Liver glycogen synthase phosphatase and phosphorylase phosphatase activities in vitro following glucose and glucagon administration

Correlation of the changes in phosphorylase a concentration with the synthase phosphatase velocity in a glycogen particle preparation in the presence of EDTA revealed that the initial synthase phosphatase rate was greatest in extracts from glucose-treated rats and least in extracts from glucagon-treated rats. In all cases the velocity increased with time and with a decrease in phosphorylase a. However, a threshold release of phosphatase activity when phosphorylase a reached a critical level was not observed. The data are compatible with either an independent regulation of synthase phosphatase by glucose and glucagon or regulation of the activity by phosphorylase over a range of phosphorylase a concentrations.     

Reversible inhibition of skeletal muscle phosphoprotein phosphatase by ATP, phosphate and fluoride.

A phosphoprotein phosphatase preparation which showed activity towards glycogen synthase, phosphorylase, phosphorylase kinase, and phosphohistones was reversibly inhibited (70–90%) by preincubation with free ATP (apparent K_i about 0.3 mM). Other nucleotides (ADP2 (apparent K_a 3μM) prior to assay. Other divalent metals (Co⁺⁺ > Zn⁺⁺ > Mg⁺⁺) were partially effective in reversing the inhibition. It is concluded that ATP by virtue of its special structure and metal binding capacity possibly removes a catalytically important metal ion from the enzyme.     

Stimulation of liver glycogen particle synthase D phosphatase activity by caffeine, AMP, and glucose 6-phosphate

Caffeine stimulates synthase and phosphorylase phosphatase activities by independent mechanisms. Both effects of caffeine are concentration-dependent with different apparent A_0.5 for each reaction. Stimulation of the synthase phosphatase reaction was independent of the initial phosphorylase a concentration, was immediate, and did not follow in sequence the depletion of phosphorylase a. Glucose 6-phosphate also was stimulatory to the synthase phosphatase reaction (A_0.5 = 0.14 mM) with little effect on phosphorylase phosphatase. In combination glucose 6-phosphate and caffeine effects were additive suggesting the existence of separate binding sites. The synthase phosphatase reaction also was stimulated by AMP (binding affinity 2.3 mm) but with no effect on phosphorylase phosphatase activity. Together caffeine and AMP effects were not additive suggesting that they share a common binding site or closely interrelated sites. The location of the AMP and caffeine site(s) has not yet been determined.     

Rat liver glycogen metabolism in the perinatal period

The correlation between blood glucose levels, the concentration of glycogen, the activities of glycogen sythase and phosphorylase and their respective kinases and phosphatases was examined in liver of rat fetuses between day 18 of gestation and one day after birth. Between day 18 and 21 there is a rapid increase in the concentration of glycogen and in the activity of synthase a and a much slower increase in the activity of phosphorylase a. The activity of the respective kinases increased rapidly during this period and reached maximun on day 21. The activity of synthase phosphatase and phosphorylase phosphatase increased after day 18, to reach a maximum on day 19 and 20, respectively, but decreased again towards day 21. The possibility that the changes in glycogen concentration and enzyme activities were related to an effect of glucose of AMP on the respective phosphatases was considered. It was found that the Km of phosphatase for glucose in the prenatal period was 5–7 mM, as in the adult. Since the level of blood glucose during this period was constant (2.8 mM), an effect of glucose on phosphatase activity seems unlikely. AMP concentration increased between day 18 and 21 from 6–15 nmol/g. In view of the low level of phosphorylase a activity during this period, the increase in AMP concentration is not considered to be important in the regulation of glycogen breakdown at this time.Immediately after birth blood glucose levels dropped to 5 mg/dl. This was accompanied by a rapid decrease in glycogen concentration and in the activity of glycogen synthase and a rise in phosphorylase activity. Blood glucose levels returned to the initial level within 1 h after birth, whereas the changes in glycogen concentration and enzyme activities continued for at least 3 h after birth. On day 22 all parameters examined had reached the level found in adult rat liver.It is suggested that the rapid changes observed immediately after birth are due to an effect of hypoglycemia mediated by hormones and cannot be ascribed to direct effects of metabolites on the enzyme systems involved.     

A protein inhibitor of rabbit liver phosphorylase phosphatase.

Extracts of rabbit liver contain a heat-stable, non-dialysable inhibitor of phosphorylase phosphatase. The inhibitory activity is destroyed by trypsin treatment or by ethanol precipitation. The kinetics of the inhibition are non-competitive with respect to phosphorylase a. The inhibitory activity is polydisperse on gel permeation chromatography. The mechanism of the inhibition is due to a direct interaction of the protein inhibitor with the enzyme.     

