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Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20
Degradation of Streptomyces Metalloprotease Inhibitor (SMPI) by Neutral Protease from Bacillus suhtilis var. amylosacchariticus a
a
a
a
Daisuke Tsuru , Yasuyuki Fujita , Shin-ya Morikawa , Kiyoshi Ito & Tadashi Yoshimoto
a
a
School of Pharmaceutical Sciences, Nagasaki University, Bunkyo-machi 1–14, Nagasaki 852, Japan Published online: 12 Jun 2014.
To cite this article: Daisuke Tsuru, Yasuyuki Fujita, Shin-ya Morikawa, Kiyoshi Ito & Tadashi Yoshimoto (1992) Degradation of Streptomyces Metalloprotease Inhibitor (SMPI) by Neutral Protease from Bacillus suhtilis var. amylosacchariticus, Bioscience, Biotechnology, and Biochemistry, 56:8, 1275-1278, DOI: 10.1271/bbb.56.1275 To link to this article: http://dx.doi.org/10.1271/bbb.56.1275
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Biosci. Biotech. Biochem., 56 (8), 1275-1278, 1992
Degradation of Streptomyces Metalloprotease Inhibitor (SMPI) by Neutral Protease from Bacillus suhtilis var. amylosacchariticus Daisuke TSURU, Yasuyuki FUJITA, Shin-ya MORIKAWA, Kiyoshi ITO, and Tadashi YOSHIMOTO School of Pharmaceutical Sciences, Nagasaki University, Bunkyo-machi 1-14, Nagasaki 852, Japan Received February 21, 1992
Downloaded by [University of Hawaii at Manoa] at 08:58 25 December 2014
The zinc-containing neutral endopeptidase (neutral protease: BANP) from Bacillus subtilis var. amylosacchariticus was inhibited by the proteinaceous metalloprotease inhibitor isolated from Streptomyces nigrescens (SMPI). The degree of inhibition was, however, significantly less than that for thermolysin (TLN). During incubation of BANP with SMPI, the inhibitor was proteolytically degraded and inactivated. Analysis of the digestion products suggested that a minor diversity in their substrate specificities between TLN and BANP affects the sensitivity to the proteinaceous metalloprotease inhibitor, SMPI.
In 1978, Murao and co-workers 1 ) isolated a potent proteinaceous metalloprotease inhibitor from the culture filtrate of Streptomyces nigrescens TK-23 and named it Streptomyces metalloprotease inhibitor, SMPI. It is a single polypeptide with a molecular mass of about 10 kDa and inhibits metalloendopeptidases such as TLN, B. subtilis YT -25 neutral protease, and Pseudomonas elastase. 2 ) Its complete amino acid sequence was analyzed by Murai et al. 3) and the reactive site was proposed to be the C ys 64-Va1 65 region. We have completely sequenced amino acids of the neutral endopeptidase from B. amylosacchariticus (BANP).4) It is a Zn-protease highly homologous to TLN in primary and three-dimensional structures. 5) However, BANP was shown to be significantly less sensitive to SMPI than B. subtilis YT-25 metalloprotease, Pseudomonas elastase or TLN. 2 ) Through the analysis of BANP-SMPI interaction, we found that SMPI was proteolytically degraded by BANP. This paper deals with comparison of the inhibitory effects of SMPI on BANP and TLN, and sequence analysis of the digestion products.
Separation of pep tides resulted by proteolytic digestion of SMPI. The reaction mixture composed of 42 J1.M SMPI and 1.9 J1.M BANP (or TLN) in 230 J1.1 of 20 mM Tris-HCI buffer containing 2 mM CaCl z, pH 7.2, was incubated at 3JOC for 5 hr. The reaction was stopped by the addition of 25 J1.1 of 99% formic acid and the mixture was analyzed by HPLC on a Vydac CI8 column (5 J1.m, 300 A, 4.6 x 250 mm, Separations group, CA, U.S.A.) using the solvent system of 0.075% trifluoroacetic acid (TFA) in water vs. acetonitrile-2-propanol (3: 1) with 0.06% TFA. Pep tides were eluted by an increasing gradient of organic solvent concentration at a flow rate of 1.0 ml/min and at room temperature. Elution of peptides was monitored by measuring absorbances at 214, 280, and/or 290 nm. Analyses of amino acid sequence and composition. The amino acids of each peptide were sequenced by the Edman degradation procedure as described by Kobayashi and Tarr 9 ) and the amino acid composition was estimated by the method of Spackman et al. IO ) as mentioned previously. I I)
Results and Discussion Comparison in inhibitory activity of SMPI on BANP and TLN Figure 1 shows the inhibitory effects of SMPI on BANP compared with that on TLN. TLN was severely inhibited by SMPI with a IC 50 value of about 10- 8 M, but BANP was less sensitive to the inhibitor (IC 50 : 6 x 10- 6 M).
