Download Phenylalanineaminopeptidase of L. pneumophila

Journal o/ Getterul Microbiology (1986), 132, 387392. Printed in Great Britain
387
Proteinases of Legionella: Phenylalanineaminopeptidase of L. pneumophila
By S E R G E I V . G U L ' N I K , ' M A R G A R I T A P . Y U S U P O V A , ?
GALINA I. LAVRENOVA,' IGOR S. TARTAKOVSKY,3
SERGEI V . PROZOROVSKY3 AND VALENTIN M. STEPANOV2*
Chemistr?, Department, Moscow State Uniuersity, Moscow, USSR
Institute of Genetics and Selection qf' Industrial Microorganisms, Moscow 113545, USSR
N . F. Ganiulepi Institute of' Epidmiology and Microbiology, Moscow, USSR
(Receitied 5 February 1985; revised 29 July 1985)
Phenylalanineaminopeptidase was isolated and purified from the culture filtrate of Legionella
pneumophila by affinity chromatography on 0-tert-butyl-L-threonyl-L-phenylalanyl-L-prolylglycyl-aminosilochrom and by gel-filtration; a 401-fold purification with a yield of 18% was
achieved. The enzyme was a metalloenzyme with a molecular weight of 35000 and a PI of 5.8. It
was stable at pH 7-9 and had an activity optimum in the range of pH 8-9.5 with L-phenylalanine
p-nitroanilide as substrate. Enzyme activity was highest towards the latter compound,
substantially lower towards L-leucine p-nitroanilide and only marginal towards other pnitroanilides. Besides phenylalanineaminopeptidase, a metalloproteinase and a serine
pro: Zinase were also detected in L . pneumophila culture filtrate.
INTRODUCTION
Bacteria in the genus Legionella possess an unusual type of metabolism characterized by the
utilization of amino acids as energy and carbon sources (George et a/., 1980). There have been
several reports of proteinase formation by Legionella, but no data are available on individual
enzymes. Thus, L . pneumophila degraded several serum proteins including proteinase inhibitors
(Muller, 1980). Enzymes in Legionella culture filtrate were reported to hydrolyse proteins
(Thompson et al., 1981 ) as well as synthetic peptide substrates (Berdal & Fossum, 1982; Berdal et
af., 1982a, b ; 1983). A cell suspension of L . pneumophila hydrolysed the synthetic substrates of
aminopeptidases (Muller, 1981). It was suggested that extracellular proteinases might be at least
partially responsible for the pathogenicity of this bacterium (Fraser et al., 1977; Muller, 1980).
This paper deals with the isolation and biochemical characterization of phenylalanineaminopeptidase (EC 3.4.1 1 .-) from culture filtrates of L . pneumophila, strain Philadelphia-1 .
METHODS
Chemicals. p-N itroanilides of L-phenylalanine (L-Phe), L-leucine @-Leu), L-proline @-Pro) and L-alanine (L-Ala)
were purchased from N PO Biokhimreaktiv, Olaine, Latvian SSR; those of L-valine @-Val), L-glutamic acid (LGlu) and L-arginine (L-Arg) were from Serva, as was bovine serum albumin, chymotrypsinogen, myoglobin,
lysozyme, cytochrome c, di-isopropyl fluorophosphate and phenylmethylsulphonyl fluoride. QAE-Sephadex A-25
and Sephadex (3-75 were from Pharmacia; Acrylex P-60 was from Reanal, Hungary and ovalbumin was from
Fluka. p-Nitroanilides of L-pyroglutamyl-L-alanyl-L-alanyl-L-leucine(L-Pyr-L-Ala-L-Ala-L-Leu) and of Lpyroglutamyl-L-phenylalanine(L-Pyr-L-Phe) (Oksenoit et al., 1982) were supplied by E. N . Lysogorskaya, I. Yu.
Filippova and E. S. Oksenoit (Chemistry Department, Moscow State University). The affinity sorbent
tetrapeptide-aminosilochrom was synthesized as described by Lyublinskaya et al. (1984).
Cultivation. L . pnruntophilu, strain Philadelphia-1, received from the Center for Disease Control, Atlanta,
Georgia, USA, was grown in shake flasks at 200 r.p.m. at 37" C in a medium composed of (g 1-l) Difco proteosepeptone 15.0; L-cysteine 0.4; K,HPO, 4.0; K H 2 P 0 , 1.0; NaCl 5.0. The cells were separated from the culture
medium by centrifugation for 15 min at 18000g. The supernatant was filtered through a Millipore filter (pore
diameter 0.3 pm) and stored frozen at - 20 "C.
