Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “PHOSPHORYLASES”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 523 records · Page 29Linked to original sources
PubMed1980

Platelet phosphorylase kinase activity and its regulation by the calcium-dependent regulatory protein, calmodulin.

Platelet phosphorylase kinase (ATP:phosphorylase phosphotransferase, EC 2.7.1.38) was found to be a Ca2+-sensitive enzyme. It was two Ka values for Ca2+ viz. 0.25 and 2.6 microM, respectively. The "calcium-dependent regulator" or calmodulin can enhance the activity of phosphorylase kinase, increasing its affinity for Ca2+. In the presence of calmodulin phosphorylase kinase has only one, high affinity binding site for Ca2+ (Ka = 0.27 microM). Platelet phosphorylase kinase can be phosphorylated by endogenous cyclic AMP-dependent protein kinase increasing its catalytic activity and this activation process is reversed by dephosphorylation. The changing level of intracellular Ca2+ and cyclic AMP may control the activity of phosphorylase kinase, regulating the mobilization of glycogen.

Blood Platelets↗
PubMed1981

Purification of chloroplast alpha-1,4-glucan phosphorylase from spinach leaves by chromatography on Sepharose-bound starch.

Chloroplast alpha-1,4-glucan phosphorylase (EC 2.4.1.1) has been purified to homogeneity from spinach leaves as revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purification procedure is composed of (NH4)2SO4 precipitation, ion-exchange chromatography, and chromatography on Sepharose-bound starch. In order to achieve binding of the chloroplast phosphorylase, a previously described Sepharose-glucan gel (Steup, M. Schächtele, C. and Latzko, E. (1980) Planta 148, 168-173) was modified by introducing hydrophobic groups in addition to the covalently bound starch. The chloroplast phosphorylase exhibited complete binding to this type of gel and could be eluted by a mixture of soluble glucan and NaCl. For the purified chloroplast phosphorylase, sodium dodecyl-sulfate polyacrylamide gel electrophoresis and pyridoxal phosphate determination resulted in a molecular weight estimation of about 110,000 per monomer. The apparent molecular weight of the native enzyme, as determined by polyacrylamide density gradient electrophoresis and gel filtration on Sephadex G-200, was 200,000 and 220,000, respectively. The data indicate that the chloroplast phosphorylase is a dimer with a molecular weight higher than that of the non-chloroplast phosphorylase.

Chromatography, Affinity↗
PubMed1990

5'-deoxy-5'-methylthioadenosine phosphorylase--V. Acycloadenosine derivatives as inhibitors of the enzyme.

Various adenosine acyclonucleoside derivatives were tested as inhibitors of 5'-deoxy-5'-methylthioadenosine (MeSAdo) phosphorylase, an enzyme involved in the salvage of adenine and methionine from MeSAdo. The 2-halogenated derivatives of acyloadenosine [9-(2-hydroxyethoxy-methyl)adenine], including the chloro-, bromo- and iodo-congeners, all inhibited murine Sarcoma 180 (S180) MeSAdo phosphorylase, with Ki values in the range of 10(-6) to 10(-5) M. Halogenated derivatives of 9-(1,3-dihydroxy-2-propoxymethyl)adenine, which more closely resemble the natural substrate, were substantially more potent inhibitors of the enzyme, with Ki values in the range of 2-7 x 10(-7) M. 5'-Methylthio and 5'-halogenated analogs of 2'-deoxy-1',2'-seco-adenosine were weak inhibitors, with Ki values of 10(-4) M or greater. 9-[(1-Hydroxy-3-iodo-2-proxy)methyl]adenine. (HIPA), the derivative with the lowest Ki values among these analogs, was a competitive inhibitor of S180 MeSAdo phosphorylase. In preliminary studies, HIPA inhibited MeSAdo phosphorylase in intact HL-60 human promyelocytic leukemia cells, as it limited the incorporation of [8-14C]MeSAdo into cellular adenine nucleotide pools. In addition, 9-(phosphonoalkyl)adenines, representing potential multisubstrate inhibitors of MeSADo phosphorylase, were synthesized. Of these the heptyl derivative was the most potent inhibitor, with a Ki of 1.5 x 10(-5) M at low (3.5 mM) phosphate concentrations. The inhibitory effects of these analogs could be ablated at high phosphate concentrations (50 mM), suggesting that they interact with the phosphate binding site on the enzyme. Some of these novel MeSAdo phosphorylase inhibitors may have a role in cancer chemotherapy as potentiators of agents that block purine de novo synthesis, e.g. antifolates and 6-methylmercaptopurine ribonucleoside.

