Purification and characterization of the glycogen-bound protein phosphatase from rat liver

Glycogen-bound protein phosphatase G from rat liver was transferred from glycogen to beta-cyclodextrin (cycloheptaamylose) linked to Sepharose 6B. After removal of the catalytic subunit and of contaminating proteins with 2 M NaCl, elution with beta-cyclodextrin yielded a single protein on native polyacrylamide gel electrophoresis and two polypeptides (161 and 54 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Several lines of evidence indicate that the latter polypeptides are subunits of the protein phosphatase G holoenzyme. First, these polypeptides were also present, together with the catalytic subunit, in the extensively purified holoenzyme. Also, polyclonal antibodies against these polypeptides were able to bind the holoenzyme. Further, while bound to cyclodextrin-Sepharose, the polypeptides were able to recombine with separately purified type-1 (AMD) catalytic subunit, but not with type-2A (PCS) catalytic subunit. The characteristics of the reconstituted enzyme resembled those of the nonpurified protein phosphatase G. At low dilutions, the spontaneous phosphorylase phosphatase activity of the reconstituted enzyme was about 10 times lower than that of the catalytic subunit, but it was about 1000-fold more resistant to inhibition by the modulator protein (inhibitor-2). In contrast with the free catalytic subunit, the reconstituted enzyme co-sedimented with glycogen, and it was able to activate purified liver glycogen synthase b. Also, the synthase phosphatase activity was synergistically increased by a cytosolic phosphatase and inhibited by physiological concentrations of phosphorylase alpha and of Ca2+.     

Phosphorylase phosphatase from skeletal muscle membranes

Microsomes containing 12-15 U/mg phosphorylase phosphatase were obtained from skeletal muscle glycogen particles following glycogen digestion and differential centrifugation. The phosphatase associated with the membranes is in an inhibited state; dilution induces dissociation and deinhibition of the enzyme. Phosphatase-depleted membranes can rebind purified phosphatase catalytic subunit but not the complex between catalytic subunit and inhibitor 2. Binding involves a receptor, deduced from saturation phenomena, which is responsible for inhibition of the bound enzyme and which is a protein, since trypsin treatment releases all bound enzyme and prevents rebinding. The phosphatase extracted from the membranes is of type 1 and is a mixture of complexes, the major ones displaying a Mr of 300,000 and 70,000. From these complexes the 35-kDa catalytic subunit can be obtained either by trypsin treatment or by acetone precipitation. Purification to homogeneity involves chromatography on polylysine and FPLC chromatography on Mono Q and Polyanion SI columns. The purified enzyme exhibits a specific activity of 26,800 U/mg (27,900 U/mg after trypsin treatment) and consists of a major protein of 38 kDa (SDS gel electrophoresis). A minor component of 33 kDa, which may represent either a proteolytic product or an isozyme, can be separated. Both 38-kDa and 33-kDa catalytic subunits form a 70-kDa inactive complex with inhibitor 2 and upon incubation of the complexes the catalytic subunit is slowly converted to the inactive conformation which can then be reactivated by either the kinase FA or trypsin and Mn2+. Alternatively the inactive catalytic subunit is reactivated by Mn2+ alone once it has been isolated by FPLC chromatography on SI. The observation that the same catalytic subunit is present at various cell locations (namely cytosol, glycogen particles and microsomes), though in different conformations, is in favour of the hypothesis that displacement of the catalytic subunit from one cell site to the other may represent a new mechanism for phosphatase regulation in skeletal muscle.     

Distinct type-1 protein phosphatases are associated with hepatic glycogen and microsomes

