Abstract
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.
Highlights
(250 kDa), andPCSL(200kDa) phosphatase according purified [6,7]two highmolecularweight protein phosphatases to the apparenmtolecular weight of the nativeenzymes from rat liver cytosol: protein phosphatase IB [6] containing in gel filtration
Taking advantage of the stimulation of phosphorylase phosphatase activity bylow concentrations of polycations, the inhibitor-1 [11] anddeinhibitor[16]phosphatase activity, and theobservation that one of them (PCS, phosphatase) is activated by a Ca2+-dependenptrotease [18],we have purified to homogeneity from rabbit skeletalmuscle four types of PCS phosphorylase phosphatases (Table I)
Phosphorylase kinase (a-subunit), casein, and myosin light chains were dephosphorylated by these enzymes. Both the PCSHl and PC&, enzymes are very specific deinhibitor protein phosphatases [16,17], acriterion that can be usedfor their purification
Summary
Taking advantage of the stimulation of phosphorylase phosphatase activity bylow concentrations of polycations, the inhibitor-1 [11] anddeinhibitor[16]phosphatase activity, and theobservation that one of them (PCS, phosphatase) is activated by a Ca2+-dependenptrotease [18],we have purified to homogeneity from rabbit skeletalmuscle four types of PCS phosphorylase phosphatases (Table I). Containing three additional components of 140, Monitoring the molecular weight of the PCSHphosphatases 95, and 72 or 75 kDa, respectively, for PCSHl and PCSM by Superose 6 chromatography during the purification steps phosphatase, all staining with lesser intensity, the enzymes showed that the apparent change in molecular weight coin- migrated after the Mono Q FPLC step as a single band in cided with the AcAgel filtration step.This reduction in polyacrylamide gel electrophoresis under nondenaturing conmolecular weight is, not due to a dissociation in ditions. The tendency to lose the 55-kDa subunit during purification was observed as early as 1977 by Lee and co-workers [3] and could represent something besides a simple conversion of one “type” of enzyme into another
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