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PubMed1986

Phosphorylase kinase from chicken skeletal muscle. Quaternary structure, regulatory properties and partial proteolysis.

Phosphorylase kinase has been purified from white and red chicken skeletal muscle to near homogeneity, as judged by sodium dodecyl sulphate (SDS) gel electrophoresis. The molecular mass of the native enzyme, estimated by chromatography on Sepharose 4B, is similar to that of rabbit skeletal muscle phosphorylase kinase, i.e. 1320 kDa. The purified enzyme both from white and red muscles showed four subunits upon polyacrylamide gel electrophoresis in the presence of SDS, corresponding to alpha', beta, gamma' and delta with molecular masses of 140 kDa, 129 kDa, 44 kDa and 17 kDa respectively. Based on the molecular mass of 1320 kDa for the native enzyme and on the molar ratio of subunits as estimated from densitometric tracings of the polyacrylamide gels, a subunit formula (alpha' beta gamma' delta)4 has been proposed. The antiserum against the mixture of the alpha' and beta subunits of chicken phosphorylase kinase gave a single precipitin line with the chicken enzyme but did not cross-react with the rabbit skeletal muscle phosphorylase kinase. The pH 6.8/8.2 activity ratio of phosphorylase kinase from chicken skeletal muscle varied from 0.3 to 0.5 for different preparations of the enzyme. Chicken phosphorylase kinase could utilize rabbit phosphorylase b as a substrate with an apparent Km value of 0.02 mM at pH 8.2. The apparent V (18 mumol min-1 mg-1) and Km values for ATP at pH 8.2 (0.20 mM) were of the same order of magnitude as that of the purified rabbit phosphorylase kinase b. The activity of chicken phosphorylase kinase was largely dependent on Ca2+. The chicken enzyme was activated 2-4-fold by calmodulin and troponin C, with concentrations for half-maximal activation of 2 nM and 0.1 microM respectively. Phosphorylation with the catalytic subunit of cAMP-dependent protein kinase (up to 2 mol 32P/mol alpha beta gamma delta monomer) and autophosphorylation (up to 8 mol 32P/mol alpha beta gamma delta monomer) increased the activity 1.5-fold and 2-fold respectively. Limited tryptic and chymotryptic hydrolysis of chicken phosphorylase kinase stimulated its activity 2-fold. Electrophoretic analysis of the products of proteolytic attack suggests some differences in the structure of the rabbit and chicken gamma subunits and some similarities in the structure of the rabbit red muscle and chicken alpha'.

Adenosine Diphosphate↗

PubMed1988

Purification and properties of inosine-guanosine phosphorylase from Escherichia coli K-12.

A xanthosine-inducible enzyme, inosine-guanosine phosphorylase, has been partially purified from a strain of Escherichia coli K-12 lacking the deo-encoded purine nucleoside phosphorylase. Inosine-guanosine phosphorylase had a particle weight of 180 kilodaltons and was rapidly inactivated by p-chloromercuriphenylsulfonic acid (p-CMB). The enzyme was not protected from inactivation by inosine (Ino), 2'-deoxyinosine (dIno), hypoxanthine (Hyp), Pi, or alpha-D-ribose-1-phosphate (Rib-1-P). Incubating the inactive enzyme with dithiothreitol restored the catalytic activity. Reaction with p-CMB did not affect the particle weight. Inosine-guanosine phosphorylase was more sensitive to thermal inactivation than purine nucleoside phosphorylase. The half-life determined at 45 degrees C between pH 5 and 8 was 5 to 9 min. Phosphate (20 mM) stabilized the enzyme to thermal inactivation, while Ino (1 mM), dIno (1 mM), xanthosine (Xao) (1 mM), Rib-1-P (2 mM), or Hyp (0.05 mM) had no effect. However, Hyp at 1 mM did stabilize the enzyme. In addition, the combination of Pi (20 mM) and Hyp (0.05 mM) stabilized this enzyme to a greater extent than did Pi alone. Apparent activation energies of 11.5 kcal/mol and 7.9 kcal/mol were determined in the phosphorolytic and synthetic direction, respectively. The pH dependence of Ino cleavage or synthesis did not vary between 6 and 8. The substrate specificity, listed in decreasing order of efficiency (V/Km), was: 2'-deoxyguanosine, dIno, guanosine, Xao, Ino, 5'-dIno, and 2',3'-dideoxyinosine. Inosine-guanosine phosphorylase differed from the deo operon-encoded purine nucleoside phosphorylase in that neither adenosine, 2'-deoxyadenosine, nor hypoxanthine arabinoside were substrates or potent inhibitors. Moreover, the E. coli inosine-guanosine phosphorylase was antigenically distinct from the purine nucleoside phosphorylase since it did not react with any of 14 monoclonal antisera or a polyvalent antiserum raised against deo-encoded purine nucleoside phosphorylase.