The type-1 protein phosphatase associated with hepatic microsomes has been distinguished from the glycogen-bound enzyme in five ways. (1) The phosphorylase phosphatase/synthase phosphatase activity ratio of the microsomal enzyme (measured using muscle phosphorylase a and glycogen synthase (labelled in sites-3) as substrates) was 50-fold higher than that of the glycogen-bound enzyme. (2) The microsomal enzyme had a greater sensitivity to inhibitors-1 and 2. (3) Release of the catalytic subunit from the microsomal type-1 phosphatase by tryptic digestion was accompanied by a 2-fold increase in synthase phosphatase activity, whereas release of the catalytic subunit from the glycogen-bound enzyme decreased synthase phosphatase activity by 60%. (4) 95% of the synthase phosphatase activity was released from the microsomes with 0.3 M NaCl, whereas little activity could be released from the glycogen fraction with salt. (5) The type-1 phosphatase separated from glycogen by anion-exchange chromatography could be rebound to glycogen, whereas the microsomal enzyme (separated from the microsomes by the same procedure, or by extraction with NaCl) could not. These findings indicate that the synthase phosphatase activity of the microsomal enzyme is not explained by contamination with glycogen-bound enzyme. The microsomal and glycogen-associated enzymes may contain a common catalytic subunit complexed to microsomal and glycogen-binding subunits, respectively. Thiophosphorylase a was a potent inhibitor of the dephosphorylation of ribosomal protein S6, HMG-CoA reductase and glycogen synthase, by the glycogen-associated type-1 protein phosphatase. By contrast, thiophosphorylase a did not inhibit the dephosphorylation of S6 or HMG-CoA reductase by the microsomal enzyme, although the dephosphorylation of glycogen synthase was inhibited. The I50 for inhibition of synthase phosphatase activity by thiophosphorylase a catalysed by either the glycogen-associated or microsomal type-1 phosphatases, or for inhibition of S6 phosphatase activity catalysed by the glycogen-associated enzyme, was decreased 20-fold to 5-10 nM in the presence of glycogen. The results suggest that the physiologically relevant inhibitor of the glycogen-associated type-1 phosphatase is the phosphorylase a-glycogen complex, and that inhibition of the microsomal type-1 phosphatase by phosphorylase a is unlikely to play a role in the hormonal control of cholesterol or protein synthesis. Protein phosphatase-1 appears to be the principal S6 phosphatase in mammalian liver acting on the serine residues phosphorylated by cyclic AMP-dependent protein kinase.     

Purification of porcine heart latent protein phosphatase Fc.M.

Latent protein phosphatase, Fc.M, was purified from porcine heart extracts by a procedure involving precipitation at pH 5.0, DEAE-Sephacel chromatography, ammonium sulfate fractionation, chromatography on phenyl-Sepharose, Biogel-A 0.5m and poly-L-lysine-agarose. The purified enzyme had a specific activity of 12,200 nanomoles of phosphate released from phosphorylase a/mg protein when assayed following activation by pretreatment with Mn++ and trypsin in the presence of 0.2 M NaCl. The enzyme is a heterodimer of 66 kDa composed of a catalytic (37 kDa) and a modulator (31 kDa) subunit.     

Isolation and characterization of a high molecular weight protein phosphatase from rabbit skeletal muscle

A high molecular weight protein phosphatase (phosphatase H-II) was isolated from rabbit skeletal muscle. The enzyme had a Mr = 260,000 as determined by gel filtration and possessed two types of subunit, of Mr = 70,000 and 35,000, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On ethanol treatment, the enzyme was dissociated to an active species of Mr = 35,000. The purified phosphatase dephosphorylated lysine-rich histone, phosphorylase a, glycogen synthase, and phosphorylase kinase. It dephosphorylated both the alpha- and beta-subunit phosphates of phosphorylase kinase, with a preference for the dephosphorylation of the alpha-subunit phosphate over the beta-subunit phosphate of phosphorylase kinase. The enzyme also dephosphorylated p-nitrophenyl phosphate at alkaline pH. Phosphatase H-II is distinct from the major phosphorylase phosphatase activities in the muscle extracts. Its enzymatic properties closely resemble that of a Mr = 33,500 protein phosphatase (protein phosphatase C-II) isolated from the same tissue. However, despite their similarity of enzymatic properties, the Mr = 35,000 subunit of phosphatase H-II is physically different from phosphatase C-II as revealed by their different sizes on sodium dodecyl sulfate-gel electrophoresis. On trypsin treatment of the enzyme, this subunit is converted to a form which is a similar size to phosphatase C-II.     

