Purification and characterization of phosphorylase B from Ascaris suum

Abstract

Glycogen phosphorylase b (EC 2.4.1.1) has been purified from the muscle of the roundworm, Ascaris suum. The 223-fold purified enzyme was shown to be homogenous by high performance liquid chromatography (HPLC), gel filtration column chromatography and sodium dodecyl sulfate (SDS) gel electrophoresis. The apparent native molecular weight of the enzyme determined by size exclusion chromatography by HPLC and gel filtration corresponded to 200 000 and 199 000, respectively. The subunit molecular weight of the enzyme was determined to be 100 000 by electrophoresis in the presence of SDS. Therefore, the enzyme appears to be a dimer with identical or near identical subunits. The enzyme contained 1 mol of pyridoxal 5′-phosphate per mol subunit and exhibited an absorbance index E 280 1% of 13.8. The apparent isoelectric point of the enzyme is 5.53. The enzyme, inactive in the absence of AMP, can be converted to the active form by rabbit muscle phosphorylase kinase and MgATP. The molecular weight of the activated form of the enzyme is 200 000. Kinetic studies showed apparent K m values of 0.17% for glycogen, 36 mM for P i and 52 mM for glucose-1-P. The apparent K a for AMP was 0.22 mM.