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Glial Fibrillary Acidic Protein: Norepinephrine Stimulated Phosphorylation in Intact C‐6 Glioma Cells
Author(s) -
Browning Edward T.,
Ruina Monica
Publication year - 1984
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 - 10.1111/j.1471-4159.1984.tb02742.x
Subject(s) - phosphoprotein , microbiology and biotechnology , peptide , biochemistry , glial fibrillary acidic protein , protease , biology , phosphorylation , chemistry , enzyme , immunology , immunohistochemistry
Coelectrophoresis in two‐dimensional gels of rat glial fibrillary acidic protein (GFA) and 32 P‐labeled whole cell extracts of rat C‐6 glioma cells showed that the GFA migrated in close proximity to a previously noted phosphoprotein, 50K‐6.1, of these cells. GFA electrophoresed as a 50K polypeptide with at least four charge variants, the most acidic of which coelectrophoresed with 50K‐6.1. Exposure of the C‐6 cultures to dibutyryl cyclic AMP (dbcAMP) for 48 h increased the relative abundance of the endogenous polypeptide associated with 50K‐6.1 by threefold, consistent with the hypothesis that 50K‐6.1 was GFA. Norepinephrine stimulated 50K‐6.1 phosphorylation 3.2‐fold in dbcAMP‐induced cultures. Peptide mapping with V8 protease and subtilisin was used to test the hypothesis that GFA and 50K‐6.1 were identical polypeptides. With V8 protease, the peptides generated from the [ 35 S]methionine labeled putative GFA spot of the C‐6 cells were indistinguishable from the stained bands derived from authentic GFA in mixed samples of the two proteins. Likewise, the 35 S‐labeled acidic satellite to the putative GFA spot also yielded a peptide map that matched that of the authentic GFA. 32 P‐labeled peptides derived from the 50K‐6.1 protein were a subset of those from authentic GFA. With three subtilisin concentrations, 32 P‐labeled 50K‐6.1 was degraded to peptides which were again a subset of the stained GFA peptides. A cytoskeletal fraction from 32 P‐labeled C‐6 cells contained a 50K phosphoprotein. Pep‐, tide mapping with V8 protease produced a 32 P‐peptide pattern which was a subset of that from authentic GFA. The pattern closely resembled the 32 P‐peptide pattern for the 50K‐6.1 protein from 2‐dimensional gels of whole cell extract. It was concluded that the protein 50K‐6.1 is a phosphorylated form of GFA and that GFA is a phosphoprotein whose phosphòrylation is stimulated by norepinephrine in C‐6 glioma cells.