Journal:

Article Title: Characterization of the Hemorrhagic Reaction Caused by Vibrio vulnificus Metalloprotease, a Member of the Thermolysin Family

doi:

Figure Lengend Snippet: (A) Hemorrhagic activity of bacterial metalloproteases. VVP, thermolysin, serralysin, or C. histolyticum collagenase (1.0 or 10 μg) was injected intradermally into the dorsal skin of a guinea pig. At 30 min postinjection, the animal was sacrificed, the dorsal skin was stripped off, and the area of the hemorrhagic spot was measured (n = 2). (B) Permeability-enhancing activity of bacterial metalloproteases. Each of the proteases (1.0 or 10 μg) was injected into the dorsal skin of a guinea pig which had previously been administered 5% Evans blue (1 ml/kg) intravenously. At 30 min postinjection, the blue spot caused by extravasation of the Evans blue-serum albumin complex was measured (n = 2).

Article Snippet: Thermolysin (35 kDa) and C. histolyticum collagenase (105 kDa) were obtained from Seikagaku Corporation (Tokyo, Japan).

Techniques: Activity Assay, Injection, Permeability

Journal:

Article Title: Characterization of the Hemorrhagic Reaction Caused by Vibrio vulnificus Metalloprotease, a Member of the Thermolysin Family

doi:

Figure Lengend Snippet: (A) Time dependence of degradation of reconstituted BM gel by bacterial metalloproteases. Each of the proteases (3 μg) was layered on the reconstituted BM gel (0.4 mg of total protein) and was allowed to incubate at 37°C. After an appropriate incubation period, the supernatant was withdrawn, and the protein released from the reconstituted gel was quantified by the ninhydrin method (n = 3). (B) Dose dependence of degradation of the reconstituted gel by bacterial metalloproteases. An appropriate amount (0 to 10 μg) of each protease was allowed to react on the reconstituted gel at 37°C for 1 h. Thereafter, the protein released from the reconstituted gel was quantified (n = 3). Symbols: •, VVP; ■, thermolysin; ▴, serralysin; ▾, C. histolyticum collagenase.

Article Snippet: Thermolysin (35 kDa) and C. histolyticum collagenase (105 kDa) were obtained from Seikagaku Corporation (Tokyo, Japan).

Techniques: Incubation

Journal:

Article Title: Characterization of the Hemorrhagic Reaction Caused by Vibrio vulnificus Metalloprotease, a Member of the Thermolysin Family

doi:

Figure Lengend Snippet: Amount of intact type IV collagen in the reconstituted BM gel which was incubated with bacterial metalloproteases. An appropriate amount (0 to 2 μg) of each protease was allowed to react on the reconstituted BM (80 μg of total protein) at 37°C for 1 h. After incubation, each of the protease-treated gels was separated into its components, and only type IV collagen was precipitated with the specific IgG antibody. Thereafter, the relative amount of intact type IV collagen precipitated was determined (n = 3). Symbols: •, VVP; ■, thermolysin; ▴, serralysin or C. histolyticum collagenase.

Article Snippet: Thermolysin (35 kDa) and C. histolyticum collagenase (105 kDa) were obtained from Seikagaku Corporation (Tokyo, Japan).

Techniques: Incubation

Journal: Journal of Structural Biology: X

Article Title: MicroED structure of the C11 cysteine protease clostripain

doi: 10.1016/j.yjsbx.2024.100107

Figure Lengend Snippet: Representative MicroED pattern of a clostripain lamella collected by continuous rotation. Inset shows a FIB/SEM image of clostripain crystals on a Quantifoil holey carbon grid. The side edge length of the crystals was approximately 1 µm.

Article Snippet: Clostripain (from C. histolyticum ) in lyophilized, pre-activated form was purchased from Abnova (Taiwan) and used without further purification.

Techniques:

Journal: Journal of Structural Biology: X

Article Title: MicroED structure of the C11 cysteine protease clostripain

doi: 10.1016/j.yjsbx.2024.100107

Figure Lengend Snippet: MicroED structure of clostripain. (A) Quaternary structure of clostripain. The light chain is shown in purple and heavy chain is shown in orange. (B) Tertiary structure of clostripain. α-Helices are shown in cyan, β-strands in yellow and the loops in gray. The N- and C-termini, α-helices and β-strands are all labelled. The helices and strands are numbered based on the sequence starting from the N-terminus.

Article Snippet: Clostripain (from C. histolyticum ) in lyophilized, pre-activated form was purchased from Abnova (Taiwan) and used without further purification.

