u b c e l l u l a r D i s t r i b u t i o n o f H y d r o l a s e Activity a g a i n s t L e u L e u - L e u a n d P h e - G l y - G l y in S u b c e l l u l a r F r a c t i o n s D e r i v e d f r o m Lactococcus lactis subsp, cremoris A M 2
nmoles of N-terminal aminoacid re~eased (min -I tota/ fraction -1)
E x t r a c e l l u l a r fluid Wash Cell wall Cell m e m b r a n e Cytoplasm
Phe-Gly-Gly
Leu-Leu-Leu
---1 540 2 800
---2 520 3 850
170
C. L. Bacon, M. Wilkinson, P V. Jennings, I. N. Fhaolain, G. O'Cuinn 1
2
3
12
-6 1-0
10 -4
~E
2 2 <
0.50 -2
A~
...Z~,~
-5
I~
tt tXl~ A
~. 0 100
I
i
2OO
30O
. . . .
Elution Volume (ml) Fig. 1. The clution of cytoplasm (predialysed into 50 mM Tris-HCl, pH 75) from Laetococcus lactis subsp, cremoris AM2 off Sephacryl S-200 in 50 mM Tris-HCL pH 7-5. Tripeptidase activity measured using P h e - G l y - G l y as substrate ( l - - - - - l ) . Aminopeptidase activity using Lys-pNA as substrate ( O - I - O ) . Protein measured by absorbance at 280 mM (A . . . . A). Fractions that released Pro and Leu from Pro-LeuGly-Gly, Leu and Trp from Leu-Trp-Met-Arg and Leu, Trp and Met from Leu-TrpMet-Arg-Phe are indicated by < . . . . >. Void volume was calculated using ferritin (mol wt. 440 000) and by measuring eluates at 280 nm: 3-ml fractions were collected at a flow rate of 16 ml h t.
TABLE 2 Purification of Aminotripeptidase from Cytoplasm of Lactococcus lactis subsp, cremoris AM2. (Aminotripeptidase Activity Assayed Using P h e - G l y - G l y as Substrate)
Total protein (rag)
Cytoplasm Post Sephacryl S-200 Post DE-52 Post calcium phosphatecellulose
Total enzyme activity (nmol Phe min -1)
Specific activity (nmol Phe min -1 mg -/)
Purification
Yield (%)
9'0 1"38
I 150 5 250
127-7 3 804
1"0 29"78
100 456
0'507 0'0585
2 340 900
4 615 15 384
36' 14 120'47
203 78
Purification and characterisation of an aminotripeptidase
171
Incubation of the purified tripeptidase with a range of possible inhibitors (Table 3) showed that no inhibition occurred with p-chloromercuribenzoate, N-ethylmaleimide, bacitracin or phenyl methyl sulphonyl fluoride. Modest inhibition was noted with benzamidine and iodoacetamide while more significant inhibition was caused by EDTA and puromycin. Bestatin and amastatin caused total inhibition. After allowing for the effect of captopril, 1,10-phenanthroline, dithiotreitol and 8-hydroxyquinoline on the L-amino acid oxidase-peroxidase system, 1,10-phenanthroline and captopril were found to significantly inhibit the activity of the tripeptidase while total inhibition was caused by dithiotreitol and 8-hydroxyquinoline. These observations were confirmed by chromatography of incubates containing each inhibitor on silica gel G plates where total inhibition of the tripeptidase was observed with dithiothreitol and 8-hydroxyquinoline while only partial inhibition occurred with 1,10-phenanthroline