Search results for: Anti-ADAM-29 (A Disintegrin And Metalloproteinase-29); Propeptide domain Antibody
#17822731 2007/07/28 To Up
Fractionation of snake venom metalloproteinases by metal ion affinity: a purified cobra metalloproteinase, Nk, from Naja kaouthia binds Ni2+-agarose.Snake venom metalloproteinases represent unique probes for analyzing platelet adhesion receptors regulating hemostasis and thrombosis. Snake venom metalloproteinase-disintegrins consist of a propeptide domain, a catalytic domain containing a metal ion-coordination sequence (HEXXHXXGXXH), a disintegrin domain, and a Cys-rich domain. Here, we investigate whether metal ion-affinity chromatography may be used to fractionate venom metalloproteinases based on the metal ion-coordination motif. First, we showed that a purified cobra metalloproteinase, Nk, from Naja kaouthia bound Ni(2+)-agarose, and was eluted by approximately 10mM imidazole, confirming the validity of the approach. Nk cleaved the platelet von Willebrand factor (VWF) receptor, glycoprotein (GP)Ibalpha, with similar activity to the previously reported cobra metalloproteinase, mocarhagin, as shown by EDTA-inhibitable Nk-dependent proteolysis of a purified GPIbalpha extracellular fragment (glycocalicin), and inhibition of (125)I-VWF binding to GPIbalpha on washed human or canine platelets. Second, crude venom from the viper, Trimeresurus albolabris, was fractionated on Ni(2+)-agarose. Samples of flow-through, wash, and imidazole-eluted (0-30mM gradient) fractions were analyzed by (i) SDS-polyacrylamide gel electrophoresis, (ii) immunoblotting with a rabbit anti-mocarhagin antibody, and (iii) assessing metalloproteinase activity using human fibrinogen as substrate. The combined results support the general concept of using Ni(2+)-agarose to fractionate snake venom metalloproteinases.
Lakshmi C Wijeyewickrema, Elizabeth E Gardiner, Yang Shen, Michael C Berndt, Robert K Andrews
2240 related Products with: Fractionation of snake venom metalloproteinases by metal ion affinity: a purified cobra metalloproteinase, Nk, from Naja kaouthia binds Ni2+-agarose.100μg100μg100ug1mg0.1mg100μg250ug0.1 mg0.1mg100μg100μg100ul
#26478757 // To Up
Measurement of ADAMTS13.ADAMTS13, encoded on chromosome 9q34, is a member of the ADAMTS (a disintegrin and metalloprotease with thrombospondin type 1 motif) metalloprotease family, containing the common domain structure of (from the amino terminus) signal peptide, propeptide, reprolysin type metalloprotease, thrombospondin type 1 motif, cysteine-rich region, and spacer domain. ADAMTS13 cleaves von Willebrand factor (VWF) in a shear stress dependent manner. Deficiency of the enzyme causes the platelet aggregation of thrombotic thrombocytopenic purpura (TTP). Inhibitory antibodies of ADAMTS13 are detected in patients with acquired TTP, while homozygous or double heterozygous mutations of the ADAMTS13 gene cause the hereditary form of the disease 1. Targeting of the ADAMTS13 gene by recombinant technology has reproduced the phenotype of human TTP in ADAMTS13-null mice 2. Despite these advances, intense controversy and confusion persist regarding the role of ADAMTS13 assays in the diagnosis of TTP. This brief review highlights some of the contentious issues and proposes steps to improve the diagnostic value of ADAMTS13 assays.
Han-Mou Tsai16 Arrays/Slide96 tests16 Arrays/Slide16 Arrays/Slide16 Arrays/Slide16 Arrays/Slide16 Arrays/Slide16 Arrays/Slide16 Arrays/Slide
#15711742 // To Up
The spacer domain of ADAMTS13 contains a major binding site for antibodies in patients with thrombotic thrombocytopenic purpura.Thrombotic thrombocytopenic purpura (TTP) is a microangiopathy often associated with a severely decreased activity of ADAMTS13. In plasma of the majority of patients with TTP, antibodies are present that inhibit the von Willebrand factor (VWF) processing activity of ADAMTS13. We describe a sensitive assay that monitors binding of recombinant ADAMTS13 to immobilized IgG derived from patient plasma. Analysis of fifteen patients with TTP and severely reduced ADAMTS13 activity revealed that in all patients antibodies directed to ADAMTS13 were present. Levels of anti-ADAMTS13 antibodies varied considerably among patients, specific antibody levels in plasma range from less than 100 ng/ml to over 1 microg/ml. Longitudinal analysis in three patients revealed that anti-ADAMTS13 antibody levels declined with different kinetics. For further characterization of anti-ADAMTS13 antibodies, we prepared a series of recombinant fragments corresponding to the various ADAMTS13 domains. All seven TTP plasma samples tested, showed reactivity of antibodies towards a fragment consisting of the disintegrin/TSR1/cysteine-rich/spacer domains. In one patient, we also observed reactivity towards the TSR2-8 repeats. No binding of antibodies to propeptide, metalloprotease and CUB domains was detected. To further delineate the binding site in the disintegrin/TSR1/cysteine-rich/spacer region, we prepared additional ADAMTS13 fragments. Antibodies directed towards the cysteine-rich/spacer fragment were found in all plasma samples analyzed. No antibodies reacting with the disintegrin/TSR1 domains were detected. A recombinant fragment comprising the spacer domain was recognized by all patients samples analyzed, suggesting that the 130-amino-acid spacer domain harbors a major binding site for anti-ADAMTS-13 antibodies.
