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PubMed1986

Expression of human malaria parasite purine nucleoside phosphorylase in host enzyme-deficient erythrocyte culture. Enzyme characterization and identification of novel inhibitors.

The intraerythrocytic human malaria parasite, Plasmodium falciparum, requires a source of hypoxanthine for nucleic acid synthesis and energy metabolism. Adenosine has been implicated as a major source for intraerythrocytic hypoxanthine production via deamination and phosphorolysis, utilizing adenosine deaminase and purine nucleoside phosphorylase, respectively. To study the expression and characteristics of human malaria purine nucleoside phosphorylase, P. falciparum was successfully cultured in purine nucleoside phosphorylase-deficient human erythrocytes to an 8% parasitemia level. Purine nucleoside phosphorylase activity was undetectable in the uninfected enzyme-deficient host red cells but after parasite infection rose to 1.5% of normal erythrocyte levels. The parasite purine nucleoside phosphorylase was not cross-reactive with antibody against human enzyme, exhibited a calculated native molecular weight of 147,000, and showed a single major electrophoretic form of pI 5.4 and substrate specificity for inosine, guanosine and deoxyguanosine but not xanthosine or adenosine. The Km values for substrates, inosine and guanosine, were 4-fold lower than that for the human erythrocyte enzyme. In these studies we have identified two novel potent inhibitors of both human erythrocyte and parasite purine nucleoside phosphorylase, 8-amino-5'-deoxy-5'-chloroguanosine and 8-amino-9-benzylguanine. These enzyme inhibitors may have some antimalarial potential by limiting hypoxanthine production in the parasite-infected erythrocyte.

Animals↗

PubMed1985

Monoclonal antibodies to rabbit skeletal muscle phosphorylase kinase. Probes for studies of subunit function.

Monoclonal antibodies to rabbit skeletal muscle phosphorylase kinase were produced by the conventional hybridoma cell technique. 90 out of 600 hybridomas were found to produce phosphorylase kinase binding antibodies from which only five secreted also phosphorylase kinase activity affecting antibodies. Three of them were cloned; two hybridomas resisted all cloning efforts. Employing immunoblot technique all monoclonal antibodies show cross-reactivity with the alpha, beta, and gamma subunits of phosphorylase kinase indicating that similar, if not identical, epitopes are present on these three subunits. No cross-reactivity with delta is observed. Monoclonal antibodies secreted by two clones which bind to the alpha subunit stimulate the Ca2+-independent A0 activity of phosphorylase kinase more than 30-fold, whereas all other monoclonal antibodies obtained are ineffective in this respect. Monoclonal antibodies binding to the beta subunit inhibit the Ca2+-dependent activities significantly. Antibody produced by one hybridoma binds to the alpha, beta, and gamma subunits with approximately the same affinity. Based on the dual function of calmodulin in phosphorylase kinase (Hessová, Z., Varsányi, M., and Heilmeyer, L.M.G., Jr. (1985) Eur. J. Biochem. 146, 107-115) we conclude that binding of anti-alpha monoclonal antibodies to a regulatory domain in the alpha subunit results in an uncoupling of the inhibitory function of the Ca2+-free delta from the holoenzyme which leads to a concomitant increase in A0 activity. Furthermore, binding of anti-beta monoclonal antibodies to the beta subunit prevents a signal transfer from the Ca2+-saturated delta to the catalytic site of the holoenzyme which inhibits the Ca2+-dependent activities.

Animals↗

PubMed1982

The role played by hormonal factors in the rapid activation of liver glycogen phosphorylase in traumatized rats.

