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PubMed1999

Characterization of trehalose phosphorylase from Schizophyllum commune.

During growth on d-glucose, the basidiomycete Schizophyllum commune produces an intracellular alpha,alpha-trehalose phosphorylase. Specific phosphorylase activity increases steadily during the exponential growth phase, up to a maximum of approx. 0.08 unit/mg of protein, and decreases after the available d-glucose in the medium has been fully depleted. The variation with time of the concentrations of intracellular alpha,alpha-trehalose and Pi is reciprocal to that of trehalose phosphorylase activity, indicating that the enzyme makes temporary use of the pool of alpha, alpha-trehalose (approx. 0.42 mmol/g dry cell) via phosphorolysis. The enzyme has been purified, 150-fold, to homogeneity in 55% yield and characterized. It is a monomeric 61 kDa protein, which seems to lack regulation at the level of enzyme activity. The enzyme catalyses the reversible phosphorolysis of alpha,alpha-trehalose into alpha-d-glucose 1-phosphate and alpha-d-glucose in the absence of cofactors, with a catalytic-centre activity at 30 degrees C of 14 s(-1). Double-reciprocal analysis of the initial velocities for trehalose phosphorolysis and synthesis yields intersecting patterns, and no exchange reaction occurs between alpha-d-glucose 1-phosphate and the phosphate analogue arsenate. Therefore trehalose phosphorylase operates by a ternary-complex, rather than a Ping-Pong, kinetic mechanism. The specificity constants (kcat/Km) of phosphate (6000 M(-1).s(-1)) and alpha-d-glucose 1-phosphate (3500 M(-1).s(-1)) compared with those of alpha,alpha-trehalose (161 M(-1).s(-1)) and d-glucose (260 M(-1).s(-1)), together with the inhibition by NaCl, which is competitive with respect to phosphate with a Ki of 67 mM, suggest an important role for ionic enzyme-phosphate interactions in the catalytic mechanism of trehalose phosphorylase. The isolated enzyme requires alpha,alpha-trehalose (0.1-0.3 M) for its conformational stability.

Enzyme Stability↗
PubMed1995

Phosphorylation/activation of phosphorylase b kinase by cAMP/Ca2(+)-independent, autophosphorylation-dependent protein kinase.

Phosphorylase b kinase from rabbit skeletal muscle can be phosphorylated and activated by a cyclic nucleotide- and Ca2(+)-independent protein kinase previously identified as an autophosphorylation-dependent multifunctional protein kinase (auto-kinase) from brain and liver (Yang et al., J. Biol. Chem. 262, 7034-7040 (1987) and Yang et al. J. Biol. Chem. 262, 9421-9427 (1987)). This independent kinase phosphorylates both alpha and beta subunits of phosphorylase b kinase and results in a approximately 5-fold activation of the kinase when 0.55 and 0.5 mol of phosphate are incorporated into the alpha and beta subunits, respectively. Activation of phosphorylase b kinase catalyzed by auto-kinase is about 70% of that observed with cAMP-dependent protein kinase. Analysis of phosphopeptide maps of alpha and beta subunits further reveals that both kinases phosphorylate almost the same sites on both alpha and beta subunits, suggesting that activation of phosphorylase b kinase by the two kinases may be through a common molecular action mechanism. Taken together with the previous result that auto-kinase can inactivate glycogen synthase, the present study provides initial evidence that a coordinate control mechanism for simultaneous regulation of glycogenolysis and glycogenesis can be modulated by autophosphorylation-dependent protein kinase in a cAMP- and Ca2(+)-independent pathway, representing a new mode of control mechanism for the regulation of glycogen metabolism in cells.

Animals↗
PubMed1986

Chemical modification of rabbit skeletal muscle phosphorylase kinase with phenylglyoxal.