Brenda M Luken, Ellen A M Turenhout, Janine J J Hulstein, Jan A Van Mourik, Rob Fijnheer, Jan Voorberg
2173 related Products with: The spacer domain of ADAMTS13 contains a major binding site for antibodies in patients with thrombotic thrombocytopenic purpura.100.00 ug100.00 ug100.00 ug100.00 ug100 μg100.00 ug100 μg0.2 mg100 μg100 μg100 μg
#14976043 2004/02/19 To Up
Epitope mapping of ADAMTS13 autoantibodies in acquired thrombotic thrombocytopenic purpura.Severe deficiency of the von Willebrand factor (VWF)-cleaving protease ADAMTS13 can lead to thrombotic thrombocytopenic purpura (TTP), a disease associated with the widespread formation of platelet-rich thrombi in many organs. Autoantibodies that inactivate ADAMTS13 are the most frequent cause of acquired TTP. Little is known about epitope specificity and reactivity of anti-ADAMTS13 antibodies. In this study, a series of ADAMTS13 domains were expressed in Escherichia coli, and the reactivity of purified recombinant fragments with anti-ADAMTS13 auto-antibodies from 25 patients with severe ADAMTS13 deficiency was evaluated in vitro. All TTP plasmas contained antibodies directed against the cysteine-rich spacer (cys-rich/spacer) domain of ADAMTS13. In the plasma of 3 patients, antibodies were detected that reacted exclusively with the cys-rich/spacer domain, underscoring the importance of this region for functional activity of ADAMTS13. In 64% of the plasmas, antibodies reacted with the 2 CUB domains, and in 56% they reacted with the isolated first thrombospondin type 1 (TSP-1) repeat and with the compound fragment consisting of the catalytic, the disintegrin-like, and the TSP1-1 domain. Less frequently, in 28% of the plasmas, antibodies reacted with the TSP1 repeats 2 to 8. Unexpectedly, antibodies reacted with the propeptide region in 20% of the plasmas. In conclusion, this study shows that even though anti-ADAMTS13 autoantibodies react with multiple domains of the protease, the cys-rich/spacer domain is consistently involved in antibody reactivity.
Christoph Klaus, Barbara Plaimauer, Jan-Dirk Studt, Friedrich Dorner, Bernhard LÃ¤mmle, Pier Mannuccio Mannucci, Friedrich Scheiflinger
1944 related Products with: Epitope mapping of ADAMTS13 autoantibodies in acquired thrombotic thrombocytopenic purpura.100 μg2 mg100 μg100 ug10 100ug Lyophilized 100 G1 Set
#12562771 2003/01/31 To Up
Identification and characterization of ADAMTS-20 defines a novel subfamily of metalloproteinases-disintegrins with multiple thrombospondin-1 repeats and a unique GON domain.We have cloned a mouse brain cDNA encoding a new protein of the ADAMTS family (a disintegrin and metalloproteinase domain, with thrombospondin type-1 repeats), which has been called ADAMTS-20. This protein shows a domain organization similar to that described for other ADAMTSs including signal sequence, propeptide, metalloproteinase domain, disintegrin domain, central TS-1 motif, cysteine-rich region, and C-terminal TS module. However, this last module is more complex than that of other ADAMTSs, being composed of a total of 14 repeats. The structural complexity of ADAMTS-20 is further increased by the presence of an additional domain 200 residues long and located immediately adjacent to the TS module. This domain has been tentatively called GON domain and can also be recognized in some ADAMTSs such as gon-1 from Caenorhabditis elegans and human and mouse ADAMTS-9. The presence of this domain is a hallmark of a novel subfamily of structurally and evolutionarily related ADAMTSs, called GON-ADAMTSs. Expression analysis demonstrated that ADAMTS-20 transcripts can be detected at low levels in several human and mouse tissues, especially in testis. This gene is also overexpressed in some human malignant tumors, including brain, colon, and breast carcinomas. Western blot analysis using polyclonal antibodies raised against recombinant ADAMTS-20 produced in Escherichia coli showed the presence of a 70-kDa band in mouse brain and testis extracts. This recombinant ADAMTS-20 hydrolyzed a synthetic peptide used for assaying matrix metalloproteinases. These data suggest that this novel enzyme may play a role in the tissue remodeling process occurring in both normal and pathological conditions.
