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PubMed1985

Multiple forms of synthase D phosphatase and phosphorylase a phosphatase in liver and regulatory effects of metabolites on their activities.

The smooth endoplasmic reticulum (ER) and cytosol fractions of liver homogenates exhibit phosphoprotein phosphatase activity towards glycogen synthase D and phosphorylase a. The following observations suggest that liver contains multiple forms of these phosphatases. Synthase phosphatase activity in either fraction was more readily inactivated by heating than phosphorylase phosphatase activity. Both synthase phosphatase and phosphorylase phosphatase activities in smooth ER were non-competitively inhibited by Mg2+, but were activated by this ion in the cytosol. Synthase phosphatase activities in cytosol and smooth ER were stimulated by a number of sugar phosphates, particularly glucose-1-phosphate, galactose-6-phosphate and fructose-6-phosphate. Erythrose-4-phosphate stimulated synthase phosphatase activity in the cytosol, but inhibited the microsomal enzyme. Phosphorylase phosphatase activities in either fraction were inhibited by most sugar phosphates. Adenosine mono-, di- and tri-phosphates inhibited phosphatase activities in both fractions. Low concentrations of AMP and ADP inhibited phosphorylase phosphatase activities to a greater extent than synthase phosphatase activities. Chromatography of the smooth ER fraction on DEAE-cellulose resulted in the separation of synthase phosphatase from phosphorylase phosphatase, as soluble proteins. The elution profile for the microsomal phosphatase was different from that for the cytosol enzymes. It is concluded that: both synthase phosphatase and phosphorylase phosphatase in liver have at least two isoenzyme forms; synthase phosphatase and phosphorylase phosphatase are separate enzymes; the different behaviour of microsomal and cytosol phosphatases towards divalent cations and sugar phosphates provides a potential mechanism for the differential regulation of these activities in liver.

Adenine Nucleotides↗
PubMed1996

Genetic deficiencies of the glycogen phosphorylase system.

Several types of glycogen storage disease attributable to a deficiency of phosphorylase or phosphorylase kinase have been described. These diseases have been divided according to clinical symptoms, mode of inheritance, and affected tissue. However, this classification is questionable, as the clinical symptoms of these different diseases are similar, the mode of inheritance is often difficult to establish, and the biochemical assays are subject to several technical problems. A better classification would be based upon the identification of mutations in the respective disease genes. The molecular heterogeneity, however, is large, and at least 10 genes are involved. Mutations have been found in the muscle phosphorylase gene in patients with muscle phosphorylase deficiency, in the gene encoding the liver alpha subunit of phosphorylase kinase in patients with X-linked liver glycogenosis, and in the gene for the muscle alpha subunit of phosphorylase kinase in a patient with muscle phosphorylase kinase deficiency. We review here the different deficiencies of the phosphorylase system.

Genes, Recessive↗
PubMed1990

Activation of glycogen phosphorylase in rat pheochromocytoma PC12 cells and isolated hepatocytes by organophosphates.

Several organophosphates including diisopropylfluorophosphonate (DPF) and a variety of compounds used as chemical warfare agents produced dose- and time-dependent increases in phosphorylase-a, the phosphorylated form of glycogen phosphorylase in rat pheochromocytoma cells, PC12, and isolated hepatocytes. Increases in phosphorylase-a did not occur in cells exposed to the carbamates, physostigmine or pyridostigmine, or to O-ethyl S-2-diisopropylaminoethylmethyl-phosphonathiolate (VX), an organophosphate which is protonated at physiological pH. When extracellular pH was increased to pH 8, VX acted like the other organophosphates and increased phosphorylase-a activity. The possibility that organophosphates increase phosphorylase-a in intact cells by releasing Ca2+ from intracellular binding sites is supported by the following findings: organophosphate-induced increases in phosphorylase-a did not correlate with changes in cyclic AMP in the two cell types studied; in PC12 cells, increases in this activity occurred in the absence of extracellular calcium and were not inhibited by the calcium channel blocker, verapamil; fluorescence of the calcium sensitive dye, Quin-2, in PC12 cells preloaded with the acetoxymethyl ester of the dye was increased by soman; finally, addition of the calcium ionophore, A23187, to PC12 cells maintained in calcium-free medium caused sarin-stimulated phosphorylase-a activity to return rapidly to basal levels. Collectively, these data argue strongly that organophosphates increase phosphorylase-a activity in intact cells via a novel mechanism involving release of calcium from intracellular binding sites.