In adult male SPF rats anaesthetized with pentobarbital and subjected to traumatization in revolving Noble-Collip drums for 2 min (= 120 revolutions) maximal increases of liver glycogen phosphorylase activity were observed. In experiments on rats with permanent arterial catheters for blood sampling no posttraumatic increase of plasma norepinephrine and an only slight increase of plasma epinephrine was observed if the animals were traumatized under anaesthesia, in contrast to the considerable increases in the plasma level of both hormones in rats subjected to the injury without anaesthesia. Time and extent of the phosphorylase response of anaesthetized rats after trauma were compared with changes in enzyme activity after i.v. administration of exogenous epinephrine or glucagon. A nearly maximal response after 1 microgram kg-1 epinephrine was present within 1 min, whereas after 0.1 micrograms kg-1 of glucagon there was comparable phosphorylase activation 2 min after administration of the hormone. The plasma renin-angiotensin activity was not increased after injury for 2 min under anaesthesia so that only the increase in plasma vasopressin fitted in with the criteria for possible activators of phosphorylase. An additional role of glucagon also cannot be excluded on the basis of data obtained by the present authors. The increase of phosphorylase activity in this type of stress is ensured by several mechanisms. Moreover, the high effectivity of these hormonal factors in evoking the phosphorylase response even without major activation of the sympathicoadrenal system is underlined.

Anesthesia↗

PubMed1982

Phosphorylation and activation of rabbit skeletal muscle phosphorylase kinase by a cyclic nucleotide- and Ca2+-independent protein kinase.

Phosphorylase kinase from rabbit skeletal muscle can be phosphorylated and activated by a cyclic nucleotide- and Ca2+-independent protein kinase previously identified as a glycogen synthase kinase (Itarte, E., and Huang, K.-P. (1979) J. Biol. Chem. 254, 4052-4057). This independent kinase phosphorylates the beta subunit of phosphorylase kinase approximately 15 times faster than it does the alpha subunit. The cAMP-dependent and -independent kinases separately catalyze the incorporation of 1 mol of phosphate into the beta subunit. Analyses of the tryptic peptides from the beta subunit phosphorylated with either kinase by isoelectric focusing and peptide mapping indicate that both kinases phosphorylate the same site on the beta subunit. Activation of phosphorylase kinase catalyzed by the independent kinase is only 60% of that observed with cAMP-dependent kinase. If phosphorylase kinase is first incubated with the independent kinase to phosphorylate the beta subunit, subsequent addition of cAMP-dependent kinase results in a predominant phosphorylation of the alpha subunit. This additional phosphorylation of the alpha subunit is accompanied by a further activation of the alpha subunit is accompanied by a further activation of phosphorylase kinase to the same extent as that achieved by cAMP-dependent kinase alone. Hence, the phosphorylation of the alpha subunit is clearly required for full activation of phosphorylase kinase, even at low [Mg2+].

Animals↗

PubMed1983

Biochemical genetic analysis of the role of methylthioadenosine phosphorylase in a murine lymphoid cell line.

The enzyme methylthioadenosine phosphorylase functions in both purine and polyamine metabolism is dividing mammalian cells. To determine the effects of the loss of this enzyme on cell growth and metabolism, we selected two methylthioadenosine phosphorylase-deficient mutant clones of the transplantable murine T lymphoma cell line R1.1. The first had 3.5% of wild type methylthioadenosine phosphorylase activity. The second was completely enzyme-deficient. The loss of the enzyme did not alter the growth rate, cloning efficiency, or tumor-forming ability of the T lymphoma cells. The methylthioadenosine phosphorylase-deficient clones excreted substantial amounts of methylthioadenosine into the culture medium (0.13 and 0.32 nmol/h/mg of protein, respectively) and were unable to utilize the methylthioadenosine phosphorylase substrate 2',5'-dideoxyadenosine as a purine source when de novo purine synthesis was blocked. Spermine levels were 10-20% lower in the enzyme-deficient clones than in wild type cells. The loss of methylthioadenosine phosphorylase rendered the mutants exquisitely sensitive to the antiproliferative effects of methylthioadenosine. Methylthioadenosine at 3-6 microM inhibited their growth by 50%. The toxic effects of methylthioadenosine were not attributable to inhibition of purine, pyrimidine, or polyamine synthesis.

Animals↗

PubMed1984

Phosphorylation of myosin light chain and phosphorylase in tracheal smooth muscle in response to KCl and carbachol.