and captopril. The enzyme was active over a wide pH range (6.8-9.1), with m a x i m u m activity at pH 8.6. The relative molecular mass of the purified tripeptidase was computed to be 105 000 and S D S - P A G E showed only one b a n d with a relative molecular mass of 55 000. The ability of the purified enzyme to hydrolyse a range of dipeptides, tripeptides and tetrapeptides was tested. Only tripeptides were hydrolysed and in each case, only the amino terminal amino acid of the tripeptide TABLE 3
The Effect of Inhibitors on the Activity of an Aminotripeptidase Purified from Cytoplasm of Lactococcus lactis subsp, cremoris AM2
Inhibitor used
1~ 10 Phenanthroline 8-Hydroxyquinoline EDTA lodoacetamide N-ethylmaleimide p-Chloromercuriben zoate Captopril Bestatin Dithiothreitol Amastatin Puromycin Benzamidine Bacitracin Phenylmethylsulphonylfluoride
Concentration of inhib#or (mM)
Remaining activiW (% of control)
0"05 1-0 1.0 5"0 1-0 0" 1 1-0 1.0 2"0 1-0 5"0 5"0 0" 1 1"0
12-5 0 32"0 72"9 100'0 100-0 46.0 0-0 0'0 3"0 62"5 83"3 100.0 100.0
172
C L. Bacon. M. Wilkinson, P. V. Jennings, I. N. Fhaolain, G. O'Cuinn
was released (Table 4). Dipeptide amides (Leu-Leu-NH2, Leu-GlyNH2) or tripeptide amides (Gly-GIy-GlyNH2, Leu-Leu-LeuNH2) were not hydrolysed. The tripeptidase released a range of amino terminal amino acids from tripeptides presented, including proline from Pro-Gly-Gly and aspartic acid from Asp-Tyr-Met. No hydrolysis was observed, however, when proline was present in the central position in a tripeptide. Km values of the purified enzyme with eight substrates (Table 5) revealed little variation in affinity for these substrates. Trp-Gly-Gly, with a Km value of 0.38 mM appeared to have least affinity for the enzyme, gca t values also did not vary notably with Met-Gly-Gly (201-6s -t) showing the highest value and Trp-Gly-Gly (117.6s -I) showing the lowest value. The purified aminotripeptidase showed no loss of activity at 25°C or 37°C over a 60 min incubation at these temperatures. Incubation at 50°C, however, resulted in a 50% loss in activity after 13 min. When the purified aminotripeptidase was
TABLE 4 Substrate Specificity of an Aminotripeptidase Purified from Cytoplasm of Lactococcus lactis subsp, cremoris A M 2 Arg-Asp Ala-Gln
-"
lie-lie-lie
+h
-
Phe-Gly-Gly
+
Ala-Ala
-
Thr-Gly-Gly
+
Ala-Gly
-
Tyr-Gly-Gly
+
Ala-Gly-Gly
+
Met-Gly-Gly
+
Ala-Gly-Gly-Gly
-
Trp-Gly-Gly
+
Ala-Gly-Gly-Gly-Gly
-
Lys-Gly-Gly
+
Tyr-Pro
-
Arg-Gly-Gly
+
Gly-Pro
-
Pro-Gly-Gly
+
Gly-Gly
-
Glu-Gly-Phe
+
-
Glu-Asp
-
+
Asp-Lys
-
Gly-GlyNH
2
Gly-Gly-Gly GIy-GIy-GIyNH
2
Gly-Gly-Gly-Gly Leu-Gly
-
Asp-Tyr-Met
+
-
Ala-Pro-Arg
-
-
Ala-Pro-Gly
-
-
Arg-Pro-Pro
-
Leu-Gly-Gly
+
Ile-Pro-lle
-
Leu-Leu
+
Pro-Pro-Pro Pro-Pro
-
-
Pro-Phe
-
Leu-GlyNH
2
Leu-Leu-Leu Leu-Leu-LeuNH Leu-Ala-Pro
2
+
" - indicates no hydrolysis. t'+ indicates cleavage of N-terminal amino acid.