Nonactivated phosphorylase kinase from rabbit skeletal muscle is inactivated by treatment with phenylglyoxal. Under mild reaction conditions, a derivative that retains 10-15% of the pH 8.2 catalytic activity is obtained. The kinetics of inactivation profile, differential effects of modification on pH 6.8 and 8.2 catalytic activities, and the insensitiveness of the modified enzyme to activation by ADP reveal that the 10-15% of catalytic activity remaining is very likely due to intrinsic catalytic activity of the derivative rather than to the presence of unmodified enzyme molecules. The kinetic results also suggest that the inactivation is correlatable with the reaction of one molecule of the reagent with the enzyme without any prior binding of phenylglyoxal. The phenylglyoxal modification reduces the autophosphorylation rate of the kinase. Autophosphorylated phosphorylase kinase is inactivated by phenylglyoxal at a much slower rate than the inactivation of nonactivated kinase. Thus, phenylglyoxal modification influences the phosphorylation and vice versa. The modified enzyme can be reactivated by treatment with trypsin or by dissociation using chatropic salts. The activity of the phenylglyoxal-modified enzyme after trypsin digestion or dissociation with LiBr reaches the same level as that of the native enzyme digested with trypsin or treated with LiBr under identical conditions. The results suggest that the effect of modification is overcome by dissociation of the subunits of phosphorylase kinase and that the catalytic site is not modified under conditions when 85% of the pH 8.2 catalytic activity is lost. Among various nucleotides and metal ions tested, only ADP, with or without Mg2+, afforded effective protection against inactivation with phenylglyoxal. At pH 6.8, 1 mM ADP afforded complete protection against inactivation. Experiments with 14C-labeled phenylglyoxal revealed that ADP seemingly protects one residue from modification. This result is in agreement with the kinetic result that the inactivation seemingly is due to reaction of one molecule of the reagent with the enzyme. The results confirm the existence of a high-affinity ADP binding site on nonactivated phosphorylase kinase and suggest the involvement of a functional arginyl residue at or near the ADP binding site in the regulation of of pH 8.2 catalytic activity of the enzyme.

Adenosine Diphosphate↗
PubMed1981

Facilitated transport of inosine and uridine in cultured mammalian cells is independent of nucleoside phosphorylases.

The zero-trans uptake of uniformly and base-labeled inosine and uridine was measured a 25 degrees C in suspensions of Novikoff rat hepatoma cells, Chinese hamster ovary cells, mouse L cells, mouse S49 lymphoma cells and a purine-nucleoside phosphorylase-deficient subline thereof (NSU-1), and in monolayer culture of mouse 3T3 and L cells. The initial velocities of uptake of both nucleosides were about the same in all cell lines investigated, regardless of the position of the label or of the substrate concentration between 3 and 300 microM or whether or not the cells possessed uridine or purine-nucleoside phosphorylase activity. The kinetic parameters for the facilitated transport of uridine and inosine were also similar in phosphorylase positive and negative cell lines (K = 120--260 microM and V = 6--40 pmol/microliters cell water per s) and the transport activities of the cells exceeded their total phosphorylase activities by at least 10-fold for uridine and 1--2-fold for inosine. Chromatographic fractionation of the intracellular contents and of the culture fluid showed that the free nucleosides appeared intracellularly prior to and more rapidly than their phosphorolysis products. During the initial 20--60 s of uptake of U-14C-labeled nucleosides the rates of intracellular appearance of ribose-1-P and base were about the same. After several minutes of incubation, on the other hand, the main intracellular component was ribose-1-P whereas the base attained a low intracellular steady-state concentration and accumulated in the medium due to exit transport. Other nucleosides, dipyridamole and nitrobenzylthioinosine, specifically inhibited the transport of uridine and inosine, and depressed the intracellular accumulation of ribose-1-P and the formation of base commensurate with that inhibition. The data indicate that the metabolism of inosine and uridine by the various cell lines can be entirely accounted for by the facilitated transport of unmodified nucleoside into the cell followed by intracellular phosphorolysis.

Animals↗
PubMed1977

Inactivation and reactivation of liver phosphorylase b kinase.

