Recombinant Mouse ACE Protein, CF

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

Recombinant Mouse ACE Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave the fluorogenic peptide substrate, Mca-RPPGFSAFK(Dnp)-OH (Catalog # ES005). The specific activity is > 500 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived mouse ACE/CD143 protein
  Leu35-Gln1264, with a C-terminal 10-His tag
• Accession #: P09470
• N-terminal Sequence: Leu35
• Structure / Form: Recombinant Mouse ACE is prone to proteolytic cleavage at C-terminus. The predominant form of the purified protein lacks the His tag.
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Ace
• 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: 143 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: 160 kDa, under 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 under reducing conditions and visualized by silver stain.
• Assay Procedure:
  • Assay Buffer: 50 mM MES, pH 6.5
  • Recombinant Mouse ACE (rmACE) (Catalog # 1513-ZN)
  • Substrate: MCA-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(DNP)-OH (Catalog # ES005)
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rmACE to 0.5 ng/µL in Assay Buffer.
  2. Dilute Substrate to 20 µM in Assay Buffer.
  3. Load 50 µL of 0.5 ng/µL rmACE in a black well plate, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing Assay Buffer in place of rmACE.
  4. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
  5. 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:
  • rmACE: 0.025 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 Mouse ACE Protein, CF

• ACE
• ACE1angiotensin converting enzyme, somatic isoform
• angiotensin I converting enzyme (peptidyl-dipeptidase A) 1
• carboxycathepsin
• CD143 antigen
• CD143
• DCP
• DCP1
• DCP1angiotensin-converting enzyme
• dipeptidyl carboxypeptidase 1
• Dipeptidyl carboxypeptidase I
• EC 3.2.1.-
• EC 3.4.15.1
• Kininase II
• MGC26566
• MVCD3
• peptidase P
• testicular ECA

Background

ACE (also known as peptidyl-dipetidase A) is a zinc metallopeptidase important for blood pressure control and water and salt metabolism (1). It cleaves the C-terminal dipeptide from angiotensin I to produce the potent vasopressor octapeptide angiotensin II and inactivates bradykinin by the sequential removal of two C-terminal dipeptides. In addition to the two physiological substrates, ACE cleaves C-terminal dipeptides from various oligopeptides with a free C-terminus. Because of its location and specificity, ACE plays additional roles in immunity, reproduction and neuropeptide regulation. For example, ACE degrades Alzheimer amyloid beta -peptide (A beta ), retards A beta aggregation, deposition, fibril formation, and inhibits cytotoxicity (2).

ACE is a type I membrane protein and exists in two isoforms (1). Somatic ACE, found in endothelial, epithelial and neuronal cells, comprises two highly similar catalytic domains called N- and C-domains. Germinal ACE, found exclusively in the testes, comprises a single catalytic domain identical to the C-domain of somatic ACE except for an N-terminal 67 residue germinal ACE-specific sequence. Physiological functions of the two tissue-specific isozymes are not interchangeable (3). For example, sperm-specific expression of the germinal ACE, not the somatic ACE, in ACE knockout male mice restored fertility.

Soluble ACE is present in many biological fluids, such as serum, seminal fluid, amniotic fluid and cerebrospinal fluid (1). The soluble ACE is derived from the membrane forms by actions of secretases or sheddases. The identities of the secretases have not been revealed, although they belong to the family of zinc metallopeptidases (4, 5).

1. Corvol, P. et al. (2004) in Handbook of Proteolytic Enzymes (Barrett, A.J. et al., eds.) p. 332, Academic Press, San Diego.
2. Hu, J. et al. (2001) J. Biol. Chem. 276:47863.
3. Kessler, S.P. et al. (2000) J. Biol. Chem. 275:26259.
4. Eyries, M. et al. (2001) J. Biol. Chem. 276:5525.
5. Alfalah, M. et al. (2001) J. Biol. Chem. 276:21105.

Publications for ACE/CD143 (1513-ZN)(1)

Publication using 1513-ZN

• Park S, Bivona BJ, Kobori H, Seth DM, Chappell MC, Lazartigues E, Harrison-Bernard LM Major role for ACE-independent intrarenal ANG II formation in type II diabetes. Am. J. Physiol. Renal Physiol., 2009-10-21;298(1):F37-48. 2009-10-21 [PMID: 19846569] (Enzyme Assay, Mouse)

Bioinformatics

• Gene Symbol: Ace
• Uniprot:
  • P09470()