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At least 793 records · Page 44Linked to original sources
PubMed1989

Glycogenolysis during recovery from muscular work. The time course of phosphorylase activity is dependent on Pi concentration in the abdominal muscle of the shrimp Crangon crangon.

1) Glycogen is degraded in the abdominal muscle of the shrimp Crangon crangon (Decapoda, Crustacea) during the recovery period following work. The regulation of post-exercise glycogen breakdown and the properties of glycogen phosphorylase (EC 2.4.1.1) have been studied: 2) Glycogen phosphorylase exists as unphosphorylated b-form and phosphorylated a-form, the latter contains 1 molecule phosphate/subunit. Both forms of phosphorylase are dimers, isoenzymes have not been detected. 3) The purified b-form is inactive in absence of AMP and has very low affinities for AMP and Pi. For half-maximum activation 0.33 +/- 0.04 mM AMP is necessary, and the Km-value for Pi at 1 mM AMP is 48 +/- 5 mM. IMP does not affect the activity of the b-form. 4) The a-form is active without effectors, its Km-value for Pi is 5.3 +/- 1.5 mM. The proportion of phosphorylase a increases in vivo, from about 25% at rest, to approximately 90% upon work and remains at this high level during the first minutes of recovery. 5) It is concluded that the glycogenolytic flux in the abdominal muscle of the shrimp even during post-exercise periods depends on the level of the a-form the activity of which is restricted in time and extent by the cytoplasmic Pi concentration (Kamp, G. & Juretschke, H. P. (1987) Biochim. Biophys. Acta 929, 121-127).

Adenosine Monophosphate↗
PubMed1986

Kinetic properties of glycogen synthase and phosphorylase and structural aspects of glycogen in the db/db mouse liver.

Kinetic studies were carried out on liver glycogen synthase and phosphorylase isolated from genetically diabetic db/db mice. Glycogen synthase a and b enzymes from diabetic mice had Vmax values 30% and 20% lower, respectively, than the enzymes from normal mice. Glycogen synthase b from diabetic mice also had a 30% lower I0.5 for Pi and ATP at physiologic concentrations of UDP-glucose (0.25 mM) compared with the normal enzyme. Kinetic studies of phosphorylase a showed that, at low glycogen concentrations (0.25 mg/ml), the Vmax of the diabetic enzyme was twofold greater than that of the normal enzyme. This was probably related to the diabetic phosphorylase a having a lower apparent Km for glycogen. This enzyme also had a slightly higher I0.5 for ATP compared with the enzyme from normal mice. Structural studies of liver glycogen isolated from these diabetic mice showed differences from normal mouse glycogen. Both the alpha- and beta-amylase limits were lower in the diabetic glycogen, and the average chain lengths, exterior chain lengths, and interior chain lengths calculated from these limits were all shorter in the glycogen from diabetic mice. Although both normal and diabetic glycogen absorbed light maximally at 430 nm when complexed with iodine, the absolute absorbance value was significantly lower for the diabetic glycogen. These data suggest an altered branching pattern of liver glycogen from the diabetic mice and it is suggested that this altered structure may ultimately influence the activities of glycogen-metabolizing enzymes. These results provide further characterization of the db/db mouse and show heretofore undescribed changes in phosphorylase a kinetics and glycogen structure that occur in diabetes.

Animals↗
PubMed1991

[Interaction of phosphorylase kinase with thin filament proteins of rabbit skeletal muscles].

The binding of phosphorylase kinase to thin filaments and their effects on the enzyme activity as well as the contribution of the enzyme to contractile protein phosphorylation have been studied. The data obtained suggest that the kinase binding to thin filaments is controlled by the regulatory proteins, troponin and tropomyosin. The bulk of the enzyme is bound to the F-actin-tropomyosin-troponin complex which activates the enzyme in a far greater degree than each of its constituent components. Ca2+ and ATP control the kinase binding to F-actin. ATP increases the enzyme binding 6-fold; Ca2+ decrease the S0.5 value for F-actin 5-fold. In acetone powder extracts phosphorylase kinase phosphorylates thin filament-bound phosphorylase b, troponin T and troponin I as well as 51-58 kDa and 114 kDa proteins. These results suggest that phosphorylase kinase plays a role in the mechanism of synchronization of glycogenolysis and muscle contraction rates.