0001-2483 0 1986 SGM
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 06 May 2017 01:27:27
388
S . V . G U L ' N I K A N D OTHERS
Enzyme assays. Serine endopeptidase assayed at pH 8.0 using L-Pyr-L-Ala-L-Ala-L-Leu-pNA (pNA represents
the p-nitroanilide group) as substrate according to the method of Lyublinskaya et a/. (1977). One unit of activity
was defined as the amount of enzyme releasing 1 pmol p-nitroalinine min-' at 37 "C. The molecular extinction
coefficient for free p-nitroaniline was assumed to be equal to 8800 at 410 nm. Specific activity values were based on
the protein content of the enzyme solution, measured as the A l s o .
Aminopeptidase was assayed at pH 8.0 or 9.0 according to the method of Ivanova et al. (1977). Under the
conditions of this assay the molecular extinction coefficient of p-nitroaniline was equal to 8200 at 410 nm.
Total endopeptidase activity was measured at pH 8.0 using azocasein as the substrate according to the method
of Dod et al. (1978).
Isolation of phenylalanineaminopeptidase. Culture filtrate (140 ml), obtained after 20 h of cultivation, was
adjusted to pH 5.6 with acetic acid; dry NaCl was then added to give a final concentration of 0.25 M. The deeply
coloured solution was applied to QAE-Sephadex (3.7 x 3 cm) on a sintered glass filter. The ion exchanger had
been pre-equilibrated with 0. I M-acetate buffer, pH 5.6, containing 0.25 M-NaC1. The sorbent was washed with the
same buffer and the fraction containing the activity towards L-Phe-pNA was collected and concentrated using an
Amicon UM-2 membrane. The ultrafiltrate (22 ml) was adjusted to pH 7.5 and its NaCl concentration was
increased to 1 M ; it was then applied to a column of tetrapeptide-aminosilochrom (1 x 17 cm) equilibrated with
50 mM-borate buffer, pH 7.5, and 1 M NaCl. The column was then washed with the same solution (10 column vols)
until the A z s o had dropped to 0.02. The active enzyme was eluted with the same buffer containing 1 M-NaC1 and
25% (v/v) propan-2-01. The eluate fractions containing aminopeptidase activity were pooled, and applied at a rate
of 32 ml h-' to an Acrylex P-60 column (2.5 x 72 cm) that had been pre-equilibrated and eluted with 50 mM-borate
buffer, pH 7.5, containing 0.1 M-NaCI. The active fractions (137-180 ml of the eluate) were concentrated to 3 ml
using a PM-10 Amicon membrane, and then applied to a Sephadex G-75 (superfine) column (1.6 x 86 cm), which
was equilibrated and eluted with 50 mM-borate buffer, pH 7.4, containing 0.1 M-NaCl. The fraction that contained
pure phenylalanineaminopeptidase (75-80 ml) was kept frozen at - 20 "C.
Molecular weight. This was determined by polyacrylamide gel electrophoresis (PAGE) in 0.1 (w/v) SDS
(Weber & Osborn, 1969), and by gel filtration (Andrews, 1965) using a Sephadex G-75 (superfine) column
equilibrated with 50 mM-borate buffer, pH 7.5, and 0.1 M-NaCI. The column was calibrated with proteins of
known molecular weights : bovine serum albumin (66000), ovalbumin (45 OOO), chymotrypsinogen (25000), and
lysozyme (14300). Electrophoresis of the enzyme was done using 7.9% polyacrylamide gel at pH 8.3 and 300 V
(Davis, 1964). The protein bands were stained with Coomassie brilliant blue G-250. To detect the band containing
phenylalanineaminopeptidase, slices were cut from the unstained gel that corresponded to the protein bands
vizualized on the control gel slab. The slices were incubated with L-Phe-pNA solution at pH 8.0 and 37 "C (Kaluger
et al., 1983). Isoelectrofocusing was done using a 'Multiphor' unit (LKB) in an Ampholine polyacrylamide gel
plate.