Adenine↗
PubMed1986

Isolation of partial cDNAs for rat liver and muscle glycogen phosphorylase isozymes.

cDNA clones for the rat liver and muscle glycogen phosphorylase isozymes have been isolated using isozyme-selective antibodies and libraries prepared in the expression vector, lambda gt11. A 1.2 kb cDNA coding for the carboxy-terminal domain of rat liver phosphorylase was found to have 82% homology with the amino acid sequence of rabbit muscle phosphorylase. Limited sequencing of rat muscle phosphorylase cDNA indicated a 95% homology with the rabbit muscle enzyme. The rat liver clone has eight additional amino acid residues at the COOH-terminus compared to the rat muscle clone. Furthermore, 17 of 26 (65%) residues between amino acids 815-840 differ between liver and muscle isozymes. The similarity in enzymatic properties and conservation of structure except at the COOH-terminus suggest that the liver and muscle phosphorylase isozymes do not exist in order to have significant differences in the regulation of glycogen breakdown in the two tissues. Rather, the phosphorylase isozymes probably evolved for tissue-specific transcriptional regulation of the genes in liver and muscle.

Animals↗
PubMed1983

Regulation of glycogen phosphorylase activity in fat body of Locusta migratoria and Periplaneta americana.

Saline extracts of the corpus cardiacum (CC) of Locusta migratoria activate glycogen phosphorylase in locust fat body. The response of phosphorylase to CC extracts and to synthetic adipokinetic hormone (AKH) suggests that the factor responsible for the activating effect of the CC on phosphorylase is AKH, supplemented to a minor degree with Compound II. Octopamine does not influence fat body phosphorylase activity in locusts, however, it elicits a rapid short-term hyperlipemia. In cockroaches, Periplaneta americana, injection of octopamine results in a strong activation of fat body phosphorylase within 1 min. Cockroach CC extract exerts a more prolonged effect on phosphorylase activity than does octopamine.

Adipose Tissue↗
PubMed1989

Activation and inactivation of glycogen phosphorylase isoenzymes purified from diabetic rat heart.

1. Hearts of diabetic rats gradually accumulate glycogen, although the activities of glycogen synthase and glycogen phosphorylase are altered in favor of a depletion of glycogen. 2. Phosphorylase in diabetic hearts has been reported to be even more activated in response to adrenaline than controls. 3. The situation is further complicated by the fact that in rat heart two isoenzymes of phosphorylase are present. Therefore we have studied the properties of phosphorylases purified from diabetic rat heart in more detail. 4. This investigation revealed that compared to controls: (A) the amount of enzyme protein which could be isolated from diabetic animals is drastically lower; (B) the affinities towards glycogen and inorganic phosphate are decreased; (C) the activation by phosphorylase kinase is delayed; and (D) the inactivation by protein phosphatase-1 is accelerated. 5. We conclude that all of the reported changes in diabetes might contribute to a phosphorylase system less able to catalyze glycogen breakdown effectively.

Animals↗
PubMed1992

Identification of a putative collagen-binding protein from chicken skeletal muscle as glycogen phosphorylase.