Actins↗
PubMed1990

The pH-dependent conformational transition in glycogen phosphorylase b. The effect of carnosine and anserine on its activity.

The conformational transition of phosphorylase b which occurs upon pH change from 6.0 to 7.0 is linked to the pH-dependent activity regulation. Skeletal muscle dipeptides, carnosine and anserine, activate rabbit and bovine phosphorylase b at pH 6.5-6.0 and inhibit it at pH 6.5-7.0. The enzyme activation constant was found to be equal to 20 mM, and the inhibition constant corresponded to 22 mM for both carnosine and anserine. The data obtained suggest that glycogen phosphorylase b adopts different conformational states at pH 6.0 and 7.0. Evidence for the pH-induced conformational transition of the enzyme was obtained using chemical modification of histidine residues. The change in the phosphorylase activity under effects of the dipeptides in the pH range of 7.0-6.0 may be due to their physiological role in muscle contraction.

Animals↗
PubMed1987

Characterization of the isolated rat flexor digitorum brevis for the study of skeletal muscle phosphorylase kinase phosphorylation.

The flexor digitorum brevis skeletal muscle, a nearly homogeneous fast-twitch oxidative glycolytic fiber type, has been examined for its suitability to explore the regulation of phosphorylase kinase by multisite phosphorylation. A characterization of the adrenergic response of glycogenolytic enzymes, together with the previous data on contractile properties (Carlsen, R. C., Larson, D. B., and Walsh, D. A. (1985) Can. J. Physiol. Pharm. 63, 958-965), has demonstrated that this muscle is stably maintained for the several hours necessary for phosphorylation studies. The phosphorylase kinase in this muscle is primarily the alpha' isozyme, suggesting that the alpha versus alpha' isozyme distribution in muscle is related more to oxidative capacity than to fiber contractile characteristics. Using this muscle system, beta-adrenergic activation of phosphorylase kinase was observed to occur with concomitant phosphorylation of both the alpha' and beta subunits, with the total in the alpha' subunit being approximately 3-fold greater. Similarly, deactivation, following initial adrenergic activation, occurred concomitantly with the dephosphorylation of the two subunits. These results are compatible with the conclusions drawn from previous studies of the isolated enzyme and of the enzyme in perfused rat cardiac muscle, that both alpha' (or alpha) and beta subunit phosphorylation regulate phosphorylase kinase activity.

Adenosine Triphosphate↗
PubMed1986

[Interaction of muscle glycogen phosphorylase B with F-actin].

The binding of rabbit muscle glycogen phosphorylase b to F-actin has been studied by sedimentation in analytical centrifuge in 10 mM Tris-acetate buffer pH 6.8 at 20 degrees C. The adsorption capacity of F-actin is equal to (7.8 +/- 0.9) X 10(-7) mole of glycogen phosphorylase b per 1 g of F-actin; the microscopic dissociation constant for the glycogen phosphorylase-F-actin complex is (5.4 +/- 0.5) X 10(-7) M. It was found that the allosteric activator, AMP, facilitates the adsorption of glycogen phosphorylase b on F-actin, whereas the substrate, Pi, and the inhibitor, ATP, cause an opposite effect.

Actins↗
PubMed1987

[Allosteric properties of phosphorylase b].

Rabbit skeletal muscle phosphorylase b was separated into two fractions by column chromatography on AMP-Sepharose. The first fraction protein was eluted by glucose-6-phosphate while the second fraction protein was eluted in an AMP concentration gradient. The bulk of the protein eluate was represented by the first fraction protein. Chromatography of phosphorylase b from bovine skeletal muscle under identical conditions also resulted in two fractions, however, with a reverse correlation: the bulk protein of this fraction was eluted by AMP. It was shown that the two phosphorylase b forms eluted by glucose-6-phosphate and AMP differ by their kinetic and physico-chemical properties as well as by the SH-group reactivity. The phosphorylase b forms eluted by the nucleotide were practically uninhibited by glucose-6-phosphate. It can thus be assumed that the equilibrium between the "active" (R) and "inactive" (T) conformations of the protein changes depending on metabolic peculiarities of a given tissue used as a source for enzyme isolation.

