Purification and Characterization of a Trypsin-Like Enzyme with Fibrinolytic Activity Present in the Abdomen of Horn Fly, Haematobia irritans irritans (Diptera: Muscidae)
Dametto, M. ; David, A. P. ; Azzolini, S. S. ; Campos, I. T. N. ; Tanaka, A. M. ; Gomes, A. ; Andreotti, R. ; Tanaka, A. S.
Springer
Published 2000
Springer
Published 2000
| ISSN: |
1573-4943
|
|---|---|
| Keywords: |
trypsin-like enzyme ; fibrinolytic activity ; protein purification ; hematophagous ; Haematobia irritans irritans
|
| Source: |
Springer Online Journal Archives 1860-2000
|
| Topics: |
Chemistry and Pharmacology
|
| Notes: |
Abstract This work describes the purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of Haematobia irritans irritans (Diptera: Muscidae). The enzyme was purified using a one-step process, consisting of affinity chromatography on SBTI-Sepharose. The purified protease showed one major active proteinase band on reverse zymography with 0.15% gelatin, corresponding to a molecular mass of 25.5 kDa, with maximum activity at pH 9.0. The purified trypsin-like enzyme preferentially hydrolyzed synthetic substrates with arginine residue at the P1 position. The K m values determined for three different substrates were 1.88 × 10−4, 1.28 × 10−4, and 1.40 × 10−4 M for H-α-benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S2222), dl-Ile-Pro-Arg-p-nitroanilide (S2288), and DL-Phe-Pip-Arg-p-nitroanilide (S2238), respectively. The enzyme was strongly inhibited by typical serine proteinase inhibitors such as SBTI (soybean trypsin inhibitor, K i = 0.19 nM) and BuXI (Bauhinia ungulata factor Xa inhibitor, K i = 0.48 nM), and less inhibited by LDTI (leech-derived tryptase inhibitor, K i = 1.5 nM) and its variants LDTI 2T and 5T (0.8 and 1.5 nM, respectively). The most effective inhibitor for this protease was r-aprotinin (r-BPTI) with a K i value of 39 pM. Synthetic serine protease inhibitors presented only weak inhibition, e.g., benzamidine with K i = 3.0 × 10−4 M and phenylmethylsulfonyl fluoride (PMSF) showed traces of inhibition. The purified trypsin-like enzyme also digested natural substrates such as fibrinogen and fibrin net. The protease showed higher activity against fibrinogen and fibrin than did bovine trypsin. These data suggest that the proteolytic enzyme of H. irritans irritans is more specific to proteins from blood than are the vertebrate digestive enzymes. This enzyme's characteristics may be an adaptation resulting from the feeding behavior of this hematophagous insect.
|
| Type of Medium: |
Electronic Resource
|
| URL: |
| _version_ | 1798296777869230080 |
|---|---|
| autor | Dametto, M. David, A. P. Azzolini, S. S. Campos, I. T. N. Tanaka, A. M. Gomes, A. Andreotti, R. Tanaka, A. S. |
| autorsonst | Dametto, M. David, A. P. Azzolini, S. S. Campos, I. T. N. Tanaka, A. M. Gomes, A. Andreotti, R. Tanaka, A. S. |
| book_url | http://dx.doi.org/10.1023/A:1026557600429 |
| datenlieferant | nat_lic_papers |
| hauptsatz | hsatz_simple |
| identnr | NLM197813186 |
| issn | 1573-4943 |
| journal_name | The protein journal |
| materialart | 1 |
| notes | Abstract This work describes the purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of Haematobia irritans irritans (Diptera: Muscidae). The enzyme was purified using a one-step process, consisting of affinity chromatography on SBTI-Sepharose. The purified protease showed one major active proteinase band on reverse zymography with 0.15% gelatin, corresponding to a molecular mass of 25.5 kDa, with maximum activity at pH 9.0. The purified trypsin-like enzyme preferentially hydrolyzed synthetic substrates with arginine residue at the P1 position. The K m values determined for three different substrates were 1.88 × 10−4, 1.28 × 10−4, and 1.40 × 10−4 M for H-α-benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S2222), dl-Ile-Pro-Arg-p-nitroanilide (S2288), and DL-Phe-Pip-Arg-p-nitroanilide (S2238), respectively. The enzyme was strongly inhibited by typical serine proteinase inhibitors such as SBTI (soybean trypsin inhibitor, K i = 0.19 nM) and BuXI (Bauhinia ungulata factor Xa inhibitor, K i = 0.48 nM), and less inhibited by LDTI (leech-derived tryptase inhibitor, K i = 1.5 nM) and its variants LDTI 2T and 5T (0.8 and 1.5 nM, respectively). The most