Recombinant Human Latexin Protein, CF

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

Recombinant Human 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 <1 nM, as measured under the described conditions.
• Source: E. coli-derived human Latexin protein
  Glu2-Glu222, with an N-terminal Met and 6-His tag
• Accession #: AAH05346
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Gene: LXN
• 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: 27 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: 30 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:
  • Assay Buffer: 50 mM Tris, 150 mM NaCl, 10 mM CaCl₂, 0.05% Brij-35, pH 7.5 (TCNB)
  • Recombinant Human Latexin (rhLatexin) (Catalog # 3246-PI)
  • Recombinant Human Carboxypeptidase A1/CPA1 (rhCPA1) (Catalog # 2856-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 rhCPA1 to 100 µg/mL with 1 µg/mL Trypsin in Assay Buffer.
  2. Incubate at room temperature for 1 hour.
  3. Dilute active rhCPA1 to 20 µg/mL in Assay Buffer.
  4. Prepare a curve of rhLatexin (MW: 26,575 Da) in assay buffer. Make the following serial dilutions: 1600, 800, 400, 200, 150, 100, 50, and 25 nM.
  5. Mix equal volumes of the rhLatexin curve dilutions and the diluted rhCPA-1. Include a control (in duplicate) containing Assay Buffer and the diluted rhCPA-1.
  6. Incubate mixtures at 37 °C for 1 hour.
  7. After incubation, dilute the mixtures by 50 fold in Assay Buffer.
  8. Combine equal volumes of Substrate and DTNB. Dilute this mixture to 200 µM Substrate and 200 µM DTNB in Assay Buffer.
  9. Load 50 µL of the incubated mixtures into the 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 rhLatexin by plotting OD/min (or specific activity) vs. concentration with 4-PL fitting.
  12. The specific activity for rhCPA1 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:
  • rhCPA1: 0.010 µg
  • rhLatexin curve: 8, 4, 2.0, 1.0, 0.75, 0.5, 0.25, and 0.125 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 Human Latexin Protein, CF

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

Background

Human 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:
  • AAH05346()