Adenosine Monophosphate↗
PubMed1982

The effect of AMP on inhibition of muscle phosphorylase a by glucose derivatives.

Kinetic studies of the inhibition of rabbit muscle phosphorylase a D-glucan:orthophosphate alpha-D-glucosyltransferase (EC 2.4.1.1) by various glucose derivatives indicate that glucose derivatives in the chair conformation (glucose, 2-deoxyglucose, glucosyl fluoride, and 1-O-methylglucose) stabilize the "T" form of phosphorylase a, whereas those in the half-chair conformation (1,5-gluconolactone and glucal) bind to the "R" form. Derivatives which stabilized the T form were generally competitive inhibitors of phosphate binding, were synergistic with caffeine, and weakened AMP binding. Derivatives which bound to the R form were noncompetitive with respect to Pi in the presence of AMP. The mechanism of inhibition of the R form by gluconolactone and glucal appears to depend upon the presence or absence of AMP. In the presence of the nucleotide, gluconolactone is probably a linear mixed-type inhibitor. However, in the absence of AMP, simple linear uncompetitive inhibition was apparent. Since gluconolactone is thought to be an analogue of the substrate transition state of the phosphorylase reaction, a major function of nucleotide in activating phosphorylase may be the formation of the transition state binding pocket in the binary enzyme-glycogen complex. In the absence of AMP, glucal was competitive with respect to phosphate. However, in the presence of nucleotide, simple intersecting linear noncompetitive inhibition was observed. The results indicate that a second function of AMP is the formation of the phosphate binding pocket in the enzyme-glucal-glycogen-AMP complex. This conclusion is consistent with the crystallographic and structural data available at present.

Adenosine Monophosphate↗
PubMed1993

Differences in activities and substrate specificity of human and murine pyrimidine nucleoside phosphorylases: implications for chemotherapy with 5-fluoropyrimidines.

Enzyme inhibition studies on extracts from human liver, mouse liver, and human placenta indicate that there are considerable differences between human and murine hepatic uridine phosphorylases (UrdPase, EC 2.4.2.3) and thymidine phosphorylases (dThdPase, EC 2.4.2.4) with regard to their specificities and roles in the phosphorolysis of natural and 5-fluoropyrimidine nucleosides. To confirm further these differences between human and murine pyrimidine nucleoside phosphorylases, UrdPase and dThdPase were isolated from human liver, mouse liver, and human placenta using diethylaminoethyl-cellulose ion exchange chromatography. The pattern of elution from the column suggests that the hydrophobicity or charges on the human enzymes at pH 8 are different from those on their murine counterparts. The amount of each enzyme present differed between tissues and species. The apparent Km, Vmax, and efficiency of catalysis (Vmax/Km) values were determined for each enzyme using uridine, thymidine, deoxyuridine, 5-fluorouridine (FUrd), 5-fluoro-2'-deoxyuridine (FdUrd), and 5'-deoxy-5-fluorouridine (5'-dFUrd) as substrates. Kinetic parameters and inhibition studies were used to ascertain the binding affinity, substrate specificity, and contributions of UrdPase and dThdPase to the phosphorolysis of the various nucleosides in the 3 tissues. The roles of UrdPase and dThdPase in human liver were quite distinct from those of their counterparts from human placenta and mouse liver. In human liver, UrdPase appears to be highly specific to uridine. Human hepatic UrdPase contributes only 15% to the cleavage of FUrd and does not contribute to the cleavage of the deoxyribosides (thymidine, deoxyuridine, FdUrd, and 5'-dFUrd). In mouse liver, UrdPase has a broader specificity as it cleaves over 85% of FUrd, 15% of FdUrd, and 25% of 5'-dFUrd. On the other hand, human hepatic dThdPase has a broader specificity than murine hepatic dThdPase. Human hepatic dThdPase cleaves all nucleosides tested including the ribosides, uridine, and FUrd. Approximately 15% of uridine and 85% of FUrd phosphorolysis in human liver is carried out by dThdPase. This contrasts with the murine hepatic dThdPase, which is more specific to deoxyribosides, as it does not contribute to the phosphorolysis of uridine, and contributes only 15% toward the cleavage of FUrd. dThdPase is the principal enzyme responsible for the phosphorolysis of 5'-dFUrd in both human and murine livers. The specificities of UrdPase and dThdPase from human placenta resembled the enzymes from the murine liver more than those from human liver. Thus, it appears that the specificities of human hepatic pyrimidine nucleoside phosphorylases are distinct from those from extrahepatic tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Glycogen phosphorylase activity in the olfactory bulb of the young rat.

