Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recombinant nidogen fragments comprising the globular domains G1 plus G2, the rod-like domain, and the rod connected to the globe G3 were prepared from the culture media of transfected human cell clones. In addition, domains G1 and G2 were separated from each other after cleavage with chymotrypsin. The purified fragments were characterized by N-terminal sequences, electrophoresis, electron microscopy, and radioimmunoassays and the cell clones by Northern hybridization. Transfection with a construct comprising a large part of domain G3 showed high mRNA levels but no secreted protein, indicating a protein folding problem. All these fragments were used as soluble and/or immobilized ligands in binding assays. This demonstrated major binding sites on domain G2 for collagen IV and heparan sulfate proteoglycan. Affinity chromatography on zinc- and cobalt-loaded columns showed binding of domains G2 and G3 and the rod. Protein binding, but not metal binding, was abolished by reduction and alkylation of nidogen. This allowed for the isolation of several zinc-binding tryptic peptides, four from G2, two from the rod, and one from the G3 domain. Most of these short peptides contained several histidines that are likely to mediate binding. Zinc inhibited efficiently G3-mediated nidogen binding to laminin at 4 degrees C (IC50 approximately 5 microM) but less at higher temperatures. Similarly, zinc inhibited binding to collagen IV and proteoglycan at low temperatures but not at high (37 degrees C) temperatures. This indicates a complex modulation of nidogen binding to other basement membrane proteins by some, but not all, transition metals. Whether the particularly striking effects shown for zinc are of biological relevance remains to be established.
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PMID:Mapping of nidogen binding sites for collagen type IV, heparan sulfate proteoglycan, and zinc. 849 53

Clusters of nicotinic acetylcholine receptor (AChR) in cultured rat myotubes are organized into rectilinear arrays of receptor-rich and receptor-poor domains. Extracellular matrix (ECM) molecules, including fibronectin, heparan sulfate proteoglycan, laminin, and type IV collagen, codistribute with AChR in these clusters. We have examined the stability of this association. We disrupted the AChR clusters in intact myotubes with sodium azide, an energy metabolism inhibitor, and with culture medium free of Ca2+. We also altered or extracted proteins from detergent-isolated AChR clusters by treating with buffers of low ionic strength or alkaline pH or with insoluble chymotrypsin. Each of these treatments dispersed AChR clusters and, simultaneously, caused fibronectin, heparan sulfate proteoglycan, laminin, and type IV collagen to disperse from AChR-rich strips of membrane. Control experiments indicated that insoluble chymotrypsin had no direct effect on the ECM at AChR clusters. It did, however, remove spectrin and the receptor-associated 58-kDa protein from the cytoplasmic surface of receptor clusters. Thus, the ECM at AChR clusters is disrupted by an agent acting at the cytoplasmic surface of the membrane. We discuss the possibility that both AChR and ECM are bound to a common membrane skeleton and the implications this may have for synaptogenesis.
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PMID:Evidence for transmembrane anchoring of extracellular matrix at acetylcholine receptor clusters. 850 May 52

Pigment epithelium-derived factor (PEDF), a neurotrophic protein, is a secreted serpin identified in extracellular matrixes. We show that PEDF extractions from the interphotoreceptor matrix are more efficient with increasing NaCl concentrations, indicating that ionic interactions mediate its association with this polyanionic matrix. We have used affinity chromatography and ultrafiltration to probe for direct binding of PEDF to glycosaminoglycans/polyanions. Correctly folded PEDF bound to immobilized heparin, chondroitin sulfate-A, -B, -C, and dextran sulfate columns and eluted from each with an increase in NaCl concentration. However, in the presence of urea, the protein lost its affinity for heparin. Binding of PEDF to heparan sulfate proteoglycan in solution was in a concentration-dependent fashion (half-maximal specific binding EC50 = 40 micrograms/mL) and was sensitive to increasing NaCl concentrations. The glycosaminoglycan-binding region was analyzed using chemical modification and limited proteolysis. PEDF chemically modified on lysine residues by biotinylation lost its capacity for interacting with heparin, implicating the involvement of PEDF lysine residues in heparin binding. Cleavage of the serpin-exposed loop with chymotrypsin did not affect the heparin-binding property. A limited proteolysis product containing residues 21-approximately 260 bound to heparin with similar affinity as the intact PEDF. Homology modeling of PEDF based on the X-ray crystal structures of antithrombin III and ovalbumin shows a region at the center of beta-sheet A-strands 2 and 3- and helix F that has a basic electrostatic surface potential and is densely populated with lysines exposed to the surface (K134, K137, K189, K191, H212, and K214) that are available to interact with various glycosaminoglycans/polyanions. This region represents a novel site for glycosaminoglycan binding in a serpin, which in PEDF, is distinct and nonoverlapping from the PEDF neurotrophic active region.
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PMID:Pigment epithelium-derived factor (PEDF) binds to glycosaminoglycans: analysis of the binding site. 969 54

HBp17 was purified by Heparin-Copper biaffinity chromatography and HPLC from conditioned medium of A431 cell. The purified HBp17 was digested by staphylococcus urcus V8 protease or chymotrypsin and the heparin-binding fragments were isolated by Heparin-Sepharose. One binding site of peptide mapping is HBp17 residues 110-145 produced by V8. Another one is HBp17 residues 82-143 which were produced by chymotrypsin digestion. Two binding sites of peptide mapping are overlap. Therefore the residues 110-143 of HBp17 are the principle heparin binding site. The basic amino acid cluster in this region may be contribute to the binding of HBp17 to heparin or heparan sulfate proteoglycan on the cell surface and extracellular matrix.
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PMID:Purification of heparin-binding protein HBp17 and identification of HBp17 heparin binding site. 978 42