Characterization qf the enzyme. The pH optimum of the enzyme was determined using L-Phe-pNA as substrate at
pH 5.6 (50 mM-acetate buffer), pH 6.85 (50 mM-phosphate buffer) and at pH 7.7-10.2 (50 mM-borate buffer). To
assess the pH dependence of enzyme stability, 150 p1 samples of phenylalanineaminopeptidase solution in 50 mMborate buffer, pH 7.5, were added to 0.8 ml of the above mentioned buffers and the mixtures incubated for 2 h at
37 "C. Residual activity was then measured at pH 9.0.
The effect of various reagents that might inhibit or enhance enzyme activity was studied by incubating the
enzyme with 1 mM-phenylmethylsulphonyl fluoride or 0.1 mM-p-hydroxymercuribenzoate for 1 h at 20 "C, and
with 1 mM-Na2EDTA or 1 mM-dithiothreitol or I mM-mercaptoethanol for 10 min at 37 "C, and subsequently
measuring activity by the usual procedure. In the control tubes the enzyme was incubated under the same
conditions in the absence of these reagents. The influence of bivalent metal ions was studied by incubating the
enzyme with L-Phe-pNA for 20 min at pH 8.0 (50 mM-Tris/HCI buffer) and 37 "C in the presence of 1.0, 0.1 and
0.01 mM-(CH3COO)zZn,CoC12, CaCI,, MnCl, and MgCI,. p-Nitroaniline liberated was then determined.
Amino acidcomposition. The enzyme was hydrolpsed in 5.7 M-HClat 105 "C for 48 h and analysed in a Durrum D500 amino acid analyser. No special attempts were made to assay cysteine or tryptophan or to check the
determination of methionine which gave unusually high values.
RESULTS
The culture filtrate of L . pneumophila, strain Philadelphia-1, was found to contain proteolytic
enzymes active against azocasein, against the p-nitroanilides of L-Pyr-L-Ala-L-Ala-L-Leuand LPyr-L-Phe (typical synthetic substrates for serine proteinases) and against L-Phe-pNA (a typical
synthetic substrate for aminopeptidases). After 48 h of bacterial growth the following specific
activities [nmol min-' ( A z s ounit)-'] were found : L-Phe-pNA, 3.2; L-Leu-pNA, 0-36; L-Pyr-LAla-L-Ala-L-Leu-pNA, 0.22 ; L-Pyr-L-Phe-pNA, 0.047.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 06 May 2017 01:27:27
Legionella phenylalanineaminopeptidase
389
100
90
40
20
10
30
Time (h)
50
5
6
8
7
9
10
11
PH
Fig. 2
Fig. I
Fig. 1 . Phenylalanineaminopeptidase accumulation in L . pneumophila culture. Culture conditions were
as specified in Methods. Phenylalanineaminopeptidase activity was measured with L-Phe-pNA as
substrate.
Fig. 2. pH-dependence of L . pneumophilu phenylalanineaminopeptidase stability and activity. A,
Activity of the enzyme against L-Phe-pNA; m, residual activity of the enzyme after 2 h incubation at
37 ' C .
No activity was detected against L-Pyr-L-Ala-L-Ala-L-Phe-L-Leu-pNA and L-Pyr-L-Phe-LLeu-pNA, substrates for papain-like thiol proteinases (Philippova et al., 1984), against L-Pyr-LAla-L-Ala-L-Pro-pNA (specific for post-proline cleaving proteinases), or against L-Pyr-L-Tyr-LPhe-pN A and L-Pyr-L-Phe-L-Phe-pNA. Hydrolysis of L-Pyr-L-Ala-L-Ala-L-Leu-pN A was
completely inhibited by di-isopropyl fluorophosphate and by p-hydroxymercuribenzoate, which
is characteristic of bacterial thiol-dependent serine proteinases (Stepanov et al., 1981). Only
20% of the total activity against azocasein was inhibited by di-isopropyl fluorophosphate,
whereas 80% of the activity was inhibited by Na2EDTA, suggesting that L. pneumophila
produces metalloproteinase(s) preferentially alongside serine (apparently thiol-dependent)
proteinase. These preliminary observations on the nature of L. pneumophila proteinases remain
to be checked by separation and thorough study of these enzymes.
