Glycogen phosphorylase of skeletal muscles

A review is given on the affinity modification of pyridoxal phosphate and AMP-binding sites as well as on the chemical modification of essential amino acid residues of phosphorylase (histidine residue of the substrate-binding site and cysteine residue of the coenzyme-binding site). The role of allosteric effectors (AMP and glucose-6-phosphate) and functionally important centers of the protein in conformational transitions of rabbit muscle phosphorylase b is discussed. The kinetic properties of rabbit and bovine muscle phosphorylase are compared. Bovine muscle phosphorylase is shown to be a partly phosphorylated form of the enzyme. Some peculiarities of the pH-dependence of kinetic behaviour of the hybrid form of the bovine muscle enzyme are discussed.     

Allosteric regulation of purine nucleoside phosphorylase.

Purine nucleoside phosphorylase (EC 2.4.2.1) from bovine spleen is allosterically regulated. With the substrate inosine the enzyme displayed complex kinetics: positive cooperativity vs inosine when this substrate was close to physiological concentrations, negative cooperativity at inosine concentrations greater than 60 microM, and substrate inhibition at inosine greater than 1 mM. No cooperativity was observed with the alternative substrate, guanosine. The activity of purine nucleoside phosphorylase toward the substrate inosine was sensitive to the presence of reducing thiols; oxidation caused a loss of cooperativity toward inosine, as well as a 10-fold decreased affinity for inosine. The enzyme also displayed negative cooperativity toward phosphate at physiological concentrations of Pi, but oxidation had no effect on either the affinity or cooperativity toward phosphate. The importance of reduced cysteines on the enzyme is thus specific for binding of the nucleoside substrate. The enzyme was modestly inhibited by the pyrimidine nucleotides CTP (Ki = 118 microM) and UTP (Ki = 164 microM), but showed greater sensitivity to 5-phosphoribosyl-1-pyrophosphate (Ki = 5.2 microM).     

Kinetic characterization of rabbit skeletal muscle phosphorylase ab hybrid

Phosphorylase ab was prepared in vitro by partial phosphorylation of rabbit skeletal muscle phosphorylase b and was isolated by DEAE-Sephacel chromatography. Its phosphorylated and non-phosphorylated subunits could not be distinguished by different affinity to substrates, activators or inhibitors, indicating their coordinated function. In the absence of nucleotide activators, the Km values for Pi and glucose-1-P were 28 mM and 18 mM, respectively. Activity in the presence of 16 mM glucose-1-P was doubled by 10(-4) M AMP or 10(-3) M IMP, mainly by lowering the Km for glucose-1-P. Half-maximum activation was exerted by 2 microM AMP or 0.1 mM IMP. Activation by these nucleotides showed no cooperativity. Glucose exerted competitive inhibition with respect to glucose-1-P, while for the inhibition by glucose-6-P an allosteric mechanism is suggested; the appropriate Ki values were 4.5 mM and 1.5 mM, respectively. The Hill coefficient for glucose-1-P binding was about 1.0, even in the presence of glucose (up to 10 mM), but 10 mM glucose-6-P lowered it to 0.47, indicating a negative heterotropic cooperativity. Effective regulation of the activity of phosphorylase ab by physiological concentrations of Pi, AMP, IMP and glucose-6-P suggests its metabolic control under in vivo condition.     

Isolation and properties of three forms of phosphorylase and phosphorylase kinase from human skeletal muscle]

