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PubMed1982

Ca2+/calmodulin-dependent phospholamban kinase from cardiac sarcoplasmic reticulum is distinct from phosphorylase kinase and forms a regulatory complex with phospholamban and the Ca2+-ATPase.

We recently reported that phospholamban, the activator of the cardiac sarcoplasmic reticulum calcium pump, is phosphorylated by both cAMP-dependent protein kinase and a membrane-bound, Ca2+/calmodulin-dependent phospholamban kinase. Phospholamban kinase and glycogen phosphorylase b kinase share the same substrate specificity. They differ however in that phospholamban kinase exhibits an absolute requirement for exogenous calmodulin. In line with the latter observation, phospholamban kinase is shown in this report to be inhibited by fluphenazine. Lower concentrations of the drug induced an activation of the kinase, presumably by hydrophobic interaction with either membrane phospholipids or integral proteins. Also, phospholamban kinase was found to be totally insensitive to antibodies elicited against phosphorylase kinase. Since antipsychotic drugs fail to inhibit the delta-subunit-dependent activity of phosphorylase kinase, the above findings confirm that the two kinases are distinct molecular entities. After detergent solubilization of the sarcoplasmic reticulum, the phospholamban-ATPase complex remains a substrate for phospholamban kinase activity, which retains the ability to catalyze the phosphorylation of exogenous phosphorylase b. However, the Ca2+ dependence is entirely lost upon solubilization and no kinase activity is retained on calmodulin-Sepharose in the presence of Ca2+ ions. Phospholamban and phosphorylase kinase activities copurify with the pump-phospholamban complex upon fractionation of the solubilized proteins by density gradient ultracentrifugation, suggesting a tight interaction between the ATPase, its activator, and the phospholamban kinase. A tentative schematic representation of this supramolecular assembly is based upon the results described in this and preceding papers.

Adenosine Triphosphatases↗
PubMed2006

Interaction of phosphorylase kinase from rabbit skeletal muscle with flavin adenine dinucleotide.

The interaction of flavin adenine dinucleotide (FAD) with rabbit skeletal muscle phosphorylase kinase has been studied. Direct evidence of binding of phosphorylase kinase with FAD has been obtained using analytical ultracentrifugation. It has been shown that FAD prevents the formation of the enzyme-glycogen complex, but exerts practically no effect on the phosphorylase kinase activity. The dependence of the relative rate of phosphorylase kinase-glycogen complex formation on the concentration of FAD has cooperative character (the Hill coefficient is 1.3). Under crowding conditions in the presence of 1 M trimethylamine-N-oxide (TMAO), FAD has an inhibitory effect on self-association of phosphorylase kinase. The data suggest that the complex of glycogen metabolism enzymes in protein-glycogen particles may function as a flavin depot in skeletal muscle.

Animals↗
PubMed1994

Early release of glycogen phosphorylase in patients with unstable angina and transient ST-T alterations.

OBJECTIVE: To determine whether transient ST-T alterations in patients with unstable angina are associated with an increase in plasma glycogen phosphorylase BB concentrations on admission to hospital. DESIGN: Prospective screening of patients with unstable angina for markers of myocardial cell damage. SETTING: Accident and emergency department of university hospital. PATIENTS: 48 consecutive patients admitted for angina pectoris (18 with transient ST-T alterations). None of the patients had acute myocardial infarction according to standard criteria. MAIN OUTCOME MEASURES: Creatine kinase and creatine kinase MB activities, creatine kinase MB mass concentration, and myoglobin, cardiac troponin T, and glycogen phosphorylase BB concentrations on admission. RESULTS: All variables except for creatine kinase and creatine kinase MB activities were significantly higher on admission in patients with unstable angina and transient ST-T alterations than in patients without. However, glycogen phosphorylase BB concentration was the only marker that was significantly (p = 0.0001) increased above its discriminator value in most patients (16). In the 18 patients with transient ST-T alterations creatine kinase MB mass concentration and troponin T and myoglobin concentrations were significantly (p = 0.0001) less commonly increased on admission (in five, three, and two patients, respectively). CONCLUSIONS: The early release of glycogen phosphorylase BB may help to identify high risk patients with unstable angina even on admission to an emergency department. Glycogen phosphorylase BB concentrations could help to guide decisions about patient management.

Angina, Unstable↗
PubMed1991

Glucose and caffeine regulation of liver glycogen phosphorylase activity in the freeze-tolerant wood frog Rana sylvatica.