Materials and Methods Materials. BANP was purified as described previously6) with some modifications. 4) TLN was obtained from Sigma, U.S.A. SMPI was a generous gift from Dr. K. Oda, Kyoto Institute of Technology and also Dr. B. Tonomura of Kyoto University. Sequencing grade chemicals were purchased from Nacalai Tesque, Kyoto. Phenylisothiocyanate and N-blocked synthetic peptide substrates were from Pierce, U.S.A. and the Peptide Institute Inc., Minoh, respectively. Assays of proteolytic activity and protein concentration. The caseinolytic activity was assayed at pH 7.2 and 37°C by the method described previously,6) and protein concentration was estimated spectrophotometrically by assuming that E~ %m at 280 nm was 13.8 and 17.7 for BANP and TLN, respectively. Proteolytic activities of BANP and TLN towards N-blocked synthetic peptides were assayed as described previously.7,8) Inhibition of metalloproteases by incubation with SMPI. The reaction mixtures were composed of 13 nM enzyme and varied concentrations of the SMPI in 200 J1.1 of 20 mM Tris-HCI buffer containing 5 mM Ca-acetate, pH 7.2. After 10 min of incubation, the residual activities were assayed by the standard method. IC so was defined as the concentration of SMPI that causes 50% inactivation of enzymes under these conditions.
Analysis of proteolytic degradation products from SM P I A mixture of SMPI and BANP in a molar ratio of about
~
100
'-'
80
:E.....
60
......... 1:..1 ~
-; 40
=
"C '(iJ a.I
20
~
0
-9
-8
-7
-6
-5
-4
log I (M) Fig. 1. Comparison in Inhibitory Activity of SMPI towards BANP and TLN. See the text for the experimental details.
Abbreviations: BANP, neutral protease from Bacillus subtilis var. amylosacchariticus; B. amylosacchariticus, Bacillus subtilis var. amylosacchariticus; DFP, diisopropyl fluorophosphate; EDTA, ethylenediamine tetraacetate; HPLC, high performance liquid chromatography; SMPI, Streptomyces metalloprotease inhibitor; TF A, trifluoroacetic acid; TLN, thermolysin.
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D. TSURU et al.
1276 Table I.
Amino Acid Composition of Peptides Derived from SMPI by Incubation with BANP
Peptide No.
N-l
N-2
N-3
N-5
N-4
N-6
N-7
N-8
N-9
N-lO
N-I2
N-ll
N-13
N-I4
N-15
1.0(1)
2.4 (2) 1.1 (1) 1.9 (3) 1.5 (1) 2.6 (2) 3.5 (4)
- - - - - - - - - - - - -..