Adenosine 5'-O(3-thiotriphosphate) in the control of phosphorylase activity

Rabbit muscle phosphorylase b (EC 2.4.1.1) is converted to a thio-analog of phosphorylase a by phosphorylase kinase, Mg²⁺ and adenosine 5′-O(3-thiotriphosphate)(ATPγS). Conversion proceeds at one-fifth the rate obtained with ATP though the extent of reaction and final level of activation of the enzyme are the same. However, the thiophosphorylase a produced is resistant to phosphorylase phosphatase and, therefore, behaves as a competitive inhibitor with a K_I of 3 μM, similar to the K_M obtained with normal phosphorylase a. ATPγS can also be utilized by protein kinase in the activation of phosphorylase kinase at a rate similar to that obtained with ATP. It is hydrolyzed at 5 to 10 times the normal rate by the sarcoplasmic reticulum ATPase. When added to a muscle glycogen-particulate complex in the presence of Ca²⁺ and Mg²⁺, ATPγS triggers an activation of phosphorylase with simultaneous inhibition of phosphorylase phosphatase as previously observed with ATP.     

Purification and properties of bovine myocardial phosphorylase phosphatase (protein phosphatase C).

Phosphorylase phosphatase was purified to homogeneity from bovine myocardium by the procedure of Brandt et al. (Brandt, H., Capulong, Z. L., and Lee, E. Y. C., (1975) J. Biol. Chem.250, 8038–8044). The purified enzyme consists of a single polypeptide chain of M_r, 34,800 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The K_m for phosphorylase a was 2.9 μm and at V_max, the enzyme had a turnover number of 11.7 mol phosphorylase a (dimer) converted/mol phosphatase/s. Phosphorylated histone and protamine were also substrates for this phosphatase. The K_m for histone was 46 μm (based on incorporated ³²P_i and at V_max a turnover number of 3.3 mol phosphate released/mol phosphatase/s was found. In general, the properties of the bovine phosphorylase phosphatase closely resemble those found for the rabbit liver enzyme.     

Glycogen phosphorylase from flight muscle of the hawk moth,Manduca sexta: purification and properties of three interconvertible forms and the effect of flight on their interconversion

Glycogen phosphorylase (EC 2.4.1.1) of Manduca sexta flight muscle was separated into three distinct peaks of activity on diethylaminoethyl-Sephacel. The three fractions of phosphorylase activity were further purified by affinity chromatography on AMP-Sepharose and shown to have the same relative molecular mass (=178000) on polyacrylamide gradient gel electrophoresis under non-denaturating conditions and to produce subunits of molecular mass =92000 on SDS gelelectrophoresis. On the basis of their kinetic properties with respect to the activator AMP and the inhibitor caffeine, the three fractions of phosphorylase activity were assigned as follows: peak 1=phosphorylase b (unphosphorylated form), peak 3=phosphorylase a (phosphorylated form); peak 2 represented a phospho-dephospho hybrid in which only one subunit of the dimeric enzyme was phosphorylated. This hypothesis was corroborated as the various forms could be interconverted in vitro by either dephosphorylation by an endogenous protein phosphatase producing the b form, or by phosphorylation catalyzed by purified phosphorylase kinase from rabbit muscle producing phosphorylase ab and a. From muscle of resting moths more phosphorylase was isolated in the b form (41%) than in the forms ab (28%) and a (31%), respectively. This proportion was changed in favour of the fully phosphorylated a form after a brief interval of flight when 68% of the phosphorylase activity was represented by the a form and only 13% by the b form. Unlike the phosphorylated forms a and ab of phosphorylase, the b form had low affinities for the substrates and for the activator AMP, and was virtually inactive if near-physiological concentrations of substrates and effectors were employed in the assays. The results demonstrate that in Manduca flight muscle three forms of phosphorylase coexist and that their interconversion is a mechanism for regulating phosphorylase activity in vivo.Abbreviations DEAE diethylaminoethyl - EDTA ethylenediamine tetraacetate - EGTA ethyleneglycol-bis(-aminoethylether)N,N-tetra-acetic acid - M _r relative molecular mass - NMR nuclear magnetic resonance - PAGGE polyacrylamide gradient gel electrophoresis - P_i morganic phosphate - SDS sodium dodecylsulphate - TRIS tris(hydroxymethyl)-aminomethane - V _max maximum activity     

Histochemical studies on phosphorylase activity in the tissues of the albino rat under normal and experimental conditions