Techniques: Sequencing

Journal: Journal of Structural Biology: X

Article Title: MicroED structure of the C11 cysteine protease clostripain

doi: 10.1016/j.yjsbx.2024.100107

Figure Lengend Snippet: (A) Structure of clostripain highlighting the catalytic dyad, His176 and Cys231. The P1 specific substrate site Asp229 is presented in cyan. Nitrogen, oxygen and sulphur atoms are colored blue, red and yellow, respectively. (B) 2F o -F c map (gray mesh) contoured at 1σ showing density for the active site residues. (C) Electrostatic surface potential of clostripain in the same orientation as A showing the same residues as A, where blue and red denote positively and negatively charged surface potential, respectively, contoured at ±10 kT/e.

Article Snippet: Clostripain (from C. histolyticum ) in lyophilized, pre-activated form was purchased from Abnova (Taiwan) and used without further purification.

Techniques:

Journal: Journal of Structural Biology: X

Article Title: MicroED structure of the C11 cysteine protease clostripain

doi: 10.1016/j.yjsbx.2024.100107

Figure Lengend Snippet: (A) Superposition of the MicroED structure (purple cartoon) and the AlphaFold model (light pink cartoon) of clostripain highlighting the linker and the loop. The linker and the loop formed by residues 452 – 457 are labelled on the proenzyme. (B) Electrostatic surface for the AlphaFold model representing the proenzyme. (C) Electrostatic surface for the active clostripain MicroED structure. Here blue and red denote positively and negatively charged surface potential, respectively, contoured at ±10 kT/e.

Article Snippet: Clostripain (from C. histolyticum ) in lyophilized, pre-activated form was purchased from Abnova (Taiwan) and used without further purification.

Techniques:

Journal: Journal of Structural Biology: X

Article Title: MicroED structure of the C11 cysteine protease clostripain

doi: 10.1016/j.yjsbx.2024.100107

Figure Lengend Snippet: Superposition of clostripain (gray cartoon) with (A) Clostripain-related protein from B. thetaiotaomicron (PDB ID: 6N9J; brown cartoon), (B) inactive zymogen C11 protease from Parabacteroides distasonis (PDB ID: 6MZO; cyan blue cartoon). The active site residues are shown in the insets.

Article Snippet: Clostripain (from C. histolyticum ) in lyophilized, pre-activated form was purchased from Abnova (Taiwan) and used without further purification.

Techniques:

Journal:

Article Title: Tissue dissociation enzymes for isolating human islets for transplantation: factors to consider in setting enzyme acceptance criteria

doi: 10.1097/TP.0b013e3181ffff7d

Figure Lengend Snippet: Domain structure of class I (Col G) and class II (Col H) collagenases from C. histolyticum. Numbers in parenthesis after label indicate number of amino acids in domain. Both the catalytic domain and at least one collagen binding domain (CBD) are required for degradation of native collagen. The linking domains does not yet have a known structure or function but in multisequence alignments has regions of homology with similarities to CBD. Spacing sequences are relatively short regions (<12 amino acids) that link domains together. The spacing sequence between the two (CBD) on class 1 collagenase was unstructured in the crystal structure reported by Wilson and co-workers (89).

Article Snippet: C1 class I collagenase from C. histolyticum C1 100kDa Degraded form of class I collagenase with molecular weight of 100 kilo Daltons C1 116kDa Intact form of class I collagenase with molecular weight of 116 kilo Daltons C2 class II collagenase from C. histolyticum C2 114kDa Intact form of class II collagenase with a molecular weight of 114 kilo Daltons CBD Collagen binding domain CDA collagen degrading activity cGMP current good manufacturing practice CHNP C. histolyticum neutral protease ECM extracellular matrix FALGPA N-[3-(2-Furyl)acryloyl]-L-leucyl-glycyl-L-prolyl-L-alanine, a peptide substrate that detects collagenase activity FDA United States Food and Drug Administration FITC fluorescein isothiocyanate FITC-fibrils fluorescein isothiocyanate coupled to type I calf skin collagen fibrils HA CIzymeTM Collagenase HA manufactured by VitaCyte LLC HI LiberaseTM HI Purified Enzyme Blend manufactured by Roche Applied Sciences HPLC High pressure liquid chromatography MTF Liberase Mammalian Tissue Free NB NB neutral protease from C. histolyticum manufactured by Serva GmbH NB-1 NB-1 purified collagenase from C. histolyticum manufactured by Serva GmbH TDE tissue dissociation enzyme TLA trypsin like activity

Techniques: Binding Assay, Sequencing

Journal:

Article Title: Tissue dissociation enzymes for isolating human islets for transplantation: factors to consider in setting enzyme acceptance criteria

doi: 10.1097/TP.0b013e3181ffff7d

Figure Lengend Snippet: Domain structure of class I (Col G) and class II (Col H) collagenases from C. histolyticum. Numbers in parenthesis after label indicate number of amino acids in domain. Both the catalytic domain and at least one collagen binding domain (CBD) are required for degradation of native collagen. The linking domains does not yet have a known structure or function but in multisequence alignments has regions of homology with similarities to CBD. Spacing sequences are relatively short regions (<12 amino acids) that link domains together. The spacing sequence between the two (CBD) on class 1 collagenase was unstructured in the crystal structure reported by Wilson and co-workers (89).

