• Purity

  >95%, by SDS-PAGE under reducing conditions and visualized by silver stain

• Endotoxin Level

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

• Activity

  Measured by its ability to cleave Recombinant Human Aggrecan G1-IGD-G2 Domains (Catalog # ). rmADAMTS1 achieves >50% substrate cleavage, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  Chinese Hamster Ovary cell line, CHO-derived Phe254-Cys725, with a C-terminal 10-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Phe254

• Structure / Form

  Monomer. Recombinant Mouse ADAMTS1 is prone to proteolytic cleavage at C-terminus. The predominant form of the purified protein lacks the His tag.

• Predicted Molecular Mass

  53 kDa

• SDS-PAGE

  65-70 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 25 mM HEPES, 0.01% (w/v) Brij-35, pH 7.0

• Recombinant Human Aggrecan Aggrecan G1-IGD-G2 Domains (rhAggrecan) (Catalog # )

• Recombinant Mouse ADAMTS1 Recombinant Mouse ADAMTS1 (rmADAMTS1) (Catalog # 5867-AD)

• SDS-PAGE and/or Western blot

1. Dilute rhAggrecan to 200 μg/mL in Assay Buffer.

2. Dilute rmADAMTS1 to 20 μg/mL in Assay Buffer.

3. Combine 25 μL of 200 μg/mL rhAggrecan with 25 μL of 20 μg/mL rmADAMTS1 in a reaction tube.

4. Incubate the reaction mixture for 24 hours at 37 °C.

5. For Controls: Combine 25 μL of rhAggrecan solution and 25 μL of Assay Buffer into two tubes. Incubate one tube at 37 °C and the other at -20 °C for 24 hours.

6. Stop the reaction by adding reduced SDS-PAGE sample buffer.

7. Analyze the cleavage by SDS PAGE followed by protein staining and/or Western blot.

Per Reaction:

• rmADAMTS1: 10 μg/mL (0.5 μg)

• rhAggrecan: 100 μg/mL (5 μg)

Background: ADAMTS1

ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin motifs 1), also known as METH1, is the founding member of the family of secreted zinc proteases with a multidomain structure (1-3). The protein precursor consists of a signal peptide and the following domains: pro, catalytic, disintegrinlike, TS type 1 motif, cysteine rich, spacer and a variable number of thrombospondin type 1 motifs. Based on their substrate specificity, ADAMTS1 and associated family members may be key enzymes in the degradation of cartilage leading to inflammation and arthritis (4). It is an active protease cleaving alpha 2macroglobulin (5), aggrecan (6), and versican (7). Compared to ADAMTS4 (aggrecanase 1) and ADAMTS5 (aggrecanase 2), the aggrecanase activity of ADAMTS1 is lower. However, its activity can be enhanced by the binding of a cofactor such as fibulin1 (8). ADAMTS1 is essential for normal growth and the structure and function of the kidneys, adrenal glands and female reproductive organs (9). It also plays an important role in atherosclerosis (10). It has been shown to inhibit endothelial cell proliferation by direct binding and sequestration of VEGF165 and to inhibit fibroblast migration at high concentrations by binding to FGF 2 (11, 12). The purified rmADAMTS1 starts at the N terminus of the catalytic domain and ends in the Cys-rich domain.

• References:

1. Vazquez, F. et al. (1999) J. Biol. Chem. 274:23349.

2. Kuno, K. et al. (1997) J. Biol. Chem. 272:556.

3. Porter, S. et al. (2005) Biochem. J. 386:15.

4. Nagase, H. and M. Kashiwagi (2003) Arthritis Res. Ther. 5:94.

5. Kuno, K. et al. (1999) J. Biol. Chem. 274:18821.

6. Kuno, K. et al. (2000) FEBS Lett. 478:241.

7. Russel, D. L. et al. (2003) J. Biol. Chem. 278:42330.

8. Lee, N. et al. (2005) J. Biol. Chem. 280:34796.

9. Shindo, T. et al. (2000) J. Clin. Invest. 105:1345.

10. Wight, T.N. (2005) Arterioscler Thromb. Vasc. Biol. 25:12.

11. Luque, A. et al. (2003) J. Biol. Chem. 278:23656.

12. Krampert, M. et al. (2005) J. Biol. Chem. 280:23844.

• Long Name:

  A Disintegrin-like and Metalloproteinase Domain with Thrombospondin Motifs 1

• Entrez Gene IDs:

  9510 (Human)

• Alternate Names:

  a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 1; ADAM metallopeptidase with thrombospondin type 1 motif, 1; ADAM-TS 1; ADAMTS1; ADAM-TS1; ADAMTS-1; C3-C5; METH1; METH-1