-
Purification and characterisation of an aminotripeptidase
173
TABLE 5
Km, Vmaxand Kcat Values Derived from the Interaction of the Purified Aminotripeptidase with Tripeptide Substrates
Substrate
Met-Gly-Gly Trp-Gly-Gly Phe-Gly-Gly Leu-Gly-Gly Leu-Ala-Pro Leu-Leu-Leu Leu-Gly-Pro Tyr-Gly-Giy
K,,,
V.no.,-
K~.a,
(raM)
(nmol min -l m1-1)
(s -t)
0"23 0"38 0'20 0.19 0' 24 0' 18 0'33 0"22
571 540 434 400 392 370 344 333
201"6 190.7 153"2 141-2 132 '4 i 30"6 12 !'5 117-6
incubated in sodium acetate, pH 5.2, containing 5% NaC1 (w/v) at 10°C, 88% of starting activity was recovered after 30 days incubation. After 67 days only 25% of the initial activity remained.
DISCUSSION The results presented in Table 1 indicate that the majority of peptidase activity against either Phe-Gly-Gly or Leu-Leu-Leu in Lactococcus lactis subsp, cremoris AM2 resided in the cytoplasmic fraction. No activity against either tripeptide was observed in the extracellular fraction, in the wash or in the cell wall fraction. Significant activity against these tripeptides was found in the cell membrane fraction. The relationship between the cytoplasmic activity and that of the cell membrane remains to be determined. When the cytoplasmic activity against Phe-Gly-Gly was chromatographed on Sephacryl S-200 and when the eluted fractions were monitored for activity against Phe-GlyGly, Lys-pNA, Pro-Leu-Gly-Gly, Leu-Trp-Met-Arg and Leu-TrpMet-Arg-Phe, two adjacent peaks of activity against Phe-Gly-Gly were observed, preceded by a minor peak of activity (Fig. 1). Peak 3 was found to contain activity against Lys-pNA, Pro-Leu-Gly-Gly, Leu-Trp-MetArg and Leu-Trp-Met-Arg-Phe, suggesting the presence of a general aminopeptidase similar to that previously described for Lactococcus lactis subsp, cremoris Wg2 (Tan & Konings, 1990). The second peak, active only against tripeptides, was selected for further purification as a possible aminotripeptidase similar to those previously described in
174
C. L. Bacon, M. Wilkinson, P. V. Jennings, I. N. Fhaolain, G. O'Cuinn
Lactococcus lactis subsp, cremoris Wg2 (Bosman etal., 1990) and in Streptococcus diacetylactis 267 (Desmazeud & Zevaco, 1979).
The purification data (Table 2) show that while chromatography on Sephacryl S-200 separated three enzymes in cytoplasm capable of hydrolysing Phe-Gly-Gly (Fig. 1), a dramatic increase was observed in Phe-Gly-Gly hydrolysing activity of the partially purified aminotripeptidase relative to the level observed in the cytoplasm. Precautions had been taken to dilute the cytoplasm such that the Phe-Gly-Gly hydrolysing activity was linear with respect to the dilution of the cytoplasm. In spite of this precaution, the increase in activity was observed consistently during repeated purifications. When the enzyme activity recovered from Sephacryl S-200 gel filtration column was incubated with aliquots of other fractions from the column no significant reduction in activity was noted. Studies on the apparent cytoplasmic inhibition of this Phe-Gly-Gly hydrolysing activity are continuing. In addition, this step separated the aminotripeptidase not only from the broad specificity peptidase which hydrolysed Phe-Gly-Gly, Lys-pNA, Pro-Leu-Gly-Gly, Leu-Trp-Met-Arg and Leu-Trp-Met-Arg-Phe but also from the minor peak of activity. The purified aminotripeptidase removed the amino terminal amino acid from all the tripeptides presented with the exception of those which contained proline in the central position, indicating the inability of the enzyme to hydrolyse the imido linkage. The inability to hydrolyse dipeptides, dipeptideamides, tripeptideamides or tetrapeptides indicates that the enzyme is a strict aminotripeptidase. In a previous study, no proline aminopeptidase activity could be detected in Lactococcus lactis subsp, cremoris AM2 using Pro-AMC as substrate (Booth et al:, 1990). The observation that the broad specificity aminopeptidase, separated on Sephacryl S-200 chromatography from the aminotripeptidase (Fig. 1), could release N-terminal proline from Pro-Leu-Gly-Gly and that the purified aminotripeptidase could release proline from Pro-Gly-Gly (Table 4) may indicate that both these enzymes supplement any apparent deficiency in classical proline aminopeptidase in this culture strain. The purified aminotripeptidase was also found to release aspartic acid from Asp-Tyr-Met and glutamic acid from Glu-Gly-Phe. In this respect, the substrate specificity overlaps with that of aminopeptidase A, an enzyme purified and assayed using Asp-pNA as substrate (Bacon, C. L., unpublished data). The inhibition of the purified enzyme by 1,10-phenanthroline, EDTA and to a lesser extent by 8-hydroxyquinoline, suggests that it is a metalloenzyme. This finding is in agreement with Bosman et al. (1990).