Adenine Nucleotides↗
PubMed1983

Limited proteolysis of glycogen phosphorylase a by subtilisin BPN'.

The limited proteolysis of rabbit skeletal muscle phosphorylase a was undertaken with subtilisin BPN' immobilized to Sepharose 4B. The effect of substrates, activators and inhibitors of phosphorylase a was investigated by monitoring the changes in phosphorylase activity in the SDS gel electrophoretic pattern and in the 32P-content of 32P-labeled phosphorylase a. Phosphorylase a loses its activity upon subtilisin treatment. All ligands tested protect phosphorylase a activity against subtilisin action, probably by inducing structural changes in the tower loop of the enzyme. Glucose-6-P significantly accelerates [32P]peptide release from phosphorylase a through altering the structure of the N-terminal tail segment. The two subunits of dimeric phosphorylase a are held together by strong interactions--deduced from the correlation of the rate of proteolysis and the disappearance of catalytic activity.

Adenosine Monophosphate↗
PubMed1986

Evidence that phosphorylase kinase exhibits phosphatidylinositol kinase activity.

Phosphorylase kinase phosphorylates the pure phospholipid phosphatidylinositol. Furthermore, it catalyzed phosphatidylinositol 4-phosphate formation using as substrate phosphatidylinositol that is associated with an isolated trypsin-treated Ca2+-transport adenosinetriphosphatase (ATPase) preparation from skeletal muscle sarcoplasmic reticulum. On this basis a fast and easy assay was developed that allows one to follow the phosphatidylinositol kinase activity during a standard phosphorylase kinase preparation. Both activities are enriched in parallel approximately to the same degree. Neither chromatography on DEAE-cellulose nor that on hydroxyapatite in the presence of 1 M KCl separates phosphatidylinositol kinase from phosphorylase kinase. The presence of a lipid kinase, phosphatidylinositol kinase, in phosphorylase kinase is not a general phenomenon; diacylglycerol kinase can be easily separated from phosphorylase kinase. Polyclonal anti-phosphorylase kinase antibodies as well as a monoclonal antibody directed specifically against the alpha subunit of phosphorylase kinase immunoprecipitate both phosphorylase kinase and phosphatidylinositol kinase.

1-Phosphatidylinositol 4-Kinase↗
PubMed1981

Interaction of phosphorylase kinase with the 2',3'-dialdehyde derivative of adenosine triphosphate. 2. Differential inactivation measured with various protein substrates.

The 2',3'-dialdehyde derivative of ATP was used as an affinity label to inactivate phsosphorylase kinase in either the presence or absence of Ca2+ and Mg2+ ions. Following inactivation, the residual activity of phorphorylase kinase toward various protein substrates was measured and compared with that retained for conversion of phosphorylase b. Three different classes of substrates were distinguished by this analysis. For the first class (glycogen synthase), inactivation proceeded at the same rate as that measured with phosphorylase conversion, regardless of whether the inactivation was carried out in the presence or absence of the metal ions. For the second class of substrates (troponin I and troponin T), inactivation of the kinase in either the presence or absence of the metals was much more rapid with phosphorylase as substrate. Phosphorylation of the third class of substrates (phosphorylase kinase itself and a synthetic tetradecapeptide) was inactivated in parallel with phosphorylase b when modification was performed in the absence of metals; however, inclusion of Ca2+ and Mg2+ ions in the inactivation mixture caused activity toward phosphorylase b to be lost more rapidly than that toward the alternative substrates. Our results are consistent with a model in which glycogen synthase and phosphorylase b are preferentially phosphorylated at one type of catalytic site in phosphorylase kinase and troponin I and troponin T at another.

Adenosine Triphosphate↗
PubMed2002

Diverse effects of two allosteric inhibitors on the phosphorylation state of glycogen phosphorylase in hepatocytes.