Escherichia coli↗

PubMed1989

Glycogen phosphorylase activation by two different alpha 1-adrenergic receptor subtypes: methoxamine selectively stimulates a putative alpha 1-adrenergic receptor subtype (alpha 1a) that couples with Ca2+ influx.

We compared the effects of methoxamine on alpha 1-adrenergic receptor-mediated phosphorylase activation in rat hepatocytes and rabbit aorta. Although methoxamine is a potent agonist in activating phosphorylase of rabbit aorta, it had little effect in rat hepatocytes. Using the phenoxybenzamine inactivation method, we found that the quantitative relationship between 125I-BE2254 (125I-BE) binding capacity and maximal norepinephrine-stimulated phosphorylase activation was nonlinear in rabbit aorta, whereas it was linear in rat hepatocytes. The potency of methoxamine in inhibiting specific 125I-BE binding is significantly (p less than 0.05) higher in rabbit aorta (Kd, 96.4 +/- 7.7 microM), compared with rat hepatocytes (Kd, 283 +/- 16 microM). However, these quantitative differences could not fully explain the blunted [Ca2+]c and phosphorylase responses to methoxamine in rat hepatocytes. Treatment with chlorethylclonidine dose dependently suppressed 125I-BE binding sites and norepinephrine-induced phosphorylase activation in rat hepatocytes, whereas in rabbit aorta it resulted in only a 31% decrease in 125I-BE binding sites, with little effect on phosphorylase activation. Furthermore, alpha 1-adrenergic receptor-mediated cellular events of phosphatidylinositol (PI) hydrolysis and phosphorylase activation were unaffected by the removal of extracellular Ca2+ in rat hepatocytes, whereas both responses were markedly attenuated in rabbit aorta. The results indicate that two different alpha 1-adrenergic receptor subtypes activate glycogen phosphorylase, through different mechanisms for increasing [Ca2+]c in the two systems. In rat hepatocytes, alpha 1 receptors are closely linked to PI hydrolysis and Ca2+ release from intracellular stores and cause phosphorylase activation. In rabbit aorta, on the other hand, activation of alpha 1 receptors increases [Ca2+]c by Ca2+ influx from the extracellular fluid as well as by Ca2+ release, and both PI hydrolysis and phosphorylase activation are caused mainly by the Ca2+ entry. Methoxamine interacts with both chlorethylclonidine-sensitive and -insensitive alpha 1 receptor subtypes but selectively stimulates the alpha 1 receptor subtype that closely couples with the Ca2+ influx.

Animals↗

PubMed1977

Studies on alpha-adrenergic activation of hepatic glucose output. Studies on role of calcium in alpha-adrenergic activation of phosphorylase.