L. pneumophila extracellular aminopeptidase was isolated in this study and, in accordance
with its specificity, was named phenylalanineaminopeptidase. This enzyme appeared in the
culture filtrate after 16-18 h of growth (Fig. 1); then its content rapidly increased reaching a
maximum after 20-24 h. About the same time intensive pigment formation occurred. Further
growth of the culture led to the fall of phenylalanineaminopeptidase activity, apparently as a
result of degradation of the enzyme by other proteinases.
For the isolation of phenylalanineaminopeptidase the culture filtrate obtained after 20-24 h
growth was used. The bulk of the pigment was adsorbed on QAE-Sephadex at pH 5.6, whereas
phenylalanineaminopeptidase passed through the sorbent. Substantial amounts of the
remaining pigment were removed by ultrafiltration. Phenylalanineaminopeptidasewas further
purified by affinity chromatography on tetrapeptide-aminosilochrom. This sorbent was
prepared as described by Lyublinskaya et al. (1984) and contained tetrapeptide ligand attached
to macroporous silica as shown.
0 - t-But
I
0-
I
0-
1
L-Thr - L-Phe - L-Pro - Gly - NH - CH2CH2CHz- Si - 0 - Si - 0 -
I
0-
I
0-
This sorbent was effective in the purification of several aminopeptidases, and in the case of L.
pneumophila phenylalanineaminopeptidase effected a 20-fold purification.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 06 May 2017 01:27:27
s. v .
390
GUL'NIK A N D OTHERS
Table 1. Isolation q f L . pneumophila phenylalanineaminopeptidase
Activity
r -
Total
protein
Step
(A2xo)
Culture filtrate
QAE-Sephadex A-25
Ultrafiltration
Affinity chromatography on
tetrapeptideaminosilochrom
Acrylex P-60
Sephadex G-75
\
~
Total
(pmol min-I)
Specific
(pmol min-'
( A 2 s o unit)-']
Yield
Purification
(fold)
18.9
21.5
15.6
1.69 x 10-'
2.71 x lo-'
4.8 x lo-'
I00
114
83
1
10.1
7.9
3.4 I
0.98
3-1
6.78
1115
792
324
10.2
2.58
0.5 I
(7,;)
53
42
18.2
1.6
2.9
58
182
40 1
Table 2. Amino acid composition (residues per molecule) of phenylalanineaminopeptidase
Amino acid
Residues
ASP
Thr
Ser
Glu
Pro
G lY
Ala
Val
Met
42
18
26
41
22
35
39
Amino acid
25
19
8
10
7
13
5
11
ND
ND
13
Residues
340
ND,
Not determined.
The enzyme was sorbed on tetrapeptide-aminosilochrom at pH 7.5 in the presence of 1 MNaCl to reduce nonspecific binding of extraneous proteins. The enzyme was desorbed by 25%
(v/v) propan-2-01, the latter apparently suppressing hydrophobic contacts between the enzyme
and the ligand. As the final steps in the purification two gel filtration procedures - through
Acrylex P-60 and Sephadex G-75 - were used (Table 1).
The homogeneity of the isolated phenylalanineaminopeptidase was confirmed by the
presence of one protein band, RF0.6 (relative to the marker dye mobility), possessing activity
against L-Phe-pNA and L-Leu-pNA, after PAGE at pH 8.3. SDS-PAGE also revealed only one
protein band corresponding to a molecular weight of 35000. The same value was found for the
native enzyme by gel filtration through Sephadex G-75 suggesting that the enzyme consists of a
single polypeptide chain. Isoelectrofocusing in polyacrylamide gel also revealed a single protein
band of PI 5.8.
Phenylalanineaminopeptidase was characterized by a rather high content of dicarboxylic
amino acids and their amides - 83 residues out of 340 - whereas only 25 basic residues were
found (Table 2). This explained the low isoelectric point of the enzyme. The enzyme was stable
within the pH range 7-9; the optimum pH for its acivity was in the range pH 8.0-9.5 (Fig. 2).