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Failure to observe significant inhibition with N-ethylmaleimide, iodoacetamide or PCMB indicates that sulphydryl groups are not involved in the active site. The possibility that the enzyme is a serine peptidase can be eliminated since phenyl methyl sulphonyl fluoride caused no inhibition. The total inhibition of activity by dithiothreitol is in agreement with results obtained with the tripeptidase from Lactococcus lactis subsp, cremoris Wg2 (Bosman et al., 1990). Amastatin, previously noted as an inhibitor of mammalian alanine aminopeptidase and glutamine aminopeptidase (Tobe et al., 1980), was found to totally inhibit the activity of the tripeptidase. Captopril, an inhibitor of mammalian peptidyl dipeptidase I, (Cushman et al., 1977), also caused significant inhibition. These unusual inhibition features may prove helpful in characterising the tripeptidase activity. A relative molecular mass of 105 000 was obtained for the present enzyme which was very similar to the values reported for Lactococcus lactis subsp, cremoris Wg2 (Bosman et al., 1990) but larger than the 75 000 reported for Streptococcus diacetylactis 267 (Desmazeud & Zevaco, 1979). A subunit relative molecular mass of 55 000 for the present enzyme was similar to the value 52 000 for the aminotripeptides, obtained from Lactococcus lactis subsp, cremoris Wg2 (Bosman et al., 1990) and suggests that the enzyme consists of two equally sized monomers. This study confirms the findings of Bosman etal. (1990) of a strict aminotripeptidase in Lactococcus lactis subsp, cremoris. It also indicates that at least one further enzyme is present in the cytoplasm which may hydrolyse Phe-Gly-Gly and that a significant proportion of the cell's capacity to hydrolyse this tripeptide is present in the cell membrane. It also shows that the tripeptidase is unable to release the amino terminal amino acid from dipeptide amides or tripeptide amides. The study demonstrated that the g m of the enzyme for tripeptide substrates (Table 5) is not greatly affected by the nature of the amino terminal amino acid. Finally, the observed inhibition with amastatin and bestatin may prove useful in distinguishing this activity from other tripeptide hydrolysingactivities in the cell.
ACKNOWLEDGEMENTS The authors wish to acknowledge financial support from Eolas, the Irish Science and Technology Agency, and the National Dairy Product Research Centre, Moorepark, Fermoy, Co. Cork.