Downloaded by [University of Hawaii at Manoa] at 08:58 25 December 2014
Asx Thr Ser Glx Gly Ala 1/2 Cys Val Met Ile Leu Tyr Phe His Lys Arg Pro Yields (%) Number in sequence
1.1 (1) 0.8 (1)
1.1 (1) 1.6 (2) 1.1 (1) 1.4 (1) 1.1 (1)
2.3 (2)
1.2 (1) 1.1 (1)
0.9 (1)
2.4 (2) 1.1 (1) 2.7 (3) 1.1 (1) 2.4 (2) 3.6 (4)
1.9 (2) 0.8 (1) 1.3 (1)
1.0 (2) 2.6 (3)
1.6 (2) 1.3 (1) 2.2 (2)
1.4 (1) 1.2 (1)
1.9 (2)
0.5 (1)
1.4 (1) 0.8 (1)
3.0 (3) 2.6 (2) 0.7 (2)
2.3 (2) 1.6 (1)
1.1 (1) 1.0 (1)
0.9 (1)
1.3 (2) 1.4 (1) 1.1 (1) 0.9 (1) 0.8 (2) 2.1 (3)
0.5 (1) 0.8 (1)
1.0 (1)
1.3 (1)
0.9 (1) 1.0 (1)
1.3 (1) 1.2 (1)
1.4 (1) 1.0 (1)
1.8 (2)
0.9 (1) 0.4 (1) 1.0 (1)
1.2 (2)
1.1 (1) 0.4 (1) 1.4 (1)
2.4 (2) 1.0 (1) 1.8 (2) 1.0 (1) 1.0 (1)
0.6 (1) 0.8 (1) 1.2 (1)
0.7 (1)
2.0 (2) 1.0 (1) 1.6 (2)
0.6 (1) 1.3 (2) 1.0 (1)
1.4 (2)
0.9 (1) 1.3 (2)
22 54 18 32 17-22 96-102 39-45 54-61
32 1-16
44 12 14 63 10 84-9.1 26-45 62-77 23-31 23-45
1.4 (1) 1.0(1)
0.8 (1) 1.0 (1)
44 56 54 30 29 78-81 52-53 11-16 23-25 46-51
1.2 (1)
1.3 (1)
0.8 (1) 0.9 (1)
1.0 (1)
1.1 (1) 0.9 (1)
1.0 (1)
- - - - - - - - - - - - - - - - " - _ . - - - - " - - - . -.. -..
_---
The numbers in parentheses are predicted values from the established sequence of SMP1.
Table II. Amino Acid Sequences of Peptide Fragments Derived from SMPI by Incubation with BANP. Peptide No.
0.3
I
'-'
N-13 'SO
,...... 40
,,
N-I N-2 N-3 N-4 N-5 N-6 N-7 N-8
Sequence Val-Ala-Pro-Gly Phe-Thr Thr-Tyr-Ser-Gly-Thr-Gly Val-Ile-Pro Phe-Ala-Asn-Gly-ThrLeu-Ser-Gly-Ala-Arg-Thr Val-Gly-Gln-Asp-LeuLeu-Pro-Ala -Ser-A1a-
Peptide No.
Sequence
N-9 N-lO N-11 N-12 N-13
Leu-Arg-Tyr-Gly-ProAla -Pro-SerLeu-Arg-Ser-Leu-ProAla-Ser-Asp-Met-GluVal-ThrVal-Arg-Phe-ProN-14 Val-Ile-Pro-Ala-SerN-15 Val-Ile-Pro-Ala-Ser-
I
....;
..
30
Peptide No., see Fig. 2 and Table 1.
~
:::c
o
f-o p..,
N-ll 20
~
zu
...,
:::c u
10 ....
0.1
N-9 N-15
N-sl N-12
\
J
10
20
30
40
N-14
so
60
RETENTION TIME (min) Fig. 2. HPLC Profile of Peptide Fragments Derived from SMPI by Incubation with BANP. About 90/lg of peptide mixture was injected. The other experimental details are described in the text.
20: 1 was incubated at 37°C, and samples were periodically removed and analyzed by HPLC. Several peptide fragments were found to increase gradually with time, and the inhibitory activity to be lost in parallel, suggesting that SMPI was susceptible to the proteolytic action of BANP. An HPLC profile of peptides from a 5 hr incubation is illustrated in Fig. 2. Fifteen peptides were separated and purified. The amino acid compositions of these peptides are shown in Table I, and complete or partial amino acid sequences are shown in Table II. Fragment 13 gave two amino terminal sequences, Val-Thr- and Val-ArgPhe-Pro-, indicating that two peptides, Va1 62 _Cys 64 and VaI 65 -Lys77, are linked through a disulfide bridge. The amino acid compositions and amino terminal sequences of these peptides well coincided with those expected from the established sequence of SMPI, with a few exceptions. Taking account of these results, we conclude that SMPI was cleaved at sixteen sites of peptide bonds by BANP under the conditions, as shown in Fig. 3. Digestion of SMPI with TLN was also examined under the same conditions. A new peak was observed and increased
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1277
Degradation of Metalloprotease Inhibitor by B. subtilis Neutral Protease
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with time, accompanying a decrease in the native SMPI (Fig. 4). This suggests that only one peptide bond was cleaved in TLN. The proteolytically modified SMPI (the front peak in Fig. 4) gave two amino terminal sequences; Ala-Pro-Ser- (the amino terminal sequence of the virgin SMPI) and Val-Arg-Phe-Pro-. This result indicates that SMPI is cleaved only at the Cys64_Va1 65 bond, which has been suggested to be a reactive site of SMPI for TLN 3 ) and that the resulted two fragments are linked together through a disulfide bridge.