It has been proposed that Ca2+-dependent myosin light chain (P-light chain) phosphorylation in smooth muscle permits cycling of myosin cross-bridges within myofibrillar elements for muscle shortening, but a second Ca2+-dependent regulatory mechanism is responsible for force generation. Accordingly, we examined P-light chain phosphorylation and another Ca2+-dependent protein phosphorylation reaction, phosphorylase a formation, in bovine tracheal smooth muscle during isometric force generation elicited by the cholinergic agonist carbachol or KCl depolarization, two stimuli thought to increase the concentration of sarcoplasmic free Ca2+ by mobilizing different pools of Ca2+. Increases in P-light chain phosphorylation reached maximal values of 0.79 and 0.59 mole of phosphate per mole of P-light chain at 1 min and then declined during maintained isometric force developed in response to 1 microM carbachol and 60 mM KCl, respectively. Carbachol elicited approximately twice the amount of force as found in the presence of KCl, and yet a more rapid rate of decline in the phosphate content of P-light chain was apparent. Decreases in maximal levels of phosphorylase a also occurred during carbachol-mediated isometric force maintenance, yet did not occur with KCl stimulation. Concentration-dependent responses with carbachol and KCl showed a positive relationship between the extent of P-light chain phosphorylation and extent of developed isometric force after 1 min of contraction with both stimuli. Under no conditions was force generated without P-light chain phosphorylation. The concentration dependence of phosphorylase a formation with KCl was similar to isometric force and P-light chain phosphorylation. However, concentrations of carbachol necessary to stimulate phosphorylase a formation were much higher than those required for stimulation of isometric force and P-light chain phosphorylation. Furthermore, carbachol attenuated the stimulation of phosphorylase a formation by isoproterenol. Thus, carbachol appears to have both an inhibitory and stimulatory effect on phosphorylase a formation in bovine tracheal smooth muscle. These results also indicate that maintained isometric force in smooth muscle may be dependent upon the maximal extent of P-light chain phosphorylation obtained during an early temporal transient in phosphorylation.

Animals↗

PubMed1976

Activity of glycogen phosphorylase in the crystalline state.

Glutaraldehyde cross-linked crystals of muscle phosphorylase a and b (alpha-1,4-glucan:orthophosphate glucosyltransferase, EC 2.4.1.1) in the tetragonal form have been shown to be catalytically active in the direction of saccharide synthesis. Precession x-ray photographs at 5.5 A resolution of a single crystal of cross-linked phosphorylase b at the hol zone indicate little change in the diffraction pattern when compared to non-cross-linked phosphorylase b under similar conditions. Non-cross-linked crystals crack and dissolve in the presence of both substrates, maltopheptaose and glucose 1-phosphate, although they are stable in the presence of each individually. These phenomena are prevented by treatment with glutaraldehyde, which causes a marked increase in mechanical stability and completely suppresses solubilization of the enzyme under our assay conditions. Diffusion of substrates into cross-linked microcrystals does not appear to be rate-limiting and assays of such crystals are linear with respect to both time and enzyme concentration. Kinetic constants for both substrates are reported. The maximal velocities of phosphorlyase a are larger than those of phosphorylase b in both the soluble and crystalline states under our assay conditions, with the above substrates. It appears that crystallization (and cross-linking) reduces maximal velocities by about 11- to 50-fold in the case of phosphorylase b and 50- to 100-fold for phosphorylase a. Little or no differences were found between the Km values for maltoheptaose or glucose 1-phosphate in the soluble or crystalline states. Kinetic data suggest that substrate binding sites are similar in both states. Although loss of catalytic efficiency points to differences in the active site of the enzyme caused by crystallization, another explanation is that the crystal is restricting a conformational change that is an essential part of the catalytic cycle.

Animals↗

PubMed1995

[Reconstruction of muscle glycogen phosphorylase b from an apoenzyme and pyridoxal-5'-phosphate and its analogs. Interaction of apophosphorylase and the reconstructed enzyme with specific ligands].

