Characterization of hyaluronan synthase from a human glioma cell line
| Autor: | Jonas Brinck, Paraskevi Heldin, Masanobu Suzuki, Michael J. Briskin, Tomas Asplund |
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| Rok vydání: | 1998 |
| Předmět: |
Biophysics
Peptide CHO Cells Xenopus Proteins Biochemistry chemistry.chemical_compound Transferases Cricetinae Tumor Cells Cultured Animals Humans Glucuronosyltransferase Hyaluronic Acid Molecular Biology chemistry.chemical_classification biology Molecular mass ATP synthase Immune Sera Cell Membrane Glycosyltransferases Membrane Proteins Glioma Molecular biology carbohydrates (lipids) Hyaluronan synthase activity Molecular Weight Hyaluronan synthase Enzyme Digitonin chemistry Solubility Polyclonal antibodies biology.protein Hyaluronan Synthases |
| Zdroj: | Biochimica et biophysica acta. 1380(3) |
| ISSN: | 0006-3002 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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