manner as the untreated enzyme. In fact, Boc-Leu-Thr-ArgMCA and Boc-Leu-Ser-Thr-Arg-MCA were hydrolyzed by BANP at the amino side of threonine residue, while TLN was completely inert toward these substrates (Table III). It seems likely that such a discrepancy in substrate specificity is closely related to diversity in the proteolytic digestion pattern of SMPI with TLN and BANP, though more detailed experiments are required to confirm this assumption. As shown previously,4) however, both Znproteases are highly homologous in their amino acid sequences of active site regions, and the reason for discrepancy in their substrate specificities between the two enzymes remains ambiguous at present. We are now
Comparison in substrate specificity between BANP and TLN Zn-containing neutral endopeptidases have been reported to preferentially split peptide bonds involving amino termini of hydrophobic L-amino acid residues. 12 14} Surprisingly, however, the CyslO-Thrl1 bond of SMPI was shown to be cleaved through SMPI-BANP interaction (Tables I and II and Fig. 3). This activity was confirmed to be an intrinsic action ofBANP, since treatment ofBANP with 1 mM EDT A completely abolished proteolytic cleavage of SMPI, while the DFP-treated enzyme degraded the inhibitor in the same I
Table III. Comparison in Hydrolytic Activity towards Synthetie Substrates between BANP and TLN The reaction mixtures were composed of 1-2 mM substrates, 1-3 fJ,M enzyme in 20 mM Tris-HCI buffer containing 2 mM Ca acetate and 5% dimethylformamide, pH 7.2. After incubation for 5 to 30 min at 37°C, the enzyme reaction was stopped by the addition of 1/10 volume of 99% formic acid. Digestion products were separated and measured by HPLC as described previously. 7,8)
110
Aia-Pro-Ser-Cys-Pro-Ala-Gly-Ser-Leu-Cys-Thr-Tyr-Ser-GIy-Thr-
~o
+
20
Activity (fJ,mol hydrolyzed/min, mg enzyme)
Cone. (mM)
Substrates
Gly-Leu-Ser-Gly-Ala-Arg-Thr -Val-Ile-Pro-Ala-Ser-Asp-Met -Glu-
+
+
4j
L Z-Gly-Leu-NH 2
LYStAla-GlY-Thr-Asp-Gly- Val-Lysteu-prO-Ala-ser-Ala-Arg-Ser50
+
I
+
*
+I
2
1.8
6.7
2
2.1
7.2
L Z-Gly-Phe-NH 2
2
0.06
2.9
2
1.9
4.2
2
6.6
5.2
2.9
4.8
L FA-Gly-Leu-NH 2 L Pyr-Phe-Leu-NH 2 L Z-Gly-Gly-Leu-pNA
70
LYSlal-Thr-Cys-Val-Arg-Phe-Pro-Cys-Tyr-Gln-Tyr-Ala-Thr-Val-
+80
90
Gly -Lys-Val- Ala-Pro-Gly -Ala-Gln-Leu- Arg -Ser-Leu -Pro-Ser -Pro-
+
+
+
100
102
GlYtA1a-Thr-Val-Thr+Val-Gly-Gln-Asp-Leu-Gly-Asp
TLN
Z-Ala-LLeu- NH2
60
Phe-Ala-Asn-Gly-Thr-His- Phe-Thr-Leu-Arg-Tyr-Gly-Pro-Ala-Arg-
BANP
Boc- Leu t-Thr- Arg- MCA
2
0.09
Not hydrolyzed
Boc - Leu -Ser tThr -Arg - MeA
2
1.6
Not hydrolyzed
Boc, t-butyloxycarbonyl; FA, furylacryloyl; Pyr, pyroglutamyl; Z, benzyloxycarbonyl; pNA, p-nitroanilide; MCA, 4-methyl-coumaryl-7amide.
Fig. 3. Amino Acid Sequence of SMPI and the Cleavage Sites by BANP.
0.12 ,-..