The activity of glycogen phosphorylase, the enzyme that controls glycogen breakdown, was histochemically mapped in the olfactory bulbs of 19-day-old rats. The effect of early odor experience on subsequent olfactory bulb phosphorylase activity was also examined. The highest level of phosphorylase staining in the bulb (and seemingly the highest in the brain) was in the glomerular layer, followed by the external plexiform, internal plexiform, granule cell, and olfactory nerve layers. Virtually no activity was visible in the large output neurons of the bulb, mitral, and tufted cells. Early peppermint odor experience, previously shown to increase metabolic activity in specific glomerular foci as measured by 2-deoxyglucose uptake, had no apparent effect on glomerular-layer phosphorylase activity. In some odor-familiar animals, however, patches of activity were seen in the internal plexiform layer in the area of the bulb where foci of high deoxyglucose uptake are seen in response to peppermint. The patches were directly in line with modified glomerular clusters often seen to underlie foci of enhanced deoxyglucose uptake. The existence of particularly heavy activity in the peripheral third of the glomerular layer, where glycogen-containing modified Schwann cells have been localized, raises the possibility that the glomerular-layer activity is at least partially glial in origin. Finally, because of its rich noradrenaline and serotonin innervation and high density of insulin receptors, the olfactory bulb is proposed as a model system to study the interaction of glycogen/glucose metabolism with neural activity in a relatively well-defined neuronal circuit.

Age Factors↗
PubMed1993

Regulation of glycogen phosphorylase activity in isolated human hepatocytes.

Hepatocytes were isolated from human liver tissue by a two-step perfusion technique. They were treated with vasopressin, angiotensin, ATP and phenylephrine, which are known to be Ca(2+)-mediated glycogenolytic agents in rat liver tissue, and as a control, they were treated with the cyclic AMP-mediated hormones glucagon and isoproterenol. All agonists induce a time-dependent activation of glycogen phosphorylase. Glucagon and isoproterenol induce a somewhat higher degree of phosphorylase activation compared with vasopressin, angiotensin, ATP and phenylephrine, which all increase inositol tris-phosphate levels and have no effect on the cyclic AMP levels. The total activity of glycogen phosphorylase (a + b), amounting to 30 to 35 mU/mg protein, is found to be much lower than that found in rat liver tissue. Because only minor differences could be found, we conclude that the regulation of glycogen phosphorylase in human liver tissue is basically the same as that found in rat liver tissue.

Adenosine Triphosphate↗
PubMed1995

Evolutionary link between glycogen phosphorylase and a DNA modifying enzyme.

We report here an unexpected similarity in three-dimensional structure between glucosyltransferases involved in very different biochemical pathways, with interesting evolutionary and functional implications. One is the DNA modifying enzyme beta-glucosyltransferase from bacteriophage T4, alias UDP-glucose:5-hydroxymethyl-cytosine beta-glucosyltransferase. The other is the metabolic enzyme glycogen phosphorylase, alias 1.4-alpha-D-glucan:orthophosphate alpha-glucosyltransferase. Structural alignment revealed that the entire structure of beta-glucosyltransferase is topographically equivalent to the catalytic core of the much larger glycogen phosphorylase. The match includes two domains in similar relative orientation and connecting helices, with a positional root-mean-square deviation of only 3.4 A for 256 C alpha atoms. An interdomain rotation seen in the R- to T-state transition of glycogen phosphorylase is similar to that observed in beta-glucosyltransferase on substrate binding. Although not a single functional residue is identical, there are striking similarities in the spatial arrangement and in the chemical nature of the substrates. The functional analogies are (beta-glucosyltransferase-glycogen phosphorylase): ribose ring of UDP-pyridoxal ring of pyridoxal phosphate co-enzyme; phosphates of UDP-phosphate of co-enzyme and reactive orthophosphate; glucose unit transferred to DNA-terminal glucose unit extracted from glycogen. We anticipate the discovery of additional structurally conserved members of the emerging glucosyltransferase superfamily derived from a common ancient evolutionary ancestor of the two enzymes.

