Premium
Purification and Initial Characterization of Rat B49 Glial Cell Line‐Derived Neurotrophic Factor
Author(s) -
Lin LeuFen H.,
Zhang Tie Jin,
Collins Frank,
Armes Lyman G.
Publication year - 1994
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1994.63020758.x
Subject(s) - glial cell line derived neurotrophic factor , neuroscience , neurotrophic factors , microbiology and biotechnology , cell culture , ciliary neurotrophic factor , biology , brain derived neurotrophic factor , line (geometry) , chemistry , biochemistry , genetics , receptor , geometry , mathematics
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 ∼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 ∼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 EC 50 in midbrain cultures at ∼40 pg/ml, or 1 p M , 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.