C
B
A
M
50 r:.; ~ ~
::c: 0
0.08
I-
Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20
Degradation of Streptomyces Metalloprotease Inhibitor (SMPI) by Neutral Protease from Bacillus suhtilis var. amylosacchariticus a
a
a
a
Daisuke Tsuru , Yasuyuki Fujita , Shin-ya Morikawa , Kiyoshi Ito & Tadashi Yoshimoto
a
a
School of Pharmaceutical Sciences, Nagasaki University, Bunkyo-machi 1–14, Nagasaki 852, Japan Published online: 12 Jun 2014.
To cite this article: Daisuke Tsuru, Yasuyuki Fujita, Shin-ya Morikawa, Kiyoshi Ito & Tadashi Yoshimoto (1992) Degradation of Streptomyces Metalloprotease Inhibitor (SMPI) by Neutral Protease from Bacillus suhtilis var. amylosacchariticus, Bioscience, Biotechnology, and Biochemistry, 56:8, 1275-1278, DOI: 10.1271/bbb.56.1275 To link to this article: http://dx.doi.org/10.1271/bbb.56.1275
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Biosci. Biotech. Biochem., 56 (8), 1275-1278, 1992
Degradation of Streptomyces Metalloprotease Inhibitor (SMPI) by Neutral Protease from Bacillus suhtilis var. amylosacchariticus Daisuke TSURU, Yasuyuki FUJITA, Shin-ya MORIKAWA, Kiyoshi ITO, and Tadashi YOSHIMOTO School of Pharmaceutical Sciences, Nagasaki University, Bunkyo-machi 1-14, Nagasaki 852, Japan Received February 21, 1992
Downloaded by [University of Hawaii at Manoa] at 08:58 25 December 2014
The zinc-containing neutral endopeptidase (neutral protease: BANP) from Bacillus subtilis var. amylosacchariticus was inhibited by the proteinaceous metalloprotease inhibitor isolated from Streptomyces nigrescens (SMPI). The degree of inhibition was, however, significantly less than that for thermolysin (TLN). During incubation of BANP with SMPI, the inhibitor was proteolytically degraded and inactivated. Analysis of the digestion products suggested that a minor diversity in their substrate specificities between TLN and BANP affects the sensitivity to the proteinaceous metalloprotease inhibitor, SMPI.
In 1978, Murao and co-workers 1 ) isolated a potent proteinaceous metalloprotease inhibitor from the culture filtrate of Streptomyces nigrescens TK-23 and named it Streptomyces metalloprotease inhibitor, SMPI. It is a single polypeptide with a molecular mass of about 10 kDa and inhibits metalloendopeptidases such as TLN, B. subtilis YT -25 neutral protease, and Pseudomonas elastase. 2 ) Its complete amino acid sequence was analyzed by Murai et al. 3) and the reactive site was proposed to be the C ys 64-Va1 65 region. We have completely sequenced amino acids of the neutral endopeptidase from B. amylosacchariticus (BANP).4) It is a Zn-protease highly homologous to TLN in primary and three-dimensional structures. 5) However, BANP was shown to be significantly less sensitive to SMPI than B. subtilis YT-25 metalloprotease, Pseudomonas elastase or TLN. 2 ) Through the analysis of BANP-SMPI interaction, we found that SMPI was proteolytically degraded by BANP. This paper deals with comparison of the inhibitory effects of SMPI on BANP and TLN, and sequence analysis of the digestion products.
Separation of pep tides resulted by proteolytic digestion of SMPI. The reaction mixture composed of 42 J1.M SMPI and 1.9 J1.M BANP (or TLN) in 230 J1.1 of 20 mM Tris-HCI buffer containing 2 mM CaCl z, pH 7.2, was incubated at 3JOC for 5 hr. The reaction was stopped by the addition of 25 J1.1 of 99% formic acid and the mixture was analyzed by HPLC on a Vydac CI8 column (5 J1.m, 300 A, 4.6 x 250 mm, Separations group, CA, U.S.A.) using the solvent system of 0.075% trifluoroacetic acid (TFA) in water vs. acetonitrile-2-propanol (3: 1) with 0.06% TFA. Pep tides were eluted by an increasing gradient of organic solvent concentration at a flow rate of 1.0 ml/min and at room temperature. Elution of peptides was monitored by measuring absorbances at 214, 280, and/or 290 nm. Analyses of amino acid sequence and composition. The amino acids of each peptide were sequenced by the Edman degradation procedure as described by Kobayashi and Tarr 9 ) and the amino acid composition was estimated by the method of Spackman et al. IO ) as mentioned previously. I I)
Results and Discussion Comparison in inhibitory activity of SMPI on BANP and TLN Figure 1 shows the inhibitory effects of SMPI on BANP compared with that on TLN. TLN was severely inhibited by SMPI with a IC 50 value of about 10- 8 M, but BANP was less sensitive to the inhibitor (IC 50 : 6 x 10- 6 M).