We have purified and generated antisera to a 95 kDa skeletal muscle protein that constitutes the largest mass fraction of gelatin-agarose binding proteins in skeletal muscle. Preliminary results indicated that this 95 kDa chicken skeletal muscle protein bound strongly to gelatin-agarose and type IV collagen-agarose, suggesting a possible function in muscle cell adhesion to collagen. However, N-terminal sequencing of proteolytic fragments of the 95 kDa protein indicates that it is the chicken skeletal muscle form of glycogen phosphorylase, the binding of which to gelatin-agarose is unlikely to be biologically relevant. Further characterization showed that the skeletal muscle form of glycogen phosphorylase is immunologically distinct from the liver and brain forms in the chicken, and suggests that, unlike mammalian skeletal muscle, chicken skeletal muscle may have two phosphorylase isoforms. Furthermore, immunolocalization data and solubility characteristics of glycogen phosphorylase in muscle extraction experiments suggest the enzyme may interact strongly with an unidentified component of the muscle cytoskeleton. Thus, this study yields a novel purification technique for skeletal muscle glycogen phosphorylase, provides new information on the distribution and isoforms of glycogen phosphorylase, and provides a caveat for using gelatin affinity chromatography as a primary step in purifying collagen-binding proteins from skeletal muscle.

Amino Acid Sequence↗
PubMed1986

Regulation of hepatic glycogen phosphorylase and glycogen synthase by calcium and diacylglycerol.

Incubation of rat hepatocytes with angiotensin II (1 nM) produced a time-dependent accumulation of 1, 2-diacylglycerol and inactivation of glycogen synthase with maximum effects at 10 min. The level of diacylglycerol then gradually declined and the activity of glycogen synthase I returned to control values at 30 min. In contrast, angiotensin II caused an increase in cytosolic Ca2+ and an activation of glycogen phosphorylase which were rapid and transient, reaching maximum values in less than 2 min and then returning to control levels at 15 min. There were excellent correlations between the changes in glycogen synthase I and diacylglycerol levels and between the changes in phosphorylase alpha and cytosolic Ca2+ in these time-course studies. However, there was no correlation between the changes in diacylglycerol and phosphorylase alpha or between the changes in cytosolic Ca2+ and glycogen synthase I. Norepinephrine also caused a slow increase in diacylglycerol and inactivation of glycogen synthase, and a rapid increase in cytosolic free Ca2+ and activation of glycogen phosphorylase. Addition of an alpha1-adrenergic blocker (prazosin or phentolamine) caused rapid decreases in cytosolic free Ca2+ and phosphorylase alpha, but only slowly reversed the inactivation of synthase and accumulation of diacylglycerol. The dose-response curves for norepinephrine and prazosin on glycogen synthase were well correlated with those on diacylglycerol. It is proposed that in liver cells, Ca2+-mobilizing hormones regulate phosphorylase a through a Ca2+-dependent mechanism and inactivate glycogen synthase through the generation of diacylglycerol, at least in part. The data provide additional support for the view that protein kinase C may be important in the regulation of glycogen synthase in liver.

Angiotensin II↗
PubMed1988

Electrophoretic analysis of liver glycogen phosphorylase activation in the freeze-tolerant wood frog.

As an adaptation for overwinter survival, the wood frog, Rana sylvatica is able to tolerate the freezing of extracellular body fluids. Tolerance is made possible by the production of very high amounts of glucose in liver which is then sent to other organs where it acts as a cryoprotectant. Cryoprotectant synthesis is under the control of glycogen phosphorylase which in turn is activated in response to ice formation. To determine the mechanism of phosphorylase activation, a quantitative analysis of phosphorylase protein concentration and enzymatic activity in liver was carried out following separation of the phosphorylated a and nonphosphorylated b forms of the enzyme on native polyacrylamide gels. The results suggest that in gels, the b form is completely inactive, even in the presence of AMP and sodium sulfate, whereas the a form is active and stimulated 3-fold by these substances. Further, phosphorylase activation appears to arise solely from conversion of the b to a form of the enzyme without an increase in phosphorylase concentration or activation of a second isozyme. The quantitative analysis presented here should prove generally useful as a simple and rapid method for examining the physiological and genetic regulation of phosphorylase in animal cells.