Two distinct allosteric inhibitors of glycogen phosphorylase, 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) and CP-91149 (an indole-2-carboxamide), were investigated for their effects on the phosphorylation state of the enzyme in hepatocytes in vitro. CP-91149 induced inactivation (dephosphorylation) of phosphorylase in the absence of hormones and partially counteracted the phosphorylation caused by glucagon. Inhibition of glycogenolysis by CP-91149 can be explained by dephosphorylation of phosphorylase a. This was associated with activation of glycogen synthase and stimulation of glycogen synthesis. DAB, in contrast, induced a small degree of phosphorylation of phosphorylase. This was associated with inactivation of glycogen synthase and inhibition of glycogen synthesis. Despite causing phosphorylation (activation) of phosphorylase, DAB is a very potent inhibitor of glycogenolysis in both the absence and presence of glucagon. This is explained by allosteric inhibition of phosphorylase a, which overrides the increase in activation state. In conclusion, two potent phosphorylase inhibitors exert different effects on glycogen metabolism in intact hepatocytes as a result of opposite effects on the phosphorylation state of both phosphorylase and glycogen synthase.

Allosteric Regulation↗
PubMed1981

Phosphorylation of McArdle phosphorylase induces activity.

In McArdle disease, myophosphorylase deficiency, enzyme activity is absent but the presence of an altered enzyme protein can frequently be demonstrated. We have found that phosphorylation of this protein in vitro can result in catalytic activity. We studied muscle of four patients; all lacked myophosphorylase activity, but myophosphorylase protein was demonstrated by immunodiffusion or gel electrophoresis. Incubation of muscle homogenate supernatants with cyclic AMP-dependent protein kinase and ATP resulted in phosphorylase activity. The activated enzyme comigrated with normal human myophosphorylase in gel electrophoresis. Incubation with [gamma-32P]ATP resulted in incorporatin of 32P into the band possessing phosphorylase activity. Activation of phosphorylase by cyclic AMP-dependent protein kinase was inhibited by antibodies to normal human myophosphorylase or by inhibitory protein to cyclic AMP-dependent protein kinase. Incubation of muscle homogenates with phosphorylase b kinase and ATP also resulted in phosphorylase activity. After the action of cyclic AMP-dependent protein kinase, the resulting activity was similar to that of phosphorylase b. However, incubation with phosphorylase kinase resulted in activity similar to that of phosphorylase a. For several reasons, it is not likely that McArdle disease is due to lack of normal phosphorylation, but restoration of activity to the mutant protein by phosphorylation may provide a clue to understanding the mechanism of this genetic defect.

Animals↗
PubMed1979

Liver phosphorylase b kinase. Cyclic-AMP-mediated activation and properties of the partially purified rat-liver enzyme.

Phosphorylase b kinase was extensively purified from rat liver. It was located in a form which could be activated 20--30-fold by a preincubation with adenosine 3':5'-monophosphate (cyclic AMP) and ATP-Mg. This activation was time-dependent, and was paralleled by a simultaneous incorporation of 32P from [gamma-32P]ATP into two polypeptides which comigrated in sodium dodecyl sulfate gel electrophoresis with the alpha and beta subunits of rabbit skeletal muscle phosphorylase b kinase. The liver enzyme was eluted from Sepharose 4B and Bio-Gel A-50m columns at the same place as muscle phosphorylase b kinase, which is indicative of a molecular weight of 1.3 x 10(6). After activation, the most purified liver preparation had a specific activity about 10-fold less than the homogeneous muscle enzyme at pH 8.2. The inactive enzyme form had a pronounced pH optimum around pH 6.0, whereas the activated form was mostly active above neutral pH. The activation of the enzyme reduced the Km for its substrate phosphorylase b severalfold. Liver phosphorylase b kinase was shown to be partially dependent on Ca2+ ions for its activity: addition of 0.5 mM [ethylenebis-(oxoethylenenitrilo)]tetraacetic acid (EGTA) to the phosphorylase b kinase assay increased the Km for phosphorylase b about twofold for both the inactive and the activated form of liver phosphorylase b kinase, but affected the V of the inactive species only.

Animals↗
PubMed1985

Effects of dinitrophenol on phosphorylase a activity, adenine nucleotide levels and tension in rabbit colon smooth muscle.

