Purification and properties of polycation-stimulated phosphorylase phosphatases from rabbit skeletal muscle.
Four types of polycation-stimulated (PCS) phosphorylase phosphatases have been isolated from rabbit skeletal muscle. They are called PCSH (390 kDa), PCSM (250 kDa), and PCSL (200 kDa) phosphatase according to the apparent molecular weight of the native enzymes in gel filtration. Two forms of PCSH phosphatase could be separated by Mono Q fast protein liquid chromatography: PCSH1 and PCSH2. In the absence of polycations, the specific activities of the PCSH1, PCSH2, PCSM, and PCSL phosphatase were 400, 680, 600, and 3000 units/mg, respectively, using phosphorylase a as a substrate. They all contain a 62-65- and a 35-kDa subunit, the latter being the catalytic subunit. In addition PCSH1 phosphatase contains a 55-kDa subunit and the PCSM phosphatase a 72-75-kDa subunit in a substoichiometric ratio. All the PCS phosphatases are insensitive to Ca2+ calmodulin, inhibitor-1, and modulator protein. They display a high specificity for the alpha-subunit of phosphorylase kinase and a broad substrate specificity. The PCSH1 and PCSH2 phosphatases, but not the catalytic subunit (PCSC phosphatase), show a high degree of specificity for the deinhibitor protein. During the purification the phosphorylase to inhibitor-1 phosphatase activity ratio (10:1) remained constant for the PCSH and PCSL enzymes but decreased for the PCSM phosphatase. The stimulation observed with low concentrations of polycations is enzyme directed. The different enzyme forms show a characteristic concentration optimum and degree of stimulation. At higher concentrations, polycations become inhibitory and a time-dependent deactivation of the phosphatases is observed.
D000818:Animals; D002413:Cations, Divalent; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010765:Phosphorylase Phosphatase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011073:Polyamines; D000071228:Polyelectrolytes; D011108:Polymers / Q000494:pharmacology*; D011817:Rabbits; D013379:Substrate Specificity
D002413:Cations, Divalent; D010749:Phosphoprotein Phosphatases; D010765:Phosphorylase Phosphatase; D011073:Polyamines; D000071228:Polyelectrolytes; D011108:Polymers