When crude rat liver preparations were incubated at 30degrees C, a gradual loss of phosphorylase kinase (ATP:phosphorylase b phosphotransferase, EC 2.7.1.38) activity was observed. This inactivation was Mg2+ dependent and was partially inhibited by sodium fluoride. Addition of Mg2+ ATP to the liver preparations, at any time throughout the incubation, caused a reactivation of the phosphorylase kinase and this was accelerated by micromolar concentrations of cyclic AMP. The reactivation process could be completely abolished by the addition of a heat stable protein kinase inhibitor, implicating cyclic AMP dependent protein kinase in the activation reaction. Both the low and the high activity forms of the enzyme required micromolar quantities of Ca2+ for full activity (KA = 0.6 micronM). The two forms exhibit quite different pH dependencies and at the physiological pH of liver (pH 7.4) their activities differed by a factor of 5-10. Conversion of the lower activity form into the higher seems to affect only the V - Km for muscle phosphorylase b (EC 2.4.1.1) was about 1 mg/ml for both enzyme forms.

Adenosine Triphosphate↗
PubMed1979

Phosphorylase kinase from human polymorphonuclear leukocytes.

Phosphorylase kinase from human polymorphonuclear leukocytes was investigated in a gel filtered crude preparation (17,000 x g supernatant). It was found to exist in two forms, one (the phosphorylated form) more active than the other (the dephosphorylated form). Interconversion between the two forms was carried out by a cyclic AMP dependent protein kinase and phosphoprotein phosphatase, respectively. The ratio of activity measured at pH 8.0 and 6.0 was 0.36 for the non-activated and 0.83 for the activated form, which is in contrast to the behaviour of phosphorylase kinase from muscle. Km app for the substrate phosphorylase b was 650 U/ml and 85 U/ml for the non-activated and activated form, respectively, whereas Km app for ATP was 0.03 mM and identical for the two forms. The non-activated form of phosphorylase kinase was activated by Ca2+ in the range 10(-7)--5 . 10(-6) M, which may have physiological importance, whereas the activated form was insensitive to variations in Ca2+ concentration between 10(-9) and 10(-3) M.

Calcium↗
PubMed1981

Effect of prostaglandins on phosphorylase b.

Prostaglandins at mM concentration are able to induce a small activation to rabbit muscle glycogen phosphorylase b (1,4-alpha-D-glucan: orthophosphate alpha-D-glucosyltransferase, EC 2.4.1.1) in the absence of AMP. The extent of this activation depends on the nature of the molecular structure of prostaglandins. Saturated and unsaturated higher fatty acids were unable to substitute for prostaglandins. The main findings of our studies can be summarized as follows: (1) Prostaglandins inhibit the AMP-induced activation of phosphorylase b. (2) Modification of the AMP binding site with 2,3-butanedione could not inhibit the activity induced by prostaglandins. (3) Enzyme activation by prostaglandins is stimulated by spermine. (4) Phosphorylase b activation by prostaglandins and the observed stimulation of this activation by spermine were found to be temperature dependent. (5) Prostaglandins affect the quaternary structure of phosphorylase b inducing a partial enzyme tetramerization which is enhanced in the presence of spermine. The extent of this tetramerization is temperature dependent.

Adenosine Monophosphate↗
PubMed1986

Inhibition of uridine phosphorylase from Escherichia coli by benzylacyclouridines.

The benzylacyclouridines, potent and specific inhibitors of mammalian uridine phosphorylase, were also found to be inhibitors of uridine phosphorylase but not thymidine phosphorylase from Escherichia coli. Competitive inhibition was observed in all cases and the most potent of these compounds was HM-BBAU (5-(3-benzyloxybenzyl)-1-[(2'-hydroxy-1'-hydroxymethyl)methyl]urac il) with a Ki value of 0.15 microM. The inhibitory potencies of these compounds parallel those obtained with enzymes from mammalian sources [Niedzwicki et al., Biochem. Pharmac. 31, 1857 (1982) and Naguib et al., manuscript in preparation] indicating that the structure of the active site of uridine phosphorylase from E. coli may resemble that of the mammalian enzyme.

Escherichia coli↗
PubMed1983

Effect of glucose-6-P on the catalytic and structural properties of glycogen phosphorylase a.