Purification of a latent protein phosphatase from rabbit skeletal muscle

A high molecular weight protein phosphatase (Mr = 260K) has been isolated from rabbit skeletal muscle. The enzyme has a very low activity towards phosphorylase a isolated from the same tissue, but its activity towards this substrate is stimulated several fold after dissociation by 2-mercaptoethanol treatment. The purified phosphatase shows one major protein staining band on non denaturing polyacrylamide gel electrophoresis, and contains four subunits with molecular weights of 95K, 75K, 65K and 38K. The catalytic activity resides in the Mr = 38K subunit and is not sensitive to inhibition by the heat stable protein phosphatase inhibitor-1 or modulator protein. Polyamines stimulate the holoenzyme in a dose dependent, biphasic manner, but inhibit the activity of the dissociated Mr = 38K catalytic subunit.     

Phosphorylase phosphatase catalytic subunit. Evidence that the Mr = 33,000 enzyme fragment is derived from a native protein of Mr = 70,000

An active form of phosphorylase phosphatase of Mr = 33,000, referred to as the catalytic subunit for over a decade, was purified to near-homogeneity from rabbit skeletal muscle. Repeated immunization of a sheep produced immunoglobulins that blocked the activity of the phosphatase. These immunoglobulins were affinity-purified on columns of immobilized phosphorylase phosphatase and used as macromolecular probes in a "Western" immunoblotting procedure with peroxidase-conjugated rabbit anti-sheep immunoglobulins. Only one protein, of Mr = 33,000, was stained in samples of the immunogen, attesting to the specificity of the probes. However, the Mr = 33,000 phosphatase protein was not detected in muscle extracts or in partially purified preparations. Instead, a single protein of Mr = 70,000 was detected. Limited proteolysis, in particular by Staphylococcus aureus V8 protease and thermolysin, converted the immunoreactive protein from Mr = 70,000 to Mr = 33,000. Coagulation of the phosphatase preparation with 80% ethanol at room temperature rendered the Mr = 70,000 protein insoluble, but allowed extraction of the Mr = 33,000 protein from the precipitate. Thus, we conclude that the immunoreactive protein of Mr = 70,000 is the "catalytic subunit" of phosphorylase phosphatase with a catalytic domain of Mr = 33,000. Previous purification schemes have yielded only the fragment of Mr = 33,000 due to its relative resistance to proteolysis and coagulation. Gel filtration chromatography of the "native" form of phosphorylase phosphatase showed Mr approximately 230,000. Both the Mr = 70,000 catalytic subunit and a Mr = 60,000 protein related to inhibitor-2 were detected by immunoblotting in the same fractions that exhibited activity after treatment with Co2+ and trypsin. Only the Mr = 60,000 protein was degraded during this activation process. We propose that the native phosphorylase phosphatase is an elongated structure with two-fold symmetry, containing one catalytic subunit of Mr = 70,000 and one regulatory subunit of Mr = 60,000.     

Regulation of protein phosphatase-1G from rabbit skeletal muscle. 1. Phosphorylation by cAMP-dependent protein kinase at site 2 releases catalytic subunit from the glycogen-bound holoenzyme

The glycogen-associated form of protein phosphatase-1 (PP-1G) is a heterodimer comprising a 37-kDa catalytic (C) subunit and a 161-kDa glycogen-binding (G) subunit, the latter being phosphorylated by cAMP-dependent protein kinase at two serine residues (site 1 and site 2). Here the amino acid sequence surrounding site 2 has been determined and this phosphoserine shown to lie 19 residues C-terminal to site 1 in the primary structure. The sequence in this region is: (sequence; see text) At physiological ionic strength, phosphorylation of glycogen-bound PP-1G was found to release all the phosphatase activity from glycogen. The released activity was free C subunit, and not PP-1G, while the phospho-G subunit remained bound to glycogen. Dissociation reflected a greater than or equal to 4000-fold decrease in affinity of C subunit for G subunit and was readily reversed by dephosphorylation. Phosphorylation and dephosphorylation of site 2 was rate-limiting for dissociation and reassociation of C subunit. Release of C subunit was also induced by the binding of anti-site-1 Fab fragments to glycogen-bound PP-1G. At near physiological ionic strength, PP-1G and glycogen concentration, site 2 was autodephosphorylated by PP-1G with a t0.5 of 2.6 min at 30 degrees C, approximately 100-fold slower than the t0.5 for dephosphorylation of glycogen phosphorylase under the same conditions. Site 2 was a good substrate for all three type-2 phosphatases (2A, 2B and 2C) with t0.5 values less than those toward the alpha subunit of phosphorylase kinase. At the levels present in skeletal muscle, the type-2A and type-2B phosphatases are potentially capable of dephosphorylating site 2 in vivo within seconds. Site 1 was at least 10-fold less effective than site 2 as a substrate for all four phosphatases. In conjunction with information presented in the following paper in this issue of this journal, the results substantiate the hypothesis that PP-1 activity towards the glycogen-metabolising enzymes is regulated in vivo by reversible phosphorylation of a targetting subunit (G) that directs the C subunit to glycogen--protein particles. The efficient dephosphorylation of site 2 by the Ca2+/calmodulin-stimulated protein phosphatase (2B) provides a potential mechanism for regulating PP-1 activity in response to Ca2+, and represents an example of a protein phosphatase cascade.     

