ADAMTS-4, recombinant, His Tagged

General description

ADAMTS (a disintegrin and metalloproteinase with thrombospondin motif) is a novel family of extrcellular proteinases (Tang, 2001). Presently nine family members have been identified in mammals (Tang, 2001). ADAMTS4 was first purified from IL-1-stimulated bovine nasal cartilage conditioned media and human ADAMTS4-cDNA was cloned from a human heart cDNA library (Tortorella, 1999). Mature ADAMTS4 consists of a prodomain which confers latency to the proenzyme, a catalytic domain, a disintegrin domain and a C-terminal sequence with a thrombospondin type-1 motif. The prodomain is most likely cleaved off by a furin-type enzyme before active ADAMTS4 is released from cells. Active ADAMTS4 consists of 625 amino acids (F213 - K837) with a calculated Mr of 67 943 (Tortorella, 1999).

ADAMTS4 hydrolyzes aggrecan, the major proteoglycan of articular cartilage (Tortorella, 1999). As aggrecan is also digested by 2 other members of the ADAMTS family, ADAMTS1 (Kuno, 2000) and ADAMTS5 (Abbaszade, 1999), aggrecan degradation products found in normal and rheumatoid and osteoarthritic joint cartilage (Lark, 1997) may arise from action of either of the 3 proteinases. Isolated ADAMTS4 hydrolyzes aggrecan at 5 different sites (Tortorella, 2000a). Four cleavage sites are located in the chondroitin sulfate-rich region between globular domains G2 and G3, while one site is placed in the rod-shaped polypeptide between globular domains G1 and G2. The thrombospondin motif in ADAMTS4 appears to be critical for aggrecan substrate recognition and cleavage (Tortorella, 2000b). ADAMTS4 hydrolyzes also other lecticans as brevican (Nakamura, 2000) and versican (Sandy, 2001).

ADAMTS4 is inhibited by TIMP 3 (Hashimoto, 2001) and by the N-terminal domain of TIMP 3 (Kashigawa, 2001) with Ki-values in the nanomolar range. Inhibition by TIMP 1,2, and 4 is much weaker (Hashimoto, 2001).

Molecular form: Recombinant human ADAMTS4 delta580-837 is produced with the baculovirus expression system and purified from insect cell culture supernatants. The protein consists of amino acids F213 to A579 of full-length ADAMTS4 and a C-terminal His6tag. The calculated Mr is 40,366 Da.



Inhibitors: ADAMTS4 is inhibited by tissue inhibitors of matrix metalloproteinases 3 (TIMP 3) and to a lesser extend by other tissue inhibitors. Enzyme activity is also suppressed by chelators of divalent captions as EDTA and by synthetic metalloproteinase inhibitors.

Applications: Recombinant ADAMTS4 is used to study the degradation of extracellular matrix proteoglycans, to screen for inhibitors of proteoglycan hydrolysis and to characterize inhibitor actions. The enzyme can also serve as standard in enzymatic and immunochemical assays.

Product Source: Recombinant human ADAMTS-4 δ580-837 is produced with the baculovirus expression system and purified from insect cell culture supernatant. The protein contains amino acids F213 ... A579 of full-length ADAMTS-4 and a C-terminal His6tag. Calculated MW is 40 kDa.

Packaging

in 25 μL

Physical form

ADAMTS4 is solublized in 50mM Tris-HCI, pH 7.5, 150 mM NaCl, 5mM CaCl2, 0.05% Brij-35.

Storage and Stability

Maintain protein at -70°C for one year from date of receipt. The enzyme can be kept at -20°C for several weeks, and on ice for several days. Repeated freezing and thawing should be avoided.

Analysis Note

Recombinant ADAMTS4 appears as a major protein of about 42,000 Da in SDS-PAGE (>90% of total protein). Due to autoproteolytic activity minor bands of ADAMTS4 may be visible in the enzyme preparation.

Specific Activity: Aggrecanase activity of ADAMTS4 is determined with recombinant His-tagged aggrecan interglobular domain (Aggrecan-IGD1 from Chemicon). ADAMTS4 hydrolyzes the "aggrecanase" site within this domain (peptide bond E373 -A374 in human aggrecan). The recombinant substrate is incubated at a concentration of 0.1 μM with 1 nM ADAMTS4 in 50 mM Tris-HCI, pH 7.5, 150 mM NaCl, 5 mM CaCl2, 1 μM leupeptin, 1 μM pepstatin, 1mM Pefabloc, 0.05% Brij 35 for 15 mins at 37°C. Cleavage at the "aggrecanase" - site is estimated from the appearance of the C-terminus ARGSVIL. Using polyclonal neoepitope antibodies to the ARGS-terminus the fragment is fixed to a microplate and quantified with anti-His-tag antibody. Under the specified conditions, recombinant ADAMTS4 hydrolyzes 0.15 nM substrate/ min per nM truncated ADAMTS4. When related to mg enzyme the value is 3.7 nmoles hydrolyzed substrate/min per mg ADAMTS4.

Legal Information

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