Materials and Methods Materials. BANP was purified as described previously6) with some modifications. 4) TLN was obtained from Sigma, U.S.A. SMPI was a generous gift from Dr. K. Oda, Kyoto Institute of Technology and also Dr. B. Tonomura of Kyoto University. Sequencing grade chemicals were purchased from Nacalai Tesque, Kyoto. Phenylisothiocyanate and N-blocked synthetic peptide substrates were from Pierce, U.S.A. and the Peptide Institute Inc., Minoh, respectively. Assays of proteolytic activity and protein concentration. The caseinolytic activity was assayed at pH 7.2 and 37°C by the method described previously,6) and protein concentration was estimated spectrophotometrically by assuming that E~ %m at 280 nm was 13.8 and 17.7 for BANP and TLN, respectively. Proteolytic activities of BANP and TLN towards N-blocked synthetic peptides were assayed as described previously.7,8) Inhibition of metalloproteases by incubation with SMPI. The reaction mixtures were composed of 13 nM enzyme and varied concentrations of the SMPI in 200 J1.1 of 20 mM Tris-HCI buffer containing 5 mM Ca-acetate, pH 7.2. After 10 min of incubation, the residual activities were assayed by the standard method. IC so was defined as the concentration of SMPI that causes 50% inactivation of enzymes under these conditions.
Analysis of proteolytic degradation products from SM P I A mixture of SMPI and BANP in a molar ratio of about
~
100
'-'
80
:E.....
60
......... 1:..1 ~
-; 40
=
"C '(iJ a.I
20
~
0
-9
-8
-7
-6
-5
-4
log I (M) Fig. 1. Comparison in Inhibitory Activity of SMPI towards BANP and TLN. See the text for the experimental details.
Abbreviations: BANP, neutral protease from Bacillus subtilis var. amylosacchariticus; B. amylosacchariticus, Bacillus subtilis var. amylosacchariticus; DFP, diisopropyl fluorophosphate; EDTA, ethylenediamine tetraacetate; HPLC, high performance liquid chromatography; SMPI, Streptomyces metalloprotease inhibitor; TF A, trifluoroacetic acid; TLN, thermolysin.
NII-Electronic Library Service
D. TSURU et al.
1276 Table I.
Amino Acid Composition of Peptides Derived from SMPI by Incubation with BANP
Peptide No.
N-l
N-2
N-3
N-5
N-4
N-6
N-7
N-8
N-9
N-lO
N-I2
N-ll
N-13
N-I4
N-15
1.0(1)
2.4 (2) 1.1 (1) 1.9 (3) 1.5 (1) 2.6 (2) 3.5 (4)
- - - - - - - - - - - - -..
