Purification and properties of phosphorylase from baker's yeast.
A rapid, reliable method for purification of phosphorylase, yielding 200-400 mg pure phosphorylase from 8 kg of pressed baker's yeast, is described. The enzyme is free of phosphorylase kinase activity but contains traces of phosphorylase phosphatase activity. Phosphorylase constitutes 0.5-0.8% of soluble protein in various strains of yeast assayed immunochemically. The subunit molecular weight (Mr) of yeast phosphorylase is around 100,000. The enzyme is composed of two subunits in various ratios, differing slightly in molecular weight and N-terminal sequence. Both are active. Only the enzyme species containing the larger subunit can form tetramers and higher oligomers. The activated enzyme is dimeric. Correlated with specific activity (1 to 110 U/mg), phosphorylase contained between less than 0.1 to 0.74 covalently bound phosphate per subunit. Inactive forms of phosphorylase could be activated by phosphorylase kinase and [gamma-32P]ATP with concomitant phosphorylation of a single threonine residue in the aminoterminal region of the large subunit. The small subunit was not labeled. The incorporated phosphate could be removed by yeast phosphorylase phosphatase, resulting in loss of activity of phosphorylase, which could be restored by ATP and phosphorylase kinase.
D004789:Enzyme Activation; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010710:Phosphates / Q000032:analysis; D006005:Phosphorylases / Q000302:isolation & purification* / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000032:analysis; D012440:Saccharomyces / Q000201:enzymology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013045:Species Specificity
D046911:Macromolecular Substances; D010710:Phosphates; D011732:Pyridoxal Phosphate; D006005:Phosphorylases