Solubilization and partial purification of hyaluronate synthetase from oligodendroglioma cells.
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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K F
KF
Department of Pediatrics, Joseph P. Kennedy Jr. Mental Retardation Research Center, University of Chicago, Illinois 60637.
Schwartz
N B
NB
D000818:Animals; D002853:Chromatography, Liquid; D004072:Digitonin / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D014453:Glucuronosyltransferase / Q000302:isolation & purification* / Q000378:metabolism; D016695:Glycosyltransferases; D000076002:Hyaluronan Synthases; D007700:Kinetics; D008565:Membrane Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D009837:Oligodendroglioma / Q000201:enzymology*; D012995:Solubility; D014166:Transferases; D014407:Tumor Cells, Cultured / Q000201:enzymology; D029867:Xenopus Proteins
D008565:Membrane Proteins; D029867:Xenopus Proteins; D014166:Transferases; D016695:Glycosyltransferases; D014453:Glucuronosyltransferase; C056944:HAS1 protein, Xenopus; D000076002:Hyaluronan Synthases; D004072:Digitonin