Amino Acid Sequence↗
PubMed1992

Multiple phosphate positions in the catalytic site of glycogen phosphorylase: structure of the pyridoxal-5'-pyrophosphate coenzyme-substrate analog.

The three-dimensional structure of an R-state conformer of glycogen phosphorylase containing the coenzyme-substrate analog pyridoxal-5'-diphosphate at the catalytic site (PLPP-GPb) has been refined by X-ray crystallography to a resolution of 2.87 A. The molecule comprises four subunits of phosphorylase related by approximate 222 symmetry. Whereas the quaternary structure of R-state PLPP-GPb is similar to that of phosphorylase crystallized in the presence of ammonium sulfate (Barford, D. & Johnson, L.N., 1989, Nature 340, 609-616), the tertiary structures differ in that the two domains of the PLPP-GPb subunits are rotated apart by 5 degrees relative to the T-state conformation. Global differences among the four subunits suggest that the major domains of the phosphorylase subunit are connected by a flexible hinge. The two different positions observed for the terminal phosphate of the PLPP are interpreted as distinct phosphate subsites that may be occupied at different points along the reaction pathway. The structural basis for the unique ability of R-state dimers to form tetramers results from the orientation of subunits with respect to the dyad axis of the dimer. Residues in opposing dimers are in proper registration to form tetramers only in the R-state.

Amino Acid Sequence↗
PubMed1993

Engineered plant phosphorylase showing extraordinarily high affinity for various alpha-glucan molecules.

alpha-Glucan phosphorylases are characterized by considerable difference in substrate specificities, even though the primary structures are well conserved among the enzymes from microorganisms, plants, and animals. The higher plant phosphorylase isozyme designated as type L exhibits low affinity for a large, highly branched glucan (glycogen), presumably due to steric hindrance caused by a unique 78-residue insertion located beside the mouth of the active-site cleft, whereas another isozyme without the insertion (designated as type H) shows very high affinity for both linear and branched glucans. Using the recombinant type L isozyme from potato tuber as a starting framework and aiming at altering its substrate specificity, we have genetically engineered the 78-residue insertion and its flanking regions. Firstly, removal of the insertion and connection of the newly formed C- and N-terminals yielded a totally inactive enzyme, although the protein was produced in Escherichia coli cells in a soluble form. Secondly, a chimeric phosphorylase, in which the 78-residue insertion and its flanking regions are replaced by the corresponding region of the type H isozyme, has been shown to exhibit high affinity for branched glucans (Mori, H., Tanizawa, K., & Fukui, T., 1993, J. Biol. Chem. 268, 5574-5581), but when two and four unconserved residues in the N-terminal flanking region of the chimeric phosphorylase were mutated back to those of the type L isozyme, the resulting mutants showed significantly lowered affinity for substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

A continuous spectrophotometric method for the determination of glycogen phosphorylase-catalyzed reaction in the direction of glycogen synthesis.

We offer a "continuous" spectrophotometric method for the determination of the glycogen phosphorylase-catalyzed reaction in the direction of glycogen synthesis. This method relies on a coupled enzyme procedure, involving purine nucleoside phosphorylase and its chromophoric substrate, 2-amino-6-mercapto-7-methyl ribonucleoside (7-methyl-6-thioguanosine (MTGuo)), for the estimation of inorganic phosphate (M. R. Webb, Proc. Natl. Acad. Sci. USA 89, 4884-4887, 1992). We have examined the effects of the reaction components on the catalytic activities of both "primary" and "coupling" enzymes. While MTGuo exhibits no effect on the glycogen phosphorylase-catalyzed reaction, glucose 1-phosphate and AMP are partially inhibitory to nucleoside phosphorylase. However, the latter effects pose no problem as long as the coupling enzyme is maintained at a relatively higher concentration in the assay system. The coupled enzyme assay system, standardized for the measurement of glycogen phosphorlase activity, has enabled us to demonstrate explicitly that the rate of the enzyme-catalyzed reaction exhibits sigmoidal dependence on both AMP and glucose 1-phosphate concentrations. We argue that these sigmoidal profiles have been observed due to the sensitivity and precision of the present assay system.