effective inhibitor for this protease was r-aprotinin (r-BPTI) with a K i value of 39 pM. Synthetic serine protease inhibitors presented only weak inhibition, e.g., benzamidine with K i = 3.0 × 10−4 M and phenylmethylsulfonyl fluoride (PMSF) showed traces of inhibition. The purified trypsin-like enzyme also digested natural substrates such as fibrinogen and fibrin net. The protease showed higher activity against fibrinogen and fibrin than did bovine trypsin. These data suggest that the proteolytic enzyme of H. irritans irritans is more specific to proteins from blood than are the vertebrate digestive enzymes. This enzyme's characteristics may be an adaptation resulting from the feeding behavior of this hematophagous insect. |
| package_name | Springer |
| publikationsjahr_anzeige | 2000 |
| publikationsjahr_facette | 2000 |
| publikationsjahr_intervall | 7999:2000-2004 |
| publikationsjahr_sort | 2000 |
| publisher | Springer |
| reference | 19 (2000), S. 515-521 |
| schlagwort | trypsin-like enzyme fibrinolytic activity protein purification hematophagous Haematobia irritans irritans |
| search_space | articles |
| shingle_author_1 | Dametto, M. David, A. P. Azzolini, S. S. Campos, I. T. N. Tanaka, A. M. Gomes, A. Andreotti, R. Tanaka, A. S. |
| shingle_author_2 | Dametto, M. David, A. P. Azzolini, S. S. Campos, I. T. N. Tanaka, A. M. Gomes, A. Andreotti, R. Tanaka, A. S. |
| shingle_author_3 | Dametto, M. David, A. P. Azzolini, S. S. Campos, I. T. N. Tanaka, A. M. Gomes, A. Andreotti, R. Tanaka, A. S. |
| shingle_author_4 | Dametto, M. David, A. P. Azzolini, S. S. Campos, I. T. N. Tanaka, A. M. Gomes, A. Andreotti, R. Tanaka, A. S. |
| shingle_catch_all_1 | Dametto, M. David, A. P. Azzolini, S. S. Campos, I. T. N. Tanaka, A. M. Gomes, A. Andreotti, R. Tanaka, A. S. Purification and Characterization of a Trypsin-Like Enzyme with Fibrinolytic Activity Present in the Abdomen of Horn Fly, Haematobia irritans irritans (Diptera: Muscidae) trypsin-like enzyme fibrinolytic activity protein purification hematophagous Haematobia irritans irritans trypsin-like enzyme fibrinolytic activity protein purification hematophagous Haematobia irritans irritans Abstract This work describes the purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of Haematobia irritans irritans (Diptera: Muscidae). The enzyme was purified using a one-step process, consisting of affinity chromatography on SBTI-Sepharose. The purified protease showed one major active proteinase band on reverse zymography with 0.15% gelatin, corresponding to a molecular mass of 25.5 kDa, with maximum activity at pH 9.0. The purified trypsin-like enzyme preferentially hydrolyzed synthetic substrates with arginine residue at the P1 position. The K m values determined for three different substrates were 1.88 × 10−4, 1.28 × 10−4, and 1.40 × 10−4 M for H-α-benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S2222), dl-Ile-Pro-Arg-p-nitroanilide (S2288), and DL-Phe-Pip-Arg-p-nitroanilide (S2238), respectively. The enzyme was strongly inhibited by typical serine proteinase inhibitors such as SBTI (soybean trypsin inhibitor, K i = 0.19 nM) and BuXI (Bauhinia ungulata factor Xa inhibitor, K i = 0.48 nM), and less inhibited by LDTI (leech-derived tryptase inhibitor, K i = 1.5 nM) and its variants LDTI 2T and 5T (0.8 and 1.5 nM, respectively). The most effective inhibitor for this protease was r-aprotinin (r-BPTI) with a K i value of 39 pM. Synthetic serine protease inhibitors presented only weak inhibition, e.g., benzamidine with K i = 3.0 × 10−4 M and phenylmethylsulfonyl fluoride (PMSF) showed traces of inhibition. The purified trypsin-like enzyme also digested natural substrates such as fibrinogen and fibrin net. The protease showed higher activity against fibrinogen and fibrin than did bovine trypsin. These data suggest that the proteolytic enzyme of H. irritans irritans is more specific to proteins from blood than are the vertebrate digestive enzymes. This enzyme's characteristics may be an adaptation resulting from the feeding behavior of this hematophagous insect. 1573-4943 15734943 Springer |
