Recombinant Human Meprin alpha Subunit/MEP1A Protein, CF

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

Recombinant Human Meprin alpha Subunit/MEP1A Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave a fluorogenic peptide substrate, Mca-YVADAPK(Dnp)-OH (Catalog # ES007). The specific activity is >500 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human Meprin alpha Subunit/MEP1A protein
  Val22-Gln601 & Leu27-Gln601, both with a C-terminal 10-His tag
• Accession #: AAA21338
• N-terminal Sequence: Val22 and Leu27
• Structure / Form: Disulfide-linked homodimer
• Protein/Peptide Type: Recombinant Enzymes
• Gene: MEP1A
• 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:
  • Enzyme Activity
• Theoretical MW: 67 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: 86 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 opening.
• Buffer: Supplied as 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
• Assay Procedure:
  • Activation Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
  • Assay Buffer: 50 mM Tris, pH 7.5
  • Recombinant Human Meprin alpha Subunit/MEP1A (rhMEP1A) (Catalog # 3220-ZN)
  • Activator: Trypsin (Sigma, Catalog # T-1426)
  • AEBSF (Tocris, Catalog # 5175), 100 mM stock in deionized water
  • Fluorogenic Peptide Substrate: MCA-Tyr-Val-Ala-Asp-Ala-Pro-Lys(DNP)-OH (Catalog # ES007)
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhMEP1A to 100 µg/mL in Activation Buffer with 0.1 µg/mL Trypsin.
  2. Incubate 100 µg/mL rhMEP-1A at 37 °C for 3 hours.
  3. Stop Trypsin activity by adding AEBSF to a final concentration of 1 mM.
  4. Dilute activated rhMEP1A to 0.4 µg/mL in Assay Buffer.
  5. Dilute Substrate to 40 µM in Assay Buffer.
  6. Load into a black well plate 50 µL of 0.4 µg/mL rhMEP1A, and start the reaction by adding 50 µL of 40 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 40 µM Substrate.
  7. Read 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:
  • rhMEP1A: 0.02 µg
  • Substrate: 20 µM

Notes

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

Alternate Names for Recombinant Human Meprin alpha Subunit/MEP1A Protein, CF

• bA268F1.1 (meprin A alpha (PABA peptide hydrolase))
• EC 3.4.24
• EC 3.4.24.18
• endopeptidase-2
• MEP1A
• meprin A subunit alpha
• meprin A, alpha (PABA peptide hydrolase)
• Meprin alpha Subunit
• N-benzoyl-L-tyrosyl-P-amino-benzoic acid hydrolase subunit alpha
• PABA peptide hydrolase
• PPH alpha
• PPHA

Background

Meprins are multimeric proteases composed of alpha and beta subunits, which are members of the astacin family of zinc endopeptidases (1, 2). Both subunits form disulfide-linked homo- or heterooligomers, which are also referred to as Meprin A (composed of alpha subunits with or without beta  subunits) and Meprin B (composed of beta subunits only) (3). Although the two subunits share 42% identity in their amino acid sequence, they differ significantly in their oligomeric structure, post-translational processing and subsequently cellular location, and substrate and peptide bond specificity (4). The 746 amino acid sequence of human meprin alpha subunit precursor consists of a signal peptide (residues 1 to 21), a pro region (residues 22 to 65), and a mature chain (residues 66 to 746) containing following domains, catalytic (residues 62 to 263), MAM (residues 264 to 433), MATH (residues 434 to 593), EGF-like (residues 670 to 710), transmembrane (residues 713 to 740), and cytoplasmic (residues 741 to 746). The pro enzyme terminating at residue 601 was expressed and the secreted protein purified from conditioned medium. The molecular masses of recombinant human MEP1A are similar to those observed for the alpha subunit of rat Meprin A (5).

1. Bond, J.S. and Beynon, R.J. (1995) Protein Sci. 4:1247.
2. Stocker, W. et al. (1995) Protein Sci. 4:823.
3. Bertenshaw, G.P., et al. (2001) J. Biol. Chem. 276:13248.
4. Ishmael, F.T. et al. (2005) J. Biol. Chem. 280:13895.
5. Bertenshaw, G.P., et al. (2003) J. Biol. Chem. 278:2522.

Publications for Meprin alpha Subunit/MEP1A (3220-ZN)(3)

Publications using 3220-ZN

• V Biasin, M Wygrecka, LM Marsh, C Becker-Pau, L Brcic, B Ghanim, W Klepetko, A Olschewski, G Kwapiszews Meprin ? contributes to collagen deposition in lung fibrosis Sci Rep, 2017-01-06;7(0):39969. 2017-01-06 [PMID: 28059112] (Bioassay, Human)
• Van Hiel , Matthias, Breugelmans , Bert, Pagel , Charles, Williams , Adam K, Varan , Aiden K, Burke , Richard, Bowles , Vernon M, Batterham , Philip The ovicidal, larvacidal and adulticidal properties of 5,5&#039;-dimethyl-2,2&#039;-bipyridyl against Drosophila melanogaster. PLoS ONE, 2012-11-20;7(11):e49961. 2012-11-20 [PMID: 23185497] (Zymography)
• Tsuchiya S, Simmer JP, Hu JC, Richardson AS, Yamakoshi F, Yamakoshi Y Astacin proteases cleave dentin sialophosphoprotein (Dspp) to generate dentin phosphoprotein (Dpp). J. Bone Miner. Res., 2011-01-01;26(0):220. 2011-01-01 [PMID: 20687161] (Bioassay, Human)

Bioinformatics

• Gene Symbol: MEP1A
• Uniprot:
  • AAA21338()