Sedimentation methods were used to study the effects of modification of the pyridoxal-5'-phosphate (PLP) molecule at the 5th position on the affinity of reconstituted muscle glycogen phosphorylase b for the substrate (glycogen) and the allosteric inhibitor (FMN) as well as on the enzyme capacity to association induced by AMP. Reconstituted phosphorylase b was obtained with PLP analogs containing at the 5th position -CH2-CH2-COOH (analog I), trans-CH=CH-COOH (analog II) or -C identical to COOH (analog III) residues. Reconstitution of phosphorylase b is accompanied by the recovery of the enzyme quaternary structure. Phosphorylase b reconstituted with PLP or analogs I, II and III is not distinguished practically from the native enzyme in its affinity for glycogen. Substitution of the native coenzyme in the phosphorylase molecule with any tested PLP analog leads to lower enzyme affinity for FMN. Microscopic dissociation constants of the FMN-enzyme complexes increase in the following order: enzyme.I < enzyme.II < enzyme.III. Phosphorylase b reconstituted with analogs I, II and III differs substantially from the native enzyme in its capacity to association in the presence of 1 mM AMP: the reconstituted enzyme is represented practically by only the tetrameric form.

Adenosine Monophosphate↗

PubMed1997

Continuous enzymatic assay for phosphorylase kinase in a monocascade enzyme system.

A turbidimetric method for continuous monitoring of the enzymatic reaction catalyzed by rabbit skeletal muscle phosphorylase kinase has been developed. The reaction mixture contained the substrates of glycogen phosphorylase a, i.e., glycogen and glucose 1-phosphate (or P(i)), in addition to the usual components of the kinase reaction. The kinetics of the cascade enzyme system were followed by the change in glycogen concentration over time, as measured by the absorbance of the reaction medium at 360 nm. The reliability of this turbidimetric method for measuring phosphorylase kinase activity was proven by comparison with a commonly used radiochemical assay. We present here a newly developed method for calculating the initial rate of phosphorylase kinase reaction in our conjugated system. We demonstrate that our procedure is applicable for investigating the hysteretic properties of phosphorylase kinase.

Animals↗

PubMed1986

Formation of partially phosphorylated phosphorylase in isoproterenol stimulated rat hearts.

Phosphorylase ab hybrid was demonstrated in perfused rat hearts and during the in vitro conversion of purified rat heart phosphorylase b. Phosphorylase ab hybrid was determined in rat heart extracts by the activating effect of AMP in the presence of caffeine. These results were confirmed by the quantitative determination of incorporated 32P in vitro and through the characteristic inhibition of ab hybrid by glucose-6-phosphate. As shown by our results, in aerobically perfused control hearts only the ab hybrid represents the active form of phosphorylase, its activity reaching about 20% of the total. In response to isoproterenol (5-1000 ng), the amount of ab hybrid rose to about 30-40%, preceding the rise of the a form, which increased in a dose-dependent manner up to 45% of the total. The great sensitivity of the ab form to AMP activation and glucose-6-phosphate inhibition supports its physiological significance in heart under in vivo conditions as well. Our results strongly suggest that the activity ratio -AMP/ + AMP reflects rather the percentage ratio of phosphorylated subunits than that of the activated (partially or totally phosphorylated) phosphorylase molecules.

Adenosine Monophosphate↗

PubMed1991

Phosphorylase kinase: development of a continuous fluorometric assay for the determination of catalytic activity.

The preferential binding of 1-anilinonaphthalene-8-sulfonate by rabbit muscle phosphorylase a is the basis of a continuous fluorometric assay for phosphorylase kinase. The maximum rate of change in fluorescence (d delta F/dt) is dependent on both the concentration of phosphorylase kinase and on conditions, such as pH and calcium ion concentration, which affect the enzyme. Parallel measurements of the increases in fluorescence and of 32P incorporation demonstrate the existence of a distinct intermediate in the conversion of phosphorylase b to a. We have used the assay to monitor the increase in calcium-independent activity which accompanies the limited chymotryptic digestion of phosphorylase kinase.

Anilino Naphthalenesulfonates↗

PubMed1986

Phosphorylase a is an allosteric inhibitor of the glycogen and microsomal forms of rat hepatic protein phosphatase-1.