Three forms of phosphorylase (I, II and III), two of which (I and II) were active in the presence of AMP and one (III) was active without AMP, were isolated from human skeletal muscles. The pI values for phosphorylases b(I) and b(II) were found to be identical (5.8-5.9). During chromatofocusing a low molecular weight protein (M(r) = 20-21 kDa, pI 4.8) was separated from phosphorylase b(II). This process was accompanied by an increase of the enzyme specific activity followed by its decline. During reconstitution of the complex the activity of phosphorylase b(II) returned to the initial level. Upon phosphorylation the amount of 32P incorporated into phosphorylase b(II) was 2 times as low as compared with rabbit phosphorylase b and human phosphorylase b(I). It may be supposed that in the human phosphorylase b(II) molecule one of the two subunits undergoes phosphorylation in vivo. This form of the enzyme is characterized by a greater affinity for glycogen and a lower sensitivity to allosteric effectors (AMP, glucose-6-phosphate, caffeine) compared with phosphorylase b(I). Thus, among the three phosphorylase forms obtained in this study, form b(II) is the most unusual one, since it is partly phosphorylated by phosphorylase kinase to form a complex with a low molecular weight protein which stabilizes its activity. A partially purified preparation of phosphorylase kinase was isolated from human skeletal muscles. The enzyme activity necessitates Ca2+ (c0.5 = 0.63 microM). At pH 6.8 the enzyme is activated by calmodulin (c0.5 = 15 microM). The enzyme activity ratio at pH 6.8/8.2 is equal to 0.18.     

Purification and characterization of a phosphoprotein phosphatase from bovine adrenal cortex.

A phosphoprotein phosphatase which is active against chemically phosphorylated protamine has been purified about 500-fold from bovine adrenal cortex. The enzyme has a pH optimum between 7.5 and 8.0, and has an apparent Km for phosphoprotamine of about 50 muM. The hydrolysis of phosphoprotamine is stimulated by salt, and by Mn2+. Hydrolysis of phosphoprotamine is inhibited by ATP, ADP, AMP, and Pi, but is not affected by AMP or cyclic GMP. The purified phosphoprotein phosphatase preparation also dephosphorylates p-nitrophenyl phosphate and phosphohistone, and catalyzes the inactivation of liver phosphorylase, the inactivation of muscle phosphorylase a (and its conversion to phosphorylase b), and the inactivation of muscle phosphorylase b kinase. Phosphatase activities against phosphoprotamine and muscle phosphorylase a copurify over the last three stages of purification. Phosphoprotamine inhibits phosphorylase phosphatase activity, and muscle phosphorylase a inhibits the dephosphorylation of phosphoprotamine. These results suggest that one enzyme possesses both phosphoprotamine phosphatase and phosphorylase phosphatase activities. The stimulation of phosphorylase phosphatase activity, but not of phosphoprotamine phosphatase activity, by caffeine and by glucose, suggests that the different activities of this phosphoprotein phosphatase may be regulated separately.     

Characteristics of the dephosphorylated form of phosphorylase purified from rat liver and measurement of its activity in crude liver preparations.

The phosphorylated form of liver glycogen phosphorylase (alpha-1,4-glucan : orthophosphate alpha-glucosyl-transferase, EC 2.4.1.1) (phosphorylase a) is active and easily measured while the dephosphorylated form (phosphorylase b), in contrast to the muscle enzyme, has been reported to be essentially inactive even in the presence of AMP. We have purified both forms of phosphorylase from rat liver and studied the characteristics of each. Phosphorylase b activity can be measured with our assay conditions. The phosphorylase b we obtained was stimulated by high concentrations of sulfate, and was a substrate for muscle phosphorylase kinase whereas phosphorylase a was inhibited by sulfate, and was a substrate for liver phosphorylase phosphatase. Substrate binding to phosphorylase b was poor (KM glycogen = 2.5 mM, glucose-1-P = 250 mM) compared to phosphorylase a (KM glycogen = 1.8 mM, KM glucose-1-P = 0.7 mM). Liver phosphorylase b was active in the absence of AMP. However, AMP lowered the KM for glucose-1-P to 80 mM for purified phosphorylase b and to 60 mM for the enzyme in crude extract (Ka = 0.5 mM). Using appropriate substrate, buffer and AMP concentrations, assay conditions have been developed which allow determination of phosphorylase a and 90% of the phosphorylase b activity in liver extracts. Interconversion of the two forms can be demonstrated in vivo (under acute stimulation) and in vitro with little change in total activity. A decrease in total phosphorylase activity has been observed after prolonged starvation and in diabetes.     

Regulation of glycogen phosphorylase. Role of the peptide region surrounding the phosphoserine residue in determining enzyme properties.