The enzyme was especially active against L-Phe-pNA with considerably less activity towards LLeu-pNA (17% of the specific activity shown for L-Phe-pNA). Its action on thep-nitroanilides of
L-Val, L-G~u,L-Ala, L-Arg and L-Pro was negligible. However, the influence of secondary
specificity broadened this specificity pattern if peptides rather than amino acid p-nitroanilides
were used as the substrates. Indeed, rather extensive cleavage of leucine, proline, valine and
isoleucine residues was observed when the following peptides were hydrolysed by L. pneurnophila
phenylalanineaminopeptidase for 18 h at pH 9.0 and 37 "C (residues removed totally are given in
bold type) : Leu-Gly-Gly ; Pro-Leu-Gly-NH, ; Val-Val ; Ile-Ser-Asp-Thr-Asn-Gln ; Ile-Val-AspThr-Gly-Thr-Ser-Leu. The glycyl peptides Gly-Asn, Gly-Ala, Gly-Met, Gly-Phe, Gly-Leu, GlyDownloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 06 May 2017 01:27:27
Legionella phenylalanineaminopeptidase
39 1
Trp, Gly-Gly, Gly-Gly-NH2and Gly-Gly-Gly were completely resistant to the enzyme. Hence,
the specificity of phenylalanineaminopeptidase is considerably broader than might be
concluded from the hydrolysis of a range of amino acid p-nitroanilides.
The enzyme was completely inhibited by 1 mM-Na2EDTA,4 mM-cysteine, 1 mM-merCaptOethanol and 1 mM-dithiothreitol. This indicated the specific role of a metal ion in enzyme
activity. Bivalent metal ions - Zn2+, Ca2+, Mg2+, Mn2+, Coz+ - did not influence enzyme
activity at concentrations up to 1OpM. Neither 1 M-NaCl nor 1 M-LiCl influenced enzyme
activity whereas 1 M-KCldecreased the activity by about 50%. p-Hydroxymercuribenzoate also
inhibited the enzyme by about 50% but phenylmethylsulphonyl fluoride had no effect.
DISCUSSION
L . pneumophila forms substantial amounts of phenylalanineaminopeptidase. The role of this
enzyme is apparently the liberation of free amino acids, especially hydrophobic ones, from the
peptides present in the growth medium. Under physiological conditions the enzyme might act
on the host intracellular peptides or the products of the host proteins digested by Legionella
secretory proteinases, including the metalloproteinase. A more precise description of the way
this bacterium produces free amino acids requires more extensive characterization of its
proteinases.
Unfortunately, the paucity of the data on microbial aminopeptidases as well as the vagueness
of Legionella taxonomy handicaps any comparison of L . pneumophila phenylalanineaminopeptidase with other microbial aminopeptidases. Several aminopeptidases secreted by bacteria are
metalloenzymes of relatively low molecular weight and are devoid of quartenary structure. Thus,
the molecular weight of Aeromonas proteolytica aminopeptidase is 29 500 (Prescott et al., 197l),
that of Vibrio SA 1 is 21 000 (Wiersma et al., 1978) and that of Bacillus lichenijormis is 37500
(Rodriguez-Absi & Prescott, 1978). The molecular weight of the L. pneumophila enzyme, at
35 000, is obviously of the same magnitude. Metal ions are apparently involved in L . pneumophila
aminopeptidase activity. Similarly, the aminopeptidases from A . proteolytica (Prescott et al.,
1971), Vibrio SA 1 (Wiersma et al., 1978) and Alteromonas B-207 (Merkef et al., 1981) also
contain metal ions in their active sites. The pH-optimum of the L . pneumophila aminopeptidase
is similar to those of the bacterial aminopeptidases mentioned above.
To our knowledge, the remarkable preference for L-Phe-pNA shown by the L . pneumophila
enzyme is not matched by other bacterial proteinases, which as a rule prefer leucine as the Nterminal amino acid (Wagner et al., 1972; Wiersma et al., 1978; Rodriguez-Absi & Prescott,
1978; Merkef et al., 1981). Aminopeptidases that preferentially cleave N-terminal phenylalanine have however, been found in several plants (Kolehmainen & Mikola, 1971; Vodkin &
Scandalious, 1980; Waters & Dalling, 1984; Ninomiya et al., 1981). These aminopeptidases are
apparently not metalloenzymes and are thus distinguished from L. pneumophila
phenylalanineaminopeptidase.
Phenylalanineaminopeptidase isolated from Trichoderma uiride (Vaganova et al., 1983) also
shows a strong preference for L-Phe-pNA. Hence, several aminopeptidases of different origin
share the same pattern of substrate specificity which is characterized by a preference for L-PhepNA. L. pneumophila phenylalanineaminopeptidase belongs to this type.
The authors are indebted to Dr E. A . Timokhina for amino acid analyses.
REFERENCES
ANDREWS.P. (1965). The gel filtration behaviour of
proteins related to their molecular weights over a
wide range. Biochemical Journul 96, 595-606.