The role of Ca2+ ions in alpha-adrenergic activation of hepatic phosphorylase was studied using isolated rat liver parenchymal cells. The activation of glucose release and phosphorylase by the alpha-adrenergic agonist phenylephrine was impaired in cells in which calcium was depleted by ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA) treatment and restored by calcium addition, whereas the effects of a glycogenolytically equivalent concentration of glucagon on these processes were unaffected. EGTA treatment also reduced basal glucose release and phosphorylase alpha activity, but did not alter the level of cAMP or the protein kinase activity ratio (-cAMP/+cAMP) or impair viability as determined by trypan blue exclusion, ATP levels, or gluconeogenic rates. The effect of EGTA on basal phosphorylase and glucose output was also rapidly reversed by Ca2+, but not by other ions. Phenylephrine potentiated the ability of low concentrations of calcium to reactivate phosphorylase in EGTA-treated cells. The divalent cation inophore A23187 rapidly increased phosphorylase alpha and glucose output without altering the cAMP level, the protein kinase activity ratio, and the levels of ATP, ADP, or AMP, The effects of the ionophore were abolished in EGTA-treated cells and restored by calcium addition. Phenylephrine rapidly stimulated 45Ca uptake and exchange in hepatocytes, but did not affect the cell content of 45Ca at late time points. A glycogenolytically equivalent concentration of glucagon did not affect these processes, whereas higher concentrations were as effective as phenylephrine. The effect of phenylephrine on 45Ca uptake was blocked by the alpha-adrenergic antagonist phenoxybenzamine, was unaffected by the beta blocker propranolol, and was not mimicked by isoproterenol. The following conclusions are drawn: (a) alpha-adrenergic activation of phosphorylase and glucose release in hepatocytes is more dependent on calcium than is glucagon activation of these processes; (b) variations in liver cell calcium can regulate phosphorylase alpha levels and glycogenolysis; (c) calcium fluxes across the plasma membrane are stimulated more by phenylephrine than by a glycogenolytically equivalent concentration of glucagon. It is proposed that alpha-adrenergic agonists activate phosphorylase by increasing the cytosolic concentration of Ca2+ ions, thus stimulating phosphorylase kinase.

Animals↗

PubMed1993

Initiation of glycogen synthesis. Control of glycogenin by glycogen phosphorylase.

Glycogen biosynthesis involves a specific initiation event, mediated by a specialized protein, glycogenin. Glycogenin undergoes self-glucosylation to generate an oligosaccharide primer, which, when long enough, supports the action of glycogen synthase to elongate the polysaccharide chain, leading ultimately to the formation of glycogen. We report that primed glycogenin is also a substrate for glycogen phosphorylase. Phosphorylase removed glucose from the oligosaccharide attached to glycogenin in a phosphorolysis reaction that required phosphate and produced glucose 1-phosphate. The phosphorylated form, phosphorylase a, was much more effective than the dephosphorylated phosphorylase b. However, in the presence of the allosteric effector AMP, phosphorylase b also catalyzed the phosphorolysis reaction. Glucose, an allosteric inhibitor of phosphorylase, inhibited the reaction. Glycogen, but not a short oligosaccharide (maltopentaose), also inhibited the reaction. Treatment of fully primed glycogenin with phosphorylase converted the glycogenin to a form with slightly lower apparent molecular weight, which was less effective as a substrate for glycogen synthase. These results suggest a novel role for phosphorylase in the control of glycogen biosynthesis. We propose that the glucosylation level of glycogenin would be determined by the balance between the self-glucosylation reaction and the opposing action of phosphorylase. The level of glucosylation would in turn determine whether or not glycogenin was an effective primer for glycogen synthase. In this way, several known controls of phosphorylase activity, such as epinephrine, glucagon, and insulin, could influence not only the elongation/degradation stage of glycogen metabolism but also its initiation.

Animals↗

PubMed1982

Rabbit muscle phosphorylase derivatives with oligosaccharides covalently bound to the glycogen storage site.

Linear maltooligosaccharides, e.g., maltoheptaose or terminal 4-O-methylmaltoheptaose, activated by cyanogen bromide, react covalently with rabbit muscle phosphorylases b and a (EC 2.4.1.1). Site-specific modification prevents further binding to glycogen and shifts the phosphorylase a tetramer-dimer equilibrium in favor of the dimer. Use was made of these properties to separate by affinity chromatography and gel filtration phosphorylase a dimers with specifically bound oligosaccharide from unspecifically modified products. The phosphorylase a-maltoheptaose derivative carries one oligosaccharide residue per monomer and can be distinguished from the native enzyme by its electrophoretic mobility in polyacrylamide gels or by affinity electrophoresis. Phosphorylase a preparations with covalently bound maltooligosaccharides are enzymatically active in the presence of a primer and alpha-D-glucopyranose 1-phosphate (glucose-1-P). Methylation of the nonreducing chain terminus of the bound oligosaccharide has no effect on glycogen synthesis. These findings exclude the participation of bound oligosaccharides in chain elongation. Purified covalent phosphorylase a-maltoheptaose complexes are stable dimers. They are no longer activated by glycogen. The properties of covalently modified phosphorylase-oligosaccharides are consistent with and provide direct evidence for the existence of a glycogen storage site in rabbit muscle phosphorylases. Covalent occupation of the storage site renders the affinity of glucose-1-P to phosphorylase a independent of modulation by glycogen, supporting the assumption that the glycogen storage site is involved in interactions with the catalytic site.

