Matrix metalloproteinases (MMPs) are Zn 2+–and Ca 2+-dependent endopeptidases which function in the turnover of extracellular matrix components (1). Main subfamilies of MMP are collagenases, gelatinases, stromelysins and membrane-type matrix metalloproteinases (2). Three homologous collagenases have been identified in human tissues: Interstitial collagenase, neutrophil collagenase and collagenase-3. These three enzymes cleave fibrillar collagens at a single site, generating fragments of approximately 3/4 and 1/4 the size of the original molecules. ProMMP-13 (Procollagenase-3) consists of 452 amino acids with a calculated Mr of 52.520(3). Due to N-linked glycosylation, the actual Mr is about 60000 Da (4). Within the protein the following domains and sequence regions can be distinguished (3,4): An N-terminal propeptide, which confers latency to the proenzyme, a Ca2+ and Zn2+-ion binding catalytic domain, a hinge region, and a C-terminal hemopexin-like domain. Latent procollagenase-3 can be activated by proteases such as stromelysin (4), gelatinase A, MT1-MMP and plasmin (5) or incubation with APMA (4). The Mr of active collagenase-3 which begins with the N-terminal sequence YNVFPRTL is 48,000 Da. Collagenase-3 hydrolyzes type II collagen 5-to 6-times faster than type I and type III collagens. The enzyme also exhibits high activity towards gelatin and it degrades SERPINS as a1-antichymotrypsin and plasminogen activator inhibitor-2 (4). Collagenase-3 is inhibited in a 1:1 stoichiometric fashion by TIMP-1, TIMP-2 and TIMP-3. Collagenase-3 is expressed during fetal bone development (6). In adult human tissues collagenase-3 has been detected only in pathological conditions: in malignant tumors (3), in chronic ulcers (7), in arthritic cartilage (8) and synovium (9). The recombinant catalytic domain of Pro-MMP-13 is useful for the study of degradation of extracellular matrix proteins.. The enzyme allows screening of matrix metalloproteinase inhibitors and characterization of inhibitor action.
Recombinant 452 amino acid polypeptide corresponding to human Pro-MMP-13, with an additional C-terminal His-tag with the sequence GVTHHHHHH expressed in E. coli and purified from periplasm. The calculated Mr is 51.681 Da. Upon activation with APMA activated MMP-13 is formed.
Specific Activity:The specific activity of activated MMP-13 is 250–300mU/mg, where 1U is the activity that hydrolyzes 1umol peptide (7-methoxycoumarin-4-yl)acetyl-Pro- Leu-Gly-Leu-Dpa-Ala-Arg) within 1 minute under the assay conditions described by Knight ,et al. ACTIVATION: An aliquot of 19.5ul procollagenase-3 is mixed with 0.5ul APMA solution (40mM p-aminophenyl mercuric acetate in DMSO) and the mixture is incubated for 30 minutes at 37°C. The mixture may be stored on ice until use for activity assays.
Inhibitors:MMP-13 is inhibited by TIMPs and by chelators of divalent cations such as EDTA or o-phenanthroline.
Storage and Stability:Maintain frozen at-70°C in undiluted aliquots. The enzyme may be stored at –20°C for several weeks without significant loss of activity. Repeated freezing and thawing should be avoided.
仕様
Size：5ug
Source Antigen：Recombinant from E. coli
Grade：Highly Purified
Purity：Pro-MMP-13 appears as a major band at about 60kD in SDS-PAGE (>95% total protein).
Form：Provided as a liquid in 50mM Tris-HCl, pH 7.5, 150mM sodium chloride, 5mM CaCl2, 0.05% Brij-35.
