Recombinant Mouse Latexin Protein, CF

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

Recombinant Mouse Latexin Protein, CF Summary

• Details of Functionality: Measured by its ability to inhibit carboxypeptidase A1 cleavage of the colorimetric peptide substrate Ac-Phe-Thiaphe-OH in the presence of 5,5’Dithio-bis (2-nitrobenzoic acid) (DTNB). Edwards, K.M. et al. (1999) J. Biol. Chem. 274:30468. The IC₅₀ value is <2.0 nM, as measured under the described conditions.
• Source: E. coli-derived mouse Latexin protein
  Glu2-Glu222, with an N-terminal Met and 6-His tag
  Accession # P70202
• Accession #: P70202
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Lxn
• 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:
  • Inhibition Activity
• Theoretical MW: 26 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: 29 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, NaCl and Glycerol.
• 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% (v/v) Brij-35, pH 7.5 (TCNB)
  • Recombinant Mouse Latexin (rmLatexin) (Catalog # 3620-PI)
  • Recombinant Mouse Carboxypeptidase A1/CPA1 (rmCPA1) (Catalog # 2765-ZN)
  • Trypsin (Sigma, Catalog # T-1426)
  • Substrate: Ac-Phe-Thiaphe-OH (Peptides International, Catalog # STP-3621-PI),10 mM stock in DMSO
  • 5,5’-dithio-bis(2-nitrobenzoic acid) (DTNB) (Sigma, Catalog # D-8130) 10 mM stock in DMSO
  • 96 well clear plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rmCPA1 to 100 µg/mL with 1.0 µg/mL Trypsin in Assay Buffer.
  2. Incubate at room temperature for 1 hour.
  3. Dilute activated rmCPA1 to 20 µg/mL in Assay Buffer.
  4. Prepare a curve of rmLatexin (MW: 26,297 Da) in Assay Buffer. Make the following serial dilutions: 2500, 1600, 800, 300, 200, 150, 100, 50, 25, and 12.5 nM.
  5. Mix equal volumes of the rmLatexin curve dilutions and the diluted rmCPA1. Include a control (in duplicate) containing Assay Buffer and the diluted rmCPA1.
  6. Incubate mixtures at 37 °C for 1 hour.
  7. After incubation, dilute the mixtures 50 fold in Assay Buffer.
  8. Dilute Substrate to 200 µM in Assay Buffer containing 200 µM DTNB.
  9. Load 50 µL of the diluted incubated mixtures to a plate, and start the reaction by adding 50 µL of 200 µM Substrate mixture.
  10. Read in kinetic mode for 5 minutes at an absorbance of 405 nm.
  11. Derive the 50% inhibition concentration (IC₅₀) for rmLatexin by plotting RFU/min (or specific activity) vs. concentration with 4‑PL fitting.
  12. The specific activity for rmCPA1 at each point may be determined using the following formula (if needed):

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (OD/min) x well volume (L) x 10^12 pmol/mol) / (ext. coeff** (M^-1cm^-1) x path corr.*** (cm) x amount of enzyme (µg))

  
  *Adjusted for Substrate Blank
  **Using the extinction coefficient 13260 M^-1cm^-1
  ***Using the path correction 0.32 cm
  Note: the output of many spectrophotometers is in mOD Per Well:
  • rmCPA1: 0.010 µg
  • rmLatexin curve: 12.5, 8, 4, 1.5, 1.0, 0.75, 0.5, 0.25, 0.125, and 0.0625 nM
  • Substrate: 100 μM
  • DTNB: 100 μM

Notes

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

Alternate Names for Recombinant Mouse Latexin Protein, CF

• ECI
• ECIMUM
• Endogenous carboxypeptidase inhibitor
• Latexin
• LXN
• TCI
• TCIProtein MUM
• Tissue carboxypeptidase inhibitor

Background

Latexin, also known as tissue carboxypeptidase inhibitor, is the only mammalian carboxypeptidase inhibitor identified so far. Latexin was initially identified as a marker of neurons in the lateral neocortex of the developing brain (hence Latexin) (1). Further studies have shown that Latexin is highly expressed in mast cells and macrophages (2, 3). It is induced in acute pancreatitis and lung inflammatory disease. In addition, it is able to inhibit carboxypeptidases from the pancreas (CPA1 and CPA2) and mast cells (CPA3) (4). Therefore, it is thought that Latexin primarily functions in inflammation and innate immunity pathways. Compared to other carboxypeptidase inhibitors from plants and parasites, the 222 amino acid Latexin is much larger and more importantly, it lacks some conserved C-terminal residues, which interact with the target carboxypeptidase in a substrate-like manner (5). This distinct feature of Latexin suggests that it has a different carboxypeptidase inhibition mechanism.

1. Arimatsu, Y. et al. (1994) Neurosci. Res. 20:131.
2. Uratani, Y. et al. (2000) Biochem. J. 346:817.
3. Aagaard A. et al. (2005) Structure 13:309.
4. Normant E. et al. (1995) Proc. Natl. Acad. Sci. USA 92:12225.
5. Reverter D. et al. (2000) Nat. Struct. Biol. 7:322.

Bioinformatics

• Gene Symbol: Lxn
• Uniprot:
  • P70202()