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)

Hyaluronate synthetase was solubilized with digitonin from crude membranes of mouse oligodendroglioma cells. Detergent extraction was carried out in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered saline with an optimal digitonin to protein ratio (w/w) of 0.7-0.8. The solubilized synthetase was partially purified approximately 230-fold by gel filtration and ion-exchange chromatography. The solubilized enzyme displayed similar properties to membrane-bound enzyme: (a) it synthesized high molecular weight hyaluronate which eluted in the void volume of a Sepharose CL-2B column; (b) the apparent Km values obtained for UDP-GlcUA and UDP-GlcNAc were 50 and 100 microM, respectively; and (c) treatment of intact cells with hyaluronidase prior to extraction with digitonin resulted in a 3-fold increase in solubilized synthetase activity. Furthermore, gel filtration chromatography of the solubilized hyaluronidase-treated synthetase complex showed that it was smaller than the solubilized untreated synthetase complex, due to shorter nascent-bound hyaluronate. The solubilized synthetase was shown to be associated with hyaluronate in the form of a complex. Both hyaluronidase-treated and -untreated synthetase-hyaluronate complexes after solubilization were adsorbed by an affinity matrix using the hyaluronate binding domain of rat chondrosarcoma proteoglycan as ligand. This solubilized active enzyme preparation should allow the identification and characterization of the components of the hyaluronate-synthetase complex.
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PMID:Solubilization and partial purification of hyaluronate synthetase from oligodendroglioma cells. 250 Dec 97

Hyaluronidase treatment of mouse oligodendroglioma cells in monolayer culture resulted in a 4-5-fold stimulation of hyaluronate synthetase, assayed in washed membrane preparations [Philipson, L., & Schwartz, N. B. (1984) J. Biol. Chem. 259, 5017-5023]. We now report studies on the mechanism of the hyaluronidase-induced increase in the specific activity of the membrane-bound synthetase complex. The stimulation was dependent on the concentration of hyaluronidase but not on the particular bond cleaved or the nature of the product generated. Analysis of chain growth during cell-free synthesis by the disaccharide ratio method suggested that substantial internal labeling of hyaluronate chains had occurred. With both treated and untreated membranes, greater than 90% of incorporated (and recovered) radioactivity appeared in unsaturated disaccharides. Further analysis showed that hyaluronidase treatment increased both the rate of elongation and the rate of release of elongated chains from the enzyme complex. Hyaluronidase treatment also caused a change in the apparent steady-state kinetic patterns of double-reciprocal plots from intersecting lines for membranes from control cells to a family of parallel lines. Both the overall stimulation of synthesis and the change in apparent kinetic pattern were reversed by brief incubation of washed cells in the absence of hyaluronidase. These results have led to the development of an explicit kinetic model for hyaluronate synthesis which suggests an explanation for the switch in apparent kinetic patterns based on changing concentrations of a postulated key intermediate.
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PMID:Effect of hyaluronidase treatment of intact cells on hyaluronate synthetase activity. 393 51