We have examined the effect of glucose and caffeine inhibition on the activity of liver glycogen phosphorylase a from the freeze-tolerant frog Rana sylvatica. Kinetic studies indicate that this enzyme exhibits similar sensitivity to glucose inhibition (glucose dissociation constant = 12.5 mM) as the mammalian enzyme. Little inhibition (less than 25%) was observed at normal glucose concentrations (1-5 mM), while significant inhibition (60-95%) occurred at glucose concentrations (50-500 mM) present in freezing-exposed animals. These results favour the hypothesis that in the normal state glucose regulates phosphorylase activity primarily through the promotion of dephosphorylation of phosphorylase a, whereas during freezing regulation is achieved through phosphorylase a inactivation. The caffeine dissociation constant (0.93 mM) and the degree of synergism between caffeine and glucose (interaction factor, alpha = 0.14) were also similar to that observed for the mammalian enzyme. Hence, if a caffeine-like ligand exists in vivo, it must be in low enough amounts during freezing to allow sufficient phosphorylase a activity for high glucose production.

Animals↗
PubMed1992

Particulate glycogen of mammalian liver: specificity in binding phosphorylase and glycogen synthase.

The glycogen particle - glycogen metabolizing enzyme complex was investigated to gain some understanding of its physiological significance. Fractionations of populations of particles from mouse liver were carried out utilising open column and high performance liquid chromatography, and based either on the molecular weight of the particles or the hydrophobic interactions of the glycogen-associated proteins. The activities of glycogen phosphorylase and glycogen synthase were measured in these fractions. Fractionations were of tissue in different stages of glycogen deposition or mobilization. In animals fed ad libitum, glycogen synthase was associated with the whole spectrum of molecular weights, while the glycogen phosphorylase distribution was skewed in favour of the lower molecular weight species. Under conditions of glycogen mobilization, the phosphorylase distribution changed to include all molecular weights. The hydrophobic interaction separations demonstrated that glycogen synthase binds to a specific subpopulation of particles that is a minor proportion of the total. In general, there was a direct relationship of the total amount of phosphorylase and synthase bound during periods of mobilization and deposition, respectively. Two notable exceptions were the large amounts of glucose-6-P dependent synthase present during the early period of glycogen mobilization and the high amounts of active phosphorylase appearing shortly after food withdrawal, in spite of interim glycogen deposition from presumably already ingested food.

Animals↗
PubMed1976

The effect of calcium oncardiac phosphorylase activation, contractile force and cyclic AMP in euthyroid and hyperthyroid rat hearts.

Calcium chloride injected into isolated perfused rat hearts produced a positive inotropic effect and increased the levels of phosphorylase a (EC 2.4.1.1). The increase in enzyme activity lagged behind the inotropic effect. Pretreatment of animals with thyroid hormone enhanced the ability of noradrenaline to activate phosphorylase but did not affect the inotropic or phosphorylase activating effect of calcium. Thyroid hormone pretreatment did enhance the chronotropic effect of calcium. Calcium did not affect the cardiac levels of cyclic AMP. It is concluded that calcium can activate phosphorylase by a mechanism other than cyclic AMP and that the enhancement of adrenergic amine-induced phosphorylase activation by thyroid hormone is not a calcium mediated event.

Animals↗
PubMed2001

Structural relationships among regulated and unregulated phosphorylases.

Species and tissue-specific isozymes of phosphorylase display differences in regulatory properties consistent with their distinct roles in particular organisms and tissues. In this review, we compare crystallographic structures of regulated and unregulated phosphorylases, including maltodextrin phosphorylase (MalP) from Escherichia coli, glycogen phosphorylase from yeast, and mammalian isozymes from muscle and liver tissues. Mutagenesis and functional studies supplement the structural work and provide insights into the structural basis for allosteric control mechanisms. MalP, a simple, unregulated enzyme, is contrasted with the more complicated yeast and mammalian phosphorylases that have evolved regulatory sites onto the basic catalytic architecture. The human liver and muscle isozymes show differences structurally in their means of invoking allosteric activation. Phosphorylation, though common to both the yeast and mammalian enzymes, occurs at different sites and activates the enzymes by surprisingly different mechanisms.

Allosteric Site↗
PubMed1997

Expression of glycogen phosphorylase isozymes in developing rat lung.