A phosphopeptide which contains 14 residues including phosphoserine and which is derived from the NH2-terminal region of skeletal muscle glycogen phosphorylase (Nolan, C., Novoa, W. B., Krebs, E. G., and Fischer, E. H. (1964) Biochemistry 3, 542-551) has been shown to induce the enzymic properties of phosphorylase a in phosphorylase b and b'. When phosphorylase b is incubated with the phosphorylated tetradecapeptide, the following changes occur: (1) the enzyme becomes partially catalytically active in the absence of AMP; (2) the allosteric interactions of the enzyme are altered, as evidenced by the fact that phosphorylase b does not bind AMP cooperatively, and is no longer inhibited by glucose-6-P; and (3) the enzyme, normally present as a dimer, associates to a tetramer. Phosphorylase b' is a modified form of phosphorylase in which the phosphorylation site has been removed by limited tryptic attack. In the presence of phosphopeptide, 86% of the total enzyme activity can be induced in the absence of AMP. The properties of phosphorylases b and b' with phosphopeptide, cited above, are all characteristics of the phosphonenzyme, phosphorylase a. In addition, evidence is presented that these effects are specific. They are not the result of the polycationic nature of the peptide since they cannot be duplicated by spermine, and the phosphate group must also be present for the peptide to effect changes on the enzyme.     

Yeast Glycogen Phosphorylase: Characterization of the Dimeric Form and Its Activation

The purification of yeast glycogen phosphorylase [EC 2.4.1.1] was improved by ethanol precipitation and affinity chromatography on a glycogen-Sepharose column. The purified enzyme gave a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and had a subunit molecular mass of 100 kDa. Gel electrophoresis also showed that the major activity of native phosphorylase was ascribed to a dimer of 203 kDa, which was agreed with the value obtained by gel filtration on Sephadex G-200. The yeast phosphorylase showed a high affinity for AMP- Sepharose, whereas the enzyme was specifically inhibited by AMP. This inhibition was competitive with respect to the substrate glucose 1-phosphate and gave a Ki value of 9.3 mm. Activation of the crude extract by phosphorylation with an endogenous phosphorylase kinase indicated that the yeast phosphorylase occurred in a mixture of phosphorylated and non-phosphorylated forms.     

Investigation of myosin substrate-binding site using phosphorylating analogs of the substrate

Affinity modification of HMM has been performed, using mixed anhydrides of AMP, epsilon AMP, ADP or IMP and sterically hindered aromatic carbonic acids. The affinity labelling site of HMM was demonstrated to be highly specific towards the adenosine fragment of the affinity analog. The number of phosphate groups and the hydrophobicity of the aromatic acid substitutents did not influence the mode of the analogs interaction with HMM. The data obtained confirms our previous suggestion that the nucleotide analogs in question modify the substrate binding site which is other than the active site of the enzyme.     

The effect of solvents on nucleotide regulation of glycogen phosphorylase.

The activity of glycogen phosphorylase is controlled by two nucleotide sites. We have found that organic solvents affect the regulatory properties of phosphorylase by altering the binding at these two sites. At the activator site, the Ka for AMP is lowered 10-fold in the presence of 10% 1,2-dimethoxyethane while, at the inhibitor site, the Ki for caffeine is increased 6-fold. The stimulation of activity by organic solvents is highly dependent on the enzyme's activity state. Phosphorylase b, which has a requirement for a nucleotide activator, loses this requirement in the presence of organic solvents, while the active form of the enzyme, phosphorylase a, is only slightly stimulated by organic solvents. The activation profile obtained with rabbit liver phosphorylase suggests that differences in the properties of this enzyme from rabbit muscle phosphorylase might be explained by a change in the relative affinity for AMP at the two nucleotide sites. The results also suggest that 1,2-dimethoxyethane may be useful to determine accurately the activities of different forms of liver phosphorylase.     

Thermodynamics of nucleotides binding to phosphorylase b.

