Purification and characterization of three thermostable alkaline fibrinolytic serine proteases from the polychaete Cirriformia tentaculata

Park, Jong Woo; Park, Jung Eun; Choi, Hyun Ki; Jung, Tae Won; Yoon, Seong Myeong; Lee, Jung Sup

doi:10.1016/j.procbio.2013.03.017

Cited by 21 publications

(8 citation statements)

References 48 publications

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“…Therefore, SVMP-MM seems to be a fibrin(ogen)ase belonging to snake venom metalloprotease family, which can digest both the Aαand the Bβ-chains of fibrinogen. Azocasein assay is routinely used for examining the proteolytic activity of most proteases (22,24). Unfortunately, SVMP-MM did not effectively cleave azocasein as a substrate.…”

Section: Discussionmentioning

confidence: 99%

“…The clotting time was monitored using a KC1 coagulometer. Protease activity was also examined using azocasein assay as previously described (22).…”

Section: Methodsmentioning

confidence: 99%

“…From the reaction mixture, 15 µl each of aliquots was withdrawn at various time periods and the reaction was terminated by the addition of 3 µl of 6X SDS-PAGE sample buffer. The samples were then boiled for 3 min and the resulting products were analyzed by SDS-PAGE as previously described (22,24).…”

Section: Proteolytic Activity Assay With a Fluorogenic Peptide Substrmentioning

confidence: 99%

“…Turbidity assay for the spontaneous polymerization of fibrin monomers. The spontaneous polymerization of fibrin monomers generated from fibrinogen by thrombin or SVMP-MM cleavage was examined by measuring the increase in turbidity as previously described (22). Typically, 90 µg of fibrinogen dissolved in standard reaction buffer were mixed with 0.02 units of thrombin, 0.04 units of plasmin or SVMP-MM enzyme (3 or 6 µg) at 37˚C and the increase in the absorbance at 350 nm was then recorded using a 96-well plate reader (Molecular Devices Corp.).…”

Section: Proteolytic Activity Assay With a Fluorogenic Peptide Substrmentioning

confidence: 99%

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Purification and biochemical characterization of a fibrin(ogen)olytic metalloprotease from Macrovipera mauritanica snake venom which induces vascular permeability

Lee

Park

et al. 2014

International Journal of Molecular Medicine

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In the present study, a novel fibrin(ogen)olytic metalloprotease from Macrovipera mauritanica snake venom was purified and characterized in terms of enzyme kinetics and substrate specificity. The purified enzyme [termed snake venom metalloprotease-Macrovipera mauritanica (SVMP‑MM)] was composed of a single polypeptide with an apparent molecular weight of 27 kDa, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminus of the enzyme was composed of NH(2)-QRFAPRYIEL-COOH, as determined by N-terminal sequencing. The Aα- and the Bβ-chains of fibrinogen were completely cleaved by SVMP-MM within 20 and 480 min, respectively. However, the γ-chain was much more resistant to digestion by the enzyme. The enzyme also exhibited proteolytic activity, cleaving the α-α polymer of cross-linked fibrin, but did not effectively digest the γ-γ polymer. To determine the kinetic parameters for SVMP-MM, a fluorescence-quenching peptide (termed o-aminobenzoic acid-HTEKLVTS-2,4-dinitrophenyl‑NH(2)) containing a K-L sequence for SVMP-MM cleavage was designed and synthesized. The optimal pH and temperature for the enzyme activity were found to be 5.5 and 37˚C, respectively, when the fluorogenic substrate was synthesized and used as a substrate. Among the various divalent cations tested, Ni(2+) and Cu(2+) showed strong inhibitory effects on enzyme activity, with an average of 69.6% inhibition. The enzyme activity was also inhibited by treatment with 1,10-phenanthroline, ethylenediaminetetraacetic acid and glycol-bis-(2‑aminoethylether)-N,N,N',N'-tetra-acetic acid, but not with aprotinin, tosyl-lysine chloromethyl ketone and tosyl-phenylalanyl chloromethyl ketone, suggesting that SVMP-MM is a metalloprotease and not a serine protease. The enzymatic parameters, including the K(M), k(cat), and k(cat)/K(M) values were estimated to be 0.015 mM, 0.031 sec(-1), and 20.67 mM(-1)sec(-1), respectively. SVMP-MM induced vascular permeability by digesting type IV collagen. The results obtained in our study demonstrate that SVMP-MM is a fibrin(ogen)olytic P-I class metalloprotease, which can induce a hemorrhagic reaction in vivo.

