Glial fibrillary acidic protein: norepinephrine stimulated phosphorylation in intact C-6 glioma cells.
Coelectrophoresis in two-dimensional gels of rat glial fibrillary acidic protein (GFA) and 32P-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 [35S]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 35S-labeled acidic satellite to the putative GFA spot also yielded a peptide map that matched that of the authentic GFA. 32P-labeled peptides derived from the 50K-6.1 protein were a subset of those from authentic GFA. With three subtilisin concentrations, 32P-labeled 50K-6.1 was degraded to peptides which were again a subset of the stained GFA peptides. A cytoskeletal fraction from 32P-labeled C-6 cells contained a 50K phosphoprotein.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005904:Glial Fibrillary Acidic Protein; D005910:Glioma / Q000378:metabolism*; D007381:Intermediate Filament Proteins / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D009638:Norepinephrine / Q000494:pharmacology*; D010446:Peptide Fragments / Q000032:analysis; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D051381:Rats; D013116:Spinal Cord / Q000032:analysis; D013462:Sulfur Radioisotopes
D005904:Glial Fibrillary Acidic Protein; D007381:Intermediate Filament Proteins; D010446:Peptide Fragments; D010761:Phosphorus Radioisotopes; D013462:Sulfur Radioisotopes; D009638:Norepinephrine