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MMP-14 (catalytic domain) (human), (recombinant)

 
ALX-201-098-C010 10 µg 663.00 USD
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Product Specification

Alternative Name:Matrix metalloproteinase 14, MT-1 MMP, Membrane-type 1 matrix metalloproteinase
 
MW:~20kDa (SDS-PAGE).
 
Source:Produced in E. coli. Mature human MMP-14 (aa 89-265).
 
EC:3.4.24.-
 
UniProt ID:P50281
 
Concentration:0.2mg/ml
 
Formulation:Liquid. In 50mM TRIS-HCl, pH 7.5, 150mM NaCl, 5mM CaCl2.
 
Purity:≥95% (SDS-PAGE)
 
Specific Activity:≥140mU/mg protein. One unit is defined as the amount of enzyme that hydrolyzes 1µmol Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 per min. at 37°C, pH 7.5.
 
Application Notes:Used to study the activation of progelatinase A (matrix metalloproteinase 2) and the degradation of proteins of the extracellular matrix. The enzyme allows screening of matrix metalloproteinase inhibitors and characterization of inhibitor action.
 
Shipping:Shipped on Dry Ice
 
Long Term Storage:-80°C
 
Handling:Avoid freeze/thaw cycles.
 
Scientific Background:MMP-14 is expressed in adult lung, placenta, kidney, ovaries, intestine, prostate and spleen. Increased amounts of the enzyme are found in tumor tissues as lung carcinoma, gastric caqrcinoma, colon, breast, head and neck carcinoma.
MMP-14 is activated by removal of its prodomain. The reaction is catalyzed by furin, a subtilisin-type serine protease, which recognizes a motif of four basic amino acid residues located between prodomain and catalytic domain.
MMP-14 activates pro-MMP-2 and pro-MMP-13 by proteolytic cleavage of their prodomains. The ability of MMP-14 to activate other MMPs provides potential for enhanced pericellular proteolysis in physiological and pathological processes. In particular, activation of pro-MMP-2 by MMP-14 is considered to contribute to local degradation of extracellular matrix during cell migration and proliferation. MMP-14 hydrolyzes also fibrillar collagens I, II and III into characteristic 3/4 and 1/4 fragments and it cleaves a number of other proteins of the extracellular matrix, among them fibronectin, vitronectin, laminin-1 and dermatan sulfate proteoglycan. The activity of MMP-14 is poorly inhibited by tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), but efficiently inhibited by TIMP-2 and TIMP-3.
 
Technical Info/Product Notes:SequenceY89 A I Q G L K W Q H N E I T F C I Q N Y T P K V G E Y A T Y E A I R K A F R V W E S A T P L R F R E V P Y A Y I R E G H E K Q A D I M I F F A E G F H G D S T P F D G E G G F L A H A Y F P G P N I G G D T H F D S A E P W T V R N E D L N G N D I F L V A V H E L G H A L G L E H S S D P S A I M A P F Y Q W M D T E N F V L P D D D R R G I Q Q L Y G G E S G265
InhibitorsThe catalytic domain of MMP-14 is inhibited by tissue inhibitors of MMP-2 and -3 (TIMP-2 and TIMP-3) and by chelators of divalent cations like EDTA or o-phenanthroline.
 
Protocol:Measurement of MT1-MMP activity

Preparation and stability of solutions
Peptide hydrolysis buffer: 50mM Tris-HCl, pH 7.5, 150mM NaCl, 5mM CaCl2, 0.025% Brij 35.
Solution is stable for several weeks at 4°C.
Stock solution of peptide substrate: 100mM solution of Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg in 20% dimethylsulfoxide.
Store solution at -20°C.
Stock solution of unquenched peptide: 10mM solution of (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-NH2 (Mca-Pro-Leu) in 20% dimethylsulfoxide.
Store solution at -20°C.

Assay protocol
The activity of MT1-MMP catalytic domain is measured fluorimetrically with a synthetic internally quenched fluorescent substrate according to Knight et al..
An excitation wavelength of 328nm and an emission wavelength of 393nm are set in an appropriate fluorimeter. The instrument is calibrated with the unquenched peptide Mca-Pro-Leu at a concentration corresponding to between 2 and 10% hydrolysis of the protease substrate. Kinetic reactions are conveniently carried out in a constant volume of 2.5ml.
The substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg is diluted in peptide hydrolysis buffer to a concentration of 0.8µM and equilibrated at a temperature of 37°C.
Aliquots of 2 to 4µl of matrix metalloproteinase are than added and the increase in fluorescence is recorded over a time interval between 2 and 12 min.

Activity units per ml enzyme solution are calculated according to the following equation:

Activity U/ml = (cMca-Pro-Leu/FMca-Pro-Leu) x (δFMca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg/venzyme) x Vtotal

cMca-Pro-Leu : Concentration of Mca-Pro-Leu used for calibration of the fluorimeter (mmoles/ml)
FMca-Pro-Leu : Fluorescence of Mca-Pro-Leu at the concentration c Mca-Pro-Leu used for fluorimeter calibration
δFMca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg: Change in fluorescence during peptide hydrolysis per min
V : Volume of peptide hydrolysis reaction (2.5 ml)
v : Volume of added enzyme (0.002 ml to 0.004 ml)

 

General Literature References

A novel coumarin-labelled peptide for sensitive continuous assays of the matrix metalloproteinases: C.G. Knight, et al.; FEBS Lett. 296, 263 (1992), Abstract;

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