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Section: Discussionmentioning

confidence: 99%

“…The clotting time was monitored using a KC1 coagulometer. Protease activity was also examined using azocasein assay as previously described (22).…”

Section: Methodsmentioning

confidence: 99%

Section: Proteolytic Activity Assay With a Fluorogenic Peptide Substrmentioning

confidence: 99%

Section: Proteolytic Activity Assay With a Fluorogenic Peptide Substrmentioning

confidence: 99%

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Purification and biochemical characterization of a fibrin(ogen)olytic metalloprotease from Macrovipera mauritanica snake venom which induces vascular permeability

Lee

Park

et al. 2014

International Journal of Molecular Medicine

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“…Previously reported data about serine proteases showed that the size of the protein varies between 20 kDa-60 kDa. Park et al (2013) 27 studied three alkaline serine proteases from the invertebrate polychaete Cirriformia tentaculata and their estimated molecular masses were found to be 28.8, 30.9, and 28.4 kDa 27 . Another study on a serine protease from the sea cucumber (Stichopus japonicus) was 34 kDa 28 .…”

Section: Puri Cation Molecular Mass Determination and Mass Spectrommentioning

confidence: 99%

Novel Recombinant keratin Degrading Subtilisin Like Serine Alkaline Protease from Bacillus Cereus Isolated from Marine Hydrothermal Vent Crabs 

Gurunathan

Huang

Ponnusamy

et al. 2020

Preprint

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Microbial secondary metabolites from extreme environments like hydrothermal vents are a promising source for industrial applications. In our study the protease gene from Bacillus cereus from shallow marine hydrothermal vents in the East China Sea was cloned, expressed and purified. The protein sequence of 38 kDa protease SLSP-k was retrieved from mass spectrometry and identified as a subtilisin serine proteinase. The novel SLSP-k is a monomeric protein with 38 amino acid signal peptides being active over wide pH (7–11) and temperature (40–80 ℃) ranges, with maximal hydrolytic activities at pH 10 and at 50 ℃ temperature. The hydrolytic activity is stimulated by Ca2+, Co2+, Mn2+, and DTT. It is inhibited by Fe2+, Cd2+, Cu2+, EDTA, and PMSF. The SLSP-k is stable in anionic, non- anionic detergents, and solvents. The ability to degrade keratin in chicken feather and hair indicate that the protein is suitable for waste management and value-added product synthesis as well as several research applications.

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Exploitation of Fibrinolytic Enzymes in Combating Blood Clotting Disorders – Recent Advances and Strategies: A Comprehensive Review

Sahoo

2020

Advances in Pharmaceutical Biotechnology

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Purification and characterization of three thermostable alkaline fibrinolytic serine proteases from the polychaete Cirriformia tentaculata

Cited by 21 publications

References 48 publications

Purification and biochemical characterization of a fibrin(ogen)olytic metalloprotease from Macrovipera mauritanica snake venom which induces vascular permeability

Purification and biochemical characterization of a fibrin(ogen)olytic metalloprotease from Macrovipera mauritanica snake venom which induces vascular permeability

Novel Recombinant keratin Degrading Subtilisin Like Serine Alkaline Protease from Bacillus Cereus Isolated from Marine Hydrothermal Vent Crabs

Exploitation of Fibrinolytic Enzymes in Combating Blood Clotting Disorders – Recent Advances and Strategies: A Comprehensive Review

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Purification and characterization of three thermostable alkaline fibrinolytic serine proteases from the polychaete Cirriformia tentaculata

Cited by 21 publications

References 48 publications

Purification and biochemical characterization of a fibrin(ogen)olytic metalloprotease from Macrovipera mauritanica snake venom which induces vascular permeability

Purification and biochemical characterization of a fibrin(ogen)olytic metalloprotease from Macrovipera mauritanica snake venom which induces vascular permeability

Novel Recombinant keratin Degrading Subtilisin Like Serine Alkaline Protease from Bacillus Cereus Isolated from Marine Hydrothermal Vent Crabs&nbsp;

Exploitation of Fibrinolytic Enzymes in Combating Blood Clotting Disorders – Recent Advances and Strategies: A Comprehensive Review

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Product

Resources

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Novel Recombinant keratin Degrading Subtilisin Like Serine Alkaline Protease from Bacillus Cereus Isolated from Marine Hydrothermal Vent Crabs