Glycogen accumulates to significant levels in epithelial cells of the developing respiratory tract. Mobilization of glycogen stores is regulated differentially along the respiratory epithelium such that glycogenolysis in the alveolar epithelium (the site of surfactant synthesis) precedes that in the bronchial and bronchiolar epithelium. The initial step in glycogen degradation is catalyzed by glycogen phosphorylase, which exists as three genetically distinct isozymes referred to as muscle, liver, and brain isoforms. The goal of this study was to characterize the temporal and spatial expression of each of the glycogen phosphorylase isozymes in developing lung to determine which isoform(s) was associated with glycogen mobilization in the fetal type II epithelial cell. RNA levels encoding glycogen phosphorylase were assessed by ribonuclease protection assay using isoform-specific antisense probes. RNAs encoding the brain and liver isozymes were detected in isolated day 20 fetal type II epithelial cells and at lower levels in adult type II cells. The muscle isoform RNA was barely detectable in fetal type II cells and was undetectable in adult type II cells. Expression of brain and liver isoform RNAs was higher in whole fetal lung than in fetal type II cells. Consistent with this result, in situ hybridization studies demonstrated widespread expression of the brain and liver isoforms in developing lung tissues; in contrast, expression of the muscle isoform was restricted to the pulmonary vein. Glycogen phosphorylase enzyme activity corresponding to the brain isoform was clearly detected in isolated fetal type II cells; however, the majority of enzyme activity migrated as two bands with distinct electrophoretic mobilities that may have been the result of isoform heterodimerization. Collectively, these results suggest that the brain and liver isoforms of glycogen phosphorylase may be involved in mobilization of type II cell glycogen during late fetal lung development.

Animals↗
PubMed1981

Proposed explanation for S-adenosylhomocysteine hydrolase deficiency in purine nucleoside phosphorylase and hypoxanthine-guanine phosphoribosyltransferase-deficient patients.

We have examined the basis for the recently reported, but unexplained deficiency of S-adenosylhomocysteine hydrolase (AdoHcyase) in the erythrocytes of patients with genetic deficiencies of purine nucleoside phosphorylase and hypoxanthine-guanine phosphoribosyltransferase. We found that a hemolysate from a patient with purine nucleoside phosphorylase deficiency had only 7% of control AdoHcyase activity, conforming the original observation. Of the purine nucleosides known to accumulate in nucleoside phosphorylase-deficient patients, inosine alone caused the phosphate-dependent, irreversible inactivation of purified human placental AdoHcyase, and of AdoHcyase in intact erythrocytes and cultured lymphoblastoid cells. Hypoxanthine did not inactivate purified AdoHcyase, but potentiated the effect of inosine in intact hypoxanthine-guanine phosphoribosyltransferase-deficient human lymphoblastoid cells. This presumably resulted from the ability of hypoxanthine to shift the equilibrium of the nucleoside phosphorylase reaction, preventing inosine breakdown. This could account for the partial AdoHcyase deficiency reported in hypoxanthine-guanine phosphoribosyltransferase-deficient patients. We have also demonstrated the AdoHycase-catalyzed synthesis of S-inosylhomocysteine from inosine and L-homocysteine, a reaction which may occur in nucleoside phosphorylase-deficient patients.

Adenosylhomocysteinase↗
PubMed1975

Electrophoretic studies on the phosphorylase isozymes.

The electrophoretic method of Davis, Schliselfeld, Wolf, Leavitt and Krebs (1967) for phosphorylase isozymes has been modified. By this method, five isozymes were separated in various organs of rat and pig and were disignated as phosphorylase L, LI, I, II and III. The L and III enzymes were the only forms found in liver and skeletal muscle, respectively, while the I enzyme was dominant in brain, uterus, lung and small intestine, which also contained some fractions of the II and III enzymes. The I enzyme was also dominant in adrenal, ovary and kidney, but these organs contained the L+II or L+LI as minor components. The L and LI were richly found in spleen and leukocytes of adult rats and pigs and in liver of newborn rats. Such organ-specific heterogeneity of phosphorylase was confirmed by the immunological tests with the antibodies prepared against phosphorylases I, III and L. The II and LI enzymes were found to be the hybrid molecules between the I and III enzymes, and between the I and L enzymes which have been previously reported as unhybridizable, respectively. In view of the above findings, it was concluded that the rat and pig possessed at least five molecular forms of phosphorylase.

Animals↗
PubMed2005

Quantification of the glycogen cascade system: the ultrasensitive responses of liver glycogen synthase and muscle phosphorylase are due to distinctive regulatory designs.