Kinetic studies of muscle phosphorylase a in cationic buffer (pH 6.8) demonstrate that glucose-6-P competitively inhibits the binding of the substrate, glucose-1-P, to the enzyme. The inhibitory effect of glucose-6-P is largely overcome by glycerol-2-P. AMP counteracts inhibition of the enzyme by glucose-6-P, while glucose and glucose-6-P can interact to produce a synergistic inhibition of phosphorylase a activity. Preincubation of phosphorylase a with glucose-6-P at 20 degrees C results in approximately 3-fold increase in activity, while ultracentrifugation experiments carried out under the same conditions showed that phosphorylase a can be converted to dimers by glucose-6-P.

Animals↗
PubMed1990

The ammonium sulfate activation of phosphorylase b.

The ammonium sulfate activation of phosphorylase b has been studied. Ammonium sulfate, when present in high concentrations, induces properties of phosphorylase a in phosphorylase b, such as an enhanced affinity for AMP, a reversal of the glucose-6-P inhibition and enzyme tetramerization. The data are consistent with the interpretation that sulfates bind to the Ser-14 site and the sulfate-protein interactions at this site are responsible for activation of phosphorylase b.

Adenosine Monophosphate↗
PubMed1988

Regulation of the dephosphorylation of phosphorylase A by glucose, AMP and polyamines.

1. The effect of glucose, caffeine, AMP and polyamines was investigated on the dephosphorylation of phosphorylase a by the catalytic subunits of protein phosphatase-1 and -2A. 2. Caffeine at 1-20 microM inhibited the dephosphorylation of the dimeric phosphorylase a at 37 degrees C using skeletal muscle enzymes; 0.1-10 mM of caffeine enhanced the rate of dephosphorylation greatly at 13 degrees C and slightly at 37 degrees C. 3. alpha-D-Glucose was more effective in accelerating both the dephosphorylation and the tryptic digestion of phosphorylase a than the beta-anomer. 4. Polyamines were found to moderate the inhibitory effect of AMP at concentrations which may occur in the tissues. In the presence of 5 mM glucose polyamines could cancel the AMP inhibition of the dephosphorylation of liver phosphorylase a by hepatic protein phosphatase-1 and -2A.

Adenosine Monophosphate↗
PubMed1988

Purification and characterization of uridine (thymidine) phosphorylase from Giardia lamblia.

Giardia lamblia is totally dependent on salvage synthesis for its pyrimidine requirements. The salvage pathway enzyme, uridine phosphorylase (pyrimidine nucleoside phosphorylase) was purified to apparent homogeneity from G. lamblia crude extracts by fast protein liquid chromatography and gel filtration on a Superose 12 column, resulting in an overall 3500 fold purification and a recovery of 7.5%. Mono P chromatofocusing gave rise to a major activity peak eluting from the column at pH 5.9, indicating that the enzyme has an isoelectric point (pI) at approximately this value. The molecular weight was found to be 43,000 +/- 2000 from the Superose 12 column, while sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the purified enzyme gave a single protein band with a subunit molecular weight of 38,000 +/- 2000, indicating that it is a monomer. The activities of uridine, deoxyuridine and thymidine phosphorylases from G. lamblia remained associated throughout the purification procedure, suggesting that one enzyme is responsible for the three enzyme activities. The ratio of activities was consistent throughout the purification procedure. In the reverse (anabolic) direction, the enzyme could use both uracil and thymine as substrates. The properties of the phosphorylase differ significantly from those of the mammalian host.

Animals↗
PubMed1984

Purification and crystallization of bovine liver phosphorylase.

We have purified and crystallized bovine liver phosphorylase a. Starting from 2.5 kg of liver, we obtain 250 mg of phosphorylase a, with a specific activity of 90 units/mg, representing 15% recovery. SDS polyacrylamide gels show three bands, a 95 kDa band with the same mobility as muscle phosphorylase, and two smaller bands of 55 kDa and 40 kDa, which are probably proteolytic fragments. These fragments remain associated and have native conformation and catalytic activity. Crystals which diffract to 2.8 A resolution, were prepared by the hanging drop method using polyethylene glycol PEG 4000 as precipitant. The crystals were prepared in the presence of activators maltotriose and phosphite and crack when placed in solutions containing the inhibitors glucose and caffeine. This suggests phosphorylase is present in an active conformation.

