Recombinant Human

Carboxypeptidase A4/CPA4 Catalog Number: 5906-ZN

DESCRIPTION Source Mouse myeloma cell line, NS0­derived Gly17­Tyr421, with a C­terminal 10­His tag Accession # Q9UI42

N­terminal Sequence Gly17 Analysis Structure / Form Monomer

Predicted Molecular 47 kDa Mass

SPECIFICATIONS SDS­PAGE 48 kDa, reducing conditions

Activity Measured by its ability to cleave the colorimetric peptide substrate Ac­Phe­Thiaphe­OH in the presence of 5,5’Dithio­bis (2­nitrobenzoic acid) (DTNB). Edwards, K.M. et al. (1999) J. Biol. Chem. 274:30468. The specific activity is >3,500 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

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

Purity >95%, by SDS­PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining. Formulation Supplied as a 0.2 μm filtered solution in Tris and NaCl. See Certificate of Analysis for details.

Activity Assay Protocol

Materials l Activation Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl , 0.05% (w/v) Brij­35, pH 7.5 (TCNB) l Assay Buffer: 50 mM Tris, pH 8.0 l Recombinant Human Carboxypeptidase A4/CPA4 (rhCPA4) (Catalog # 5906­ZN) l Recombinant Human Active Trypsin 3/PRSS3 (rhTrypsin 3) (Catalog # 3714­SE) l AEBSF (Catalog # EI001), 100 mM in deionized water l Substrate: N­Acetyl­L­Phenylalanyl­L­3­Thiaphenylalanine (Peptides International, Catalog # STP­3621­PI),10 mM stock in DMSO l 5,5’­dithio­bis(2­nitrobenzoic acid) (DTNB) (Sigma, Catalog # D­8130), 10 mM stock in DMSO l 96 well clear plate (Costar, Catalog # 92592) l Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

Assay 1. Dilute rhCPA4 to 100 µg/mL with 1.0 µg/mL rhTrypsin 3 in Activation Buffer. 2. Incubate at 37 °C for 2.5 hours. 3. Stop rhTrypsin 3 activity by adding AEBSF at a final concentration of 1 mM. 4. Incubate at room temperature for 15 minutes. 5. Dilute activated rhCPA4 to 0.2 µg/mL in Assay Buffer. 6. Dilute Substrate to 200 µM with 200 µM DTNB in Assay Buffer (prepare immediately before use). 7. Load 50 µL of the 0.2 µg/mL rhCPA4 into a plate, and start the reaction by adding 50 µL of Substrate/DTNB mixture. Include a Substrate Blank with 50 µL of Assay Buffer and 50 µL of Substrate/DTNB mixture. 8. Read in kinetic mode for 5 minutes at an absorbance of 405 nm. 9. Calculate specific activity using the following formula: Adjusted V * (OD/min) x well volume (L) x 1012 pmol/mol Specific Activity (pmol/min/µg) = max ext. coeff** (M­1cm­1) x path corr.*** (cm) x amount of enzyme (µg)

*Adjusted for Substrate Blank **Using the extinction coefficient 13260 M­1cm­1 ***Using the path correction 0.32 cm Note: the output of many spectrophotometers is in mOD Final Assay Per Well: Conditions l rhCPA4: 0.01 µg l Substrate: 100 µM l DTNB: 100 µM

PREPARATION AND STORAGE Shipping The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below. Stability & Storage Use a manual defrost freezer and avoid repeated freeze­thaw cycles. l 6 months from date of receipt, ­20 to ­70 °C as supplied. l 3 months, ­20 to ­70 °C under sterile conditions after opening.

Rev. 6/9/2016 Page 1 of 2

Recombinant Human

Carboxypeptidase A4/CPA4 Catalog Number: 5906-ZN

BACKGROUND Carboxypeptidase A4, also known as CPA4, is a secreted, zinc­dependent metallocarboxypeptidase that removes the C­terminal amino acid from peptides having a free C­terminal carboxyl group. CPA4 can hydrolyze both amide and ester bonds and has a preference for cleavage at the amino side of hydrophobic residues. CPA4 is inhibited by latexin, an endogenous inhibitor of carboxypeptidases (1). The deduced amino acid sequence of human CPA4 consists of a signal peptide (residues 1­ 16), a pro region (17­113), and a mature chain (residues 114­421). The CPA4 gene has been associated with prostate cancer aggressiveness and is involved in the histone hyperacetylation signaling pathway (2, 3). Recent studies show that coding variation in the CPA4 gene is linked to high risk prostate cancer among younger patients (4).

References: 1. Pallares, I. et al. (2005) Proc. Natl. Acad. Sci. USA 102:3978. 2. Kayashima, T. et al. (2003) Hum. Genet. 3:220. 3. Huang, H. et al. (1999) Cancer Research. 59:2981. 4. Ross, P.L. et al. (2009) BMC Cancer. 9:69.

Rev. 6/9/2016 Page 2 of 2