BACKGROUND: Signaling pathways include intricate networks of reversible covalent modification cycles. Such multicyclic enzyme cascades amplify the input stimulus, cause integration of multiple signals and exhibit sensitive output responses. Regulation of glycogen synthase and phosphorylase by reversible covalent modification cycles exemplifies signal transduction by enzyme cascades. Although this system for regulating glycogen synthesis and breakdown appears similar in all tissues, subtle differences have been identified. For example, phosphatase-1, a dephosphorylating enzyme of the system, is regulated quite differently in muscle and liver. Do these small differences in regulatory architecture affect the overall performance of the glycogen cascade in a specific tissue? We address this question by analyzing the regulatory structure of the glycogen cascade system in liver and muscle cells at steady state. RESULTS: The glycogen cascade system in liver and muscle cells was analyzed at steady state and the results were compared with literature data. We found that the cascade system exhibits highly sensitive switch-like responses to changes in cyclic AMP concentration and the outputs are surprisingly different in the two tissues. In muscle, glycogen phosphorylase is more sensitive than glycogen synthase to cyclic AMP, while the opposite is observed in liver. Furthermore, when the liver undergoes a transition from starved to fed-state, the futile cycle of simultaneous glycogen synthesis and degradation switches to reciprocal regulation. Under such a transition, different proportions of active glycogen synthase and phosphorylase can coexist due to the varying inhibition of glycogen-synthase phosphatase by active phosphorylase. CONCLUSION: The highly sensitive responses of glycogen synthase in liver and phosphorylase in muscle to primary stimuli can be attributed to distinctive regulatory designs in the glycogen cascade system. The different sensitivities of these two enzymes may exemplify the adaptive strategies employed by liver and muscle cells to meet specific cellular demands.

Animals↗
PubMed1981

Glycogenosis due to liver and muscle phosphorylase kinase deficiency.

A four-year-old Israeli Arab boy was found to have glycogen accumulation in both liver and muscle without clinical symptoms. Liver phosphorylase kinase (PK) activity was 20% of normal, resulting in undetectable activity of phosphorylase a. Muscle PK activity was about 25% of normal, resulting in a marked decrease of phosphorylase a activity. Two sisters showed a similar pattern, whereas one brother had normal PK activity. The patient's liver protein kinase activity was normal Addition of exogenous protein kinase did not affect PK activity, whereas exogenous PK restored phosphorylase activity to normal. These findings indicate that these patients are affected by a rare variant of PK deficiency, which involves both muscle and liver and which apparently is not sex linked. It is possible that this defect represents an unusual mutation of a subunit of the phosphorylase kinase enzyme.

Child↗
PubMed1987

Regulation of rat liver glycogen phosphorylase concentration by in vivo relative levels of glucagon and insulin.

The concentrations of glycogen phosphorylase protein were determined by rocket immunoelectrophoresis in liver extracts from rats that had artificially induced altered hormonal patterns. These levels were compared with measurements of total phosphorylase activity. Minipump-induced chronic hyperglucagonemia and streptozotocin-induced diabetes resulted in 47% and 67% decreases, respectively, in total phosphorylase activity along with corresponding 52% and 68% drop, respectively, in phosphorylase protein levels. Insulin replacement in diabetic rats returned both parameters to control values. Minipump-induced hyperinsulinemia or injection of glucagon antiserum, T3, or propylthiouracil had no effect. The results of this study indicate that conditions which lead to an elevation of the glucagon to insulin molar ratio to values higher than 1.0 cause a significant decrease in the liver phosphorylase protein level.

Animals↗
PubMed1979

Phosphorylase isoenzymes in normal and myophosphorylase-deficient human heart.

Phosphorylase isoenzymes were studied by acrylamide-slab electrophoresis in normal tissues and in the heart of a child with a fatal infantile form of myophosphorylase deficiency. Of the three bands present in normal human heart, two were missing in the patient's heart: the slow "muscle" isoenzyme and the intermediate band. Only the fast "cardiac" isoenzyme remained. When extracts of normal skeletal muscle and the patient's heart were mixed in appropriate conditions, the intermediate band reappeared in the electropherogram. Phosphorylase activity in extracts of the patient's heart was not inhibited by antibodies against purified enzyme from mature human muscle, whereas normal human heart phosphorylase was inhibited by approximately 50%. These results suggest that the intermediate band of human heart phosphorylase is a hybrid of skeletal and cardiac muscle isoenzymes. Retained activity of the cardiac isoenzyme may explain why patients genetically lacking skeletal muscle phosphorylase do not have clinical heart disease.

Adolescent↗
PubMed1979

McArdle disease: phosphorylase activity in regenerating muscle fibers.

Phosphorylase activity was found histochemically in regenerating muscle fibers in biopsied muscle from a patient with otherwise typical McArdle disease. Phosphorylase activity, shortly after episodes of muscle necrosis, was identified in this patient and in others reported in the literature. Detection of phosphorylase activity accompanied histologic evidence of regenerating muscle fibers and excessive glycogen accumulation. The biopsied muscle had virtually no biochemical enzyme activity. The present study supports a recently introduced hypothesis stating that in McArdle disease there is a lack of "mature" phosphorylase, but skeletal muscle cells are able to manufacture "fetal" phosphorylase isoenzyme during muscle regeneration.