Glycogen phosphorylase a activity, the contents of adenine nucleotides and isometric tension were measured in rabbit colon smooth muscle after exposure to 2,4-dinitrophenol (DNP). DNP caused a dose-dependent increase in phosphorylase a activity, with an ED50 value of 1 X 10(-4)M. Since adenine nucleotides, especially 5-AMP by inhibiting the phosphorylase a to b conversion, might increase the phosphorylase a activity, a time-response study was undertaken in order to analyse the time course of changes in the adenine nucleotide content and phosphorylase a activity after addition of DNP. The latter activity was increased after only 1 min. of incubation. At this time no changes were found in the contents of ATP, ADP, 5-AMP or CP. Not until the phosphorylase a activity had reached its maximum after 5-7.5 min. did the 5-AMP content increase. The phosphorylase a activity then started to decline, but the 5-AMP content continued to rise. The effect of DNP on isometric tension was also studied to test whether the cytoplasmic Ca2+ concentration was increased. An increase in tension was observed 5 min. after administration of the drug and was maximal at 15 min. The results seem to dispute against 5-AMP as being a mediator for the DNP-stimulated increased in phosphorylase a activity in smooth muscle. The finding that DNP elicited a contraction of rabbit colon supports the earlier suggestion that the cytoplasmic Ca2+ concentration was increased (Pettersson 1983).

Adenine Nucleotides↗
PubMed1988

Phosphorylase activity in needle biopsy samples--factors influencing transformation.

Phosphorylase was determined in biopsy samples frozen immediately or after a delay of 10 s to 6 min. Muscle biopsies were performed at rest without and with propranolol, or adrenalin infusion and after electrical stimulation. The phosphorylase a fraction was 36% (28-44) in resting samples frozen immediately and 12% (12-13) after 10 s delay and remained at the same level when the freezing was further delayed (up to 6 min). It is suggested that an increase in [Ca2+] in the cytoplasm due to the insertion of the needle in muscle or cutting of tissue membranes may cause transformation of phosphorylase from b to a form, a transformation which is restored when Ca2+ is pumped back during the delay. Also the increased phosphorylase a fraction observed in biopsy samples obtained during adrenalin infusion reverted partially back when freezing was delayed for 10 s and 30 s, respectively. In muscle samples taken during contraction the mole fraction of phosphorylase a decreased from 53 to 12% when freezing was delayed for 10 s. The lowest value of the phosphorylase a mole fraction was observed in resting muscle after beta-blockade when the tissue samples were frozen 10 s after sampling and corresponded to 10% of the total phosphorylase. It is concluded that both muscle sampling and circulating adrenalin will increase phosphorylase a fraction in resting muscle and probably also augment the effect of adrenalin infusion.

Adult↗
PubMed1983

The effects of isoproterenol and ouabain on oxygen consumption, lactate production, and the activation of phosphorylase in coronary artery smooth muscle.

In previous work, oxidative metabolism in vascular smooth muscle was found to be correlated with isometric force; aerobic lactate production, however, was found to be substantial and correlated with Na-K transport processes (Paul et al., 1979; Paul, 1980). In this work, the role of glycogen phosphorylase in this unusual functional compartmentalization of of vascular energy metabolism was investigated. Isometric force, oxygen consumption, lactate production, and the level of phosphorylase activity were measured in porcine coronary arterial segments. For comparison with previous studies, phosphorylase activity was also measured in rabbit aorta. Added potassium chloride induced a contracture in which oxygen consumption, lactate production, and phosphorylase activity all increased. Ouabain also induced a contracture, and an increase in oxygen consumption and phosphorylase activity. However, lactate production was inhibited. Isoproterenol, when added to a potassium chloride-induced contracture, elicited a relaxation in isometric force, and oxygen consumption returned to basal levels; however, lactate production and phosphorylase activity remained at the previously elevated levels. Isoproterenol alone had only marginal effects on all parameters studied. The results indicate that the role of phosphorylase in vascular metabolism is complex and unlikely to be a rate-limiting factor for the observed aerobic glycolysis. The high levels of phosphorylase activity observed in the absence of actomyosin interaction support a mechanism of beta-adrenergic relaxation in which the sensitivity of actin-myosin interaction to calcium ion is decreased.