Animals↗
PubMed1989

Kinetic study on the dimer-tetramer interconversion of phosphorylase b by a stopped-flow X-ray scattering method.

The dimer-tetramer interconversion of phosphorylase b induced by the binding of AMP and Mg2+ was monitored using a stopped-flow X-ray scattering method. The rate constants of this second-order reaction have been determined by a nonlinear least-squares method. Burst phases in both radii of gyration and zero-angle intensities were detected at the initial step of the reaction. This suggests that rapid association might take place, followed by a slow association process of which the kinetics were measured in the present study. The radius of gyration of tetrameric phosphorylase b was determined and found to be in excellent agreement with that of phosphorylase a, but different from that of phosphorylase b reported elsewhere (G. Puchwein, O. Kratky, C. F. Golker and E. Helmreich, Biochemistry 9 (1970) 4691). The reason for this inconsistency is discussed.

Animals↗
PubMed1988

S-thiolation of creatine kinase and glycogen phosphorylase b initiated by partially reduced oxygen species.

S-thiolation of cardiac creatine kinase and skeletal muscle glycogen phosphorylase b was initiated by reduced oxygen species in reaction mixtures containing reduced glutathione. Both proteins were extensively modified at similar rates under conditions in which the oxidation of glutathione was inadequate to cause S-thiolation by thiol-disulfide exchange. Creatine kinase was both S-thiolated and non-reducibly oxidized at the same time at low glutathione concentration. The amount of each modification was decreased by adding additional reduced glutathione, and with adequate glutathione oxidation was prevented while S-thiolation was still very active. S-thiolation of glycogen phosphorylase b was not significantly affected by glutathione concentration and non-reducible oxidation of glycogen phosphorylase b was not observed. These experiments suggest that oxyradical or H2O2-initiated processes may be an important mechanism of protein S-thiolation during oxidative stress, and that the cellular concentration of glutathione may be an important factor in S-thiolation of different proteins. Both creatine kinase and glycogen phosphorylase b competed favorably with ferricytochrome c for superoxide anion in the standard xanthine oxidase system for the generation of oxyradicals and H2O2. These proteins were as effective as ascorbate and much more effective than reduced glutathione in this regard. Ascorbate was also an effective inhibitor of oxyradical-initiated S-thiolation of creatine kinase, suggesting a role of superoxide anion in protein S-thiolation. Other experiments showed that both catalase and superoxide dismutase could partially inhibit protein S-thiolation. Thus, reduced oxygen species may react with protein sulfhydryls resulting in S-thiolation by a mechanism that involves the reaction of an activated protein thiol with reduced glutathione.

Animals↗
PubMed1982

Comparative study of the conformational transitions of frog and rabbit phosphorylases B.

1. Conformational motility of the purified muscle glycogen phosphorylase B from two species of vertebrates (rabbit and frog) was investigated by the Hydrogen-Exchange method and Infrared Spectometry. 2. The experimental results of the 1H-2H exchange were expressed in terms of the probability P of exposure to isotopic solvent of phosphorylase peptide groups and in terms of the corresponding changes in standard free energy delta Go. 3. The combined methods used didn't show considerable differences of the protein conformations in the physiological pH region but rabbit phosphorylase was only characterized by rather more compact structure in comparison with frog phosphorylase.

Animals↗
PubMed1985

31P NMR relaxation studies of the activation of the coenzyme phosphate of glycogen phosphorylase. The role of motion of the bound phosphate.