Summary Various substrate mixtures have been tested for the histochemical demonstration of phosphorylase in tissue blocks. These studies indicate that phosphorylase activity, cytological detail and localization of the reaction product are optimally demonstrated when tissue blocks are incubated for one hour or longer in a medium containing high concentrations of G-1-P, ATP and PVP.Abbreviations AMP adenosine-5-monophosphate - ATP adenosine-5-triphosphate - EDTA ethylenediamine-tetraacetic acid - G-1-P glucose-1-phosphate - PPa active phosphorylase - PPb inactive phosphorylase - PVP polyvinyl pyrrolidone     

Regulatory properties of rabbit liver phosphorylase kinase

• 1.1. Purified native rabbit liver phosphorylase kinase becomes activated during the assay of its activity while low molecular weight forms of the same enzyme do not.
• 2.2. The activation requires ATP and maganesium ions, suggesting the phosphorylation of the enzyme by a protein kinase as the mechanism involved.
• 3.3. The activation of the enzyme can be reverted by the action of a type 1 protein phosphatase isolated from the same tissue.
• 4.4. The activation can also be catalyzed by the catalytic subunit of cAMP-dependent protein kinase in a process that requires a much lower ATP concentration to proceed.
• 5.5. The activation is believed to be due to an autocatalytic phosphorylation of phosphorylase kinase itself. In support of this hypothesis are the regulation of the process through calcium ions, the low levels of endogenous protein kinase detected in the purified preparation, the high ATP concentrations required in the absence of cAMP dependent protein kinase and the fact that the process cannot be blocked by an excess of the heat stable inhibitor specific for the later enzyme.
• 6.6. The low molecular weight forms of the enzyme on their side are not affected by the action of neither protein phosphatase 1 nor cyclic AMP dependent protein kinase.
• 7.7. Both activated and nonactivated phosphorylase kinase are partially dependent on calcium ions, the affinity of the former being higher than that of the latter. The low molecular forms do not require calcium ions to express their activity.

     

Lack of Types 1 and 2A Protein Serine(P)/Threonine(P) Phosphatase Activities in Chloroplasts

Protein phosphatase activity in crude leaf extracts and in purified intact chloroplasts of wheat (Triticum aestivum) and pea (Pisum sativum) was analyzed using exogenously supplied phosphoproteins or endogenous thylakoid proteins. Leaf extracts contain readily detectable amounts of protein phosphatase activity measured with either phosphohistone or phosphorylase a, substrates of mammalian protein phosphatases. No significant chloroplast protein phosphatase activity was detected using these exogenous phosphoproteins. The dephosphorylation of endogenous thylakoid light-harvesting chlorophyll a/b binding proteins in situ was inhibited by fluoride, but not by microcystin-LR or okadaic acid, diagnostic inhibitors of mammalian types 1 and 2A protein phosphatases. Additionally, no evidence for a pea chloroplast alkaline phosphatase activity was found using β-glycerolphosphate or 4-methylum-belliferyl phosphate as substrates. From these results, we conclude that phosphohistone and phosphorylase a are not useful substrates for chloroplast thylakoid protein phosphatase activity and that the chloroplast enzymes may not fit into one of the canonical classifications currently used for protein phosphatases.     

Dephosphorylation of skeletal muscle phosphorylase,glycogen synthase,and phosphorylase kinase β-subunit by a Mn2+-activated protein phosphatase

An Mn²⁺-activated phosphoprotein phosphatase of M_r = 80,000 from rabbit muscle catalyzes the dephosphorylation of skeletal muscle proteins that are phosphorylated by either phosphorylase kinase or cAMP-dependent protein kinase. Phosphorylase or glycogen synthase labeled by phosphorylase kinase at seryl residues 14 or 7, respectively, are both dephosphorylated by the phosphatase. Phosphorylase a and glycogen synthase compete with one another for the phosphatase. The phosphatase discriminates between different sites labeled by the cAMP-dependent protein kinase: glycogen synthase phosphorylated either to 1.0 or 1.8 mol phosphate/mol, or phosphorylase kinase phosphorylated on its β-subunit serve as substrates for the phosphatase, but the phosphorylase kinase α-subunit, the phosphorylated phosphatase inhibitor 1, or casein do not. Histone fraction IIA, phosphorylated by the catalytic subunit, was a poor substrate even at a concentration of 100 μm. Phosphorylation of the α-subunit of phosphorylase kinase had no influence on the kinetics of dephosphorylation of the β-subunit. Thus, the M_r = 80,000 phosphatase meets the functional definition of a protein phosphatase 1 [Cohen, P. (1978) Curr. Top. Cell. Regul.14, 117–196]. Furthermore, from a comparison of the known phosphorylated sites of these proteins, it appears that the phosphatase discriminates between different sites present in the phosphoproteins tested on the basis of the K_m values for the reactions. It displays a preferential activity toward proteins with a primary structure wherein basic residues are two positions amino-terminal from the phosphoserine, AgrLysX-YSer(P) or LysArgX-YSer(P), rather and one residue away, ArgArgX-Ser(P).     