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C L. Bacon. M. Wilkinson. P V. Jennings. 1. N. Fhaolain. G. O'Cuinn
REFERENCES Abrams, A. (1965). The release of bound adenosine triphosphate from isolated bacterial membranes and the properties of the solubilised enzymes. J. Biol. Chem., 240, 3657-81. Aidoo, K. E., Hendry, R. & Wood, B. J. B. (1981). Estimation of fungal growth in a solid state fermentation system. Eur. J. App. Microbiol. Biotechnol.. 12, 6-9. Barranger, J. A., Geiger, P. J., Nuzino, A. & Bossman, S. (1972). lsozymes of rat phenylalanine hydroxylase. Science, 175, 903-5. Bockelmann, W., Monnet, V., Geis, A., Teuber, M. & Gripon, J. C. (1989). Comparison of cell wall proteinases from Lactococcus lactis subsp, cremoris AC1 and Lactococcus lactis subsp, lactis NCDO763. Specificity towards casein. App. Microbiol. Biotechnol., 31, 112-8. Booth, M., Donnelly, W. J., Ni Fhaolain, I., Jennings, P. V. & O'Cuinn, G. (1989). Preparation of subcellular fractions from strains of Streptococcus crernoris AM2. Biochem. Soc. Trans., 17, 385-6. Booth, M., Donnelly, W. J., Ni Fhaolain, I., Jennings, P. V. & O'Cuinn, G. (1990). Proline specific peptidases of Streptococcus cremoris AM2. J. Dairy Res., 57, 79-88. Bosman, B. W., Tan, P. S. T. & Konings, W. N. (1990). Purification and characterisation of a tripeptidase from Lactococcus lactis subsp, cremoris Wg2. App. Environ. Microbiol., 56, 1839-43. Cushman, D. W., Cheung, H. S., Sabo, E. F. & Ondetti, M. A. (1977). Design of a potent competitive inhibitor of angiotensin converting enzyme. Carboxy alkanoyl and mercapto alkanoyl amino acids. Biochem., 16, 5484-91. Desmazeaud, M. J. & Zevaco, C. (1979). Isolation and general properties of two intracellular aminopeptidases of Streptococcus diacetylactis. Milchwissenschaft, 34, 606-10. Donlon, J. & Kaufman, S. (1980). Relationship between the multiple forms of rat hepatic phenylalanine hydroxylase and degree of phosphorylation. J. Biol. Chem., 255, 2146-52. Kaminogawa, S., Ninomiya, T. & Yamauchi, K. (1984). Aminopeptidase profiles of lactic streptococci. J. Dairy Sci., 67, 2483-92. King, E. J. (1932). The coiorimetric determination of phosphorus. Biochem. J., 26, 292-7. Kolstad, J. & Law, B. A. (1985). Comparative peptide specificity of cell wall, membrane and intracellular peptidases of group N streptococci. J. App. Bacteriol.. 58, 449-56. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227, 680-5. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). Protein measurement with the Folin reagent. J. Biol. Chem., 193, 265-75. Nicholson, J. A. & Peters, T. J. (1978). Fluorimetric assay for intestinal peptidases. Anal, Biochem.. 87, 418-24. Rondle, C. J. M. & Morgan, W. T. J. (1955). The determination of glucosamine and galactosamine. Biochem. J., 61,586-9. Tan, P. S. T. & Konings, W. N. (1990). Purification and characterisation of an
Purification and characterisation of an aminotripeptidase
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aminopeptidase from Lactococcus lactis subsp, cremoris Wg2. App. Environ. Microbiol., 56, 526-32. Thomas, T. D. & Mills, O. E. (1981). Proteolytic enzymes of starter bacteria. Netherlands Milk and Dairy J., 35, 255-73. Thomas, T. D. & Pritchard, G. G. (1987). Proteolytic enzymes of dairy starter cultures. FEMS Microbiology Reviews, 46, 245-68. Tobe, H., Kojima, F., Aoyagi, T. & Umezawa, H. (1980). Purification by affinity chromatography using amastatin and properties of aminopeptidase A from pig kidney. Biochimica et Biophysica Acta, 613, 459-68. Wittenberger, C. L. & Angelo, N. L. (1970). Purification and properties of a fructose 1,6 diphosphate activated lactate dehydrogenase from Streptococcus faecalis. J. Bacteriol., 26, 717-24.