Identification of a glycogen synthase phosphatase from yeast Saccharomyces cerevisiae as protein phosphatase 2A.

A glycogen synthase phosphatase was purified from the yeast Saccharomyces cerevisiae. The purified yeast phosphatase displayed one major protein band which coincided with phosphatase activity on nondenaturing polyacrylamide gel electrophoresis. This phosphatase had a molecular mass of about 160,000 Da determined by gel filtration and was comprised of three subunits, termed A, B, and C. The subunit molecular weights estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 60,000 (A), 53,000 (B), and 37,000 (C), indicating that this yeast glycogen synthase phosphatase is a heterotrimer. On ethanol treatment, the enzyme was dissociated to an active species with a molecular weight of 37,000 estimated by gel filtration. The yeast phosphatase dephosphorylated yeast glycogen synthase, rabbit muscle glycogen phosphorylase, casein, and the alpha subunit of rabbit muscle phosphorylase kinase, was not sensitive to heat-stable protein phosphatase inhibitor 2, and was inhibited 90% by 1 nM okadaic acid. Dephosphorylation of glycogen synthase, phosphorylase, and phosphorylase kinase by this yeast enzyme could be stimulated by histone H1 and polylysines. Divalent cations (Mg2+ and Ca2+) and chelators (EDTA and EGTA) had no effect on dephosphorylation of glycogen synthase or phosphorylase while Mn2+ stimulated enzyme activity by approximately 50%. The specific activity and kinetics for phosphorylase resembled those of mammalian phosphatase 2A. An antibody against a synthetic peptide corresponding to the carboxyl terminus of the catalytic subunit of rabbit skeletal muscle protein phosphatase 2A reacted with subunit C of purified yeast phosphatase on immunoblots, whereas the analogous peptide antibody against phosphatase 1 did not. These data show that this yeast glycogen synthase phosphatase has structural and catalytic similarity to protein phosphatase 2A found in mammalian tissues.     

Phosphorylase phosphatase complex from skeletal muscle. Activation of one of two catalytic subunits by manganese ions

Sarcoplasmic phosphorylase phosphatase extracted from ground skeletal muscle was recovered in a high molecular weight from (Mr = 250000). This enzyme has been purified from extracts by anion-exchange and gel chromatography to yield a preparation with three major protein components of Mr 83000, 72000, and 32000 by sodium dodecyl sulfate gel electrophoresis. The phosphorylase phosphatase activity of the complex form was activated more than 10-fold by Mn2+, with a K0.5 of 10(-5) M, but not by Mg2+ or Ca2+. Manganese activation occurred over a period of several minutes and resulted primarily in an increase in Vmax of a phosphatase that was sensitive to trypsin. Activation persisted after gel filtration, and the active form of the enzyme did not contain bound manganese measured by using 54Mn2+. A contaminating p-nitrophenylphosphatase was activated by either Mn2+ (K0.5 of 10(-4) M) or Mg2+ (K0.5 of 10(-3) M). Unlike the protein phosphatase this enzyme was inactive following removal of the metal ions by gel filtration. The phosphatase complex could be dissociated into its component subunits by precipitation with 50% acetone at 20 degrees C in the presence of an inert divalent cation, reducing agent, and bovine serum albumin. Two catalytic subunits were quantitatively recovered; one of Mr 83000 was a trypsin-sensitive manganese-activated phosphatase and the second of Mr 32000 was trypsin-stable and metal ion dependent. Both enzymes were effective in catalyzing the dephosphorylation of either phosphorylase a or the regulatory subunit of adenosine cyclic 3',5'-phosphate (cAMP) dependent protein kinase, but neither subunit possessed p-nitrophenylphosphatase activity.     

