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
AnalysisPhe254
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
5867-AD |
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Formulation Supplied as a 0.2 μm filtered solution in Sodium Acetate, CaCl 2 and NaCl. | ||
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 freeze-thaw cycles.
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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
Dilute rhAggrecan to 200 µg/mL in Assay Buffer.
Dilute rmADAMTS1 to 20 µg/mL in Assay Buffer.
Combine 25 µL of 200 µg/mL rhAggrecan with 25 µL of 20 µg/mL rmADAMTS1 in a reaction tube.
Incubate the reaction mixture for 24 hours at 37 °C.
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.
Stop the reaction by adding reduced SDS-PAGE sample buffer.
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:
Vazquez, F. et al. (1999) J. Biol. Chem. 274:23349.
Kuno, K. et al. (1997) J. Biol. Chem. 272:556.
Porter, S. et al. (2005) Biochem. J. 386:15.
Nagase, H. and M. Kashiwagi (2003) Arthritis Res. Ther. 5:94.
Kuno, K. et al. (1999) J. Biol. Chem. 274:18821.
Kuno, K. et al. (2000) FEBS Lett. 478:241.
Russel, D. L. et al. (2003) J. Biol. Chem. 278:42330.
Lee, N. et al. (2005) J. Biol. Chem. 280:34796.
Shindo, T. et al. (2000) J. Clin. Invest. 105:1345.
Wight, T.N. (2005) Arterioscler Thromb. Vasc. Biol. 25:12.
Luque, A. et al. (2003) J. Biol. Chem. 278:23656.
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