Animals↗

PubMed1983

Effect of oxyanions of the early transition metals on rabbit skeletal muscle phosphorylase.

The differential effects of the oxyanions of the early transition metals ions V(V), W(VI), and Mo(VI) on the catalytic activity and coenzyme binding of rabbit skeletal muscle phosphorylase are studied. The oligoanions of V(V), W(VI), and Mo(VI) are potent inhibitors of phosphorylase. Kinetic studies revealed that oligovanadates inhibit pyridoxal-reconstituted phosphorylase b by competing with both the substrate, glucose 1-phosphate, and the activator, phosphite, with K1 values of 4 microM and 6 microM, respectively. Oligovanadates in the millimolar concentration range inhibit phosphorylases a and b by competing with glucose 1-phosphate binding. The polymeric decavanadate and paratungstates caused time-dependent inactivation of phosphorylase. Spectral studies with tungstate and phosphorylase b revealed that the inactivation is due to deformation of the coenzyme site. Kinetic studies and the protective effects of substrate and effectors on inactivation and deformation by tungstate or vanadate suggest that deformation and inactivation is caused by a primary binding of the oligoanions at the glucose 1-phosphate site. Nuclear magnetic resonance (NMR) studies of vanadate-phosphorylase complexes and vanadate solutions under different conditions were carried out to ascertain the nature of vanadate ions interacting with functional groups in phosphorylase. The results suggest that decavanadate is the major protein-bound species. NMR studies also showed that guanidino groups react with decavanadate and suggest that arginine residues in phosphorylase are potential functional groups that can interact with decavanadate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1978

The susceptibility of muscle phosphorylases a and b to digestion by a neutral proteinase from rat intestinal muscle. Comparison with the effects produced by pancreatic trypsin and chymotrypsin.

1. Phosphorylase b was inactivated three times more rapidly than phosphorylase a by a neutral, trypsin-like proteinase from rat intestinal muscle. Digestion of phosphorylase a produced a modified form which was deactivated by AMP. Removal of the pyridoxal phosphate cofactor increased the rate of inactivation of the b form by about 3-fold but the subceptibility of apophosphorylase a was no different from the holo form. 2. The extent of proteolysis of both holoenzyme forms, as guaged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, was limited and similar digestion patterns were obtained in both cases. 3. With (32)P-labelled phosphorylase a as substrate, the initial event in the inactivation was the release of a trichloroacetic acid-soluble peptide from the N-terminus of the enzyme, leaving the original 100000 subunit form essentially unchanged. Subsequent proteolysis was restricted, producing derivatives of mol.wt. 85000, 70000 and 65000, none of which contained any radioactive label. 4. By treatment of inactivated phosphorylase b with carboxypeptidase B, it was shown that the intestinal muscle proteinase had cleaved approximately 3 -Lys-X and 3 -Arg-X bonds in the polypeptide. 5. The protective effects of various allosteric modulators of phosphorylase on the inactivation of the a and b forms were generally in agreement with the known roles of the modifiers. Glucose increased the susceptibility of phosphorylase a. 6. Inactivation of phosphorylase b by trypsin and chymotrypsin also resulted in limited proteolysis but, in both cases, the digestion patterns obtained on sodium dodecyl sulphate/polyacrylamide gels were different from each other and from the pattern obtained with the intestinal muscle proteinase. 7. Inactivation of phosphorylase b by the muscle proteinase is about 100 times more rapid than the effects produced by trypsin or chymotrypsin when the activities are compared on an equimolar basis. 8. Consideration is given to regulation of the rate of enzyme degradation intracellularly by modulation of the conformation and susceptibility of the enzyme via factors such as covalent modification, allosteric ligands and state of aggregation.