Article Snippet: C1 class I collagenase from C. histolyticum C1 100kDa Degraded form of class I collagenase with molecular weight of 100 kilo Daltons C1 116kDa Intact form of class I collagenase with molecular weight of 116 kilo Daltons C2 class II collagenase from C. histolyticum C2 114kDa Intact form of class II collagenase with a molecular weight of 114 kilo Daltons CBD Collagen binding domain CDA collagen degrading activity cGMP current good manufacturing practice CHNP C. histolyticum neutral protease ECM extracellular matrix FALGPA N-[3-(2-Furyl)acryloyl]-L-leucyl-glycyl-L-prolyl-L-alanine, a peptide substrate that detects collagenase activity FDA United States Food and Drug Administration FITC fluorescein isothiocyanate FITC-fibrils fluorescein isothiocyanate coupled to type I calf skin collagen fibrils HA CIzymeTM Collagenase HA manufactured by VitaCyte LLC HI LiberaseTM HI Purified Enzyme Blend manufactured by Roche Applied Sciences HPLC High pressure liquid chromatography MTF Liberase Mammalian Tissue Free NB NB neutral protease from C. histolyticum manufactured by Serva GmbH NB-1 NB-1 purified collagenase from C. histolyticum manufactured by Serva GmbH TDE tissue dissociation enzyme TLA trypsin like activity

Techniques: Binding Assay, Sequencing

Journal:

Article Title: Tissue dissociation enzymes for isolating human islets for transplantation: factors to consider in setting enzyme acceptance criteria

doi: 10.1097/TP.0b013e3181ffff7d

Figure Lengend Snippet: Assays used to assess collagenase enzymatic activity Enzymatic assays

Article Snippet: C1 class I collagenase from C. histolyticum C1 100kDa Degraded form of class I collagenase with molecular weight of 100 kilo Daltons C1 116kDa Intact form of class I collagenase with molecular weight of 116 kilo Daltons C2 class II collagenase from C. histolyticum C2 114kDa Intact form of class II collagenase with a molecular weight of 114 kilo Daltons CBD Collagen binding domain CDA collagen degrading activity cGMP current good manufacturing practice CHNP C. histolyticum neutral protease ECM extracellular matrix FALGPA N-[3-(2-Furyl)acryloyl]-L-leucyl-glycyl-L-prolyl-L-alanine, a peptide substrate that detects collagenase activity FDA United States Food and Drug Administration FITC fluorescein isothiocyanate FITC-fibrils fluorescein isothiocyanate coupled to type I calf skin collagen fibrils HA CIzymeTM Collagenase HA manufactured by VitaCyte LLC HI LiberaseTM HI Purified Enzyme Blend manufactured by Roche Applied Sciences HPLC High pressure liquid chromatography MTF Liberase Mammalian Tissue Free NB NB neutral protease from C. histolyticum manufactured by Serva GmbH NB-1 NB-1 purified collagenase from C. histolyticum manufactured by Serva GmbH TDE tissue dissociation enzyme TLA trypsin like activity

Techniques: Activity Assay, Fluorescence

Journal:

Article Title: Tissue dissociation enzymes for isolating human islets for transplantation: factors to consider in setting enzyme acceptance criteria

doi: 10.1097/TP.0b013e3181ffff7d

Figure Lengend Snippet: Physical assays used to assess collagenase quality

Article Snippet: C1 class I collagenase from C. histolyticum C1 100kDa Degraded form of class I collagenase with molecular weight of 100 kilo Daltons C1 116kDa Intact form of class I collagenase with molecular weight of 116 kilo Daltons C2 class II collagenase from C. histolyticum C2 114kDa Intact form of class II collagenase with a molecular weight of 114 kilo Daltons CBD Collagen binding domain CDA collagen degrading activity cGMP current good manufacturing practice CHNP C. histolyticum neutral protease ECM extracellular matrix FALGPA N-[3-(2-Furyl)acryloyl]-L-leucyl-glycyl-L-prolyl-L-alanine, a peptide substrate that detects collagenase activity FDA United States Food and Drug Administration FITC fluorescein isothiocyanate FITC-fibrils fluorescein isothiocyanate coupled to type I calf skin collagen fibrils HA CIzymeTM Collagenase HA manufactured by VitaCyte LLC HI LiberaseTM HI Purified Enzyme Blend manufactured by Roche Applied Sciences HPLC High pressure liquid chromatography MTF Liberase Mammalian Tissue Free NB NB neutral protease from C. histolyticum manufactured by Serva GmbH NB-1 NB-1 purified collagenase from C. histolyticum manufactured by Serva GmbH TDE tissue dissociation enzyme TLA trypsin like activity

Techniques: Electrophoresis