Animals↗
PubMed1982

Differences between male and female rats in the regulation of hepatic glycogenolysis. The relative role of calcium and cAMP in phosphorylase activation by catecholamines.

The relative importance of alpha- and beta-adrenergic pathways and of their respective intracellular mediators, calcium and cAMP, in the stimulation of phosphorylase alpha induced by catecholamines was studied in hepatocytes isolated from mature male and female rats. The fractional efflux of 45Ca was used as an index of intracellular calcium activity. Our results show that, in females: 1) the activation of phosphorylase alpha induced by 10(-8) to 10(-5) M epinephrine correlates with a rise in cellular cAMP as well as with an increase in 45Ca fractional efflux, 2) both alpha- and beta-agonists stimulate phosphorylase, 3) neither alpha- nor beta-antagonists effectively block the rise in phosphorylase caused by epinephrine, and 4) propranolol suppresses the rise in cAMP while phenoxybenzamine blocks the rise in calcium efflux. On the other hand, we found that in the male: 1) phosphorylase alpha activity is exclusively correlated with a rise in fractional calcium efflux, 2) epinephrine (10(-8) to 10(-7) M) does not increase cAMP and it causes a greater rise in calcium efflux than in the female at all concentrations, 3) phenylephrine increases calcium efflux and phosphorylase activity without affecting cAMP, 4) phenoxybenzamine totally blocks epinephrine action, and 5) beta-agonists and beta-antagonists are without effects. We conclude that, in females, epinephrine utilizes both alpha- and beta-adrenergic pathways which activate phosphorylase by calcium or cAMP, respectively, while, in adult male rats, epinephrine increases phosphorylase alpha activity by an alpha-mediated, calcium-dependent and cAMP-independent pathway.

Animals↗
PubMed1982

A comparison of the activator sites of liver and muscle glycogen phosphorylase b.

Characteristics of the activator sites of liver and muscle phosphorylase b were probed by using AMP and AMP analogs in kinetic studies, by quantitative affinity chromatography, and by reaction with an affinity-labeling reagent. Activation of liver phosphorylase b by N6-(6-aminohexyl)AMP in comparison with AMP and other analogs is explained by preferential binding to the activator site. The KM value for glucose-1-P of liver phosphorylase b activated with N6-(6-aminohexyl)AMP is considerably higher than that of muscle phosphorylase b. Affinity chromatography utilizing AMP-Sepharose suggests that the activator site is less well formed in liver phosphorylase than in muscle phosphorylase. Reaction with 8-[m-(m-fluorosulfonylbenzamido)benzylthio]adenine activates liver phosphorylase b and is consistent with the reaction at the activator site. The results suggest that part of the reason that liver phosphorylase b is not activated by AMP and AMP analogs is due to a poor coupling between the activator and active sites. Lack of good activation by AMP also can be explained by binding at the inhibitor site.

Adenosine Monophosphate↗
PubMed1981

Liver glycogenosis caused by a defective phosphorylase system: hemolysate analysis.

Investigated were 24 cases of glycogenosis caused by a reduction in liver phosphorylase activity. The intravenous glucagon tolerance test could not discriminate between phosphorylase kinase deficiency [glycogen storage disease (GSD) IX] and phosphorylase deficiency (GSD VI). These two subgroups were distinguished by hemolysate enzyme assays: (1) GSD IX was characterized by a residual phosphorylase kinase activity, a low activation curve for endogenous phosphorylase b and increased amylo-1,6-glucosidase activity. (2) GSD VI was characterized by a normal or increased phosphorylase kinase activity, a slight activation of endogenous phosphorylase b and a normal amylo-1,6-glucosidase activity.

Child↗
PubMed1991

Resolution of glycogen phosphorylase isoenzymes in precast PhastSystem polyacrylamide gels.