Adenosine Monophosphate↗

PubMed1992

Glycogen phosphorylase isoenzymes from hepatoma 3924A and from a non-tumorigenic liver cell line. Comparison with the liver and brain enzymes.

Glycogen phosphorylase isoenzymes were isolated from normal rat liver, rat brain, the glycogen-poor Morris hepatoma (MH) 3924A, and the glycogen-rich non-tumorigenic liver cell line C1I. Electrophoretic and immunological characterization of the enzymes showed that tumour and C1I cells expressed a phosphorylase isoform similar to the brain type; the liver type was not detectable. All enzymes were obtained as dimers; the Mr of the subunits was 96,000 (liver), 93,000 (brain and MH 3924A) and 92,000 (C1I). Isoelectric focusing revealed a main band of pI 6.34 for liver phosphorylase a, pI 5.67 for the enzymes from MH 3924A and brain, and pI 5.68 for C1I phosphorylase. Partial kinetic characterization of the AMP-independent forms of the isoenzymes yielded Km values for glucose 1-phosphate of 3.5 +/- 0.5 mM (liver), 3.9 mM (brain), 1.9 +/- 0.3 mM (MH 3924A) and 2.5 +/- 0.5 mM (C1I); Km values for glycogen were 0.4 mM (liver) and 0.3 mM (MH 3924A and C1I), calculated as glucose equivalents. The AMP-independent phosphorylase was inhibited by glucose 6-phosphate (Glc6P) with Ki values of 0.32 +/- 0.03 mM (C1I), 0.50 +/- 0.04 mM (MH 3924A) and approximately 5 mM (brain). The inhibition could be abolished by 1 mM-AMP, indicating that AMP and Glc6P may partially compete for the same site on the protein. Liver phosphorylase a was not inhibited by up to 25 mM-Glc6P. In contrast with liver and brain isoenzymes, phosphorylase from the cell lines was not affected by NaF and Na2SO4. The data show that both the hepatocellular carcinoma and the non-malignant immortalized liver cells express a phosphorylase isoform different from the liver type. Furthermore, there is some evidence that the enzyme from MH 3924A and C1I cells is distinct from brain phosphorylase a, in spite of electrophoretic and immunological resemblance, and that this isoenzyme is subject to altered metabolic regulation.

Animals↗

PubMed1984

Dissociation of phosphorylase a activation and contractile activity in rat portal vein.

Isometric force, glycogen phosphorylase activity and lactate production were measured under conditions known to alter intracellular Ca2+ and cAMP to assess the role of these messengers in the coordination of metabolism with contractility in rat portal vein. Total phosphorylase (a + b) activity, was independent of treatment. The activity ratio phosphorylase activity ratio in the presence of isoproterenol and papaverine was dependent on or high-K+ medium, and 0.57 after 20 min treatment with 10(-5) M isoproterenol + 10(-4) M papaverine. Under both of these conditions the muscle was totally relaxed. The phosphorylase activity ratio in the presence of isoproterenol and papaverine was dependent on extracellular Ca2+, both in normal and depolarizing medium. This suggests a lower Ca2+ sensitivity of the contractile than the phosphorylase system under these conditions, known to be associated with raised intracellular cAMP. During spontaneous activity and high-K+ induced contractures phosphorylase activity was increased compared to the relaxed state in Ca2+-free medium. A high level of phosphorylase activity (0.48) was elicited by the addition of 300 mM sucrose, which induces a contracture in Ca2+-free medium. Lactate production was in general parallel to phosphorylase activity, except for a relative increase in anoxia. The results suggest that in the intact cell the Ca2+-mediated linkage of contraction and phosphorylase may be modified by cAMP changing the Ca2+ sensitivities of the two systems in opposite directions.

Animals↗

PubMed1978

Phosphorylase a in human skeletal muscle during exercise and electrical stimulation.

