Recombinant Human MMP-3 Protein, CF

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

Recombinant Human MMP-3 Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave the fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH₂ (Catalog # ES002). The specific activity is >150 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human MMP-3 protein
  Tyr18-Cys477 (Lys45Glu)
• Accession #: P08254
• N-terminal Sequence: Tyr18
• Structure / Form: Pro form
• Protein/Peptide Type: Recombinant Enzymes
• Gene: MMP3
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 52 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: 54-56 kDa doublet, 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 opening.
• Buffer: Supplied as a 0.2 μm filtered solution in MES, NaCl, CaCl₂, Glycerol and Brij-35.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
  • Recombinant Human MMP-3 (rhMMP-3) (Catalog # 513-MP)
  • Chymotrypsin (Sigma, Catalog # C-3142), 1 mg/mL stock in 1 mM HCl
  • Phenylmethyl Sulfonyl Fluoride (PMSF) (Sigma, Catalog # P-7626), 0.2 M stock in 2-Propanol
  • Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH₂ (Catalog # ES002) , 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. Activate rhMMP-3 at 20 µg/mL in Assay Buffer containing 5 µg/mL Chymotrypsin.
  2. Incubate reaction at 37 °C for 30 minutes.
  3. Stop activation with 2 mM PMSF. Pre-warm the PMSF to 37 °C prior to adding to sample.
  4. Dilute activated rhMMP-3 to 2.5 ng/µL in Assay Buffer.
  5. Dilute Substrate to 20 µM in Assay Buffer.
  6. In a plate load 50 µL of 2.5 ng/µL rhMMP-3, and start the reaction by adding 50 µL of 20 µM Substrate to wells. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 20 µM Substrate.
  7. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
  8. Calculate specific activity

  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-3: 0.125 µg
  • Substrate: 10 µM

Notes

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

Alternate Names for Recombinant Human MMP-3 Protein, CF

• CHDS6
• EC 3.4.24
• EC 3.4.24.17
• matrix metallopeptidase 3 (stromelysin 1, progelatinase)
• matrix metalloproteinase 3 (stromelysin 1, progelatinase)
• Matrix metalloproteinase-3
• MGC126102
• MGC126103
• MMP3
• MMP-3
• proteoglycanase
• SL-1
• STMY
• STMY1MGC126104
• STR1
• Stromelysin 1
• stromelysin-1
• transin-1

Background

Matrix metalloproteinases are a family of zinc and calcium dependent endopeptidases with the combined ability to degrade all the components of the extracellular matrix. MMP-3 (stromelysin-1), can degrade a broad range of substrates including collagen alpha  chains, aggrecan, laminin, fibronectin, elastin, casein, alpha -1 antitrypsin, myelin basic protein, IL-1 beta , IGFBP-3, pro MMP-1, pro MMP-7, pro MMP-8, pro MMP-9 and pro MMP-13. MMP-3 does not cleave the triple helical region of interstitial collagens, a characteristic which distinguishes the stromelysins from the collagenases. The MMP-3 substrate repertoire extends beyond extracellular matrix proteins and implicates MMP-3 in roles other than direct tissue remodelling, for instance, enzyme cascades and cytokine regulation. MMP-3 is expressed by fibroblasts, chrondrocytes, osteoblasts, endothelial cells, smooth muscle cells and macrophages. Structurally, MMP-3 may be divided into several distinct domains; a pro-domain which is cleaved upon activation; a catalytic domain containing the zinc binding site; a short hinge region and a carboxyl terminal (hemopexin-like) domain.