The effects of several chemical modifications in the AMP molecule on its interaction with phosphorylase b are examined by microcalorimetry, equilibrium dialysis, light scattering and ultracentrifuge experiments. In this work we report the results obtained for eight AMP analogues corresponding to different substituents in the puric base or in the ribose, or to different positions of the phosphate. The thermodynamic properties of the interaction between the phosphorylase b and the above mentioned nucleotides are also reported. The following conclusions were obtained: a) Except for IMP and 2'dIMP all the nucleotides studied clearly show two types of binding sites in the enzyme. b) The interaction of the nucleotide with its weaker affinity binding site is highly dependent upon chemical alterations in the puric base. c) Both the amino group in C(6) and the N(1) of the adenine in the AMP seem to play an important role in the conformational transitions induced by the nucleotide on the enzyme. d) The tetramerization of phosphorylase b in the presence of 10(-2) M AMP and in the conditions of the ultracentrifuge experiments is drastically affected by modifications in the ribose-phosphate part of the AMP molecule.     

Human liver glycogen phosphorylase. Kinetic properties and assay in biopsy specimens.

1.The two forms of glycogen phosphorylase were purified from human liver, and some kinetic properties were examined in the direction of glycogen synthesis. The b form has a limited catalytic capacity, resembling that of the rabbit liver enzyme. It is characterized by a low affinity for glucose 1-phosphate, which is unaffected by AMP, and a low V, which becomes equal to that of the a form in the presence of the nucleotide. Lyotropic anions stimulate phosphorylase b and inhibit phosphorylase a by modifying the affinity for glucose 1-phosphate. Both enzyme forms are easily saturated with glycogen. 2. These kinetic properties have allowed us to design a simple assay method for total (a + b) phosphorylase in human liver. It requires only 0.5 mg of tissue, and its average efficiency is 90% when the enzyme is predominantly in the b form. 3. The assay of total phosphorylase allows the unequivocal diagnosis of hepatic glycogen-storage disease caused by phosphorylase deficiency. One patient with a complete deficiency is reported. 4. The assay of human liver phosphorylase a is based on the preferential inhibition of the b form by caffeine. The a form displays the same activity when measured by either of the two assays.     

Effect of osmolytes on the interaction of flavin adenine dinucleotide with muscle glycogen phosphorylase b

The effect of three osmolytes, trimethylamine N-oxide (TMAO), betaine and proline, on the interaction of muscle glycogen phosphorylase b with allosteric inhibitor FAD has been examined. In the absence of osmolyte, the interaction is described by a single intrinsic dissociation constant (17.8 microM) for two equivalent and independent binding sites on the dimeric enzyme. However, the addition of osmolytes gives rise to sigmoidal dependencies of fractional enzyme-site saturation upon free inhibitor concentration. The source of this cooperativity has been shown by difference sedimentation velocity to be an osmolyte-mediated isomerization of phosphorylase b to a smaller dimeric state with decreased affinity for FAD. These results thus have substantiated a previous inference that the tendency for osmolyte-enhanced self-association of dimeric glycogen phosphorylase b in the presence of AMP was being countered by the corresponding effect of molecular crowding on an isomerization of dimer to a smaller, nonassociating state.     

Allosteric effectors and trehalose protect larval Manduca sexta fat body glycogen phosphorylase B against thermal denaturation

In this paper we assessed the ability of modulators of the activity of glycogen phosphorylase b from the fat body of larval Manduca sexta to stabilize the enzyme against thermal denaturation. This approach has allowed us to distinguish between modulators that stabilize the enzyme, presumably through some conformational effect, from those that do not affect thermal stability. For example, 5'-AMP and 5'-IMP are both positive modulators of the enzyme and the K(m)s for AMP and IMP were similar, 0.71 and 1.09 mM, respectively. However, the V(max) for AMP (123 nmol/mg/min) was 10 times higher than the value found for IMP (12.5 nmol/mg/min) and AMP increased the thermal stability of glycogen phosphorylase b, however IMP did not increase the enzyme's thermal stability. Indeed, IMP decreased both the allosteric activation of the enzyme by AMP and the thermal protection conferred by AMP. The allosteric inhibitors ADP and ATP, which in vertebrate phosphorylase bind to the same site as AMP, both increased the thermal stability of the enzyme, however with less efficiency than AMP. Inorganic phosphate increased thermal stability, but glycogen and amylose did not. Glycerol, at 600 mM, protected the enzyme against thermal inactivation, whereas sorbitol at the same concentration did not show any effect. Among the polyols tested, trehalose was the most effective in conferring thermal stability. In fact, in the presence of 20 mM AMP and 600 mM trehalose, 90% of the enzyme activity remained after 20 min at 60 degrees C.     

