| Description | Clostripain (Endoproteinase-Arg-C) is a two chain proteinase associated with collagenase and isolated from Clostridium histolyticum. It is highly specific for the carboxyl peptide bond of arginine. Clostripain has a sulfhydryl requirement; it is activated by dithiothreitol, cysteine, or other Clostripain (Endoproteinase-Arg-C) is a two chain proteinase associated with collagenase and isolated from Clostridium histolyticum. It is highly specific for the carboxyl peptide bond of arginine. Clostripain has a sulfhydryl requirement; it is activated by dithiothreitol, cysteine, or other sulfhydryl containing reagents. The presence of calcium ions is essential. The enzyme is inhibited by oxidizing agents and sulfhydryl reactants and by Co²⁺, Cu²⁺, Cd²⁺, and heavy metal ions. Citrate, borate, and Tris anions are less inhibitory.Clostripain is a cysteine-activated protease found, along with collagenase and other proteases, in culture filtrates of Clostridium histolyticum. It is unique in its specificity for the carboxyl peptide bond of arginine and its dependence on thiol and calcium ions.SpecificityClostripain selectively hydrolyzes arginyl bonds and lysyl bonds at a lower rate. It can also act as a transpeptidase with maximal activity at pH 7.6-9.0.CompositionClostripain is a heterodimer. The mature chain is composed of 526 residues. The two chains are held together by strong noncovalent forces. The catalytic sulfhydryl residue of the active site is believed to be Cys41 (heavy chain residue). The precursor contains a 27 amino acid putative signal peptide, a 23 amino acid propeptide, a 131 amino acid light chain subunit, a 9 amino acid linker peptide, and a 336 amino acid heavy chain subunit.Molecular CharacteristicsBoth the heavy and light chains are encoded by a single gene with a 1581 nucleotide open reading frame (ORF). Upon expression of the gene, the entire ORF (the signal region, proregion, and 9 amino acid peptide linker) is transcribed. Postranslational processing produces the heterodimeric active enzyme.ApplicationsPeptide mappingSequence analysisCell isolationHydrolysis/condensation of amide bondsPeptide synthesisCharacteristicsMolecular Weight: 53.0 kDa (Theoretical); Light chain: 12.5 kDa, Heavy chain: 45 kDaOptimal pH: 7.4-7.8 (activity against a-benzoyl-arginine ethyl ester)Isoelectric point: 4.8-4.9Extinction Coefficient: 87,890 cm⁻¹ M⁻¹ (Theoretical); E1%,280= 16.57 (Theoretical)Active Site Residues: Cysteine (C41, heavy chain)Activators: Sulfhydryl requirement: dithiothreitol, cysteine, or other reducing agents, Calcium ion is essential, Reducing agentsInhibitors: EDTA, Oxidizing agents, Sulfhydryl reagents (such as TLCK), Co2+, Cu2+, Cd2+, and heavy metal ions, Citrate, borate and Tris anions partially inhibitAssayMethodThe reaction velocity is measured as an increase in absorbance at 253 nm resulting from the hydrolysis of N-benzoyl-L-arginine ethyl ester. One unit hydrolyzes one micromole of BAEE per minute at 25°C and pH 7.6 under the conditions specified.Reagents0.075 M Sodium phosphate buffer, pH 7.67.5 mM Dithiothreitol (DTT)0.75 mM N-Benzoyl-L-arginine ethyl ester (BAEE)1.0 mM Calcium acetate containing 2.5 mM dithiothreitol (activation solution)EnzymeDissolve or dilute the enzyme at a concentration of 1 mg/ml in water. Immediately prior to assay, dilute the enzyme further in 1.0 mM Calcium acetate containing 2.5 mM dithiothreitol to a concentration of 0.2-0.8 units/ml.ProcedureAdjust spectrophotometer to 253 nm and 25°C.Pipette into each cuvette as follows:0.075 M phosphate buffer, pH 7.61.0 ml7.5 mM DTT1.0 ml0.75 mM BAEE1.0 mlIncubate in spectrophotometer for 3-5 minutes to achieve temperature equilibrium and establish blank rate, if any. At zero time, add 0.1 ml of appropriately diluted enzyme and record A253 for 4-5 minutes. Determine ΔA253/minute from the linear portion of the curve. Note: The reaction appears to be most linear with respect to enzyme concentration when ΔA253/min is between 0.007 and 0.030.Calculationwhere 1150 is the extinction coefficient of BAEE at 253 nm... Read More | Inquire | Purity>95% SDS-PAGE.FunctionThe soluble form is chemotactic for T-cells and monocytes, but not for neutrophils. The membrane-bound form promotes adhesion of those leukocytes to endothelial cells. May play a role in regulating leukocyte adhesion and migration processes at the endothelium. Binds toPurity>95% SDS-PAGE.FunctionThe soluble form is chemotactic for T-cells and monocytes, but not for neutrophils. The membrane-bound form promotes adhesion of those leukocytes to endothelial cells. May play a role in regulating leukocyte adhesion and migration processes at the endothelium. Binds to CX3CR1.Post-translationalA soluble short 95 kDa form may be released by proteolytic cleavage from the long membrane-anchored form. O-glycosylated with core 1 or possibly core 8 glycans... Read More | Purity>95% by SDS-PAGE and HPLC analyses.FunctionLigand for IL17RA and IL17RC (PubMed:17911633). The heterodimer formed by IL17A and IL17F is a ligand for the heterodimeric complex formed by IL17RA and IL17RC (PubMed:18684971). Involved in stimulating the production of other cytokines such as IL6Purity>95% by SDS-PAGE and HPLC analyses.FunctionLigand for IL17RA and IL17RC (PubMed:17911633). The heterodimer formed by IL17A and IL17F is a ligand for the heterodimeric complex formed by IL17RA and IL17RC (PubMed:18684971). Involved in stimulating the production of other cytokines such as IL6, IL8 and CSF2, and in regulation of cartilage matrix turnover (PubMed:11591732, PubMed:11591768, PubMed:11574464). Also involved in stimulating the proliferation of peripheral blood mononuclear cells and T-cells and in inhibition of angiogenesis (PubMed:11591732). Plays a role in the induction of neutrophilia in the lungs and in the exacerbation of antigen-induced pulmonary allergic inflammation... Read More | Purity>97% by SDS-PAGE and HPLC analyses.Additional sequence informationFunction N-terminal glycine. Full-length mature chain lacking the signal peptideFunctionHas chemotactic activity for neutrophils. May play a role in inflammation and exerts its effects on endothelial cells in an autocrine Purity>97% by SDS-PAGE and HPLC analyses.Additional sequence informationFunction N-terminal glycine. Full-length mature chain lacking the signal peptideFunctionHas chemotactic activity for neutrophils. May play a role in inflammation and exerts its effects on endothelial cells in an autocrine fashion. In vitro, the processed forms GRO-alpha(4-73), GRO-alpha(5-73) and GRO-alpha(6-73) show a 30-fold higher chemotactic activity.Post-translationalN-terminal processed forms GRO-alpha(4-73), GRO-alpha(5-73) and GRO-alpha(6-73) are produced by proteolytic cleavage after secretion from peripheral blood monocytes... Read More |