Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:4.2.2.7 (heparinase)
 1,270 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human skin fibroblasts in different growth states were incubated with [3H]glucosamine and/or Na(2)35SO4 and extracted with Triton X-100 for various periods of time. Free heparan-sulphate oligosaccharides and protein-bound heparan-sulphate chains were separated by chromatography on octyl-Sepharose and analyzed. A pool of endogenously produced oligosaccharides, present in the cultured cells and isolated after brief extraction, contained fragments of uniform size (approximately 7-10 kDa corresponding to approximately 14-20 disaccharides). Analysis by heparinase I and heparinase III degradations followed by electrophoretic separation (oligosaccharide mapping) showed that the oligosaccharides were rich in glucuronic acid but had a few sulphated iduronic acid residues at the periphery of each molecule. These results indicated that endoheparanase cleavage points were located close to linkages between N-sulphated glucosamine and sulphated iduronic acid, generating fragments that comprise a major portion of the unmodified segments and a minor portion of the highly modified segments. Prolonged extraction (24-48 h) of cells with Triton X-100 at 4 degrees C in the presence of proteinase inhibitors resulted in further degradation. There was an increase in the amount of heparan-sulphate oligosaccharides and a concomitant decrease in the amount of protein-bound heparan-sulphate chains present in the same extract. The heparan-sulphate oligosaccharides obtained after prolonged extraction were more heterogeneous in size comprising, in addition to the major species of approximately 7-10 kDa, intermediate and larger fragments of approximately 17 kDa and 30-40 kDa. This observation suggests that endoheparanase acted at periodically appearing, specific regions in the intact heparan-sulphate chain. Furthermore, the enzyme and substrate should remain closely associated during cold Triton X-100 extraction. To determine if the endogenously produced heparan-sulphate oligosaccharides were derived from a particular heparan-sulphate species degraded during the growth phase, proteoglycan-derived heparan-sulphate chains obtained from proliferating or quiescent fibroblasts were also examined. These chains showed similar oligosaccharide maps, except for a small increase in the amount of glucuronic acid as cell growth was arrested. Hence, an endoheparanase with restricted specificity may generate slightly different oligosaccharides in the various growth states.
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PMID:Analysis of heparan-sulphate chains and oligosaccharides from proliferating and quiescent fibroblasts. A proposed model for endoheparanase activity. 803 94

Recombinant collagen-binding domain (rCBD) comprising the three fibronectin type II-like modules of human gelatinase A was found to compete the zymogen form of this matrix metalloproteinase from the cell surface of normal human fibroblasts in culture. Upon concanavalin A treatment of cells, the induced cellular activation of gelatinase A was markedly elevated in the presence of the rCBD. Therefore, the mechanistic aspects of gelatinase A binding to cells by this domain were further studied using cell attachment assays. Fibroblasts attached to rCBD-coated microplate wells in a manner that was inhibited by soluble rCBD, blocking antibodies to the beta1-integrin subunit but not the alpha2-integrin subunit, and bacterial collagenase treatment. Addition of soluble collagen rescued the attachment of collagenase-treated cells to the rCBD. As a probe on ligand blots of octyl-beta-D-thioglucopyranoside-solubilized cell membrane extracts, the rCBD bound 140- and 160-kDa protein bands. Their identities were likely procollagen chains being both bacterial collagenase-sensitive and also converted upon pepsin digestion to 112- and 126-kDa bands that co-migrated with collagen alpha1(I) and alpha2(I) chains. A rCBD mutant protein (Lys263 --> Ala) with reduced collagen affinity showed less cell attachment, whereas a heparin-binding deficient mutant (Lys357 --> Ala), heparinase treatment, or heparin addition did not alter attachment. Thus, a cell-binding mechanism for gelatinase A is revealed that does not involve the hemopexin COOH domain. Instead, an attachment complex comprising gelatinase A-native type I collagen-beta1-integrin forms as a result of interactions involving the collagen-binding domain of the enzyme. Moreover, this distinct pool of cell collagen-bound proenzyme appears recalcitrant to cellular activation.
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PMID:The involvement of the fibronectin type II-like modules of human gelatinase A in cell surface localization and activation. 968 20