The dephosphorylation of glycogen synthase by protein phosphatase-1 in hepatic glycogen and microsomes was inhibited by nanomolar concentrations of phosphorylase a. The I50 for phosphorylase a was 1000-fold lower than its Km as a substrate, while tryptic digestion increased the I50 1000-fold without affecting Km. Protein phosphatase-1 from skeletal muscle and protein phosphatase-2A from liver were only inhibited at 1000-fold higher concentrations. Protein phosphatase-1 became desensitized to phosphorylase a when released from hepatic microsomes, but sensitivity was partially restored by readdition of the solubilized enzyme to the microsomes. The results demonstrate that phosphorylase a is a potent allosteric inhibitor of hepatic protein phosphatase-1 and suggest that inhibition may be conferred by a novel phosphorylase a-binding subunit.

Allosteric Regulation↗

PubMed1983

Interaction of calmodulin and glycogen phosphorylase.

We have demonstrated the interaction of 125I-labeled calmodulin with glycogen phosphorylase by four techniques: polyacrylamide gel overlay, sucrose density centrifugation, gel filtration chromatography, and affinity chromatography. Phosphorylase b has more affinity for calmodulin than does phosphorylase a. Under all conditions tested, the presence of calmodulin affects neither the enzymatic activity nor any kinetic characteristics of phosphorylase a or b. We present these results as evidence that while binding between calmodulin and phosphorylase clearly exists, it may not have a physiological role.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1990

Binary affinity chromatography for the purification of glycogen phosphorylase.

A binary affinity chromatography medium was prepared and found to be useful for the purification and quantitative isolation of glycogen phosphorylase from rabbit skeletal muscle and liver. Glycogen is used as the binary ligand as it has affinity toward both the column matrix and the enzyme. Agarose beads derivatized with concanavalin A bound glycogen to the level of 35 mg/ml. The glycogen-impregnated beads were able to bind 9 mg/ml of phosphorylase a or b. The phosphorylase is tightly bound so that the column can be washed free of contaminants before quantitative elution of the phosphorylase by 2 M glucose, which releases the glycogen-phosphorylase complex. It appears that binary affinity chromatography may have general utility for the isolation and purification of enzymes and other specific binding agents.

Animals↗

PubMed1983

Acidotic depression of cyclic AMP accumulation and phosphorylase b to a transformation in skeletal muscle of man.

Intravenous infusion of adrenaline was performed in three healthy subjects on two occasions. In one case subjects performed a maximal isometric contraction before infusion. Biopsies were taken from the quadriceps femoris muscle before and after infusion for 0.5 and 2 min, and analysed for muscle pH, cyclic AMP, metabolites and activities of glycogen phosphorylase and synthetase. Isometric contraction resulted in a decrease of muscle pH to 6.60 (normal value at rest 7.0-7.1). By this experimental procedure the effect of adrenaline infusion could be studied on a muscle with normal pH and one with low pH. Cyclic AMP increased from 3 to about 9.5 mumol per kg dry weight after 0.5 min of adrenaline infusion. When isometric contraction preceded the infusion, cyclic AMP increased more slowly and was about 5.5 mumol per kg dry weight after the same time of infusion. Phosphorylase a constituted about 22% of total phosphorylase in resting muscle but increased rapidly to 80% after 0.5 min infusion. When exercise preceded infusion phosphorylase a decreased and was still lower after 2 min infusion. The results can be explained by inhibition of adenylcyclase and phosphorylase b kinase at low muscle pH.

Acid-Base Equilibrium↗

PubMed2003

Contraction-mediated glycogenolysis in mouse skeletal muscle lacking creatine kinase: the role of phosphorylase b activation.