Kinase FA-mediated regulation of rabbit skeletal muscle protein phosphatase. Reversible phosphorylation of the modulator subunit

A mechanism of activation of the ATP.Mg-dependent protein phosphatase (FC.M) has been proposed (Jurgensen, S., Shacter, E., Huang, C. Y., Chock, P. B., Yang, S.-D., Vandenheede, J. R., and Merlevede, W. (1984) J. Biol. Chem. 259, 5864-5870) in which a transient phosphorylation by the kinase FA of the modulator subunit (M) is the driving force for the transition of the inactive catalytic subunit (FC) into its active conformation. Incubation of FC.M with kinase FA and Mg2+ and adenosine 5'-(gamma-thio)triphosphate results in thiophosphorylation of M and also a conformational change in the phosphatase catalytic subunit; however, the enzyme remains inactive. Proteolysis of this inactive, thiophosphorylated complex causes proteolytic destruction of the modulator subunit and yields an active phosphorylase phosphatase species. Similar treatment of the native inactive enzyme does not yield active phosphatase. Evidence is presented, suggesting that a molecule of modulator is bound at an "inhibitory site" on the native enzyme. This modulator does not prevent the conformational change in the phosphatase catalytic subunit upon incubation with kinase FA and ATP.Mg but does partially inhibit the expression of the phosphorylase phosphatase activity.     

The protein phosphatases involved in cellular regulation. Evidence that dephosphorylation of glycogen phosphorylase and glycogen synthase in the glycogen and microsomal fractions of rat liver are catalysed by the same enzyme: protein phosphatase-1

Glycogen synthase (labelled in sites-3) and glycogen phosphorylase from rabbit skeletal muscle were used as substrates to investigate the nature of the protein phosphatases that act on these proteins in the glycogen and microsomal fractions of rat liver. Under the assay conditions employed, glycogen synthase phosphatase and phosphorylase phosphatase activities in both subcellular fractions could be inhibited 80-90% by inhibitor-1 or inhibitor-2, and the concentrations required for half-maximal inhibition were similar. Glycogen synthase phosphatase and phosphorylase phosphatase activities coeluted from Sephadex G-100 as broad peaks, stretching from the void volume to an apparent molecular mass of about 50 kDa. Incubation with trypsin decreased the apparent molecular mass of both activities to about 35 kDa, and decreased their I50 for inhibitors-1 and -2 in an identical manner. After tryptic digestion, the I50 values for inhibitors-1 and -2 were very similar to those of the catalytic subunit of protein phosphatase-1 from rabbit skeletal muscle. The glycogen and microsomal fractions of rat liver dephosphorylated the beta-subunit of phosphorylase kinase much faster than the alpha-subunit and dephosphorylation of the beta-subunit was prevented by the same concentrations of inhibitor-1 and inhibitor-2 that were required to inhibit the dephosphorylation of phosphorylase. The same experiments performed with the glycogen plus microsomal fraction from rabbit skeletal muscle revealed that the properties of glycogen synthase phosphatase and phosphorylase phosphatase were very similar to the corresponding activities in the hepatic glycogen fraction, except that the two activities coeluted as sharp peaks near the void volume of Sephadex G-100 (before tryptic digestion). Tryptic digestion of the hepatic glycogen and microsomal fractions increased phosphorylase phosphatase about threefold, but decreased glycogen synthase phosphatase activity. Similar results were obtained with the glycogen plus microsomal fraction from rabbit skeletal muscle or the glycogen-bound form of protein phosphatase-1 purified to homogeneity from the same tissue. Therefore the divergent effects of trypsin on glycogen synthase phosphatase and phosphorylase phosphatase activities are an intrinsic property of protein phosphatase-1. It is concluded that the major protein phosphatase in both the glycogen and microsomal fractions of rat liver is a form of protein phosphatase-1, and that this enzyme accounts for virtually all the glycogen synthase phosphatase and phosphorylase phosphatase activity associated with these subcellular fractions.     

Fluorine compounds inhibit the conversion of active type-1 protein phosphatases into the ATPMg-dependent form.