Downloaded by [University of Hawaii at Manoa] at 08:58 25 December 2014
Asx Thr Ser Glx Gly Ala 1/2 Cys Val Met Ile Leu Tyr Phe His Lys Arg Pro Yields (%) Number in sequence
1.1 (1) 0.8 (1)
1.1 (1) 1.6 (2) 1.1 (1) 1.4 (1) 1.1 (1)
2.3 (2)
1.2 (1) 1.1 (1)
0.9 (1)
2.4 (2) 1.1 (1) 2.7 (3) 1.1 (1) 2.4 (2) 3.6 (4)
1.9 (2) 0.8 (1) 1.3 (1)
1.0 (2) 2.6 (3)
1.6 (2) 1.3 (1) 2.2 (2)
1.4 (1) 1.2 (1)
1.9 (2)
0.5 (1)
1.4 (1) 0.8 (1)
3.0 (3) 2.6 (2) 0.7 (2)
2.3 (2) 1.6 (1)
1.1 (1) 1.0 (1)
0.9 (1)
1.3 (2) 1.4 (1) 1.1 (1) 0.9 (1) 0.8 (2) 2.1 (3)
0.5 (1) 0.8 (1)
1.0 (1)
1.3 (1)
0.9 (1) 1.0 (1)
1.3 (1) 1.2 (1)
1.4 (1) 1.0 (1)
1.8 (2)
0.9 (1) 0.4 (1) 1.0 (1)
1.2 (2)
1.1 (1) 0.4 (1) 1.4 (1)
2.4 (2) 1.0 (1) 1.8 (2) 1.0 (1) 1.0 (1)
0.6 (1) 0.8 (1) 1.2 (1)
0.7 (1)
2.0 (2) 1.0 (1) 1.6 (2)
0.6 (1) 1.3 (2) 1.0 (1)
1.4 (2)
0.9 (1) 1.3 (2)
22 54 18 32 17-22 96-102 39-45 54-61
32 1-16
44 12 14 63 10 84-9.1 26-45 62-77 23-31 23-45
1.4 (1) 1.0(1)
0.8 (1) 1.0 (1)
44 56 54 30 29 78-81 52-53 11-16 23-25 46-51
1.2 (1)
1.3 (1)
0.8 (1) 0.9 (1)
1.0 (1)
1.1 (1) 0.9 (1)
1.0 (1)
- - - - - - - - - - - - - - - - " - _ . - - - - " - - - . -.. -..
_---
The numbers in parentheses are predicted values from the established sequence of SMP1.
Table II. Amino Acid Sequences of Peptide Fragments Derived from SMPI by Incubation with BANP. Peptide No.
0.3
I
'-'
N-13 'SO
,...... 40
,,
N-I N-2 N-3 N-4 N-5 N-6 N-7 N-8
Sequence Val-Ala-Pro-Gly Phe-Thr Thr-Tyr-Ser-Gly-Thr-Gly Val-Ile-Pro Phe-Ala-Asn-Gly-ThrLeu-Ser-Gly-Ala-Arg-Thr Val-Gly-Gln-Asp-LeuLeu-Pro-Ala -Ser-A1a-
Peptide No.
Sequence
N-9 N-lO N-11 N-12 N-13
Leu-Arg-Tyr-Gly-ProAla -Pro-SerLeu-Arg-Ser-Leu-ProAla-Ser-Asp-Met-GluVal-ThrVal-Arg-Phe-ProN-14 Val-Ile-Pro-Ala-SerN-15 Val-Ile-Pro-Ala-Ser-
I
....;
..
30
Peptide No., see Fig. 2 and Table 1.
~
:::c
o
f-o p..,
N-ll 20
~
zu
...,
:::c u
10 ....
0.1
N-9 N-15
N-sl N-12
\
J
10
20
30
40
N-14
so
60
RETENTION TIME (min) Fig. 2. HPLC Profile of Peptide Fragments Derived from SMPI by Incubation with BANP. About 90/lg of peptide mixture was injected. The other experimental details are described in the text.
20: 1 was incubated at 37°C, and samples were periodically removed and analyzed by HPLC. Several peptide fragments were found to increase gradually with time, and the inhibitory activity to be lost in parallel, suggesting that SMPI was susceptible to the proteolytic action of BANP. An HPLC profile of peptides from a 5 hr incubation is illustrated in Fig. 2. Fifteen peptides were separated and purified. The amino acid compositions of these peptides are shown in Table I, and complete or partial amino acid sequences are shown in Table II. Fragment 13 gave two amino terminal sequences, Val-Thr- and Val-ArgPhe-Pro-, indicating that two peptides, Va1 62 _Cys 64 and VaI 65 -Lys77, are linked through a disulfide bridge. The amino acid compositions and amino terminal sequences of these peptides well coincided with those expected from the established sequence of SMPI, with a few exceptions. Taking account of these results, we conclude that SMPI was cleaved at sixteen sites of peptide bonds by BANP under the conditions, as shown in Fig. 3. Digestion of SMPI with TLN was also examined under the same conditions. A new peak was observed and increased
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Degradation of Metalloprotease Inhibitor by B. subtilis Neutral Protease
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with time, accompanying a decrease in the native SMPI (Fig. 4). This suggests that only one peptide bond was cleaved in TLN. The proteolytically modified SMPI (the front peak in Fig. 4) gave two amino terminal sequences; Ala-Pro-Ser- (the amino terminal sequence of the virgin SMPI) and Val-Arg-Phe-Pro-. This result indicates that SMPI is cleaved only at the Cys64_Va1 65 bond, which has been suggested to be a reactive site of SMPI for TLN 3 ) and that the resulted two fragments are linked together through a disulfide bridge.
