Login

Forgot your password?
New User?
Remember me
banner banner

You are here:Home » Molecular Biology » MB-Matrix Metalloproteinases » ADAMTS4, Recombinant, His-Tag (Aggrecanase I, A Disintegrin And Metalloproteinase with ThromboSpondin-3 motif)

ADAMTS4, Recombinant, His-Tag (Aggrecanase I, A Disintegrin And Metalloproteinase with
ThromboSpondin-3 motif)

Pricing

  For pricing information, USA customers sign in.
  Outside USA? Please contact your distributor for pricing.

Specifications

ADAMTS (a disintegrin and metalloproteinase with thrombospondin motif) is a novel family of extrcellular proteinases (1). Presently nine family members have been identified in mammals (1). ADAMTS4 was first purified from I1-1 (stimulated bovine nasal cartilage conditioned media) and human ADAMTS4 (cDNA was cloned from a human heart cDNA library) (2). 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 (2).ADAMTS4 hydrolyzes aggrecan, the major proteoglycan of articular cartilage (2). As aggrecan is also digested by 2 other members of the ADAMTS family, ADAMTS1(3) and ADAMTS5 (4), aggrecan degradation products found in normal and rheumatoid and osteoarthritic joint cartilage (5) may arise from action of either of the 3 proteinases. Isolated ADAMTS4 hydrolyzes aggrecan at 5 different sites (6). 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 (7). ADAMTS4 hydrolyzes also other lecticans as brevican (8) and versican (9).
Catalog #A0859-60
ApplicationsRecombinant 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.
ActivityAggrecanase activity of ADAMTS4 is determined with recombinant His-tagged aggrecan interglobular domain. ADAMTS4 hydrolyzes the “aggrecanase” site within this domain (peptide bond E373-A374 in human aggrecan). The recombinant substrate is incubated at a concentration of 1uM with 5nM ADAMTS4 in 50mM Tris-HCI, pH 7.5, 150mM sodium chloride, 5mM calcium chloride, 1uM leupeptin, 1uM pepstatin, 1mM PMSF, 0.02% Brij 35 for 1hr 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 0.1nmoles substrate/ml • h. The specific activity is accordingly 1.8 nmoles hydrolyzed substrate/min • mg enzyme. Activities of different enzyme lots may vary by up to 50%.
InhibitorsADAMTS4 is inhibited by tissue inhibitors of matrix metalloproteinases 3 (TIMP 3) (10), by the N-terminal domain of TIMP 3 (11) with Ki-values in the nanomolar range, 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.
Amino Acid SequenceThe protein consists of amino acids F213-A579 of full-length ADAMTS4 and a C-terminal His6tag: FASLSRFVETLVVADDKMAAFHG AGLKRYLLTVMAAAAKAFKHPSIRNPVSLV VTRLVILGSGEEGPQVGPSAAQTLRSFCA WQRGLNTPEDSDPDHFDTAILFTRQDLC GVSTCDTLGMADVGTVCDPARSCAIVEDD GLQSAFTAAHELGHVFNMLHDNSKPCISL NGPLSTSRHVMAPVMAHVDPEEPWSPCS ARFITDFLDNGYGHCLLDKPEAPLHLPVTF GKDYDADRQCQLTFGPDSRHCPQLPPP CAALWCSGHLNGHAMCQTKHSPWADG TPCGPAQACMGGRCLHMDQLQDFNIPQ AGGWGPWGPWGDCSRTCGGGVQFSSR DCTRPVPRNGGKYCEGRRTRFRSCNTEDC PTGSA-(H)6.
Storage and StabilityAliquot to avoid repeated freezing and thawing and freeze at -70°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Aliquots are stable for at least 12 months.
SourceRecombinant human baculovirus insect cell.
Purity90%. Appears as a major band of 42kD in SDS-PAGE. Due to autoproteolytic activity minor bands of ADAMTS4 may be visiable in the enzymatic preparation.
Concentration~0.2mg/ml
FormSupplied as a liquid in 50mM Tris-HCl, pH 7.5, 150mM sodium chloride, 5mM calcium chloride, 0.05% Brij-35.
Important NoteThis product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.


External Links