Modification of essential carboxyl group in rabbit muscle phosphorylase by water-soluble carbodiimide

Water-soluble carbodiimide (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (EDC) and glycine ethyl ester (GEE) as a nucleophile were used to modify the essential carboxyl group of phosphorylases. The inactive b form of the muscle phosphorylase was modified faster than the active a form and potato phosphorylases. Use of N,N,N',N'-tetramethyl-ethylenediamine (TEMED)-HCl buffer system (pH 6.2) resulted in a remarkable difference from the previous results obtained with phosphate and beta-glycerophosphate buffer systems. That is, the substrate glucose 1-phosphate gave the best protection of the three phosphorylase activities. Glucose and glycogen were also effective to retard the inactivation of muscle phosphorylases, though glycogen was not effective for the potato enzyme. The EDC-GEE-modified phosphorylase b retained the affinity for AMP-Sepharose, though partially modified enzyme completely lost the homotropic cooperativity. Phosphorylase b was subjected to differential labeling with [14C]GEE. A labeled peptide was obtained after CNBr cleavage and peptic digestions, and corresponded to the catalytic site sequence surrounding the GEE-substituted Asp 661 and Glu 664. Either or both of these EDC-modified carboxyl residues may have an important role in the catalytic reaction.     

Relation between Mg2+ activation and AMP inhibition of fructose-1,6-diphosphatase from rabbit skeletal muscles

It was shown that AMP, an allosteric inhibitor of fructose-1.6-bisphosphatase, decreases the apparent affinity of the enzyme for the activating cation, Mg2+, which is accompanied by a decrease of the kinetic cooperativity between the Mg2+-binding sites. In its turn, the Mg2+ increase diminishes the enzyme sensitivity to the inhibiting effect of AMP and decreases the cooperativity of the inhibitor binding. The heterotropic interactions between the allosteric inhibitor and activator binding centers are consistent with the predictions of the Monod-Wyman-Changeux model which involves two conformational states of the enzyme (of which one is catalytically inactive) differing in their affinity for the ligands. An increase in pH from 7.4 to 9.0 increases the enzyme affinity for Mg2+ and causes an equilibrium shift towards the catalytically active state of the enzyme.     

Purine nucleoside phosphorylase. Allosteric regulation of a dissociating enzyme

Purine nucleoside phosphorylase (EC 2.4.2.1) from bovine spleen is a trimeric enzyme that readily dissociates to the monomer. Dilution of enzyme from 20 to 0.02 microgram of protein/ml is accompanied by a greater than 50-fold increase in the specific activity (vtrimer = 0.23 nmol/min/microgram; vmonomer = 12.5 nmol/min/micrograms). Gel permeation chromatography in the presence of the substrate phosphate shows the enzyme to be predominantly trimeric at 50 mM Pi and predominantly monomeric at 100 mM Pi, when experiments are done at 24 degrees C. No significant dissociation was observed at 4 degrees C with Pi or at either temperature with the substrate inosine. As measured by dissociation, the L0.5 for Pi is 88 mM and thus significantly higher than the Km of 3.1 mM for Pi. Enzyme activity as a function of phosphate concentration showed negative cooperativity, but the conformational response measured by the change in native Mr during dissociation showed positive cooperatively toward Pi. These data support a model for two separate phosphate binding sites on the enzyme. The activity and stability of purine nucleoside phosphorylase are quite sensitive to the concentration of the enzyme as well as appropriate substrates. Although the monomer is interpreted as being a fully active form of the enzyme, the data in general are most consistent with the enzyme functioning in vivo as a regulated trimer.     