| shingle_catch_all_2 | Dametto, M. David, A. P. Azzolini, S. S. Campos, I. T. N. Tanaka, A. M. Gomes, A. Andreotti, R. Tanaka, A. S. Purification and Characterization of a Trypsin-Like Enzyme with Fibrinolytic Activity Present in the Abdomen of Horn Fly, Haematobia irritans irritans (Diptera: Muscidae) trypsin-like enzyme fibrinolytic activity protein purification hematophagous Haematobia irritans irritans trypsin-like enzyme fibrinolytic activity protein purification hematophagous Haematobia irritans irritans Abstract This work describes the purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of Haematobia irritans irritans (Diptera: Muscidae). The enzyme was purified using a one-step process, consisting of affinity chromatography on SBTI-Sepharose. The purified protease showed one major active proteinase band on reverse zymography with 0.15% gelatin, corresponding to a molecular mass of 25.5 kDa, with maximum activity at pH 9.0. The purified trypsin-like enzyme preferentially hydrolyzed synthetic substrates with arginine residue at the P1 position. The K m values determined for three different substrates were 1.88 × 10−4, 1.28 × 10−4, and 1.40 × 10−4 M for H-α-benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S2222), dl-Ile-Pro-Arg-p-nitroanilide (S2288), and DL-Phe-Pip-Arg-p-nitroanilide (S2238), respectively. The enzyme was strongly inhibited by typical serine proteinase inhibitors such as SBTI (soybean trypsin inhibitor, K i = 0.19 nM) and BuXI (Bauhinia ungulata factor Xa inhibitor, K i = 0.48 nM), and less inhibited by LDTI (leech-derived tryptase inhibitor, K i = 1.5 nM) and its variants LDTI 2T and 5T (0.8 and 1.5 nM, respectively). The most effective inhibitor for this protease was r-aprotinin (r-BPTI) with a K i value of 39 pM. Synthetic serine protease inhibitors presented only weak inhibition, e.g., benzamidine with K i = 3.0 × 10−4 M and phenylmethylsulfonyl fluoride (PMSF) showed traces of inhibition. The purified trypsin-like enzyme also digested natural substrates such as fibrinogen and fibrin net. The protease showed higher activity against fibrinogen and fibrin than did bovine trypsin. These data suggest that the proteolytic enzyme of H. irritans irritans is more specific to proteins from blood than are the vertebrate digestive enzymes. This enzyme's characteristics may be an adaptation resulting from the feeding behavior of this hematophagous insect. 1573-4943 15734943 Springer |
| shingle_catch_all_3 | Dametto, M. David, A. P. Azzolini, S. S. Campos, I. T. N. Tanaka, A. M. Gomes, A. Andreotti, R. Tanaka, A. S. Purification and Characterization of a Trypsin-Like Enzyme with Fibrinolytic Activity Present in the Abdomen of Horn Fly, Haematobia irritans irritans (Diptera: Muscidae) trypsin-like enzyme fibrinolytic activity protein purification hematophagous Haematobia irritans irritans trypsin-like enzyme fibrinolytic activity protein purification hematophagous Haematobia irritans irritans Abstract This work describes the purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of Haematobia irritans irritans (Diptera: Muscidae). The enzyme was purified using a one-step process, consisting of affinity chromatography on SBTI-Sepharose. The purified protease showed one major active proteinase band on reverse zymography with 0.15% gelatin, corresponding to a molecular mass of 25.5 kDa, with maximum activity at pH 9.0. The purified trypsin-like enzyme preferentially hydrolyzed synthetic substrates with arginine residue at the P1 position. The K m values determined for three different substrates were 1.88 × 10−4, 1.28 × 10−4, and 1.40 × 10−4 M for H-α-benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S2222), dl-Ile-Pro-Arg-p-nitroanilide (S2288), and DL-Phe-Pip-Arg-p-nitroanilide (S2238), respectively. The enzyme was strongly inhibited by typical serine proteinase inhibitors such as SBTI (soybean trypsin inhibitor, K i = 0.19 nM) and BuXI (Bauhinia ungulata factor Xa inhibitor, K i = 0.48 nM), and less inhibited by LDTI (leech-derived tryptase inhibitor, K i = 1.5 nM) and its variants LDTI 2T and 5T (0.8 and 1.5 nM, respectively). The most effective inhibitor for this protease was r-aprotinin (r-BPTI) with a K i value of 39 pM. Synthetic serine protease inhibitors presented only weak inhibition, e.g., benzamidine with K i = 3.0 × 10−4 M and phenylmethylsulfonyl fluoride (PMSF) showed traces of inhibition. The purified trypsin-like enzyme also digested natural substrates such as fibrinogen and fibrin net. The protease showed higher activity against fibrinogen and fibrin than did bovine trypsin. These data suggest that the proteolytic enzyme of H. irritans irritans is more specific to proteins from blood than are the vertebrate digestive enzymes. This enzyme's characteristics may be an adaptation resulting from the feeding behavior of this hematophagous insect. 1573-4943 15734943 Springer |
