Purification and initial characterization of rat B49 glial cell line-derived neurotrophic factor
| Autor: | Tie Jin Zhang, Leu-Fen H. Lin, Lyman G. Armes, Frank H. Collins |
|---|---|
| Rok vydání: | 1994 |
| Předmět: |
Serotonin
Dopamine Nerve Tissue Proteins Biochemistry Chromatography Affinity Reuptake Cell Line Rats Sprague-Dawley Cellular and Molecular Neuroscience Neurotrophic factors Mesencephalon Pregnancy Culture Techniques Glial cell line-derived neurotrophic factor Animals Humans Glial Cell Line-Derived Neurotrophic Factor Nerve Growth Factors Polyacrylamide gel electrophoresis Cells Cultured Chromatography High Pressure Liquid gamma-Aminobutyric Acid Gel electrophoresis biology Sepharose Embryo Mammalian Recombinant Proteins Rats Molecular Weight Oligodendroglia nervous system Cell culture biology.protein Chromatography Gel Biological Assay Electrophoresis Polyacrylamide Gel Female Rat Protein GDNF family of ligands Neuroglia Chromatography Liquid |
| Zdroj: | Journal of neurochemistry. 63(2) |
| ISSN: | 0022-3042 |
| Popis: | The rat glial cell line B49 releases into its culture medium a potent neurotrophic factor that exhibits relative specificity for the dopaminergic neurons in dissociated cultures of rat embryonic midbrain. This factor is a heparin-binding, basic protein that is heterogeneously glycosylated and migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and on molecular sieve chromatography with an apparent mass of approximately 33-45 kDa. The factor behaves like a disulfide-bonded homodimer, whose biological activity is destroyed by reduction of disulfide bonds but not by SDS-PAGE or reversed-phase (RP)-HPLC. The apparent mass of the monomer is approximately 16 kDa after deglycosylation with N-Glycanase. This factor has been purified 34,000-fold to apparent homogeneity by a combination of heparin-affinity chromatography, molecular sieving chromatography, SDS-PAGE, and RP-HPLC. The purified rat protein promotes the survival, morphological differentiation, and high-affinity dopamine reuptake of dopaminergic neurons in midbrain cultures, without obvious effects on total neurons or glia and without increasing high-affinity GABA or serotonin reuptake. The purified protein exhibits an EC50 in midbrain cultures at approximately 40 pg/ml, or 1 pM, and has unique amino-terminal and internal amino acid sequences. The sequences provide a basis for cloning and expression of the gene for rat and human glial cell line-derived neurotrophic factor (GDNF), confirming that the protein purified as reported here is GDNF. |
| Databáze: | OpenAIRE |
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