Characterization of hyaluronan synthase from a human glioma cell line.
In the present study we describe a method to prepare membranes with high hyaluronan synthase activity from human glioma cells by pretreatment of the cells with both testicular hyaluronidase and 4-phorbol 12-myristate 13-acetate (PMA). A 23-fold increase in hyaluronan synthase activity was detected in comparison to untreated cells. Using isolated membranes as a source of hyaluronan synthase activity we demonstrate that chain elongation occurs at the reducing end of the hyaluronan molecule. We also present a method to solubilize hyaluronan synthase in active form with 1% digitonin. The solubilized synthase synthesized shorter hyaluronan chains than the membrane bound enzyme. Partial purification of the solubilized enzyme on a Superdex-200 column revealed a 12-fold increase in specific activity. Affinity purified polyclonal antibodies, raised against a synthetic peptide corresponding to the carboxy-terminus of the deduced protein sequence of human hyaluronan synthase recognized a 66 kDa component in the purified preparations. The elution position of the solubilized hyaluronan synthesizing activity immediately after V0 corresponding to a molecular mass of about 600 kDa, suggested that the 66 kDa enzyme forms a complex with other components which may have accessory or regulatory roles during hyaluronan synthesis.
Asplund
T
T
Department of Medical and Physiological Chemistry, Uppsala University, Biomedical Centre, Box 575, S-75123 Uppsala, Sweden.
Brinck
J
J
Suzuki
M
M
Briskin
M J
MJ
Heldin
P
P
D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000201:enzymology; D006224:Cricetinae; D005910:Glioma / Q000201:enzymology*; D014453:Glucuronosyltransferase / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D016695:Glycosyltransferases; D006801:Humans; D000076002:Hyaluronan Synthases; D006820:Hyaluronic Acid / Q000096:biosynthesis; D007106:Immune Sera / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry; D008970:Molecular Weight; D012995:Solubility; D014166:Transferases; D014407:Tumor Cells, Cultured; D029867:Xenopus Proteins
D007106:Immune Sera; D008565:Membrane Proteins; D029867:Xenopus Proteins; D006820:Hyaluronic Acid; D014166:Transferases; D016695:Glycosyltransferases; D014453:Glucuronosyltransferase; C056944:HAS1 protein, Xenopus; D000076002:Hyaluronan Synthases