Glycogen phosphorylase from Neurospora crassa: purification of a high-specific-activity, non-phosphorylated form.
A highly active glycogen phosphorylase was purified from Neurospora crassa by polyethylene glycol fractionation at pH 6.16 combined with standard techniques (chromatography and salt fractionation). The final preparation  had a specific activity of 65 +/- 5 U/mg of protein (synthetic direction, pH 6.1, 30 degrees C) and was homogeneous by the criteria of gel electrophoresis, amino-terminal analysis, gel filtration, and double immunodiffusion in two dimensions. The enzyme had a native molecular weight of 180,000 +/- 10,000 (by calibrated gel filtration and gel electrophoresis) and a subunit molecular weight of 90,000 +/- 5,000 (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Each subunit contained one molecule of pyridoxal phosphate. No phosphoserine or phosphothreonine was detected by amino acid analysis optimized for phosphoamino acid detection. The enzyme isolated from cells grown on high-specific-activity 32Pi (as sole source of phosphorus) contained one atom of 32P per subunit. All the radioactivity was removed by procedures that removed pyridoxal phosphate. Thus, the enzyme could not be classified as an a type (phosphorylated, active in the absence of a cofactor) or as a b type (non-phosphorylated, inactive in the absence of a cofactor). The level of phosphorylase was markedly increased in mycelium taken from older cultures in which the carbon source (glucose or sucrose) had been depleted. The polyethylene glycol fractionation scheme applied at pH 7.5 to mycelial extracts of younger cultures (taken before depletion of the sugar) resulted in co-purification of glycogen phosphorylase and glycogen synthetase.
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000596:Amino Acids / Q000032:analysis; D004789:Enzyme Activation; D006006:Glycogen Synthase / Q000302:isolation & purification; D008970:Molecular Weight; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D010758:Phosphorus / Q000032:analysis; D006005:Phosphorylases / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000032:analysis
D000596:Amino Acids; D010758:Phosphorus; D000249:Adenosine Monophosphate; D011732:Pyridoxal Phosphate; D006005:Phosphorylases; D006006:Glycogen Synthase