Purification and initial characterization of rat B49 glial cell line-derived neurotrophic factor.
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.
Lin
L F
LF
Synergen, Inc, Boulder, Colorado 80301.
Zhang
T J
TJ
Collins
F
F
Armes
L G
LG
D000818:Animals; D001681:Biological Assay; D002460:Cell Line; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D046508:Culture Techniques / Q000379:methods; D004298:Dopamine / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004622:Embryo, Mammalian; D005260:Female; D051100:Glial Cell Line-Derived Neurotrophic Factor; D006801:Humans; D008636:Mesencephalon / Q000166:cytology*; D008970:Molecular Weight; D009414:Nerve Growth Factors / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000494:pharmacology; D009457:Neuroglia / Q000166:cytology / Q000378:metabolism*; D009836:Oligodendroglia / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000494:pharmacology; D012685:Sepharose / Q000031:analogs & derivatives; D012701:Serotonin / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
C495039:GDNF protein, human; C495041:Gdnf protein, rat; D051100:Glial Cell Line-Derived Neurotrophic Factor; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C037405:heparin-sepharose; D012701:Serotonin; D005680:gamma-Aminobutyric Acid; D012685:Sepharose; D004298:Dopamine