Resolution of glycogen phosphorylase isoenzymes in precast PhastSystem polyacrylamide gels.
Homogeneous (7.5%) and gradient (10-15%) ultrathin nondenaturating miniaturized polyacrylamide gels (Pharmacia PhastGel media) were used to separate glycogen phosphorylase isoforms from rabbit muscle, rat liver and brain, MH 3924A cells, a dedifferentiated hepatocellular carcinoma of the rat, and C1I cells, a nontumorigenic epithelial rat liver cell line. The enzymes were detected by in situ phosphorylase assay and by immunoblotting. Phosphorylase proteins from the brain, MH 3924A, and C1I exhibited similar electrophoretic mobility, which was different from that of the enzymes from the muscle and normal liver. Molecular weight determination from sodium dodecyl sulfate gels yielded similar data for the subunits of muscle and liver enzymes (98,000 and 96,000), respectively, on one hand, and brain, MH 3924A tumor, and nontumorigenic C1I cells (93,000, 93,000 and 92,000), respectively, on the other. In the native gels the enzymes migrated as dimers: for muscle phosphorylase a, a tetramer was also observed. The a and b forms of the enzymes could not be resolved. An antibody raised against rat liver phosphorylase reacted only with the liver enzyme, whereas an antibody raised against brain phosphorylase stained the brain enzyme and the enzymes from MH 3924A and C1I cells. This indicates that hepatoma cells and immortalized nontumorigenic epithelial liver cells express a phosphorylase isoenzyme that is different from the liver type but similar to the brain type. The PhastSystem provides a rapid, sensitive, and highly reproducible method to resolve the different isoenzymes of glycogen phosphorylase.
Mayer
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Abteilung Cytopathologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
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D000818:Animals; D001921:Brain / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D015151:Immunoblotting; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification*; D008099:Liver / Q000201:enzymology; D008114:Liver Neoplasms, Experimental / Q000201:enzymology; D046911:Macromolecular Substances; D008297:Male; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology; D006005:Phosphorylases / Q000737:chemistry / Q000302:isolation & purification*; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D014407:Tumor Cells, Cultured
D007527:Isoenzymes; D046911:Macromolecular Substances; D006005:Phosphorylases