Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.36 (hyaluronidase)
 4,606 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Primary cultures of rabbit articular chondrocytes have been maintained for 10 days and labeled with [35S]sulfate, [3H]leucine, and [35S]cysteine in pulse-chase protocols to study the structure and hyaluronate binding properties of newly synthesized proteoglycan monomers. Radiolabeled monomers were purified from medium and cell-layer fractions by dissociative CsCl gradient centrifugation with bovine carrier monomer, and analyzed for hyaluronate binding affinity on Sepharose CL-2B in 0.5 M Na acetate, 0.1% Triton X-100, pH 6.8. Detergent was necessary to prevent self-association of newly synthesized monomers during chromatography. Monomers secreted during a 30-min pulse labeling with [35S]sulfate had a low affinity relative to carrier. Those molecules released into the medium during the first 12 h of chase (about 40% of the total) remained in the low affinity form whereas those retained by the cell layer rapidly acquired high affinity. In cultures where more than 90% of the preformed cell-layer proteoglycan was removed by hyaluronidase digestion before radiolabeling the newly synthesized low affinity monomers also rapidly acquired high affinity if retained in the cell layer. Cultures labeled with amino acid precursors were used to establish the purity of monomer preparations and to isolate core proteins for study. Leucine- or cysteine-labeled core proteins derived from either low or high affinity monomer preparations migrated as a single major species on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with electrophoretic mobility very similar to that of core protein derived from extracted proteoglycan monomer. Purified low affinity monomers were converted to the high affinity form by treatment at pH 8.6; however, this change was prevented by guanidinium-HCl at concentrations above 0.8 M. Conversion to high affinity was also achieved by incubation of monomers in aggregate with hyaluronic acid (HA) at pH 6.8 followed by dissociative reisolation of monomer. At both pH 6.8 and 8.6 the conversion process was slow, requiring up to 48 h for the maximum increase in affinity. It is suggested that the slow increase in HA binding affinity seen during extracellular processing of proteoglycans in cartilage and chondrocyte cultures is the result of an irreversible structural change in the HA binding domain following the binding of monomer to hyaluronate. The available evidence suggests that this change involves the formation or rearrangement of disulfide bonds.
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PMID:Studies on the hyaluronate binding properties of newly synthesized proteoglycans purified from articular chondrocyte cultures. 249 59

In the preovulatory follicle, the oocyte is surrounded by approximately 1000 closely associated cumulus cells forming the compact form of the cumulus cell-oocyte complex (COC). In response to the gonadotropin surge, the COC in a follicle destined for ovulation undergoes expansion when the cumulus cells synthesize and organize an extensive extracellular matrix enriched in hyaluronan. Successful expansion of the COC appears to be essential for ovulation and ultimately for fertilization. We studied this process in vitro by isolating compact COCs from preovulatory mouse follicles and incubating them under conditions which promote COC expansion by retention of newly synthesized hyaluronan (HA in the extracellular matrix around the cells. [3H]-Leucine and [35S]sulfate were used as precursors to label macromolecules synthesized by the cells that may be necessary for organizing the HA in this matrix. After labeling, expanded COCs were washed to remove medium and any labeled molecules that were not associated with the matrix. Macromolecules selectively associated with the matrix were then solubilized by digesting the expanded COCs briefly with Streptomyces hyaluronidase, an enzyme that specifically cleaves HA. Cells were removed by centrifugation, and the digest supernate was analyzed by molecular sieve chromatography and SDS-PAGE. A dermatan sulfate proteoglycan of large hydrodynamic size ( > 1 million Da) and a approximately 46-kDa protein were the predominant labeled species identified. The proteoglycan has properties similar to proteoglycans such as aggrecan and versican which interact specifically with HA. The approximately 46-kDa protein has the same molecular size as the link protein which interacts with HA and HA-binding proteoglycans to form stable ternary complexes in a variety of extracellular matrices. We propose that the dermatan sulfate proteoglycan and the approximately 46-kDa protein synthesized by the cumulus cells form similar ternary complexes that are necessary for retaining HA in the COC matrix and hence are required for successful COC expansion.
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PMID:Proteoglycans and proteins in the extracellular matrix of mouse cumulus cell-oocyte complexes. 856 97