Animals↗
PubMed1991

Phosphorylase kinase from bovine stomach smooth muscle: a Ca2(+)-dependent protein kinase associated with an actin-like molecule.

Phosphorylase kinase was purified (110-fold) from bovine stomach smooth muscle by a procedure involving DEAE-cellulose chromatography, ammonium sulfate fractionation and glycerol density ultracentrifugation. On sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) the final enzyme preparation shows a single protein band of 43 kDa. The purified protein exhibits a close similarity with bovine aortic actin, as revealed by amino acid analysis and sequencing of a tryptic decapeptide fragment, although it differs widely from actin in several respects. In our effort to separate phosphorylase kinase activity from the 43 kDa protein we used a variety of chromatographic procedures, but in all cases the catalytic activity (when eluted) was accompanied by the 43 kDa protein band. Bovine stomach phosphorylase kinase exhibits an apparent molecular mass of 950 kDa, it shows a low Vmax value for phosphorylase b (85 nmol.min-1.mg-1), a pH 6.8/8.2 activity ratio of 0.23, it has an absolute requirement for Ca2+ and it is activated 1.8-fold by Ca2+/calmodulin. Furthermore, the protein kinase activity is neither inhibited by antibodies against rabbit skeletal muscle phosphorylase kinase nor activated by protein phosphorylation. These results suggest that bovine stomach phosphorylase kinase is tightly bound to an aggregate of actin-like molecules.

Actins↗
PubMed1975

Activation and inactivation of phosphorylase and glycogen synthetase during perfusion of rat liver as influenced by epinephrine, glucagon and hydrocortisone.

1. The changes in phosphorylase activity and glycogen synthetase I (active form) activity during perfusion of rat liver were studied together with their responses to added epinephrine and glucagon. 2. Phosphorylase activity of the liver from fed or fasted rats fell rapidly during perfusion, regardless of whether the perfusate was added with glucose or not. The addition of epinephrine or glucagon at the start or at 60 min of perfusion caused a prompt restoration of the initial high activity. Both glycogen breakdown and glucose liberation proceeded in parallel with the changes in phosphorylase activity. 3. The I-form of glycogen synthetase in the liver from fed rats increased rapidly when the concentration of perfusate glucose was raised to near 10 mM. This increase was promptly prevented by the addition of epinephrine or glucagon. In contrast, glycogen synthetase of the liver from fasted rats responded to neither glucose nor epinephrine (or glucagon) during perfusion. 4. Phosphorylase in the liver of fasted, adrenalectomized rats did not respond to a low concentration of epinephrine (3 - 10(-8) M) or glucagon (5 - 10(-9) M), but was increased by higher concentrations of the hormones. The treatment of adrenalectomized rats with hydrocortisone restored the response of liver phosphorylase to the low concentrations of the hormones. Thus, glucocorticoid plays a "permissive" role by increasing the affinity of liver phosphorylase to epinephrine or glucagon.

Animals↗
PubMed1980

The stimulatory effect of testosterone propionate and 17 beta-estradiol on 5'-methylthioadenosine phosphorylase activity in rat target tissues.

The effect of castration and subsequent administration of 17 beta-estradiol and testosterone propionate on 5'-methylthioadenosine phosphorylase activity in rat target tissues was studied. Castration 34 days earlier resulted in a 95% reduction in ventral prostate 5'-methylthioadenosine phosphorylase activity and 16 days earlier in a 67% reduction in uterine 5'-methylthioadenosine phorphorylase activity. Four days of testosterone propionate administration stimulated ventral prostate 5'-methylthioadenosine phosphorylase activity 32% above castrate levels, which represented more than 50% of the intact control levels. 17 beta-Estradiol on the other hand stimulated uterine 5'-methylthioadenosine phosphorylase activity 35% above castrate controls within 24 h and with 3 days of continuous hormone treatment to within 97% of the intact control levels. However, castration and subsequent 17 beta-estradiol administration did not affect 5'-methylthioadenosine phosphorylase activity in rat liver and lung. Both prostate and uterine 5'-methylthioadenosine phosphorylase were shown to metabolize 5'-methylthioadenosine to 5-methylthioribose through a 5'-methylthioribose 1-phosphate intermediate. The data suggest aht 5'-methylthioadenosine is not allowed to accumulate in rat target tissues even under conditions which are known to stimulate polyamine synthesis.