1. The modulator protein slowly converts the glycogen-bound protein phosphatase from liver, as well as its catalytic subunit, into an inactive form that requires protein kinase FA and MgATP for reactivation. The inactivation process could be completely prevented by addition of either 0.3 mM-NaF or 0.3 mM-phenylmethanesulphonyl fluoride (PMSF). The effectiveness of the proteinase inhibitor was not due to production of free fluoride. With the catalytic subunit a half-maximal effect of either fluorine compound was obtained at 25-50 microM. 2. The inactivation process was instantaneously blocked by the addition of NaF or PMSF at any moment during the incubation of the catalytic subunit with modulator. This fluoride effect was reversible. It did not result from a decreased affinity of modulator for the catalytic subunit. The use of analogues of PMSF showed that the fluorine atom was essential, but structural aspects were also an important determinant. 3. The relative efficiency of fluorine compounds in preventing the inactivation of the catalytic subunit by modulator corresponded to their relative potency as inhibitors of the phosphorylase phosphatase activity, but the latter effect required at least 20-fold higher effector concentrations. Incubation of the catalytic subunit with 10 mM-PMSF or -NaF caused an irreversible inhibition of the enzyme. 4. It is possible to prepare stable complexes of catalytic subunit and modulator, either active or ATPMg-dependent. Both species displayed the same molecular size during gel filtration. The inactive complex could be reactivated by incubation with MgATP and protein kinase FA. NaF and PMSF increased the final extent of re-activation at limiting concentrations of the protein kinase.     

Catalytic subunit of the polycation-stimulated protein phosphatase. Effect of proteolysis on polycation stimulation

The phosphorylase phosphatase activity of the holoenzyme form of phosphatase 2A isolated from extracts of porcine renal cortex or bovine heart was stimulated 600% and 500%, respectively, by the addition of histone H1. After conversion of the phosphatase to the catalytic subunit form by treatment with ethanol at room temperature, histone H1 stimulated activity by about 150% only. Purification of the catalytic subunit from porcine renal cortex yielded two forms of the enzyme which were separated by heparin-Sepharose chromatography. These forms were designated peak 1 and peak 2 according to their order of elution from the column. Peak 1 catalytic subunit was stimulated by more than 400% by histone H1, whereas peak 2 was stimulated by about 50% only. Based on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, peak 2 had a slightly higher Mr value than peak 1 (35,500 vs. 35,000). Incubation of the peak 2 phosphatase with trypsin converted it to a form that was similar to peak 1 with respect to Mr and stimulation by histone H1. When the catalytic subunit of phosphatase 2A was purified from bovine heart only one form was obtained. Bovine heart enzyme was similar to renal peak 2 in that it had an apparent Mr of 35,500 and was only slightly stimulated by histone H1. Treatment of the bovine heart catalytic subunit with trypsin, chymotrypsin or type 2 Ca2+-dependent proteinase decreased the apparent Mr by about 500 and increased histone H1 stimulation to about 500%. Thus, when a small peptide was removed by proteolysis, histone H1 stimulation of the 'nicked' catalytic subunit was similar to that obtained with the holoenzyme.     

Phosphorylation of the glycogen-binding subunit of protein phosphatase-1G by cyclic-AMP-dependent protein kinase promotes translocation of the phosphatase from glycogen to cytosol in rabbit skeletal muscle

The glycogen-bound form of protein phosphatase-1 (termed protein phosphatase-1G) is composed of the catalytic (C) subunit complexed to a glycogen-binding (G) subunit that anchors the enzyme to glycogen [Str?lfors et al. (1985) Eur. J. Biochem. 149, 295-303]. Incubation of purified protein phosphatase-1G with cyclic-AMP-dependent protein kinase and MgATP, which leads to stoichiometric phosphorylation of the G-subunit [Caudwell et al. (1986) FEBS Lett. 194, 85-90], was found to promote the release of the phosphatase from glycogen; similar observations were made using glycogen-protein particle preparations. An intravenous injection of adrenaline decreased protein phosphatase-1 activity associated with the glycogen-protein particles by 50% with a corresponding increase in the amount present in the cytosol. By contrast, adrenaline did not affect the distribution of glycogen synthase or glycogen phosphorylase which remained entirely bound to glycogen in these experiments. The specific release of protein phosphatase-1 from glycogen may facilitate its inactivation by inhibitor-1 in the cytosol, thereby preventing dephosphorylation of the glycogen metabolising enzymes. Translocation of protein phosphatase-1 may represent a novel mechanism for the activation of glycogenolysis and inhibition of glycogen synthesis by adrenaline.     

