Molecular studies of glycogen phosphorylase b from bovine liver.
Two active isoforms of bovine liver phosphorylase with distinct subunit composition have previously been purified (Cámara Artigas, A., Barón, C. and Parody-Morreale, A. Prot. Express. Purif. 1994, 5, 157), one showing three SDS-PAGE polypeptide bands (molecular mass = 97, 55 and 40 kDa) and the other showing just one (molecular mass = 97 kDa). A molecular mass of 200 kDa has been determined for the native enzymes by gel filtration. Amino acid analyses have been performed in both cases, giving the same results which are similar to those obtained with other phosphorylases. SDS-PAGE experiments at different concentrations of the three-band enzyme have suggested a 1:1:1 stoichiometry between the polypeptides. The pyridoxal-5'-phosphate site is located in the 55 kDa polypeptide and the phosphorylation site in the 40 kDa one. These polypeptides can be generated from the three-band enzyme by tryptic attack in the presence of glycogen without loss of enzyme activity. In the absence of glycogen, 55 kDa and 38 kDa polypeptides are generated, with a significant decrease in activity. We conclude that the three-band enzyme is a dimer composed of an intact monomer and a broken one. The lyotropic salt activation site of the enzyme is near the amino terminal group.
Artigas
A C
AC
Departamento de Química-Física, Bioquímica y Química Inorgánica, Facultad de Ciencias Experimentales, Universidad de Almería, Spain.
Barón
C
C
Parody-Morreale
A
A
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D006005:Phosphorylases / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D011732:Pyridoxal Phosphate / Q000032:analysis; D013431:Sulfates / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D010446:Peptide Fragments; D013431:Sulfates; D011732:Pyridoxal Phosphate; D006005:Phosphorylases; D014357:Trypsin