Recombinant Human TIMP-3 Protein, CF

• Reactivity: Hu
• Applications: Inhibition Activity
• Format: Carrier-Free

Recombinant Human TIMP-3 Protein, CF Summary

• Details of Functionality: Measured by its ability to inhibit human MMP-2 cleavage of a fluorogenic peptide substrate Mca-PLGL-Dpa-AR-NH₂ (Catalog # ES001). The IC₅₀ value is approximately 3 nM, under conditions the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human TIMP-3 protein
  Cys24-Pro211
• Accession #: P35625
• N-terminal Sequence: Cys24
• Protein/Peptide Type: Recombinant Enzymes
• Gene: TIMP3
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Inhibition Activity
• Theoretical MW: 22 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 26 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Reconstitution Instructions: Reconstitute at 100 μg/mL in sterile, deionized water.
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% Brij-35 (v/v), pH 7.5 (TCNB)
  • Recombinant Human TIMP-3 (rhTIMP-3) (Catalog # 973-TM)
  • Recombinant Human MMP‑2 (rhMMP‑2) (Catalog # 902-MP)
  • 4-Aminophenylmercuric acetate (APMA), 100 mM stock in DMSO
  • Substrate: MCA-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH₂ ((Catalog # ES001) ), 2 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhMMP-2 to 100 µg/mL in Assay Buffer.
  2. Activate 100 µg/mL rhMMP-2 with 1 mM APMA.
  3. Incubate at 37 °C for 1 hour.
  4. Prepare a curve of rhTIMP-3 (MW: 21,700 Da) in Assay Buffer. Make serial dilutions of: 5,000, 2,000, 1,000, 500, 300, 200, 150, 100, 20, and 2 nM.
  5. After activation, dilute 100 µg/mL rhMMP-2 to 12.5 µg/mL in Assay Buffer.
  6. Mix 16 µL of rhTIMP-3 curve dilutions, 25.6 µL of diluted rhMMP-2, and 118.4 µL of Assay Buffer.
  7. Include a control (in duplicate) containing Assay Buffer and the diluted rhMMP-2.
  8. Incubate reactions for 2 hours at 37 °C.
  9. After incubation, dilute the mixtures 5 fold in Assay Buffer.
  10. Dilute Substrate to 10 µM in Assay Buffer.
  11. Load 50 µL of the diluted incubated mixtures in a plate, and start the reaction by adding 50 µL of 10 µM Substrate.
  12. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively in kinetic mode for 5 minutes.
  13. Derive the IC₅₀ value for rhTIMP-3 from the curve.
  14. Calculate specific activity for each point using the following formula (if needed):

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)) / (amount of enzyme (µg))

  *Adjusted for Substrate Blank

  **Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).

  Per Well:
  • rhMMP-2: 0.020 µg
  • Substrate: 5 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human TIMP-3 Protein, CF

• HSMRK222
• K222
• K222TA2
• metalloproteinase inhibitor 3
• MIG-5 protein
• Protein MIG-5
• pseudoinflammatory)
• SFD
• TIMP metallopeptidase inhibitor 3
• TIMP3
• TIMP-3
• tissue inhibitor of metalloproteinase 3 (Sorsby fundus dystrophy
• Tissue inhibitor of metalloproteinases 3

Background

Tissue inhibitors of metalloproteinases (TIMPs) are a family of proteins that regulate the activation and proteolytic activity of the zinc enzymes known as matrix metalloproteinases (MMPs). There are four members of the family, TIMP-1, TIMP-2, TIMP-3 and TIMP-4. TIMP-3 is a glycoprotein with a molecular mass of 30 kDa produced by a wide range of cell types. TIMP-3 inhibits active MMP-mediated proteolysis by forming a non-covalent binary complex with the MMP active site through its N-terminal domain. In addition, TIMP-3 is the only known member of the TIMP family that is an effective inhibitor of ADAMs such as TACE (1).

TIMP-3 is unique among the TIMPs because of its high affinity for binding to the extracellular matrix (2). Point mutations in the TIMP-3 C-terminal domain have been reported to result in Sorsby's fundus dystrophy, a disease leading to macular degeneration and loss of vision.

