Analysis of cell-growth-phase-related variations in hyaluronate synthase activity of isolated plasma-membrane fractions of cultured human skin fibroblasts.
Hyaluronate synthase activity is localized exclusively in plasma-membrane fractions of cultured human skin fibroblasts. The enzyme activity of plasma membranes prepared from exponential-growth-phase cells was about 6.5 times that of stationary-growth-phase cells. Hyaluronate synthase from exponential-growth-phase cells exhibited lower Km and higher Vmax. values for both UDP-N-acetylglucosamine and UDP-glucuronic acid and higher rate of elongation of hyaluronate chains compared with the enzyme from stationary-growth-phase cells. Hyaluronate synthase exhibited an extremely short half-life, 2.2 h and 3.8 h respectively when cells were treated with cycloheximide and actinomycin D. The cell-growth-phase-dependent variations in hyaluronate synthase activity appear to be due to its high turnover rate as well as due to some post-translational modification of the enzyme protein as cells progress from early exponential to stationary growth phase. The isolated plasma membranes contained a protein (Mr approx. 450,000) that was selectively autophosphorylated from [gamma-32P]ATP in vitro in the presence of hyaluronate precursors in the reaction mixture and that also exhibited some hyaluronate-synthesis-related properties. The 32P-labelled protein isolated from plasma membranes of exponentially growing cells expressed an efficient UDP-[14C]glucuronic acid- and UDP-N-acetyl[3H]glucosamine-binding activity and was able to synthesize oligosaccharides (Mr 5000) of [14C]glucuronic acid and N-acetyl[3H]glucosamine residues. The corresponding protein of stationary-growth-phase cells, which expressed much higher nucleotide-sugar-precursor-binding activity, appeared to have lost its oligosaccharide-synthesizing activity.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D002455:Cell Division; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D005347:Fibroblasts / Q000201:enzymology*; D014453:Glucuronosyltransferase / Q000378:metabolism*; D016695:Glycosyltransferases; D006801:Humans; D000076002:Hyaluronan Synthases; D006820:Hyaluronic Acid / Q000096:biosynthesis; D008565:Membrane Proteins / Q000378:metabolism; D008970:Molecular Weight; D010442:Peptide Chain Initiation, Translational; D010443:Peptide Chain Termination, Translational; D010766:Phosphorylation; D014166:Transferases; D029867:Xenopus Proteins
D008565:Membrane Proteins; D029867:Xenopus Proteins; D000255:Adenosine Triphosphate; D006820:Hyaluronic Acid; D014166:Transferases; D016695:Glycosyltransferases; D014453:Glucuronosyltransferase; C056944:HAS1 protein, Xenopus; D000076002:Hyaluronan Synthases