A filter paper assay for hyaluronic acid synthetase: application to the enzyme from Swiss 3T3 fibroblasts.
An improved assay for hyaluronic acid (HA) synthetase is described that is suitable for rapid processing of large numbers of samples. High background levels of unincorporated radioactivity are removed by passage of the reaction through a Sephadex G-50 spin column. The labeled HA product is then precipitated onto glass fiber filters with cetylpyridinium chloride. Apparent Km values for HA synthetase from Swiss 3T3 fibroblasts are 10.8 and 58.4 microM for UDP-glucuronic acid and UDP-N-acetylglucosamine, respectively. HA synthetase activity of quiescent cells is 4.5% of that found in actively growing cells and is stimulated in response to 10% calf serum. There is a greater than 10-fold increase in HA synthetase activity when cells are harvested with hyaluronidase as compared with trypsin.
Malinowski
N M
NM
Laboratory of Medicinal Chemistry, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Cysyk
R L
RL
August
E M
EM
D016475:3T3 Cells / Q000201:enzymology*; D000818:Animals; D002250:Carbon Radioisotopes; D002417:Cattle; D002594:Cetylpyridinium / Q000737:chemistry; D011232:Chemical Precipitation; D002845:Chromatography; D005374:Filtration / Q000379:methods*; D014453:Glucuronosyltransferase / Q000737:chemistry* / Q000502:physiology; D016695:Glycosyltransferases; D000076002:Hyaluronan Synthases; D007700:Kinetics; D008565:Membrane Proteins; D051379:Mice; D013997:Time Factors; D014166:Transferases; D014535:Uridine Diphosphate Glucuronic Acid / Q000737:chemistry; D029867:Xenopus Proteins
D002250:Carbon Radioisotopes; D008565:Membrane Proteins; D029867:Xenopus Proteins; D014535:Uridine Diphosphate Glucuronic Acid; D002594:Cetylpyridinium; D014166:Transferases; D016695:Glycosyltransferases; D014453:Glucuronosyltransferase; C056944:HAS1 protein, Xenopus; D000076002:Hyaluronan Synthases