• Purity

  >95%, by SDS-PAGE under reducing conditions and visualized by silver stain

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to cleave a fluorogenic peptide substrate, Mca-YVADAPK(Dnp)-OH (Catalog # ). The specific activity is >400 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  Spodoptera frugiperda,     Sf 21 (baculovirus)-derived Val34-Arg615, with a C-terminal 10-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Val34

• Structure / Form

  Pro form    
   

• Predicted Molecular Mass

  68 kDa

• SDS-PAGE

  80 kDa, reducing conditions

Assay Procedure

Materials

• 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, 1 M NaCl, pH 9.0

• Recombinant Mouse Meprin alpha Subunit/MEP1A (rmMEP1A) (Catalog # 4007-ZN)

• Trypsin (Sigma, Catalog # T-1426)

• AEBSF (Catalog # ), 100 mM stock in deionized water

• Substrate: MCA-Tyr-Val-Ala-Asp-Ala-Pro-Lys(DNP)-OH (Catalog # )

• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)

• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rmMEP1A to 100 µg/mL in Activation Buffer with Trypsin at 0.1 µg/mL.

2. Incubate for 45 minutes at 37 °C.

3. Stop Trypsin activation by adding an equal volume of AEBSF at 2 mM in Activation Buffer and mixing well.
   Dilute Substrate to 40 µM in Assay Buffer.

4. Dilute activated rmMEP1A to 0.8 µg/mL in Assay Buffer.

5. Load in a plate 50 µL of  0.8 µg/mL rmMEP1A, 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.

6. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.

7. Calculate specific activity:

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

Per Well:

• rmMEP1A: 0.04 µg

• Substrate: 20 µM

Background: Meprin alpha Subunit/MEP1A

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 hetero‑oligomers, 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 760 amino acid sequence of mouse meprin alpha subunit precursor consists of a signal peptide (residues 1‑33), a pro region (residues 34‑77), and a mature chain (residues 78‑760) containing the following domains, catalytic (residues 78‑275), MAM (residues 276‑445), MATH (residues 447‑607), EGF‑like (residues 684‑724), transmembrane (residues 727‑754), and cytoplasmic (residues 755‑760) (5). The pro enzyme terminating at residue 615 was expressed and the secreted protein purified from conditioned medium. The molecular masses of recombinant mouse MEP1A are similar to those observed for the alpha subunit of rat Meprin A (6).

• References:

1. Bond, J.S. and R.J. Beynon (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. Jiang, W. et al. (1992) J. Biol. Chem. 267:9185.

6. Bertenshaw, G.P. et al. (2003) J. Biol. Chem. 278:2522.

• Entrez Gene IDs:

  4224 (Human); 17287 (Mouse); 25684 (Rat)

• Alternate Names:

  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