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  • 1
    Electronic Resource
    Electronic Resource
    Purification and Characterization of a Trypsin-Like Enzyme with Fibrinolytic Activity Present in the Abdomen of Horn Fly, Haematobia irritans irritans (Diptera: Muscidae) (2000)
    Springer
    The protein journal 19 (2000), S. 515-521 
    Details
    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: http://dx.doi.org/10.1023/A:1026557600429
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  • 2
    Electronic Resource
    Electronic Resource
    Sequence of a new Bowman-Birk inhibitor fromTorresea acreana seeds and comparison withTorresea cearensis trypsin inhibitor (TcTI2) (1996)
    Springer
    The protein journal 15 (1996), S. 553-560 
    Details
    ISSN: 1573-4943
    Keywords: Leguminosae ; protein sequencing ; Torresea acreana, serine proteinase inhibitor, Bowman-Birk inhibitor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract TaTI (Torresea acreana trypsin inhibitor), a new member of the Bowman-Birk trypsin inhibitor family, was purified from seeds ofTorresea acreana, one of the two known species ofTorresea, a Brazilian native Leguminosae of the Papilionoideae subfamily. Purification was performed by acetone fractionation, anion-exchange chromatography, and gel filtration. The TaTI appears asM r 7000 in SDS-PAGE under reducing conditions. There are 63 amino acid residues present in the TaTI sequence, which was confirmed by mass spectrometry (8388 daltons). The putative reactive sites residues were Lys-15 and Arg-42 at the first and second site, respectively. The antibodies raised against TcTI2,Torresea cearensis trypsin inhibitor 2, showed a cross-reaction with TaTI, but not with other Bowman-Birk inhibitors purified from Leguminosae. The inhibition constants of TaTI and TcTI2 were comparable when measured against trypsin, chymotrypsin, and factor XIIa, but not on plasmin. The latter was tenfold more effectively inhibited by TcTI2 then by TaTI. Neither TaTI nor TcTI2 affects thrombin, plasma kallikrein, or factor Xa.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01908537
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    Paper (German National Licenses)
    Fulltext
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