Maria Llamazares, Santiago Cal, VÃctor Quesada, Carlos LÃ³pez-OtÃn
2333 related Products with: Identification and characterization of ADAMTS-20 defines a novel subfamily of metalloproteinases-disintegrins with multiple thrombospondin-1 repeats and a unique GON domain.100μg100μg100μg100μg100μg100μg1000 tests200ul10 mg100μg100ug
#12372841 2002/10/07 To Up
The metalloprotease disintegrin ADAM8. Processing by autocatalysis is required for proteolytic activity and cell adhesion.ADAMs (a disintegrin and metalloprotease domains) are metalloprotease and disintegrin domain-containing transmembrane glycoproteins with proteolytic, cell adhesion, cell fusion, and cell signaling properties. ADAM8 was originally cloned from monocytic cells, and its distinct expression pattern indicates possible roles in both immunology and neuropathology. Here we describe our analysis of its biochemical properties. In transfected COS-7 cells, ADAM8 is localized to the plasma membrane and processed into two forms derived either by prodomain removal or as remnant protein comprising the extracellular region with the disintegrin domain at the N terminus. Proteolytic removal of the ADAM8 propeptide was completely blocked in mutant ADAM8 with a Glu(330) to Gln exchange (EQ-A8) in the Zn(2+) binding motif (HE(330)LGHNLGMSHD), arguing for autocatalytic prodomain removal. In co-transfection experiments, the ectodomain but not the entire MP domain of ADAM8 was able to remove the prodomain from EQ-ADAM8. With cells expressing ADAM8, cell adhesion to a substrate-bound recombinant ADAM8 disintegrin/Cys-rich domain was observed in the absence of serum, blocked by an antibody directed against the ADAM8 disintegrin domain. Soluble ADAM8 protease, consisting of either the metalloprotease domain or the complete ectodomain, cleaved myelin basic protein and a fluorogenic peptide substrate, and was inhibited by batimastat (BB-94, IC(50) approximately 50 nm) but not by recombinant tissue inhibitor of matrix metalloproteinases 1, 2, 3, and 4. Our findings demonstrate that ADAM8 processing by autocatalysis leads to a potential sheddase and to a form of ADAM8 with a function in cell adhesion.
Uwe Schlomann, Dirk Wildeboer, Ailsa Webster, Olga Antropova, Dagmar Zeuschner, C Graham Knight, Andrew J P Docherty, Marc Lambert, Lisa Skelton, Harald Jockusch, JÃ¶rg W Bartsch
1943 related Products with: The metalloprotease disintegrin ADAM8. Processing by autocatalysis is required for proteolytic activity and cell adhesion.100ug Lyophilized100ug1 kit96tests250 ml100ug Lyophilized0.1ml (1mg/ml)100ug25 µg
#11843286 // To Up
Von Willebrand factor-cleaving protease and Upshaw-Schulman syndrome.Vascular endothelial cell (EC)-produced plasma von Willebrand factor (vWF) plays a critical role in primary hemostasis through its action of anchoring platelets onto the injured denuded subendothelial matrices under high shear stress. Unusually large vWF multimers (UL-vWFMs), present in plasma immediately after release from ECs, are most biologically active, but they are soon cleaved and degraded into smaller vWFMs by a specific plasma protease, termed vWF-cleaving protease (vWF-CPase), in normal circulation. Recent studies on the relationship between UL-vWFMs and vWF-CPase, together with its autoantibody (inhibitor) have brought about a clear discrimination between thrombotic thrombocytopenic purpura and hemolytic uremic syndrome. Furthermore, a congenital deficiency of this enzyme activity has been shown to cause Upshaw-Schulman syndrome, a complex constitutional bleeding diathesis. Successful purification of vWF-CPase revealed that this enzyme is composed of a single polypeptide with a molecular mass of approximately 190 kd, and its complementary DNA cloning unambiguously indicated that it is uniquely produced in the liver and its gene is located on chromosome 9q34. The messenger RNA of vWF-CPase had a span of 4.6 kb, and its enzyme was designated ADAMTS 13. The predicted complete amino acid sequence of this enzyme consisted of 1427 residues, including a signal peptide, a short propeptide terminating in the sequence RQRR, a reprolysin-like metalloprotease domain, a disintegrin-like domain, a thrombospondin-1 repeat (TSP1), a cysteine-rich domain, an ADAMTS spacer, 7 additional TSP1 repeats, and 2 CUB domains.
Yoshihiro Fujimura, Masanori Matsumoto, Hideo Yagi, Akira Yoshioka, Taei Matsui, Koiti Titani100μg100μg100μg100 ug100 ug100 ug100 ug1.0mg0.1 mg96T50 IU1 mg
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