Homogeneous (7.5%) and gradient (10-15%) ultrathin nondenaturating miniaturized polyacrylamide gels (Pharmacia PhastGel media) were used to separate glycogen phosphorylase isoforms from rabbit muscle, rat liver and brain, MH 3924A cells, a dedifferentiated hepatocellular carcinoma of the rat, and C1I cells, a nontumorigenic epithelial rat liver cell line. The enzymes were detected by in situ phosphorylase assay and by immunoblotting. Phosphorylase proteins from the brain, MH 3924A, and C1I exhibited similar electrophoretic mobility, which was different from that of the enzymes from the muscle and normal liver. Molecular weight determination from sodium dodecyl sulfate gels yielded similar data for the subunits of muscle and liver enzymes (98,000 and 96,000), respectively, on one hand, and brain, MH 3924A tumor, and nontumorigenic C1I cells (93,000, 93,000 and 92,000), respectively, on the other. In the native gels the enzymes migrated as dimers: for muscle phosphorylase a, a tetramer was also observed. The a and b forms of the enzymes could not be resolved. An antibody raised against rat liver phosphorylase reacted only with the liver enzyme, whereas an antibody raised against brain phosphorylase stained the brain enzyme and the enzymes from MH 3924A and C1I cells. This indicates that hepatoma cells and immortalized nontumorigenic epithelial liver cells express a phosphorylase isoenzyme that is different from the liver type but similar to the brain type. The PhastSystem provides a rapid, sensitive, and highly reproducible method to resolve the different isoenzymes of glycogen phosphorylase.

Animals↗
PubMed1995

In vitro hormonal regulation of glycogen phosphorylase activity in fat body of the tropical cockroach, Blaberus discoidalis.

Blaberus hypertrehalosemic hormone (Bld-HTH)-dependent glycogen phosphorylase activation was investigated using in vitro fat bodies from the cockroach, Blaberus discoidalis. Resting levels of active phosphorylase were decreased by the presence of trehalose and glucose. Phosphorylase activation was dose-responsive to Bld-HTH and increased ca. 3-fold over a range of 0.02 to 2 nM Bld-HTH. Maximum phosphorylase activation required only 5-min exposure to Bld-HTH; reversion to the inactive state began within 15 min after Bld-HTH removal and was completed by 60 min. Octopamine also activated phosphorylase but required 10(3)-fold higher concentrations than did Bld-HTH. Concentrations of Bld-HTH and octopamine that increased active phosphorylase did not elevate fat body cAMP levels, although a high concentration of octopamine increased tissue cAMP levels. cAMP did not increase phosphorylase activity, but Ca2+ was important for both Bld-HTH- and octopamine-dependent phosphorylase activation.

Animals↗
PubMed1993

Evolution of allosteric control in glycogen phosphorylase.

In relation to the primary sequence and three-dimensional structure of rabbit muscle glycogen phosphorylase, we have carried out a comparative sequence analysis of phosphorylases from human, rat, Dictyostelium, yeast, potato and Escherichia coli. Based on sequence similarity, a large region of the protein is shared by these enzymes extending from alpha-helix-1 to the last alpha-helix-33. Conserved residues are equally distributed between the N and C-terminal domains and occur primarily in buried residues. Phylogenetic analysis indicates that the two isozymes within either E. coli, potato or Dictyostelium are more closely related to each other than they are to other phosphorylases. Yeast phosphorylase is most closely related to the Dictyostelium isozymes. Mammalian muscle and brain isozymes are more closely related to each other than to the liver isozyme and the muscle isozyme is evolving at the slowest rate. All phosphorylases exhibit high conservation of active site and pyridoxal phosphate binding residues. Most phosphorylases also exhibit high conservation of sugar binding residues in the glycogen storage site. Phosphorylation and AMP binding site residues are poorly conserved in non-mammalian phosphorylases. In contrast, glucose-6-P binding residues are highly conserved in four of the seven non-mammalian enzymes. Analysis of interacting pairs of dimer contact residues indicates that they can be grouped into three relatively independent networks. One network contains phosphorylation and AMP binding residues and is poorly conserved in non-mammalian enzymes. A second network contains glucose-6-P binding residues and is highly conserved in enzymes containing a conserved glucose-6-P binding site. A third, conserved network contains residues within the tower helix and gate loop. A model for the evolution of allostery in phosphorylase is proposed, suggesting that glucose-6-P inhibition was an early control mechanism. The later creation of primarily distinct ligand binding sites for AMP/phosphorylation control may have allowed the establishment of a separate dimer contact network for propagating conformational changes leading to activation rather than inhibition of enzyme activity.

Allosteric Regulation↗