Experiments were conducted to examine the conversions of phosphorylase b to phosphorylase a in human skeletal muscle during bicycle exercise or isometric contractions. Muscle biopsies were obtained from the vastus lateralis with the needle technique at rest and either during or immediately after activity and frozen in liquid nitrogen within 2--4 s. Total phosphorylase and phosphorylase a activities were differentiated by measurement in the presence and absence of AMP, respectively. At rest 8.5% of the total phosphorylase activity existed in the a form. Little or no change in the percent of phosphorylase in the a form occurred during voluntary dynamic or static muscular activity that produced muscle lactate concentrations in excess of 18 mmol.kg-1 wet muscle. Electrical stimulation of the vastus lateralis muscle also failed to produce an increase in the percentage of phosphorylase a. These data suggest that during exercise the conversion of phosphorylase to the a form is of minor importance. An increased activity of phosphorylase b due to changes in muscle concentrations of ATP, AMP, and inorganic phosphate may regulate glycogenolysis during voluntary exercise in man.

Adenosine Monophosphate↗

PubMed1981

Rate dependence of isoproterenol-stimulated phosphorylase a formation in mouse heart.

In mouse right ventricular strips, field-stimulated to contract isometrically in an oxygenated bicarbonate-buffered physiological salt solution at 22--24 degrees C, isoproterenol (3 microM) stimulation of phosphorylase a accumulation was approximately a linear function of the rate of contraction from 0.2 to 5 Hz. In muscles incubated in the absence of isoproterenol, the phosphorylase a activity did not increase when the contraction frequency was varied over this range. At a low frequency of stimulation (0.2 Hz), phosphorylase a activity was not increased after a 5-min exposure to 3 microM isoproterenol, as compared to a 4-fold increase in phosphorylase a activity at a high frequency (3.-3 Hz). Isoproterenol (3 microM) increased tissue cyclic AMP content and the activated form of phosphorylase kinase activity to similar extents at both frequencies. N6,O2'-dibutyryl cyclic AMP increased the phosphorylase a activity at both frequencies but the increase at 3.3 Hz was approximately 3-fold greater than at 0.2 Hz. Verapamil did not block isoproterenol-stimulated phosphorylase a activity at 3.3 Hz at a concentration (0.6 microM) that inhibited the increased sensitivity to the inotropic action of isoproterenol seen at high frequencies of contraction. Isoproterenol stimulation of phosphorylase a accumulation did not correlate with developed tension. It is proposed that the difference in isoproterenol stimulation of phosphorylation b to a conversion at 0.2 and 3.3 Hz primarily results from a difference in Ca++ control of the activity of the activated form of phosphorylase kinase.

Animals↗

PubMed1980

Specificity of pyrimidine nucleoside phosphorylases and the phosphorolysis of 5-fluoro-2'-deoxyuridine.

Isoelectric focusing and studies with 1-(2'-deoxy-beta-D-glucopyranosyl)thymine (GPT), a specific inhibitor of uridine phosphorylase activity, were used to determine the substrate specificities of mammalian pyrimidine nucleoside phosphorylases and their cleavage of 5-fluoro-2'-deoxyuridine (FdUrd). Isoelectric focusing profiles for the cytosol fractions from Ehrlich ascites cells and from Novikoff hepatoma cells each consisted essentially of one peak of nucleoside phosphorylase activity [isoelectric points (pl) 5.4 and 5.8, respectively] that cleaved both uridine and thymidine (dThd), as well as FdUrd. By contrast, cytosol fractions from HeLa (S3) cells, mouse liver, and normal human leukocytes each exhibited a major peak of activity (pl 4.6, 6.5, and 4.9, respectively) that cleaved only dThd and FdUrd, while mouse liver exhibited a second peak (pl 5.2) that cleaved primarily uridine. To distinguish clearly between (a) uridine phosphorylases that cleave primarily uridine and that are inhibited by GPT and (b)dThd phosphorylases that cleave only deoxynucleosides and that are not inhibited by GPT, we propose the term "uridine-deoxyuridine phosphorylases" to define those pyrimidine nucleoside phosphorylases that cleave both uridine and dThd and that are inhibited by GPT. On the basis of this definition and studies with GPT in nonfocused cytosol preparations, we conclude that FdUrd is cleaved to 5-fluorouracil by uridine-deoxyuridine phosphorylase activity in Ehrlich ascites cells and in Novikoff hepatoma cells, and by dThd phosphorylases in mouse liver, in normal human leukocytes, and in HeLa (S3) cells.

