CC1047

MMP-13, human, proform, his-tagged, E. coli recombinant

• Synonym(s): Procollagenase 3
• UNSPSC Code: 12352204
• NACRES: NA.77
• eCl@ss: 32160405
• Specific activity: 250-300 mU/mg
• Assay: >95% (total protein)
• Biological source: human
• Concentration: 0.2 μg/μL

Properties

• biological source: human
• Quality Level: 100
• assay: >95% (total protein)
• form: liquid
• specific activity: 250-300 mU/mg
• manufacturer/tradename: Chemicon^®
• concentration: 0.2 μg/μL
• NCBI accession no.: NM_002427.2
• UniProt accession no.: P45452
• shipped in: dry ice
• Gene Information: human ... MMP13(4322)

Description

General description

CC1047 is a 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 of the recombinant protein is 51.681 kDa. Upon activation with APMA activated MMP-13 is formed.

Matrix metalloproteinases (MMPs) are Zn2+- and Ca2+-dependent endopeptidases which function in the turnover of extracellular matrix components [Matrisian, 1992]. Main subfamilies of MMP are collagenases, gelatinases, stromelysins and membrane-type matrix metalloproteinases [Nagase, 1997]. 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 &frac34 and &frac14 the size of the original molecules.

ProMMP-13 [Procollagenase-3] consists of 452 amino acids with a calculated Mr of 52.520 [Freije et al., 1994]. Due to N-linked glycosylation, the actual Mr is about 60000 Da [Knauper et al., 1996]. Within the protein the following domains and sequence regions can be distinguished [Freije et al., 1994; Knauper et al., 1996]: 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 [ Knauper et al., 1996], gelatinase A, MT1-MMP and plasmin [ Knauper et al., 1996] or incubation with APMA Knauper et al., 1996]. 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 [Knauper et al., 1996]. 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 [Stahlebackdahl, 1997]. In adult human tissues collagenase-3 has been detected only in pathological conditions: in malignant tumors [Freije et al., 1994], in chronic ulcers [Valaamo et al., 1997], in arthritic cartilage [Mitchell et al., 1996] and synovium [Wernicke et al., 1996].

Application

ACTIVATION:

An aliquot of 19.5 μL procollagenase-3 is mixed with 0.5 μL APMA solution (40 mM 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.

Physical form

Provided as a liquid in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM CaCl2, 0.05% Brij-35.

Preparation Note

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.

Analysis Note

Pro-MMP-13 appears as a major band at about 60 kDa in SDS-PAGE

Other Notes

Specific Activity: where 1 U is the activity that hydrolyzes 1 μmol peptide (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-Dpa-Ala-Arg) within 1 minute (Knight, 1992).

Legal Information

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

Disclaimer

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Safety Information

• Storage Class: 12 - Non Combustible Liquids
• wgk: WGK 1