manner as the untreated enzyme. In fact, Boc-Leu-Thr-ArgMCA and Boc-Leu-Ser-Thr-Arg-MCA were hydrolyzed by BANP at the amino side of threonine residue, while TLN was completely inert toward these substrates (Table III). It seems likely that such a discrepancy in substrate specificity is closely related to diversity in the proteolytic digestion pattern of SMPI with TLN and BANP, though more detailed experiments are required to confirm this assumption. As shown previously,4) however, both Znproteases are highly homologous in their amino acid sequences of active site regions, and the reason for discrepancy in their substrate specificities between the two enzymes remains ambiguous at present. We are now
Comparison in substrate specificity between BANP and TLN Zn-containing neutral endopeptidases have been reported to preferentially split peptide bonds involving amino termini of hydrophobic L-amino acid residues. 12 14} Surprisingly, however, the CyslO-Thrl1 bond of SMPI was shown to be cleaved through SMPI-BANP interaction (Tables I and II and Fig. 3). This activity was confirmed to be an intrinsic action ofBANP, since treatment ofBANP with 1 mM EDT A completely abolished proteolytic cleavage of SMPI, while the DFP-treated enzyme degraded the inhibitor in the same I
Table III. Comparison in Hydrolytic Activity towards Synthetie Substrates between BANP and TLN The reaction mixtures were composed of 1-2 mM substrates, 1-3 fJ,M enzyme in 20 mM Tris-HCI buffer containing 2 mM Ca acetate and 5% dimethylformamide, pH 7.2. After incubation for 5 to 30 min at 37°C, the enzyme reaction was stopped by the addition of 1/10 volume of 99% formic acid. Digestion products were separated and measured by HPLC as described previously. 7,8)
110
Aia-Pro-Ser-Cys-Pro-Ala-Gly-Ser-Leu-Cys-Thr-Tyr-Ser-GIy-Thr-
~o
+
20
Activity (fJ,mol hydrolyzed/min, mg enzyme)
Cone. (mM)
Substrates
Gly-Leu-Ser-Gly-Ala-Arg-Thr -Val-Ile-Pro-Ala-Ser-Asp-Met -Glu-
+
+
4j
L Z-Gly-Leu-NH 2
LYStAla-GlY-Thr-Asp-Gly- Val-Lysteu-prO-Ala-ser-Ala-Arg-Ser50
+
I
+
*
+I
2
1.8
6.7
2
2.1
7.2
L Z-Gly-Phe-NH 2
2
0.06
2.9
2
1.9
4.2
2
6.6
5.2
2.9
4.8
L FA-Gly-Leu-NH 2 L Pyr-Phe-Leu-NH 2 L Z-Gly-Gly-Leu-pNA
70
LYSlal-Thr-Cys-Val-Arg-Phe-Pro-Cys-Tyr-Gln-Tyr-Ala-Thr-Val-
+80
90
Gly -Lys-Val- Ala-Pro-Gly -Ala-Gln-Leu- Arg -Ser-Leu -Pro-Ser -Pro-
+
+
+
100
102
GlYtA1a-Thr-Val-Thr+Val-Gly-Gln-Asp-Leu-Gly-Asp
TLN
Z-Ala-LLeu- NH2
60
Phe-Ala-Asn-Gly-Thr-His- Phe-Thr-Leu-Arg-Tyr-Gly-Pro-Ala-Arg-
BANP
Boc- Leu t-Thr- Arg- MCA
2
0.09
Not hydrolyzed
Boc - Leu -Ser tThr -Arg - MeA
2
1.6
Not hydrolyzed
Boc, t-butyloxycarbonyl; FA, furylacryloyl; Pyr, pyroglutamyl; Z, benzyloxycarbonyl; pNA, p-nitroanilide; MCA, 4-methyl-coumaryl-7amide.
Fig. 3. Amino Acid Sequence of SMPI and the Cleavage Sites by BANP.
0.12 ,-..
C
B
A
M
50 r:.; ~ ~
::c: 0
0.08
I-