| shingle_catch_all_4 | Dametto, M. David, A. P. Azzolini, S. S. Campos, I. T. N. Tanaka, A. M. Gomes, A. Andreotti, R. Tanaka, A. S. Purification and Characterization of a Trypsin-Like Enzyme with Fibrinolytic Activity Present in the Abdomen of Horn Fly, Haematobia irritans irritans (Diptera: Muscidae) trypsin-like enzyme fibrinolytic activity protein purification hematophagous Haematobia irritans irritans trypsin-like enzyme fibrinolytic activity protein purification hematophagous Haematobia irritans irritans Abstract This work describes the purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of Haematobia irritans irritans (Diptera: Muscidae). The enzyme was purified using a one-step process, consisting of affinity chromatography on SBTI-Sepharose. The purified protease showed one major active proteinase band on reverse zymography with 0.15% gelatin, corresponding to a molecular mass of 25.5 kDa, with maximum activity at pH 9.0. The purified trypsin-like enzyme preferentially hydrolyzed synthetic substrates with arginine residue at the P1 position. The K m values determined for three different substrates were 1.88 × 10−4, 1.28 × 10−4, and 1.40 × 10−4 M for H-α-benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S2222), dl-Ile-Pro-Arg-p-nitroanilide (S2288), and DL-Phe-Pip-Arg-p-nitroanilide (S2238), respectively. The enzyme was strongly inhibited by typical serine proteinase inhibitors such as SBTI (soybean trypsin inhibitor, K i = 0.19 nM) and BuXI (Bauhinia ungulata factor Xa inhibitor, K i = 0.48 nM), and less inhibited by LDTI (leech-derived tryptase inhibitor, K i = 1.5 nM) and its variants LDTI 2T and 5T (0.8 and 1.5 nM, respectively). The most effective inhibitor for this protease was r-aprotinin (r-BPTI) with a K i value of 39 pM. Synthetic serine protease inhibitors presented only weak inhibition, e.g., benzamidine with K i = 3.0 × 10−4 M and phenylmethylsulfonyl fluoride (PMSF) showed traces of inhibition. The purified trypsin-like enzyme also digested natural substrates such as fibrinogen and fibrin net. The protease showed higher activity against fibrinogen and fibrin than did bovine trypsin. These data suggest that the proteolytic enzyme of H. irritans irritans is more specific to proteins from blood than are the vertebrate digestive enzymes. This enzyme's characteristics may be an adaptation resulting from the feeding behavior of this hematophagous insect. 1573-4943 15734943 Springer |
| shingle_title_1 | Purification and Characterization of a Trypsin-Like Enzyme with Fibrinolytic Activity Present in the Abdomen of Horn Fly, Haematobia irritans irritans (Diptera: Muscidae) |
| shingle_title_2 | Purification and Characterization of a Trypsin-Like Enzyme with Fibrinolytic Activity Present in the Abdomen of Horn Fly, Haematobia irritans irritans (Diptera: Muscidae) |
| shingle_title_3 | Purification and Characterization of a Trypsin-Like Enzyme with Fibrinolytic Activity Present in the Abdomen of Horn Fly, Haematobia irritans irritans (Diptera: Muscidae) |
| shingle_title_4 | Purification and Characterization of a Trypsin-Like Enzyme with Fibrinolytic Activity Present in the Abdomen of Horn Fly, Haematobia irritans irritans (Diptera: Muscidae) |
| sigel_instance_filter | dkfz geomar wilbert ipn albert fhp |
| source_archive | Springer Online Journal Archives 1860-2000 |
| timestamp | 2024-05-06T09:57:30.092Z |
| titel | Purification and Characterization of a Trypsin-Like Enzyme with Fibrinolytic Activity Present in the Abdomen of Horn Fly, Haematobia irritans irritans (Diptera: Muscidae) |
| titel_suche | Purification and Characterization of a Trypsin-Like Enzyme with Fibrinolytic Activity Present in the Abdomen of Horn Fly, Haematobia irritans irritans (Diptera: Muscidae) |
| topic | V |
| uid | nat_lic_papers_NLM197813186 |