• Purity

  >90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie? Blue stain at 5 μg per lane

• Endotoxin Level

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

• Activity

  Measured by the stimulation of procollagen type I processing by BMP-1. >50% of the full-length procollagen, in the presence of recombinant mouse PCPE, is processed by BMP-1, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  Mouse myeloma cell line, NS0-derived Met1-Ala468, with a C-terminal 10-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  No result, Gln25 predicted.

• Predicted Molecular Mass

  49 kDa

• SDS-PAGE

  52-57 kDa, reducing conditions

Assay Procedure

Materials

• Dilution Buffer: 50 mM Tris, 5 mM CaCl₂, pH 7.5

• Assay Buffer: 50 mM Tris, 5 mM CaCl₂, 150 mM NaCl, pH 7.5 (TCN)

• Recombinant Mouse PCPE?1 (rmPCPE-1) (Catalog # 2239-PE)

• Recombinant Human Pro-Collagen I  alpha 1 (rhPro?COL1A1) (Catalog # )

• Recombinant Human BMP?1/PCP (rhBMP-1) (Catalog # )

• Reducing SDS-PAGE Sample Buffer

• SDS-PAGE or Western Blot

1. Dilute rhPro-COL1A1 to 75 μg/mL in Dilution Buffer.

2. Dilute rmPCPE-1 to 15 μg/mL in Assay Buffer.

3. Combine 20 μL of diluted rhPro-COL1A1 and 20 μL of diluted rmPCPE-1. Prepare a rhPro-COL1A1 control by combining 20 μL of rhPro?COL1A1 and 20 μL of Assay Buffer. Prepare one rmPCPE-1 control by combining 20 μL of rmPCPE-1 and 20 μL of Assay Buffer (optional).

4. Incubate reaction vials and controls at 37 °C for 30 minutes.

5. Dilute rhBMP-1 to 1.875 μg/mL in Assay Buffer.

6. Add 20 μL of diluted rhBMP-1 to each reaction vial, excluding the rhPro-COL1A1 control. Add 20 μL of Assay Buffer to the rhPro?COL1A1 control in place of rhBMP-1.

7. Incubate reaction vials and controls at 37 °C for 30 minutes.

8. After incubation, combine 30 μL of each reaction mixture and control with 30 μL of reducing SDS-PAGE gel buffer. Mix and incubate samples at 95-100 °C for 3-5 minutes to stop reactions.

9. Load 40 μL (0.5 μg of rhPro-COL1A1) per lane and analyze the cleavage by SDS-PAGE followed by protein staining and/or Western blot.

10. Activity calculation:

Per Lane

• rmPCPE-1: 0.1 μg

• rhPro-COL1A1: 0.5 μg

• rhBMP-1: 0.0125 μg

Background: PCPE-1

Procollagen C-endopeptidase enhancers, known as PCPEs or PCOLCEs, are secreted extracellular matrix glycoproteins that consist of two CUB domains and one NTR domain. They are known to stimulate enzymatic cleavage of procollagens I?III by the BMP-1/tolloid family of metalloproteases, also known as procollagen C?proteinases (1). PCPE?1 is expressed primarily by interstitial connective tissues such as tendons, calvaria, and skin (2). Although BMP-1/tolloid proteinases are involved in processing of multiple extracellular proteins, the enhancer activity of PCPE-1 is specific to procollagens since it has no effect on BMP?1/tolloid cleavage of other substrates (3). It is thought that PCPE-1 enhances cleavage of procollagens by binding to the substrate, inducing a conformation change in the substrate (3), although interaction between PCPE-1 and full-length BMP?1/tolloid proteinases has also been reported (4).

• References:

1. Steiglitz, B.M. et al. (2002) J. Biol. Chem. 277:49820.

2. Kessler, E. et al. (1990) Biochem. Biophys. Res. Commun. 173:81.

3. Moali, C. et al. (2005) J. Biol. Chem. 280:24188.

4. Ge, G. et al. (2006) J. Biol. Chem. 281:10786.

• Long Name:

  Procollagen C-Proteinase Enhancer-1

• Entrez Gene IDs:

  5118 (Human); 18542 (Mouse)

• Alternate Names:

  PCOLCE; PCPE; PCPE1; PCPE-1; PCPE1Type I procollagen COOH-terminal proteinase enhancer; procollagen C-endopeptidase enhancer 1; procollagen C-endopeptidase enhancer; Procollagen COOH-terminal proteinase enhancer 1; Procollagen C-proteinase enhancer 1; procollagen, type 1, COOH-terminal proteinase enhancer; Type 1 procollagen C-proteinase enhancer protein