Animals↗
PubMed1993

Glycogen phosphorylase activity in the liver of the frog Rana esculenta.

1. Phosphorylase activity has been assayed in liver extracts of the frog, Rana esculenta, during the winter period. In native conditions, most of the phosphorylase is present as AMP-independent activity and shows properties similar to those of the a form of the liver enzyme from other vertebrates. 2. It is suggested that regulation of phosphorylase activity is through interconversion between a and b forms operated by endogenous phosphorylase kinase and phosphatase. 3. Kinetic studies show hyperbolic saturation curves for glycogen with apparent Km of 2.91 mM and 9.67 mM for a and b forms, respectively. 4. A hyperbolic saturation curve is also observed for glucose 1-P in the case of phosphorylase a, with an apparent Km of 3.95 mM, whereas a sigmoidal kinetic is shown by the b form for the same substrate; from Hill plots an S0.5 of 24.2 mM was derived. 5. Hyperbolic responses were observed in the case of AMP, and Ka of 70 microM and 0.31 mM were calculated for phosphorylase a and b, respectively.

Adenosine Monophosphate↗
PubMed1984

Axotomy increases glycogen phosphorylase activity in motoneurones.

The relative distribution of glycogen phosphorylase a and b in the lumbar spinal cord of the adult rat following either transection or crush of the sciatic nerve has been studied. The activity of the glycogen phosphorylase was measured histochemically by its capacity to convert glucose-1-phosphate to glycogen which was then stained with iodine. Prior to any treatment, the enzyme was largely in its inactive b form. Sciatic section and crush produced a transient (24 h) decrease in the amount of the active glycogen phosphorylase a in the sciatic motoneurone pool. Fourteen days post-transection, but not crush, a marked increase in the level of the active glycogen phosphorylase a form of the enzyme could be detected in the axotomised motoneurones which persisted for up to 6 weeks. No equivalent changes occurred in the axotomized dorsal root ganglion cells. Glycogen phosphorylase although normally present in neurones in its inactive b form can be converted to the active a form by calcium or adenosine 3':5'-phosphate. The substantial increase in the level of glycogen phosphorylase a in axotomized motoneurones may be a reflection of an increased calcium influx into these cells due to the development of abnormally hyperexcitable membranes and the appearance of dendritic spikes that is known to occur in these motoneurones.

Adenosine Monophosphate↗
PubMed1979

Study of the interaction between phosphorylase and hydrophobic groups by means of affinity electrophoresis.

A homologous series of water-soluble alkyl-dextrans varying in the length of their hydrocarbon side-chain [-NH-(CH2)n-CH3; n = 1-5] were synthesized. When alkyl-dextrans were entrapped in polyacrylamide gel, the electrophoretic mobility of phosphorylase was retarded by hydrophobic interaction between phosphorylase and the immobilized alkyl groups. The dissociation constants of rabbit brain phosphorylase, rabbit skeletal muscle phosphorylase a and b and potato glycogen and starch phosphorylases were calculated from the extent of the retardation of mobility as a function of the concentration of the alkyl groups. As the length of the hydrocarbon side-chains of alkyl groups increased, the affinity of the phosphorylases for the alkyl groups increased. The introduction of a hydroxyl or an amino group at the terminal position of the hydrocarbon side-chain diminished the affinity.

Animals↗
PubMed1976

Site of action and biphasic effect of neutral salts in the phosphorylase kinase reaction.