1. Amour, A. et al. (1998) FEBS Lett. 435:39.
2. Leco, K.J. et al. (1994) J. Biol. Chem. 269:9352.

Publications for TIMP-3 (973-TM)(24)

Publications using 973-TM

• AN Mwaura, MA Riaz, JB Maoga, E Mecha, COA Omwandho, G Scheiner-B, I Meinhold-H, L Konrad Role of Betaglycan in TGF-beta Signaling and Wound Healing in Human Endometriotic Epithelial Cells and in Endometriosis Biology, 2022;11(4):. 2022 [PMID: 35453712] (Bioassay, Human)
• AM Abu El-Asr, A Ahmad, MI Nawaz, MM Siddiquei, A De Zutter, L Vanbrabant, PW Gikandi, G Opdenakker, S Struyf Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation Frontiers in Physiology, 2021;12(0):807747. 2021 [PMID: 35082694] (Bioassay, Human)
• S Santamaria, DR Martin, X Dong, K Yamamoto, SS Apte, J Ahnström Post-Translational Regulation And Proteolytic Activity Of The Metalloproteinase Adamts8 The Journal of Biological Chemistry, 2021;0(0):101323. 2021 [PMID: 34687701] (enzyme activity, Human)
• J Cassuto, A Folestad, J Göthlin, H Malchau, J Kärrholm Concerted actions by MMPs, ADAMTS and serine proteases during remodeling of the cartilage callus into bone during osseointegration of hip implants Bone Rep, 2020;13(0):100715. 2020 [PMID: 32995386] (ELISA Detection, Human)
• J Mitlöhner, R Kaushik, H Niekisch, A Blondiaux, CE Gee, MFK Happel, E Gundelfing, A Dityatev, R Frischknec, C Seidenbech Dopamine Receptor Activation Modulates the Integrity of the Perisynaptic Extracellular Matrix at Excitatory Synapses Cells, 2020;9(2):. 2020 [PMID: 31972963] (Cell Culture, Rat)
• CW Su, YC Chang, MH Chien, YH Hsieh, MK Chen, CW Lin, SF Yang Loss of TIMP3 by promoter methylation of Sp1 binding site promotes oral cancer metastasis Cell Death Dis, 2019;10(11):793. 2019 [PMID: 31624299] (Cell Culture, Human)
• MC Nielsen, MN Andersen, N Rittig, S Rødgaard-H, H Grønbaek, SK Moestrup, HJ Møller, A Etzerodt The macrophage-related biomarkers sCD163 and sCD206 are released by different shedding mechanisms J. Leukoc. Biol., 2019;0(0):. 2019 [PMID: 31242338] (Bioassay, Human)
• G Ruiz-Gómez, S Vogel, S Möller, MT Pisabarro, U Hempel Glycosaminoglycans influence enzyme activity of MMP2 and MMP2/TIMP3 complex formation - Insights at cellular and molecular level Sci Rep, 2019;9(1):4905. 2019 [PMID: 30894640] (Enzyme Assay, Human)
• H Zhai, X Qi, Z Li, W Zhang, C Li, L Ji, K Xu, H Zhong TIMP?3 suppresses the proliferation and migration of SMCs from the aortic neck of atherosclerotic AAA in rabbits, via decreased MMP?2 and MMP?9 activity, and reduced TNF?? expression Mol Med Rep, 2018;18(2):2061-2067. 2018 [PMID: 29956789] (Bioassay, Rabbit)
• JM Dave, T Mirabella, SD Weatherbee, DM Greif Pericyte ALK5/TIMP3 Axis Contributes to Endothelial Morphogenesis in the Developing Brain Dev. Cell, 2018;0(0):. 2018 [PMID: 29456135] (In Vivo, Mouse)
• Cathepsin Protease Controls Copper and Cisplatin Accumulation via Cleavage of the Ctr1 Metal-binding Ecto-domain J Biol Chem, 2016;0(0):. 2016 [PMID: 27143361] (Bioassay, Mouse)
• KK Wong, F Zhu, I Khatri, Q Huo, DE Spaner, RM Gorczynski Characterization of CD200 Ectodomain Shedding PLoS ONE, 2016;11(4):e0152073. 2016 [PMID: 27111430] (Bioassay, Human)