Adenosine Monophosphate↗
PubMed1999

Structural and functional characterization of the Drosophila glycogen phosphorylase gene.

We identified a P element insertional mutant of the Drosophila glycogen phosphorylase (DGPH) gene. Glycogen phosphorylase protein concentration and enzyme activity are decreased while glycogen content is increased in flies homozygous for the mutant allele. The DGPH gene has been cloned and sequenced; its open reading frame codes for a protein of 844 amino acids with a predicted molecular mass of 97 kDa. Comparison of the conceptual amino acid sequence of the Drosophila glycogen phosphorylase with glycogen phosphorylase sequences from other organisms shows a high degree of homology to mammalian enzymes. All the residues of the allosteric effector binding sites, the active site, and the site of phosphorylation are exactly conserved, but some of the residues of the glycogen storage site are not.

Amino Acid Sequence↗
PubMed1994

An epitope proximal to the carboxyl terminus of the alpha-subunit is located near the lobe tips of the phosphorylase kinase hexadecamer.

An epitope of the alpha-subunit of phosphorylase kinase from fast-twitch skeletal muscle was localized to the tips of the bilobal kinase molecule by two types of immunoelectron microscopy. This is the first direct evidence identifying the location of any of the enzyme's 16 subunits within the phosphorylase kinase molecule. Negatively stained complexes of phosphorylase kinase with an immunoglobulin G monoclonal antibody specific for the alpha-subunit (mAb 157) were observed by conventional transmission electron microscopy, and complexes of the unstained enzyme with undecagold-labeled Fab' fragments derived from mAb 157 were visualized by scanning transmission electron microscopy. Images from both techniques indicate a symmetrical arrangement of the epitope, consistent with a "head-to-head" packing arrangement of the four alpha-subunits. In Western blots, mAb 157 crossreacted with comigrating fragments obtained by digesting non-denatured phosphorylase kinase with a variety of proteases, suggesting that the epitope for the anti-alpha mAb is contained within a protease-resistant domain. Partial sequencing of a 24.1 kDa immunoreactive chymotryptic fragment narrowed the epitope to somewhere within the carboxyl-terminal one-sixth of the alpha-subunit. Studies of the crossreactivity of mAb 157 with the holoenzyme in the presence of calmodulin, after phosphorylation or with different isoforms (all with known alpha-subunit sequence targets or differences), suggest that the epitope is even more proximal to the carboxyl terminus. This epitope was not implicated in any known function or activity of the enzyme, suggesting that the region proximal to the carboxyl terminus of the alpha-subunit, and thus to the lobe tips of the hexadecamer, may have a role other than catalytic or regulatory.

Amino Acid Sequence↗
PubMed2001

Direct visualization of the calmodulin subunit of phosphorylase kinase via electron microscopy following subunit exchange.

Calmodulin is a tightly bound, intrinsic subunit (delta) of the hexadecameric phosphorylase-b kinase holoenzyme, (alphabetagammadelta)4. To introduce specifically labeled calmodulin into the phosphorylase-b kinase complex for its eventual visualization by electron microscopy, we have developed a method for rapidly exchanging exogenous calmodulin for the intrinsic delta subunit. This method exploits previous findings that low concentrations of urea in the absence of Ca(2+) ions cause the specific dissociation of only the delta subunit from the holoenzyme [Paudel, H. K., and Carlson, G. M. (1990) Biochem. J. 268, 393-399]. In the current study, phosphorylase-b kinase was incubated with excess exogenous calmodulin and a threshold concentration of urea to promote exchange of its delta subunit with the exogenous calmodulin. Size exclusion HPLC was then used to remove the excess calmodulin from the holoenzyme containing exchanged delta subunits. Using metabolically labeled [35S]calmodulin to allow quantification and optimization of exchange conditions, we achieved exchange of approximately 10% of all delta subunits within 1 h, with the exchanged holoenzyme retaining full catalytic activity. Calmodulins derivatized with Nanogold for visualization by scanning transmission electron microscopy were then exchanged for delta, which for the first time allowed localization of the delta subunit within the bridged, bilobal phosphorylase b kinase holoenzyme complex. The delta subunits were determined to be near the edge of the lobes, just distal to the interlobal bridges and proximal to a previously identified region of the enzyme's catalytic gamma subunit.

Animals↗