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MMP2 Inhibitor Screening Assay Kit (Colorimetric) (ab139446)

Key features and details

  • Assay type: Enzyme activity
  • Detection method: Colorimetric
  • Platform: Microplate reader
  • Sample type: Inhibitor compounds

Overview

  • Product name

    MMP2 Inhibitor Screening Assay Kit (Colorimetric)
    See all MMP2 kits

  • Detection method

    Colorimetric

  • Sample type

    Inhibitor compounds

  • Assay type

    Enzyme activity

  • Product overview

    MMP2 Inhibitor Screening Assay Kit (Colorimetric) (ab139446) is a complete assay system designed to screen MMP2 inhibitors using a thiopeptide as a chromogenic substrate (Ac-PLG-[2-mercapto-4-methyl-pentanoyl]-LG-OC2H5). The MMP cleavage site peptide bond is replaced by a thioester bond in the thiopeptide. Hydrolysis of this bond by an MMP produces a sulfhydryl group, which reacts with DTNB [5,5’-dithiobis(2-nitrobenzoic acid), Ellman’s reagent] to form 2-nitro-5-thiobenzoic acid, which can be detected by its absorbance at 412 nm (ε=13,600 M-1cm-1 at pH 6.0 and above). The assays are performed in a convenient 96-well microplate format.

  • Notes

    This kit is useful to screen inhibitors of MMP2, a potential therapeutic target. The MMP inhibitor NNGH is also included as a prototypic control inhibitor.

    Thiol inhibitors should not be used with this kit, as they may interfere with the colorimetric assay.

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Please refer to protocols.
  • Components 1 x 96 tests
    96-well Clear Microplate (1/2 Volume) 1 unit
    Colorimetric Assay Buffer 1 x 20ml
    MMP Inhibitor 1 x 50µl
    MMP Substrate 1 x 50µl
    MMP2 Enzyme (Human, Recombinant) 1 x 45.7µl

  • Research areas

  • Function

    Ubiquitinous metalloproteinase that is involved in diverse functions such as remodeling of the vasculature, angiogenesis, tissue repair, tumor invasion, inflammation, and atherosclerotic plaque rupture. As well as degrading extracellular matrix proteins, can also act on several nonmatrix proteins such as big endothelial 1 and beta-type CGRP promoting vasoconstriction. Also cleaves KISS at a Gly-
    -Leu bond. Appears to have a role in myocardial cell death pathways. Contributes to myocardial oxidative stress by regulating the activity of GSK3beta. Cleaves GSK3beta in vitro.
    PEX, the C-terminal non-catalytic fragment of MMP2, posseses anti-angiogenic and anti-tumor properties and inhibits cell migration and cell adhesion to FGF2 and vitronectin. Ligand for integrinv/beta3 on the surface of blood vessels.

  • Tissue specificity

    Produced by normal skin fibroblasts. PEX is expressed in a number of tumors including gliomas, breast and prostate.

  • Involvement in disease

    Defects in MMP2 are the cause of Torg-Winchester syndrome (TWS) [MIM:259600]; also known as multicentric osteolysis nodulosis and arthropathy (MONA). TWS is an autosomal recessive osteolysis syndrome. It is severe with generalized osteolysis and osteopenia. Subcutaneous nodules are usually absent. Torg-Winchester syndrome has been associated with a number of additional features including coarse face, corneal opacities, patches of thickened, hyperpigmented skin, hypertrichosis and gum hypertrophy. However, these features are not always present and have occasionally been observed in other osteolysis syndromes.

  • Sequence similarities

    Belongs to the peptidase M10A family.
    Contains 3 fibronectin type-II domains.
    Contains 4 hemopexin-like domains.

  • Domain

    The conserved cysteine present in the cysteine-switch motif binds the catalytic zinc ion, thus inhibiting the enzyme. The dissociation of the cysteine from the zinc ion upon the activation-peptide release activates the enzyme.

  • Post-translational
    modifications

    Phosphorylation on multiple sites modulates enzymatic activity. Phosphorylated by PKC in vitro.
    The propeptide is processed by MMP14 (MT-MMP1) and MMP16 (MT-MMP3). Autocatalytic cleavage in the C-terminal produces the anti-angiogenic peptide, PEX. This processing appears to be facilitated by binding integrinv/beta3.

  • Cellular localization

    Secreted > extracellular space > extracellular matrix. Membrane. Nucleus. Colocalizes with integrin alphaV/beta3 at the membrane surface in angiogenic blood vessels and melanomas. Found in mitochondria, along microfibrils, and in nuclei of cardiomyocytes.
  • Information by UniProt

  • Alternative names

    • 72 kDa gelatinase
    • 72kD type IV collagenase
    • CLG 4
    • CLG 4A
    • CLG4
    • CLG4A
    • Collagenase Type 4 alpha
    • Collagenase type IV A
    • Gelatinase A
    • Gelatinase alpha
    • Gelatinase neutrophil
    • Matrix metallopeptidase 2 gelatinase A 72kDa gelatinase 72kDa type IV collagenase
    • Matrix Metalloproteinase 2
    • Matrix metalloproteinase 2 (gelatinase A, 72kDa gelatinase, 72kDa type IV collagenase)
    • Matrix metalloproteinase II
    • Matrix metalloproteinase-2
    • MMP 2
    • MMP II
    • MMP-2
    • MMP2
    • MMP2_HUMAN
    • MONA
    • Neutrophil gelatinase
    • PEX
    • TBE 1
    • TBE-1
    see all

Images

  • Plot of OD vs time
    Plot of OD vs time
  • Inhibition of MMP3 by NNGH
    Inhibition of MMP3 by NNGH

    Control slope = 1.65E-02 OD/min

    Inhibitor slope = 9.80E-04 OD/min

    Iinhibitor % activity remaining = (9.80E-04/1.65E-02) x 100 = 5.94%

  • Inhibition of MMP3 by NNGH
    Inhibition of MMP3 by NNGH
  • Example Graph for Km and Vmax Determination
    Example Graph for Km and Vmax Determination

    Km=105 µM

    Vmax=3.9 pmol/sec

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