• Sanz R, Ferraro G, Fournier A IgLON cell adhesion molecules are shed from the cell surface of cortical neurons to promote neuronal growth. J Biol Chem, 2015;290(7):4330-42. 2015 [PMID: 25538237]
• Uchikawa S, Yoda M, Tohmonda T, Kanaji A, Matsumoto M, Toyama Y, Horiuchi K ADAM17 regulates IL-1 signaling by selectively releasing IL-1 receptor type 2 from the cell surface. Cytokine, 2015;71(2):238-45. 2015 [PMID: 25461404] (Bioassay, Primate - Chlorocebus pygerythrus (Vervet Monkey))
• Lajoie L, Congy-Jolivet N, Bolzec A, Gouilleux-Gruart V, Sicard E, Sung H, Peiretti F, Moreau T, Vie H, Clemenceau B, Thibault G ADAM17-mediated shedding of FcgammaRIIIA on human NK cells: identification of the cleavage site and relationship with activation. J Immunol, 2014;192(2):741-51. 2014 [PMID: 24337742] (Bioassay, Human)
• Frohlich C, Klitgaard M, Noer J, Kotzsch A, Nehammer C, Kronqvist P, Berthelsen J, Blobel C, Kveiborg M, Albrechtsen R, Wewer U ADAM12 is expressed in the tumour vasculature and mediates ectodomain shedding of several membrane-anchored endothelial proteins. Biochem J, 2013;452(1):97-109. 2013 [PMID: 23458101] (Bioassay, Human)
• Golubkov V, Strongin A Insights into ectodomain shedding and processing of protein-tyrosine pseudokinase 7 (PTK7). J Biol Chem, 2012;287(50):42009-18. 2012 [PMID: 23095747] (Bioassay, Human)
• Krstic D, Rodriguez M, Knuesel I Regulated proteolytic processing of Reelin through interplay of tissue plasminogen activator (tPA), ADAMTS-4, ADAMTS-5, and their modulators. PLoS ONE, 2012;7(10):e47793. 2012 [PMID: 23082219] (Enzyme Assay, Human)
• Dierker T, Dreier R, Petersen A, Bordych C, Grobe K Heparan sulfate-modulated, metalloprotease-mediated sonic hedgehog release from producing cells. J. Biol. Chem., 2009;284(12):8013-22. 2009 [PMID: 19176481] (Bioassay, Chicken)
• Sun Q, Weber CR, Sohail A, Bernardo MM, Toth M, Zhao H, Turner JR, Fridman R MMP25 (MT6-MMP) is highly expressed in human colon cancer, promotes tumor growth, and exhibits unique biochemical properties. J. Biol. Chem., 2007;282(30):21998-2010. 2007 [PMID: 17513868] (Bioassay, Human)
• Fisher KE, Pop A, Koh W Tumor cell invasion of collagen matrices requires coordinate lipid agonist-induced G-protein and membrane-type matrix metalloproteinase-1-dependent signaling. Mol. Cancer, 2006;5(0):69. 2006 [PMID: 17156449] (Bioassay, Human)
• Saunders WB, Bohnsack BL, Faske JB, Anthis NJ, Bayless KJ, Hirschi KK, Davis GE Coregulation of vascular tube stabilization by endothelial cell TIMP-2 and pericyte TIMP-3. J. Cell Biol., 2006;175(1):179-91. 2006 [PMID: 17030988] (Bioassay, Human)
• Tellier E, Rebsomen L The shedding activity of ADAM17 is sequestered in lipid rafts. Exp. Cell Res., 2006;312(20):3969-80. 2006 [PMID: 17010968] (Bioassay, Human)
• von Tresckow B, Kallen KJ, von Strandmann EP, Borchmann P, Lange H, Engert A, Hansen HP Depletion of cellular cholesterol and lipid rafts increases shedding of CD30. J. Immunol., 2004;172(7):4324-31. 2004 [PMID: 15034047] (Bioassay, Human)

Bioinformatics

• Gene Symbol: TIMP3
• Uniprot:
  • P35625()