Adult↗
PubMed1990

Studies on responsiveness of hepatoma cells to catecholamines. V. Loss of adrenergic response of glycogen phosphorylase in rat ascites hepatoma AH130 cells.

The beta-adrenoceptor-cyclic adenosine monophosphate (AMP) dependent glycogenolytic cascade was examined in normal rat hepatocytes and rat ascites hepatoma AH130 cells. The cyclic AMP content in AH130 cells was half of that in normal hepatocytes, and the cyclic AMP levels in both kinds of cells were clearly increased by isoproterenol (IPN). Cyclic AMP-dependent protein kinase activity was higher in AH130 cells than in normal hepatocytes. Phosphorylase kinase activities in 10000 x g supernatant of normal hepatocytes and AH130 cells were also increased in the presence of cyclic AMP. Phosphorylase a activities in the supernatant of both kinds of cells gradually decreased during incubation with 40 mM glucose at 37 degrees C, and the enzyme activity of normal hepatocytes was completely restored by the addition of Mg2(+)-adenosine triphosphate (ATP), but in the case of the hepatoma cells the recovery was small. The decreased phosphorylase a activity in the hepatoma cells was increased by additional glycogen but did not exceed the level before the incubation. In the case of normal hepatocytes it was not affected by glycogen. This indicates that glycogen contained in the cells influences the activation of phosphorylase; the glycogen content in AH130 cells was far less than in normal hepatocytes. On the other hand, when intact cells were incubated with a high concentration of glucose, phosphorylase a activity in the homogenate of normal hepatocytes was decreased and could be restored by IPN and dibutyryl cyclic AMP, but the enzyme activity in the homogenate of AH130 cells was very low and hardly changed after the incubation and treatment with these agents.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Hormonal regulation of glycogen synthase and phosphorylase activities in human polymorphonuclear leukocytes.

Hormonal regulation of glycogen synthase and phosphorylase activities were studied in human polymorphonuclear leukocytes. Polymorphonuclear leukocytes from normal subjects were incubated with glucose, insulin, D,L-isoproterenol and L-thyroxine, either independently or in different combinations, and changes of the enzyme activity ratios of glycogen synthase (active form (I)/total activity (T)) and glycogen phosphorylase (active form (a)/total activity (T)) were assessed. Neither glucose nor insulin changed the glycogen synthase activity ratio. However, the proportion of the active form (I) of glycogen synthase was increased by the simultaneous addition of glucose and insulin to the incubation mixture, but D,L-isoproterenol or L-thyroxine diminished this effect and caused a decrease in the proportion of the active form of glycogen synthase. Insulin had no effect on the glycogen phosphorylase activity ratio. Glucose decreased the proportion of phosphorylase in the a form. The simultaneous addition of glucose and insulin caused no further changes, whereas in the presence of D,L-isoproterenol or L-thyroxine, this glucose effect was abolished and the proportion of phosphorylase a increased. These results show that both thyroid hormone and a beta-agonist alter glycogen metabolism to reduce glycogen storage in polymorphonuclear leukocytes.

Drug Interactions↗
PubMed1994

Glycogen phosphorylase isoenzyme BB mass release after coronary artery bypass grafting.

Glycogen phosphorylase isoenzyme BB mass release was studied in 20 patients undergoing coronary artery bypass grafting. In 16 patients with uneventful coronary artery bypass grafting, glycogen phosphorylase isoenzyme BB mass concentrations showed a significant, transient increase in the post cross-clamping period and decreased to baseline values within 20 hours (peak concentrations ranged from 12.7 micrograms/l to 47.5 micrograms/l, median 40 micrograms/l). One patient did not fulfil criteria for perioperative myocardial infarction, but clinical data indicated myocardial injury after aortic unclamping. In this patient only glycogen phosphorylase isoenzyme BB mass concentration and not creatine kinase isoenzyme MB catalytic concentration was increased, compared with uneventful patients. In 2 patients with emergency coronary artery bypass grafting for evolving myocardial infarction, glycogen phosphorylase isoenzyme BB mass concentrations, but not creatine kinase isoenzyme MB catalytic concentrations, correlated with clinical evidence of myocardial ischaemia. Our data indicate that glycogen phosphorylase isoenzyme BB mass concentration is a very sensitive laboratory marker of perioperative myocardial injury in patients undergoing coronary artery bypass grafting.

Aged↗