Publications for MMP-3 (513-MP)(10)

Publications using 513-MP

• Levengood, MR;Carosino, CM;Zhang, X;Lucas, S;Ortiz, DJ;Westendorf, L;Chin, AP;Martin, AD;Wong, A;Hengel, SM;Sun, H;Zeng, W;Yumul, R;Dominguez, MM;Chen, Y;Zheng, JH;Karlsson, CAB;Trang, VH;Senter, PD;Gardai, SJ; Preclinical development of SGN-CD47M: Protease-activated antibody technology enables selective tumor targeting of the innate immune checkpoint receptor CD47 Molecular cancer therapeutics 2024-10-28 [PMID: 39463068] (Bioassay, N/A)
• KJ Roberts, MF Cubitt, TM Carlton, L Rodrigues-, L Maggiore, R Chai, S Clare, K Harcourt, TT MacDonald, KP Ray, A Vossenkämp, MR West, JS Crowe Preclinical development of a bispecific TNFalpha/IL-23 neutralising domain antibody as a novel oral treatment for inflammatory bowel disease Scientific Reports, 2021-09-30;11(1):19422. 2021-09-30 [PMID: 34593832] (Bioassay, Human)
• Y Alania, J Creighton, LT Trevelin, CA Zamperini, AK Bedran-Rus Regional contribution of proteoglycans to the fracture toughness of the dentin extracellular matrix J Biomech, 2020-01-16;0(0):109633. 2020-01-16 [PMID: 32035660] (Enzyme Assay, Human)
• L Devel, G Almer, C Cabella, F Beau, M Bernes, P Oliva, F Navarro, R Prassl, H Mangge, I Texier Biodistribution of Nanostructured Lipid Carriers in Mice Atherosclerotic Model Molecules, 2019-09-26;24(19):. 2019-09-26 [PMID: 31561608] (Bioassay, Human)
• JS Crowe, KJ Roberts, TM Carlton, L Maggiore, MF Cubitt, S Clare, K Harcourt, J Reckless, TT MacDonald, KP Ray, A Vossenkämp, MR West Preclinical Development of a Novel, Orally-Administered Anti-Tumour Necrosis Factor Domain Antibody for the Treatment of Inflammatory Bowel Disease Sci Rep, 2018-03-21;8(1):4941. 2018-03-21 [PMID: 29563546] (Bioassay)
• Senescent peritoneal mesothelium creates a niche for ovarian cancer metastases Cell Death Dis, 2016-12-29;7(12):e2565. 2016-12-29 [PMID: 28032864] (Bioassay, Human)
• Djokic J, Fagotto-Kaufmann C, Bartels R, Nelea V, Reinhardt D Fibulin-3, -4, and -5 are highly susceptible to proteolysis, interact with cells and heparin, and form multimers. J Biol Chem, 2013-06-19;288(31):22821-35. 2013-06-19 [PMID: 23782690] (Enzyme Assay, Human)
• Devel L, Beau F, Amoura M, Vera L, Cassar-Lajeunesse E, Garcia S, Czarny B, Stura E, Dive V Simple pseudo-dipeptides with a P2&#039; glutamate: a novel inhibitor family of matrix metalloproteases and other metzincins. J Biol Chem, 2012-06-11;287(32):26647-56. 2012-06-11 [PMID: 22689580] (Enzyme Assay)
• Zhao X, Qureshi F, Eastman PS, Manning WC, Alexander C, Robinson WH, Hesterberg LK Pre-analytical effects of blood sampling and handling in quantitative immunoassays for rheumatoid arthritis. J. Immunol. Methods, 2012-02-17;378(1):72-80. 2012-02-17 [PMID: 22366959] (ELISA (Standard))
• Zhou BB, Peyton M, He B, Liu C, Girard L, Caudler E, Lo Y, Baribaud F, Mikami I, Reguart N, Yang G, Li Y, Yao W, Vaddi K, Gazdar AF, Friedman SM, Jablons DM, Newton RC, Fridman JS, Minna JD, Scherle PA Targeting ADAM-mediated ligand cleavage to inhibit HER3 and EGFR pathways in non-small cell lung cancer. Cancer Cell, 2006-07-01;10(1):39-50. 2006-07-01 [PMID: 16843264] (Enzyme Assay, Human)

Bioinformatics

• Gene Symbol: MMP3
• Uniprot:
  • P08254()