Skeletal muscle that is deficient in creatine kinase (CK-/-) exhibits accelerated glycogenolysis during contraction. Understanding this phenomenon could provide insight into the control of glycogenolysis during contraction. Therefore, glycogen breakdown was investigated in isolated extensor digitorum longus CK-/- muscle. Muscles were stimulated to produce repeated tetani for 20 s in the presence of sodium cyanide to block mitochondrial respiration. Accumulation of lactate after stimulation was similar in wild-type (WT) and CK-/- muscles, whereas accumulation of glucose-6-phosphate was twofold higher in CK-/- muscles, indicating greater glycogenolysis in CK-/- muscles. Total phosphorylase activity was decreased by almost 30 % in CK-/- muscle (P < 0.001). Phosphorylase fractional activity (-/+ 3.3 mM AMP) was similar in both groups in the basal state (about 10 %), but increased to a smaller extent in CK-/- muscles after stimulation (39 +/- 4 % vs. 52 +/- 4 % in WT, P < 0.05). Inorganic phosphate, the substrate for phosphorylase, increased marginally in CK-/- muscles after stimulation (basal = 25.3 +/- 2.2 micromol (g dry muscle)-1; stimulated = 33.9 +/- 2.3 micromol (g dry muscle)-1), but substantially in WT muscles (basal = 11.4 +/- 0.7 micromol (g dry muscle)-1; stimulated = 54.2 +/- 4.5 micromol (g dry muscle)-1). Kinetic studies of phosphorylase b (dephosphorylated enzyme) from muscle extracts in vitro demonstrated higher relative activities in CK-/- muscles (60-135 %) in response to low AMP concentrations (up to 50 microM) in both the basal state and after stimulation (P < 0.05), whereas no differences in activity between CK-/- and WT muscles were observed at high AMP concentrations (> 100 microM). These data indicate that allosteric activation of phosphorylase b accounts for the accelerated glycogenolysis in CK-/- muscle during contraction.

Adenosine Diphosphate↗

PubMed1979

Reversal of phosphorylase activation in muscle despite continued contractile activity.

During studies of the regulation of phosphorylase activity and glycogenolysis in contracting muscle, it was found that conversion of phosphorlyase beta to alpha is transient. Reversal of phosphorylase activation during both continuous and intermittent stimulation in the plantaris might, in part, have been due to development of fatigue. However, a complete reversal of phosphorylase activation was also evident within 5 min in the absence of fatigue in soleus muscles stimulated tetanically with 100-ms-long trains at a rate of 60/min. These muscles showed no significant decline in contractile force. Glycogen breakdown stopped in the soleus when phosphorylase reverted to the beta form, providing evidence that phosphorylase beta was not active. This lack of activity is probably explained by the finding that ATP and AMP concentrations changed little, while glucose 6-phosphate increased. Reversal of phosphorlyase activation soon after the onset of steady-state work may be a mechanism for conserving glycogen when the supply of other substrates is adequate to meet the muscles' energy needs.

Adenosine Monophosphate↗

PubMed2006

The experimental type 2 diabetes therapy glycogen phosphorylase inhibition can impair aerobic muscle function during prolonged contraction.

Glycogen phosphorylase inhibition represents a promising strategy to suppress inappropriate hepatic glucose output, while muscle glycogen is a major source of fuel during contraction. Glycogen phosphorylase inhibitors (GPi) currently being investigated for the treatment of type 2 diabetes do not demonstrate hepatic versus muscle glycogen phosphorylase isoform selectivity and may therefore impair patient aerobic exercise capabilities. Skeletal muscle energy metabolism and function are not impaired by GPi during high-intensity contraction in rat skeletal muscle; however, it is unknown whether glycogen phosphorylase inhibitors would impair function during prolonged lower-intensity contraction. Utilizing a novel red cell-perfused rodent gastrocnemius-plantaris-soleus system, muscle was pretreated for 60 min with either 3 micromol/l free drug GPi (n=8) or vehicle control (n=7). During 60 min of aerobic contraction, GPi treatment resulted in approximately 35% greater fatigue. Muscle glycogen phosphorylase a form (P<0.01) and maximal activity (P<0.01) were reduced in the GPi group, and postcontraction glycogen (121.8 +/- 16.1 vs. 168.3 +/- 8.5 mmol/kg dry muscle, P<0.05) was greater. Furthermore, lower muscle lactate efflux and glucose uptake (P<0.01), yet higher muscle Vo(2), support the conclusion that carbohydrate utilization was impaired during contraction. Our data provide new confirmation that muscle glycogen plays an essential role during submaximal contraction. Given the critical role of exercise prescription in the treatment of type 2 diabetes, it will be important to monitor endurance capacity during the clinical evaluation of nonselective GPi. Alternatively, greater effort should be devoted toward the discovery of hepatic-selective GPi, hepatic-specific drug delivery strategies, and/or alternative strategies for controlling excess hepatic glucose production in type 2 diabetes.

Animals↗