BERDAL,B. P. & FOSSUM,K . (1982). Occurrence and
immunogenicity of proteinases from Lt~gionelluspecies. European Jourrtd of Cliriicul Microhiologr 1, 711
BERDAL,B. P . , HUSHOVD,O., OLSVIK,
O., ODECARD,
0. R . & BERGAN,T. ( 1 9 8 2 ~ ) .Demonstration of
extracellular proteolytic enzymes from Lrgioncdla
species strains by using synthetic chromogenic
peptide substrates. Act0 purhologica rt microhiologica
scandinacica B90, 1 19-1 23.
BERDAL,B. P., OLSVIK,O., MYHRE,
S. & OMLAND,T.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 06 May 2017 01:27:27
392
S. V . GUL'NIK AND OTHERS
(1982h). Demonstration of extracellular chymotrypsin-like activity from various Legiont4lu species.
Journul of' Clinicul Microhiologj- 16, 452-457.
BERDAL,B. P., BOVRE, K . , OLSVIK,
0. & OMLAND,
T.
( 1 983). Patterns of extracellular proline-specific
endopeptidases in Legiunrlku and F l u r d m t e r i u m
species demonstrated by use of chromogenic peptides. Journal of' Clinicul Mic*rohiology 17. 970-974.
DAVIS,B. J. (1964). Disc electrophoresis. 11. Methods
and application to human serum proteins. Annals oj'
the New York Academy yf Sciences 121, 404-427.
DOD,B. J., BALASSA,G., RAULET.E. & JEANNODA,
V.
(1978). Spore control (Sco) mutations in Bucillus
subtilis. 11. Sporulation and the production of extracellular proteases and a-amyliise by Sco mutants.
Moleculur and General Genc.tic.s 163. 45-56.
FRASER,
D. W., TSAI,T. F., ORENSTEIN,
W . & THE
FIELDINVESTIGATION TEAM.( 1977). Legionnaires
disease: description of an epidemic of pneunionia.
New England Journul ot' Medicinc 297, 1 189- I I96
GEORGE.J . R., PINE, L., REEVES,M. W . & HARRELL,
W . K . (1980). Aminoacid requirementsof Leggronellu
pneumophila. Journul yJ' Cliniiwl Micwhiologj- 1 I ,
286-29 I .
IVANOVA. N. M., VAGANOVA.
T. I., STRONGIN,A . YA.
& STEPANOV,
V. M . (1977). Isoliition and properties
of 1euc i nea m i no pep t id ase from A.spcJrgi/lu.s(q ' x e .
Biokhiniiju 42. 843-849.
KALUGER.S. V., BOROVIKOVA,
V. P., LAVRENOVA,
G. I., STEPANOV,
V. M.. SHPOKENE.
A . P.. GUKEEVA,
M. P. & UZHKURENAS,
A . P. (1983). Extracellular
serine proteinase A of StroptonI~*c.e.srirrgtJrsrnsis.
Biokhimijw 48, 1483- 1490.
KOLEHMAINEN.
L. & MIKOLA, J . (1971). Partial
purification and enrymatic properties of a n aminourid
peptidase from barley. Archiw.s (!/ Bioi~honii.strj~
Biophj*sic.v 145, 633 -642.
LYUBLINSKAYA.
L. A , , YAKUSHEVA.
L. D. & STEPANOV,
V. M. (1977). Synthesis of subtilisin peptide subst rit t es ii nd the i r a n a logs, BioorRrrtiiL.hi..skul'u khiniijw
3, 273-279.
LYUBLINSKAYA,
L. A.. YUSUPOVA,M. P., VAGANOVA,
T. I., IVANOVA, N . M . & STEPANOV,
V. M. (1984).
Solid-phase synthesis of biospecific sorbent for
am i nope p t id ases. Bioorg(rtii~,~i~isk
ujw k hirnij.u 10,
1490- 1495
MERKEF,J . R.. LEE, C. C . & FREUND.
T. S. (1981). A
d i m e r ic ex t race I I u I ii r hea I st ;i ble ii m i nope p t i d a se
produced by a marine pseudomonad. Biochiniicu et
hiophysicu uctrr 661, 32 -38.
M ~ L L E RH., E. (1980). Proteolytic action of Legiunc4u
pneuniophilu. I n fcctioti rrtitl I n i n i i r n i ! , ~ 34, 299- 302.