Liver phosphorylase phosphatase

Summary Recent progress in studies on the properties and regulation of liver phosphorylase phosphatase can be divided into four stages. First, isolation from multiple molecular forms of phosphorylase phosphatase, of a single form of catalytic subunit (Mr = 32 000-35 000) which is active toward phosphorylase a and also toward a variety of protein substrates phosphorylated by either cyclic AMP-dependent protein kinase or other protein kinases. This was achieved by rather drastic procedures such as treatment with 80% ethanol at room temperature, incubation with 6 M urea, freeze-thawing in the presence of 0.2 M mercaptoethanol, or digestion by trypsin. These treatments caused concomitantly large enhancement of phosphorylase phosphatase activity, and the hypothesis was proposed that an inactive form of phosphorylase phosphatase existed as complexes of a catalytic subunit and inhibitory proteins. Second, it was discovered that liver and muscle extracts contain trypsin-labile proteins which, after heating at 90 °C, inhibited the catalytic subunit of phosphorylase phosphatase. Two types of protein inhibitors were identified: inhibitor-I was phosphorylated and activated by cyclic AMP-dependent protein kinase, whereas inhibitor-2 was not phosphorylated. Although inhibitor-1 has been implicated in hormonal regulation of glycogen metabolism in skeletal muscle, a similar role of protein inhibitors in the regulation of phosphorylase phosphatase in the liver has not been demonstrated and the physiological role of the inhibitor is questionable.Third, it has been demonstrated that liver phosphorylase phosphatase is reversibly inactivated and regulated by glutathione disulfide (GSSG) at the catalytic subunit level. Liver phosphorylase phosphatase contains, per mole of catalytic subunit, two sulfhydryl groups, one of which reacts with GSSG to form mixed disulfide with consequent inactivation of the enzyme. The inactivated enzyme can be reactivated by glutathione(GSH) or other sulfhydryl compounds through a reverse reaction. Injection of GSSG into the portal vein of rabbits caused a rapid increase in phosphorylase-a activity in the liver, suggesting that GSSG is involved in regulation of phosphorylase activity in vivo.Finally, current evidence suggests that liver phosphorylase phosphatase exists in the native state in a high molecular weight form which consists of the catalytic subunit and other regulatory subunits. One such enzyme species could be a 260 000-dalton protein composed of three different types of subunit, termed , and , or a 160 000-dalton protein composed of and subunits. The a subunit (Mr = 35 000) appears to be identical to the multifunctional catalytic subunit, whereas the (Mr = 69 000) and (Mr = 58 000) subunits are catalytically inactive but can modify the catalytic a subunit. It seems likely that the substrate specificity and catalytic activity of the subunit is considerably altered when it is part of larger complexes with other regulatory subunits ( and ). It has also been suggested that in addition to the native form of phosphorylase phosphatase, liver contains a considerably large amount of latent phosphorylase phosphatase, the catalytic activity of which could be revealed only by treatment with trypsin or ethanol.     

Inactivation and reactivation of phosphoprotein phosphatase

Summary The catalytic subunit of phosphoprotein phosphatase (Mr = 35 000) is inactivated by phosphate compounds such as trimetaphosphate, PPi, and ATP. The inactivation of phosphoprotein phosphatase by these phosphate compounds is time- and concentration-dependent, is not reversed by dilution or gel filtration and is protected by Pi. A dissociation constant for the enzyme-trimetaphosphate complex and a rate constant for the reaction were calculated to be 4.6 × 10^–4 M and 0.29 min ¹, respectively. The inactivation of phosphatase by PPi and ATP shows more complex kinetics than that by trimetaphosphate. The addition of EDTA to PPi and ATP exhibits more potent inactivation, even though EDTA alone does not inactivate phosphatase. This phosphoprotein phosphatase is not labeled by [-³²P]ATP. The inactivation of phosphatase by PPi or ATP can only be reversed by Mn²⁺ or Co²⁺, among all other metals or cationic compounds tried. The reactivation also requires sulfhydryl compounds. The effectiveness of sulfhydryl compounds follows the order: dithioerythritol > mercaptoethanol > cysteine. Glutathione was without effect. Metal analysis of the catalytic subunit did not reveal any significant amounts of Ca, Cd, Co, Cu, Fe, Mg, Mn, Ni, Sn, or Zn. Phosphoprotein phosphatase activity from zinc-deficient rat livers also eliminated the possibility of this phosphatase being a zinc metalloenzyme. Inactivation does not seem to be due to a loss of a critical metal ion. Other mechanisms for inactivation are presented.Abbreviations used EDTA Ethylenediamine tetraacetic acid - PPi Inorganic pyrophosphate - DTE Dithioerythritol. To whom requests for reprints should be addressed.     

