Subunit structure and properties of the glycogen-bound phosphoprotein phosphatase from skeletal muscle.
A high molecular weight phosphoprotein phosphatase was purified approximately 11,000-fold from the glycogen-protein complex of rabbit skeletal muscle. Polyacrylamide gel electrophoresis of the preparation in the absence of sodium dodecyl sulfate showed a major protein band which contained the activity of the enzyme. Gel electrophoresis in the presence of sodium dodecyl sulfate also showed a major protein band migrating at 38,000 daltons. The sedimentation coefficient, Stokes radius, and frictional ratio of the enzyme were determined to be 4.4 S, 4.4 nm, and 1.53, respectively. Based on these values the molecular weight of the enzyme was calculated to be 83,000. The high molecular weight phosphatase was dissociated upon chromatography on a reactive red-120 agarose column. The sedimentation coefficient, Stokes radius, and frictional ratio of the dissociated enzyme (termed monomer) were determined to be 4.1 S, 2.4 nm, and 1.05, respectively. The molecular weight of the monomer enzyme was determined to be 38,000 by polyacrylamide gel electrophoresis. Incubation of the high molecular weight phosphatase with a cleavable cross-linking reagent, 3,3'-dithiobis(sulfosuccinimidyl propionate), showed the formation of a cross-linked complex. The molecular weight of the cross-linked complex was determined to be 85,000 and a second dimension gel electrophoresis of the cleaved cross-linked complex showed that the latter contained only 38,000-dalton bands. Limited trypsinization of the enzyme released a approximately 4,000-dalton peptide from the monomers and dissociated the high molecular weight phosphatase into 34,000-dalton monomers. It is proposed that the catalytic activity of the native glycogen-bound phosphatase resides in a dimer of 38,000-dalton subunits.
D000818:Animals; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006003:Glycogen / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification* / Q000378:metabolism; D011487:Protein Conformation; D011817:Rabbits
D046911:Macromolecular Substances; D006003:Glycogen; D010749:Phosphoprotein Phosphatases; D008274:Magnesium