Spin-lattice and spin-spin relaxation rates (1/T1 and 1/T2) have been determined for the catalytically essential coenzyme phosphate at the active site of glycogen phosphorylase in both activated (R state) and inactive (T state) conformations of the enzyme. Dipolar contributions to 31P relaxation due to exchangeable protons on the phosphate group have been determined by measurement of relaxation rates at different concentrations of H2O and D2O, and field dependence studies have been performed to estimate the contribution of chemical shift anisotropy to the remaining 31P relaxation in D2O. At 109 MHz, dipolar relaxation from exchangeable protons was found to account for 50% of the spin-lattice relaxation for activated phosphorylase in 75% H2O, the remainder being due to chemical shift anisotropy. The spin-lattice relaxation rates in D2O for R-state glycogen phosphorylase are very similar to those measured for other proteins of very different size such as actin (Brauer, M., and B. D. Sykes, 1981, Biochemistry. 20:6767-6775), alkaline phosphatase (Coleman, J. E., I. D. Armitage, J. F. Chlebowski, J. D. Otvos, and A. J. M. S. Uiterkamp, 1979), and phosphoglucomutase (Rhyu, G. I., W. J. Ray, Jr., and J. L. Markley, 1984, Biochemistry. 23:252-260). In inactive (T state) phosphorylase the spin-lattice relaxation rates were almost an order of magnitude slower, while the spin-spin relaxation rates were essentially identical. These results have been analyzed by calculating the theoretically expected 31P relaxation rates in the presence of internal motions that are included in the relaxation calculation using the model-free approach of Lipari and Szabo (1982, J. Am. Chem. Soc. 104:4564-4559). The analysis suggests the coenzyme phosphate is relatively immobilized in the activated enzymic conformation, but in the inactive (Tstate) conformation it is considerably more mobile with a rotational correlation time one to two orders of magnitude smaller. Since the spin-lattice relaxation rate for the active R-state (immobilized) phosphate is similar to that observed in other phosphoenzymes of different size it is suggested that a librational motion on the nanosecond time scale may constitute a common spin-lattice relaxation pathway for phosphates in macromolecules. The consequences of phosphate motion in terms of recent suggestions concerning the environment and the catalytic role of the coenzyme phosphate are discussed.

Allosteric Regulation↗
PubMed1984

Substrate-cofactor interactions for glycogen phosphorylase b: a binding study in the crystal with heptenitol and heptulose 2-phosphate.

The structural relationships between substrate and pyridoxal phosphate in glycogen phosphorylase b (EC 2.4.1.1) have been studied by X-ray diffraction experiments at 3-A resolution. Recent work [Klein, H. W., Im, M. J., & Helmreich, E. J. M. (1984) in Chemical and Biological Aspects of Vitamin B6 Catalysis (Evangelopoulos, A. E., Ed.) pp 147-160, Liss, New York] has shown that phosphorylase in the presence of inorganic phosphate catalyzes the conversion of heptenitol to heptulose 2-phosphate. The latter compound is a dead-end product and a most potent inhibitor (Ki = 14 microM). The X-ray diffraction studies show that heptenitol binds at the catalytic site of phosphorylase in a position essentially identical with that observed for the glucopyranose moiety of glucose 1-phosphate. Incubation of a phosphorylase b crystal for 50 h in a solution containing the substrates heptenitol and inorganic phosphate and the activators AMP and maltohetaose resulted in the formation of a phosphorylated product bound at the active site. The structure of this product, as analyzed by a difference Fourier synthesis at 3 A, is consistent with that of heptulose 2-phosphate. Analysis of the surrounding soak solution by thin-layer chromatography showed that heptulose 2-phosphate was produced under these conditions. Heptulose 2-phosphate binds with its glucopyranose moiety in the same position as that for glucose 1-phosphate, but there is a marked difference in phosphate positions. The presence of the methyl group in the beta-configuration in heptulose 2-phosphate forces a change in the torsion angle O5-C1-O1-P from 117 degrees as observe in glucose 1-phosphate to -136 degrees in heptulose 2-phosphate. The "down" position of the phosphate (with respect to the crystallographic z axis) results in a change in the distance between the 5'-phosphorus atom of the pyridoxal phosphate and the phosphorus atom of the substrate from 6.8 (with glucose 1-phosphate) to 4.5 A (with heptulose 2-phosphate). The closest distance between the phosphate oxygen of the cofactor and a phosphate oxygen of heptulose 2-phosphate is 2.7 A, and it is assumed that there must be a hydrogen bond between them. These observations are consistent with the NMR experiments reported in the preceding paper in which sharing of a proton between heptulose 2-phosphate and pyridoxal 5'-phosphate is observed [Klein, H.W., Im, M. J., Palm, D., & Helmreich, E. J. M. (1984) Biochemistry (preceding paper in this issue)].(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Monophosphate↗