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Beschreibung
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Molecular form: Recombinant human ADAMTS1 delta618-968 is produced with the baculovirus expression system and purified from insect cell culture supernatants. The protein consists of amino acids F 253 .N 617 of full-length ADAMTS1 and a C-terminal His 6 -tag: F V S S H R Y V E T M L V A D Q S M A E F H G S G L K H Y L L T L F S V A A R L Y K H P S I R N S V S L V V V K I L V I H D E Q K G P E V T S N A A L T L R N F C N W Q K Q H N P P S D R D A E H Y D T A I L F T R Q D L C G S Q T C D T L G M A D V G T V C D P S R S C S V I E D D G L Q A A F T T A H E L G H V F N M P H D D A K Q C A S L N G V N Q D S H M M A S M L S N L D H S Q P W S P C S A Y M I T S F L D N G H G E C L M D K P H N P I Q L P G D L P G T S Y D A N R Q C Q F T F G E D S K H C P D A A S T C S T L W C T G T S G G V L V C Q T K H F P W A D G T S C G E G K W C I N G K C V N K T D R K H F D T P F H G N W G M W G P W G D C S R T C G G G V Q Y T M R E C D N P V P K N G G K Y C E G K R V R Y R S C N L E D C P D N (H) 6 The calculated M r is 41 017 Da. ADAMTS1 is solublized in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM CaCl 2 , 0.05 % Brij-35. Specific activity: Activity of ADAMTS1 is determined with recombinant aggrecan interglobular domain (Aggrecan-IGD from Invitek). ADAMTS1 hydrolyzes the "aggrecanase" site within this domain (peptide bond E 373 -A 374 in human aggrecan). The recombinant substrate is incubated at a concentration of 0.1 uM with 0.2-3.0 nM ADAMTS1 in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM CaCl 2 , 1 uM leupeptin, 1 uM pepstatin, 1 mM Pefabloc, 0.05 % Brij 35 for 15 min at 37 o C. Cleavage at the "aggrecanase"-site is estimated from the appearance of the hydrolysis fragment with the novel N-terminus ARGSVIL. The fragment is quantified with two monoclonal antibodies, one directed against the neoepitope ARGSVIL, the other against the sequence C-terminal to the neoepitope (InviLISA Aggrecanase Activity Assay). Under the specified conditions the hydrolysis rate is > 0.06 nM hydrolysed substrate/min x nM truncated ADAMTS1. When related to mg enzyme, the value is > 1.4 nmoles hydrolyzed substrate/min x mg ADAMTS1. Inhibitors: ADAMTS1 is inhibited by chelators of divalent cations as EDTA and by catechin gallate esters. Strucutre and function: ADAMTS1 ADAMTS (a disintegrin and metalloproteinase with thrombospondin motif) is a family of secreted proteinases related to, but distinct from ADAM (a disintegrin and metalloproteinase) [1]. Presently eighteen family members have been identified in the human genome [2]. Mouse ADAMTS1 was first described by Kuno et al. [3] and the human orthologue was cloned by V
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