The inhibition of phosphorylase kinase catalytic activity by 0.1 M neutral salts was predicted by the Hofmeister series of anions. The site of action of the salts was determined by the following evidence to be on the phosphorylase kinase molecule directly, rather than on its protein substrate. (1) Nonactivated kinase was more sensitive to salt inhibition than the activated form. (2) Ca2+ partially overcame the inhibition of nonactivated kinase. (3) Inhibition by Cl- occurred with either phosphorylase or a tetradecapeptide containing the convertible seryl residue as substrate. (4) Phosphorylation of nonactivated phosphorylase kinase by protein kinase was markedly inhibited by NaNO3, but this salt had little effect on the phosphorylation of histone by protein kinase. The influence of neutral salts on phosphorylase kinase activity was biphasic. Although activity was inhibited at low salt concentrations, it actually was stimulated as the salt concentration was increased. A similar biphasic response to various salt concentrations was observed in the velocities of autophosphorylation of phosphorylase kinase. The lag in the rate of product formation seen during the activity assay was less pronounced at inhibitory salt concentrations and was abolished at stimulatory salt concentrations. How the influence of salts relates to autophosphorylation and the lag is considered.

Animals↗
PubMed1976

Evidence for negative cooperativity in the adsorption of phosphorylase b on hydrophobic agaroses.

The concept of cooperativity appears to be the key to the understanding of the complex mechanisms underlying the adsorption of proteins to agaroses substituted with hydrophobic alpha-aminoalkanes. The adsorption of phosphorylase b occurs through the positive cooperative interaction of a critical number of approximately 3-5 butyl and a higher number of methyl residues with ocrresponding sites on the enzyme. The amount of adsorbed phosphorylase b per millileter of packed gel (methyl-, butyl-Sepharose) in the absence and presence of 1.1 M ammonium sulfate at temperatures between 0 and 34 degrees C is a power function of the free solute equilibrium concentration (Freundlich isotherm). In contrast, the adsorption of cyanmyoglobin to phosphocellulose is described by the Langmuir equation. The surface coverage dependent isosteric heats of adsorption for phosphorylase b indicate an endothermic reaction only on the butyl-Sepharose in the presence of high salt concentrations. Scatchard plots of the Freundlich isotherms of phosphorylase b are concave upwards, typical of negative cooperativity. Hill plots of these isotherms (5-70% saturation) yield coefficients between nH = 0.39 and 0.71. At high surface coverages, the Hill coefficients approach unity. Apparent association constants (K0.5) of 4-39 X 10(4) M-1 are calculated for the adsorption of phosphorylase b, as compared to 2-9 X 10(4) M-1 for the adsorption of cyanmyoblobin. In general, negative cooperativity of binding may be explained by changes in the affinity of the ligand for the matrix, due to the sequential multivalent adsorption, and competition of phosphorylase b molecules for the critical number of alkyl residues (nonindependence of binding) on one side and to variations in the configuration of binding and entropy on the other.

Adsorption↗
PubMed1975

The effects of glucose and of potassium ions on the interconversion of the two forms of glycogen phosphorylase and of glycogen synthetase in isolated rat liver preparations.

In the isolated perfused rat liver, increasing glucose concentration from 5.5 to 55 mm in the perfusion medium caused a sequential inactivation of glycogen phosphorylase and activation of glycogen synthetase. The latter change was preceded by a lag period which corresponded to the time required to inactivate the major part of the phosphorylase. 2. The same sequence of events was observed in isolated rat hepatocytes incubated at 37C. In this preparation, the rate of phosphorylase inactivation was greatly increased by increasing the concentration of glucose and/or of K+ ions in the external medium. The same agents also caused the activation of glycogen synthetase, but this effect was secondary to the inactivation of phosphorylase. 3. In both types of preparations, the rate of synthetase activation was modulated by the residual amount of phosphorylase a that remained after the initial phase of rapid inactivation and was independent of glucose concentration. 4. In isolated hepatocytes, the rate of conversion of glucose into glycogen was propotional to the activity of synthetase a in the preparation. This conversion was preceded by a lag period which could be shortened by increasing either glucose or K+ concentration in the medium. The incorporation of labelled glucose into glycogen was simultaneous with a glycogenolytic process which could not be attributed to the activity of phosphorylase a.

Animals↗