Phosphorylase phosphatase activities of rat liver in streptozotocin-diabetes

Protein phosphatase-1 and 2A, accounting for all the hepatic activity regulating phosphorylase, were assayed in streptozotocin-induced (8 weeks) diabetic Wistar rats. Cytosolic protein phosphatase-1 and 2A were distinguished by chromatography on heparin-Sepharose and by inhibition with inhibitor-2. Approx. 25-35% increases in type-1 phosphorylase phosphatase activity measured in cytosols were registered in diabetic rats when compared with control and 24 h fasting animals. The enrichment of protein phosphatase-1 in the cytosol of streptozotocin-treated rat livers could not be attributed to the reduced glycogen content with the onset of diabetes, since this elevated level of type-1 phosphatase was not observed in fasting rats with low glycogen content. The translocation of type-1 phosphatase from the particulate fraction into the cytosol was also recorded in trypsin-treated samples of diabetic rat livers. The apparent molecular weight of type-1 phosphatase in the cytosol of control and fasted rats was 160,000 as judged by gel filtration. The type-1 phosphatase activity that was released from the particulate fraction by streptozotocin-induced diabetes identified a further enzyme species (Mr 110,000) in the cytosol. Our data imply that the higher levels of cytosolic protein phosphatase-1 in diabetic rat liver could be a consequence of the dissociation of the catalytic subunit of protein phosphatase-1 and the glycogen-binding subunit in rat livers.     

Regulation of protein phosphatase-1G from rabbit skeletal muscle. 2. Catalytic subunit translocation is a mechanism for reversible inhibition of activity toward glycogen-bound substrates

The glycogen-associated form of protein phosphatase-1 (PP-1G) comprises a 37-kDa catalytic (C) subunit and a 161-kDa glycogen-binding (G) subunit. In the preceding paper in this issue of the journal we showed that the C subunit is released from PP-1G in response to phosphorylation of the G subunit by cAMP-dependent protein kinase. We now show that at 0.15-0.2 M KCl the phosphorylase phosphatase activity of glycogen-bound PP-1G is 5-8 times higher than that of released C subunit or unbound PP-1G, which are strongly inhibited at these ionic strengths. The activity of glycogen-bound PP-1G towards glycogen synthase was about 5-fold higher than that of released C subunit at 0.15M KCl. Studies with glycogen-bound substrates and myosin P-light chain (which does not interact with glycogen) indicated that PP-1G activity is only enhanced compared to free C subunit at near physiological ionic strength and when both PP-1G and substrate are glycogen-associated. The inhibition by increasing ionic strength and enhanced activity upon binding to glycogen reflected changes in K'm, but not Vmax. From the determined specificity constant, k'cat/K'm approximately 4 x 10(6) s-1 M-1, it was calculated that at physiological levels of glycogen-bound PP-1G (200 nM) and phosphorylase (70 microM), dephosphorylation of the latter could occur with a half time of 15 s, sufficient to account for inactivation rates in vivo. The much higher catalytic efficiency of glycogen-bound PP-1G toward the glycogen-metabolising enzymes at physiological ionic strength compared to free C subunit substantiates the role of PP-1G in the regulation of these substrates, and establishes a novel mechanism for selectively regulating their phosphorylation states in response to adrenalin and other factors affecting phosphorylation of the G subunit.     

Purification and partial characterization of bovine heart phosphorylase kinase

Bovine heart phosphorylase kinase has been isolated by a procedure involving precipitation with polyethylene glycol, DEAE-Sephacel chromatography and calmodulin-Sepharose affinity chromatography. The isolated enzyme had a specific activity of 8.3 IU/mg of protein at pH 8.2 at 30 degrees C in the presence of 1% glycogen. The native enzyme had a sedimentation coefficient of 23 S and the Mr of the alpha', beta, gamma, and delta subunits, were 140,000, 130,000, 46,000, and 18,000, respectively. Activation of the phosphorylase kinase by the catalytic subunit of bovine heart cAMP-dependent protein kinase increases the pH 6.8/8.2 activity ratio from 0.01 to 0.32-0.38. Glycogen (1%) decreased the Km of the activated phosphorylase kinase at pH 6.8 for phosphorylase b from 5.5 to 1.25 mg/ml. Trypsin treatment increased the pH 6.8 activity but decreased the pH 8.2 activity. During this process the alpha' subunit was converted to a Mr 110,000 polypeptide and the enzyme activity was converted essentially to a 5.9 S species having an apparent Mr of 100,000 as determined by gel filtration. On extended trypsin treatment only one major polypeptide corresponding to the beta subunit remained. The same polypeptide was present in the active fractions following gel filtration of the trypsinized kinase.     