Animals↗

PubMedDate not supplied

[Comparative study of the circular dichroism of rabbit liver and muscle glycogen phosphorylases a and b].

Circular dichroism (CD) spectra of glycogen phosphorylase a and b from rabbit liver have been measured in the presence of various ligands in the near- and far-ultraviolet regions. Positive circular dichroism was detected in the absorption band of protein-bound pyridoxal phosphate (333 nm). The mean residue ellipticity of this dichroic band (35 deg cm2dmol-1) is of the same order for muscle and liver phosphorylase a and b and does not change upon binding of glucose-1-phosphate and AMP. Only glucose induces small changes in the ellipticity in this region. The CD spectra of muscle and liver phosphorylase a and b in the 250-300 nm region have at least five positive dichroic bands namely at 259, 264, 273, 281 and 288 nm and have strong resemblances for all these forms of the enzyme in spite of the fundamental differences in their properties. The binding of AMP and glucose to phosphorylase from both sources induces distinct perturbations in CD spectra; the changes are much larger for muscle and liver phosphorylase a than for phosphorylase b which indicates that conformational perturbations induced by binding of activator and inhibitor to the inactive form of phosphorylase are probably more local than for the active form. The CD spectra in far-ultraviolet region are similar for all forms of phosphorylase. The percent of alpha-helices calculated according to Chen is about 50; this value coincides very well with the value 51% received for muscle phosphorylase a by X-ray crystallographic analysis at 2.5 A resolution.

Adenosine Monophosphate↗

PubMed1990

Phosphorylase and creatine kinase modification by thiol-disulfide exchange and by xanthine oxidase-initiated S-thiolation.

The reaction of glycogen phosphorylase b and creatine kinase with glutathione disulfide, cystine, and cystamine was compared by direct analysis on electrofocusing gels. This method was useful for individual proteins or for mixtures of the proteins. Millimolar concentrations of glutathione disulfide were required for both proteins and the rate of modification of each protein was similar. The reaction of glutathione disulfide with creatine kinase was inhibited by reduced glutathione (GSH), but the effect on the reaction with phosphorylase was minimal. Cystine and cystamine were required in micromolar amounts to effectively form the disulfide adducts. Both proteins were modified by cystine but cystamine reacted only with phosphorylase. Cystamine (10 microM) was an effective inhibitor of the reaction of phosphorylase b with 2 mM glutathione disulfide. S-thiolation of creatine kinase inactivated the enzyme and a direct assay of the enzyme activity could be used to quantitate S-thiolation of this protein by each of the disulfides. The effect of each disulfide on enzyme activity confirmed the results obtained by gel electrofocusing. Glutathione disulfide and cystine both inactivated the enzyme while cystamine had no effect on the activity. S-thiolation of phosphorylase had no observable effect on any activity parameter, but it effectively prevented binding of phosphorylase to high-molecular-weight glycogen, probably at the glycogen storage site of phosphorylase. The rate of S-thiolation of a mixture of phosphorylase and creatine kinase by thiol-disulfide exchange with glutathione disulfide was compared to the rate of S-thiolation of these proteins by a xanthine oxidase-initiated process (presumably due to protein sulfhydryl activation by reactive oxygen species). The xanthine oxidase-initiated mechanism was somewhat faster than thiol-disulfide exchange with both proteins. It was shown that GSH inhibited S-thiolation of creatine kinase by this mechanism as well as by thiol-disulfide exchange. It is suggested that both mechanisms may play a role in protein S-thiolation in vivo. For proteins that are typified by creatine kinase, the concentration of GSH in the cells may determine whether the S-thiolated form of the protein accumulates. For proteins typified by phosphorylase b, the accumulation of S-thiolated forms may be more independent of GSH.

Animals↗

PubMed1984

Effects of age on glycogen synthase and phosphorylase activities in rat liver.