The roles of glucose and AMP in regulating the conversion of phosphorylase a into phosphorylase b

The ability of glucose to assist the action of phosphorylase a phosphatase on phosphorylase a, appears to be mediated through conformational changes in the phosphorylase. The system is an example of control of enzyme activity by regulation of the conformation of the substrate, and parallels the similar effect of AMP in inhibiting the dephosphorylation.     

Role of ADP in the control of actomyosin superprecipitation and ATPase activity.

ATP, in the presence of 0.05–0.15 m KCl and greater than 50 μm Mg²⁺, induces dissociation (clearing) followed by superprecipitation of skeletal muscle actomyosin. Superprecipitation has been studied as a model of muscle contraction, and ATP depletion has been associated with the onset of superprecipitation. Recent studies [Puszkin and Rubin (1975) Science188, 1319–1320] indicate that ADP stimulates superprecipitation without increasing the rate of ATP hydrolysis. We confirm that ADP stimulates superprecipitation; however, contrary to the experience of these investigators, ADP does stimulate ATP hydrolysis in the system studied here. We present evidence that superprecipitation is associated with generation of a critical ADP:ATP ratio but it appears that this ratio is an indirect measure of an associated but uncharacterized phenomenon which signals the onset of superprecipitation. Added ADP decreased the extent and duration of clearing, increased the rate of ATP hydrolysis, and increased the extent of superprecipitation of rat skeletal muscle actomyosin in the presence of excess Mg²⁺. The ADP effect was not mimicked by EDTA or AMP. The duration of clearing was related not to the time required to attain a specific level of any nucleotide phosphate, but to the time required to generate an ADP:ATP ratio of approximately 3.6. Apparently only that ADP generated in the system by ATP hydrolysis was involved in the critical ADP:ATP ratio. Added ADP stimulated myosin ATPase activity in 1.6 or 3.2 mm Mg²⁺. This effect was not mimicked by EDTA or AMP. The results are used to relate studies by others of myosin sulfhydryl modification to a recent model [Burke et al. (1973) Proc. Nat. Acad. Sci. USA70, 3793–3796] in which myosin MgATPase activity is inhibited by formation of a stable cyclic complex of MgATP and the S₁ and S₂ sites of heavy meromyosin.     

Chromatographic characteristics and subcellular localization of synthase phosphatase,phosphorylase phosphatase and histone phosphatase in human polymorphonuclear leukocytes

Summary Synthase phosphatase, phosphorylase phosphatase and histone phosphatase activity in a leukocyte homogenate were found to have different sedimentation charcteristics: both synthase phosphatase and phosphorylase phosphatase activity are associated with the microsomal fraction, while the majority of histone phosphatase activity (75–85%) was found in the cytosol. Synthase phosphatase, phosphorylase phosphatase and histone phosphatase activities accompanying the microsomal fraction are readily solubilized by 0.3% Triton X-100.When the solubilized microsomal enzymes were chromatographed on Sephadex G-200, the majority of synthase phosphatase, phosphorylase phosphatase and histone phosphatase activity migrated in single peaks corresponding to apparent molecular weights of 380 000, 250 000 and 68 000, respectively. A minor peak of 30 000, which had phosphatase activity against all three substrates was also obtained.Ethanol treatment resulted in solubilization and dissociation of the three phosphatase activities. It was found that although ethanol treatment resulted in a 4-fold increase of phosphorylase phosphatase activity, histone phosphatase activity was decreased (by 60%), while synthase phosphatase activity remained stable. Similar results were obtained when ethanol treatment was performed on the 17 000 × g supernatant.Chromatography of the ethanol-treated microsomes (or homogenate) on Sephadex G-200 showed that the phosphatase activity towards synthase D, phosphorylase a and phosphohistone coincided a M_r 30 000 species. Heat treatment of the M_r 30 000 peak resulted in dissociation of synthase phosphatase and phosphorylase phosphatase activity.Synthase phosphatase was inhibited by phosphorylase a in a kinetically non-competitive manner while histone phosphatase activity was notinhibited by synthase D (8.5 unit/ ml) orby phosphorylase a(12 unit/ ml).