Purification and characterization of the polycation-stimulated protein phosphatase catalytic subunit from porcine renal cortex

The predominant form of phosphorylase phosphatase activity in porcine renal cortical extracts was a polycation-stimulated protein phosphatase. This activity was present in extracts in a high-molecular-weight form which could be converted to a free catalytic subunit by treatment with ethanol, urea, or freezing and thawing in the presence of beta-mercaptoethanol. The catalytic subunit of the polycation-stimulated phosphatase was purified by chromatography on DEAE-Sephacel, heparin-Sepharose, and Sephadex G-75. The phosphatase appeared to be homogeneous on SDS-polyacrylamide gel electrophoresis. The enzyme had an apparent Mr of 35 000 on gel filtration and SDS-polyacrylamide gel electrophoresis. The purified phosphatase could be stimulated by histone H1, protamine, poly(D-lysine), poly(L-lysine) or polybrene utilizing phosphorylase a as the substrate. It preferentially dephosphorylated the alpha-subunit of phosphorylase kinase. The phosphatase was highly sensitive to inhibition by ATP. These results suggest that the renal polycation-stimulated phosphatase catalytic subunit is very similar to or identical with the skeletal muscle phosphatase form which has been previously designated phosphatase-2Ac.     

The catalytic subunits of protein phosphatase-1 and protein phosphatase 2A are distinct gene products

Three forms of protein phosphatase-1 were isolated from rabbit skeletal muscle that had Mr values of 37 000, 34 000 and 33 000 determined by sodium dodecyl sulphate (SDS) gel electrophoresis. Each species dephosphorylated the beta-subunit of phosphorylase kinase very much faster than the alpha-subunit, was inhibited by inhibitors 1 and 2 with equal potency, and was converted to a form dependent on glycogen synthase kinase-3 and Mg-ATP for activity by incubation with inhibitor-2. Digestion with cyanogen bromide or Staphylococcus aureus proteinase followed by SDS gel electrophoresis showed a very similar pattern of cleavage products for all three forms. The Mr-37 000 and Mr-34 000 species were converted to the Mr-33 000 form by incubation with chymotrypsin. It is concluded that the Mr-33 000 and Mr-34 000 forms are derived from the Mr-37 000 component by limited proteolysis. Conversion of the Mr-37 000 to the Mr-33 000 form was accompanied by a two-fold increase in activity, indicating that an Mr-4000 fragment at one end of the polypeptide is an inhibitory domain that decreases enzyme activity. The catalytic subunit of protein phosphatase 2A from rabbit skeletal muscle had an Mr of 36 000 determined by SDS gel electrophoresis and its specific activity (3 kU/mg) was much lower than that of the Mr-37 000 (15-20 kU/mg) or Mr-33/34 000 (40-50 kU/mg) forms of protein phosphatase-1. It dephosphorylated the alpha-subunit of phosphorylase kinase 4-5-fold faster than the beta-subunit, was unaffected by inhibitor-1 or inhibitor-2, and preincubation with the latter protein did not result in the production of a glycogen synthase kinase-3 and Mg-ATP-dependent form of the enzyme. Digestion with chymotrypsin did not alter the electrophoretic mobility of protein phosphatase 2A under conditions that caused quantitative conversion of the Mr-37 000 form of protein phosphatase-1 to the Mr-33 000 species. Digestion with cyanogen bromide or S. aureus proteinase, followed by SDS gel electrophoresis, showed a quite different pattern of cleavage products to those observed with protein phosphatase 1. Antibody to protein phosphatase-2A raised in sheep did not cross-react with any of the forms of protein phosphatase-1, as judged by immunoelectrophoretic and immunotitration experiments. It is concluded that protein phosphatase-1 and protein phosphatase-2A are distinct gene products.