The activities of glycogen synthase and phosphorylase were determined in homogenates and subcellular fractions of liver from young adult (6 months' old), adult (12 months' old) and aged (24 months' old) rats. The specific activities (enzyme activity expressed as units per mg protein) of the active form of glycogen synthase (synthase a) as well as of total synthase (synthase a + b) were significantly lower in liver homogenates of aged compared to young adult or adult rats. The age-associated decrease in the specific activity of synthase a was most marked in the 10 000 g and 100 000 g particulate fractions of liver. The specific activities of the active form of phosphorylase (phosphorylase a) as well as of total phosphorylase (phosphorylase a + b) were also significantly lower in liver homogenates of aged compared to young adult or adult animals. The age-related decrease in the specific activity of phosphorylase a was most pronounced in the 10 000 g particulate fraction of liver. Analysis of the specific activity ratios of glycogen synthase a/glycogen synthase a + b indicated a disproportionately greater age-related decrement in the active form of this enzyme. No such age-related difference was evident in the specific activity ratios of phosphorylase a/phosphorylase a + b. The age-associated decrease in the activities of glycogen synthase (a or a + b) and phosphorylase (a or a + b) was also discernible when the enzyme activities were expressed as units per g liver; this decrement in enzyme activity was reflected in all subcellular fractions examined. The levels of plasma insulin, plasma glucose and liver glycogen were not significantly different in rats of the three age groups used in these studies. The reduced activities of liver glycogen synthase and phosphorylase in aged rats is indicative of a likely diminution in the turnover of glycogen in liver during aging. Such an age-associated deficit in liver glycogen metabolism may contribute, at least in part, to the generally observed glucose intolerance upon aging.

Aging↗

PubMed1995

Interaction between glycogen phosphorylase and sarcoplasmic reticulum membranes and its functional implications.

Skeletal muscle glycogen phosphorylase b binds to sarcoplasmic reticulum (SR) membranes with a dissociation constant of 1.7 +/- 0.6 mg of phosphorylase/ml at 25 degrees C at physiological pH and ionic strength. Raising the temperature to 37 degrees C produced a 2-3-fold decrease in the dissociation constant. The SR membranes could bind up to 1.1 +/- 0.1 mg of glycogen phosphorylase b/mg of SR protein, whereas liposomes prepared with endogenous SR lipids and reconstituted Ca(2+)-ATPase were unable to bind glycogen phosphorylase. Binding of glycogen phosphorylase b to SR membranes is accompanied by inhibition of its activity in the presence of AMP. The Vmax for glycogen phosphorylase b associated with SR membranes is 40 +/- 5% of that for purified glycogen phosphorylase and shows a decreased affinity for its allosteric activators, AMP and IMP. These kinetic effects are also observed with purified glycogen phosphorylase b when starch or alpha-amylose is used as substrate instead of glycogen. Treatment of SR membranes with alpha-amylase produced dissociation of glycogen phosphorylase b from the SR membranes. Thus, linear polysaccharide fragments of glycogen bound to the SR membranes are likely mediating the binding of glycogen phosphorylase b to these membranes.

Animals↗

PubMed2001

Hepatic glycogen synthesis is highly sensitive to phosphorylase activity: evidence from metabolic control analysis.

We used metabolic control analysis to determine the flux control coefficient of phosphorylase on glycogen synthesis in hepatocytes by titration with a specific phosphorylase inhibitor (CP-91149) or by expression of muscle phosphorylase using recombinant adenovirus. The muscle isoform was used because it is catalytically active in the b-state. CP-91149 inactivated phosphorylase with sequential activation of glycogen synthase. It increased glycogen synthesis by 7-fold at 5 mm glucose and by 2-fold at 20 mm glucose with a decrease in the concentration of glucose causing half-maximal rate (S(0.5)) from 26 to 19 mm. Muscle phosphorylase was expressed in hepatocytes mainly in the b-state. Low levels of phosphorylase expression inhibited glycogen synthesis by 50%, with little further inhibition at higher enzyme expression, and caused inactivation of glycogen synthase that was reversed by CP-91149. At endogenous activity, phosphorylase has a very high (greater than unity) negative control coefficient on glycogen synthesis, regardless of whether it is determined by enzyme inactivation or overexpression. This high control is attenuated by glucokinase overexpression, indicating dependence on other enzymes with high control. The high control coefficient of phosphorylase on glycogen synthesis affirms that phosphorylase is a strong candidate target for controlling hyperglycemia in type 2 diabetes in both the absorptive and postabsorptive states.

Adenosine Monophosphate↗