MCLLER,H . E. ( I98 1 ). Enzymatic profile of Lr~giurrdlu
pnc~imrophilu. Journcrl of C'linii*irl MicrohioloKj- 13,
423- 426.
NINOMIYA,
K., TANAKA,
SH., KAWATA,SH. & MAKISUMI, S. (1981). Purification and properties of a n
aminopeptidase from seeds of Japanese apricot.
Journal of' Biochemistry 89, 193-20 I .
OKSENOIT,
E . S., PETROVA,E . N., FILIPPOVA,
I. Yu..
LAVRENOVA,
G . I., TARASOVA,
N . I. & STEPANOV,
V. M. (1983). Chromogenic a-chymotrypsin substrate -~ pyroglutamyl-phenylalanine p-nitroanilide.
Khimiyu prirodnjv.h soedineni 1, 122- 123.
PHILIPPOVA,
1. Yu., LYSOGORSKAYA,
E. N . , OKSENOIT,
E. S., RUDENSKAYA,
G . N . & STEPANOV,V. M.
( 1984). L-Pyroglutamyl-L-phenylalanyl-L-leucine-pnitroanilide - a chromogenic substrate tor thiol
proteinase assay. Anulyricul Biuchemisrrj- 142, 293297.
PRESCOTT.J . M., WILKES. S. H., WAGNER,F. w. &
WILSAN,K . J . ( I97 I ). Am)nionu.s aminopeptidase.
Improved isolation and some physical properties.
Jorrrnrrl
BioloKicrrl ( ' h c w i s t r j . 246, 1 756 1164.
RODRIGUEZ-ABSI,
J . & PRESCOTT, J . M . (1978).
Isolation iind properties of ;in aminopeptidase from
Btr cillus lich r ti If0 rm is. A rchires of' B iochcw is t ry unil
Bioph.r.sic.s 186. 383 391 .
STEPANOV,V. M., CHESTUKHINA,
G. G., RUDENSKAYA,
G. N., EPREMYAN,A. S., OSTERMAN,
A . L.,
KHODOVA,0. M. & BELYANOVA.
L. P . (1981). A new
subfamily of microbial serine proteinases? Structural
similarities of Bacillus thurirtgiensis and Thrrniouctinoniyces r dgaris ex t race1I u la r se r i ne protein ases.
Biochemical and Biophysicul Reseurch Comniuniculions 100, 1680-1687.
THOMPSON,
M. R., MILLER,R. D . & IGLEWSKI,B. H.
(1981). In vitro production of an extracellular
protease by Lxgionella pneumophilu. InJkctiun und
Immunity 34, 299-302.
VAGANOVA,
T. I., IVANOVA, N. M., YUSVPOVA,M . P. &
STEPANOV,
V. M . (1983). Exopeptidases of mycelial
fungi. In M j w l i u l Fungi ( Phj..siologj., Biochcwiistrj~.
Biotc~c~liriologj~),
pp. I 16 I 17. Edited by E. L. Golovlyev. Puschino: Nauchny Centr Biologicheskykh
Issledovany .
VODKIN,L. 0.& SCANDALIOUS,
J. G . (1980). Comparative properties of genetically defined peptidases in
maize. Biochemistrj* 19, 4660--4667.
WAGNER,F. W.. WILKES.S. H. & PRESCOTT.J . M.
(1972). Specificity of Arromonos aminopeptidase
toward amino acid amides and dipeptides. Journdof
Biologicd Ch~~niistry
247, 1208- I2 10.
WATERS,S. P. & DALLING,
M. J. (1984). Isolation and
some properties of an aminopeptidase from the
primary leaf of wheat (Triticum aestirum L. ). Plunt
Physiology 75, I 18- 1 24.
WEBER.K . & OSBORN,
M . (1969). The reliability of
molecular weight determination by dodecyl sulfate
polyacrylamide gel electrophoresis. Journul yf Biulogicd Chuniistrj~224, 4406-44 I 2.
WIERSMA.M., VERSTEEGH,
G., ASSINK,H . A., WELLING, G . W. & HARBER,W . (1978). Purification and
some properties of two extracellular proteolytic
enzymes, produced by C'ihrio SA I . Antonir run
Lwu~ttwhork44, I57 169.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 06 May 2017 01:27:27