Fructose 2,6-bisphosphate and AMP increase the affinity of the Ascaris suum phosphofructokinase for fructose 6-phosphate in a process separate from the relief of ATP inhibition

Kinetic data have been collected suggesting that heterotropic activation by fructose 2,6-bisphosphate and AMP is a result not only of the relief of allosteric inhibition by ATP but is also the result of an increase in the affinity of phosphofructokinase for fructose 6-phosphate. Modification of the Ascaris suum phosphofructokinase at the ATP inhibitory site produces a form of the enzyme that no longer has hysteretic time courses or homotropic positive (fructose 6-phosphate) cooperativity or substrate inhibition (ATP) (Rao, G.S. J., Wariso, B.A., Cook, P.F., Hofer, H.W., and Harris, B.G. (1987a) J. Biol. Chem. 262, 14068-14073). This form of phosphofructokinase is Michaelis-Menten in its kinetic behavior but is still activated by fructose 2,6-bisphosphate and AMP and by phosphorylation using the catalytic subunit of cyclic AMP-dependent protein kinase (cAPK). Fructose 2,6-bisphosphate activates by decreasing KF-6-P by about 15-fold and has an activation constant of 92 nM, while AMP decreases KF-6-P about 6-fold and has an activation constant of 93 microM. Double activation experiments suggest that fructose 2,6-bisphosphate and AMP are synergistic in their activation. The desensitized form of the enzyme is phosphorylated by cAPK and has an increased affinity for fructose 6-phosphate in the absence of MgATP. The increased affinity results in a change in the order of addition of reactants from that with MgATP adding first for the nonphosphorylated enzyme to addition of fructose 6-phosphate first for the phosphorylated enzyme. The phosphorylated form of the enzyme is also still activated by fructose 2,6-bisphosphate and AMP.     

Kinetic study on the dimer-tetramer interconversion of glycogen phosphorylase a.

Kinetic theory of dissociating enzyme systems has been applied to a study of the dimer-tetramer interconversion of glycogen phosphorylase a. All kinetic constants for the dissociating-associating reaction of phosphorylase a have been determined. The results indicate that (a) the presence of glucose-1-phosphate has no influence on either the rate of dissociation or the rate of association, and hence does not shift the dimer-tetramer equilibrium of phosphorylase a; (b) the binding og glycogen to the enzyme decreases the association rate of the dimer to form the tetramer, but has no effect on the dissociation rate of the tetramer; (c) both the dimeric and tetrameric form of phosphorylase a can bind glycogen, but the tetrameric form has a lower affinity for glycogen and is catalytically inactive.     

A novel human phosphotransferase highly specific for adenosine.

A novel nucleoside phosphotransferase, referred to as adenosine phosphotransferase (Ado Ptase), was partially purified 1230-fold from human placenta. This enzyme differed from other known nucleoside phosphotransferases in its substrate specificity. Using AMP as the phosphate donor, it readily phosphorylated Ado. Changes in the sugar moiety were tolerated. dAdo and ddAdo were phosphate acceptors and dAMP was a donor. No other nucleotide or nucleoside common in nature displayed appreciable activity as donor or acceptor substrate, respectively. In the absence of nucleoside, the enzyme catalyzed the hydrolysis of AMP, typical of other nucleoside phosphotransferases. However, in the presence of Ado, little, if any, hydrolysis occurred. Ado Ptase had an absolute requirement for a metal cation, with Mg2+ and, to a lesser extent, Mn2+ fulfilling this requisite. The apparent Km for Ado was 0.2 mM. However, the donor AMP displayed cooperativity in both transfer and hydrolytic reactions. This cooperativity was eliminated by nucleotides, 2,3-diphosphoglycerate, and inorganic phosphate. ADP and 2,3-diphosphoglycerate were especially potent. In the presence of these effectors, the apparent Km for AMP was 3.0 mM in the transfer reaction and 4.0 mM in the hydrolytic reaction. Kinetic data suggest that there are two nucleotide binding sites on Ado Ptase, one for the donor, the other for an effector. AMP appeared to bind to both sites. Although this novel enzyme might play a role in the anabolism of nucleoside analogues, the normal physiological role of this nucleoside phosphotransferase is not understood.