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PubMed1985

Use of 6-fluoroderivatives of pyridoxal and pyridoxal phosphate in the study of the coenzyme function in glycogen phosphorylase.

6-Fluoropyridoxal phosphate (6-FPLP) has been synthesized. Its properties were studied, and it was used, along with 6-fluoropyridoxal (6-FPAL), to reconstitute apophosphorylase b. Kinetic studies of the resulting enzymes showed that phosphorylases reconstituted with 6-FPLP and 6-FPAL have characteristics similar to those of native and pyridoxal enzymes, respectively, except that the former two enzymes have lower Vmax values. 19F NMR and UV spectra of 6-FPLP phosphorylase showed that the coenzyme forms a neutral enolimine Schiff base. Because the UV and fluorescence spectra of 6-FPLP phosphorylase are comparable to those obtained with native phosphorylase, it further confirms the postulate that pyridoxal phosphate forms a neutral enolimine Schiff base in phosphorylase. The results suggest that the 3-OH group is protonated and the pyridine nitrogen unprotonated in both 6-FPLP phosphorylase and native enzyme. 19F NMR study of 6-FPLP- and 6-FPAL-reconstituted phosphorylases in the inactive and active states indicates that the protein structure near the coenzyme binding site undergoes certain changes when these enzymes are activated by the substrates and AMP. The comparison of the properties of 6-FPLP-reconstituted and native phosphorylases implies that the ring nitrogen of the coenzyme PLP in phosphorylase may interact with the protein during catalysis, and this interaction is important for efficient catalysis by phosphorylase.

Animals↗

PubMedDate not supplied

Effects of chronic ethanol ingestion on male and female rat liver glycogen phosphorylase phosphatase.

The effects of chronic ethanol ingestion on the interconversion of the active to the inactive form of glycogen phosphorylase by phosphorylase phosphatase was studied. Male and female rats also were compared. Chronic ethanol feeding decreased phosphorylase a and total phosphorylase activity in male rats. In females, no change was observed in phosphorylase a, whereas total phosphorylase activity was increased 73%. This was found to correlate with the relative activities of phosphorylase phosphatase. The data show differences between the two sexes with regard to the AMP inhibition of phosphorylase phosphatase and the caffeine stimulation of the phosphatase. Ethanol markedly enhanced the AMP inhibition of the phosphatase in males but had no effect in females. Further studies in females showed that ethanol completely obliterated the well documented stimulation of the phosphatase by caffeine; however, it did not alter the caffeine effect in males. These data suggest possible alterations in the tertiary structure of phosphorylase a.

Adenosine Monophosphate↗

PubMed1981

Regulation of the dephosphorylation of glycogen phosphorylase a and synthase b by glucose and caffeine in isolated hepatocytes.

Synergistic regulation of glycogen phosphorylase a by the competitive inhibitors glucose and caffeine in vitro indicates a possible physiological role for the negative effector site which binds caffeine (nucleoside site). In intact viable hepatocytes glucose promotes the phosphorylase a to be conversion by phosphorylase phosphatase. This conversion is considered to be a necessary prelude to the activation of glycogen synthase by phosphatase and of importance in hepatic regulation of glucose homeostasis. The effects of glucose and(or) caffeine on the conversion of phosphorylase a to b and synthase b to a were studied. Assays of phosphorylase a were used which limited synergistic inhibition (in the assay) by these ligands. Such an approach is necessary to achieve an accurate measure of phosphatase activity in the viable hepatocyte when the combination of ligands is used. The data indicate that in the presence of caffeine and glucose together, the rate of loss of phosphorylase a is significantly increased (1.7-fold) over that in the presence of glucose alone. Phosphorylase phosphatase is activated. The sequential activation of glycogen synthase was also accelerated in the presence of both ligands. The results are consistent with an in vivo function for the nucleoside site, similar to that of glucose. A controlling role or phosphorylase in the regulation of glycogen metabolism by glucose is supported. Although the existence and nature of an intracellular effector is as yet unknown, crystallographic analyses of phosphorylase a crystals soaked in perchloric acid extracts of liver demonstrate that the negative effector site binds a natural metabolite.

Animals↗

PubMed1996

Allosteric regulation of liver phosphorylase a: revisited under approximated physiological conditions.

Phosphorylase removes glucosyl units from the terminal branches of glycogen through phosphorolysis, forming glucose-1-P. It is present in two interconvertible forms, phosphorylase a and b. The a form is the active form and is rate limiting in glycogen degradation. The activities of phosphorylase a and of total phosphorylase as conventionally measured exceed the activities of glycogen synthase R (active form) and of total synthase by approximately 10- and 20-fold. Thus, unless phosphorylase a is inhibited or compartmentalized or its substrates are exceedingly low in vivo, net glycogen synthesis could not occur. In addition, following an administered dose of glucose, phosphorylase a activity changes little when glycogen is being synthesized, is stable, or is being degraded, suggesting an important role for allosteric effectors in regulation. Therefore, we have determined the effect of potential modifiers of enzyme activity at estimated intracellular concentrations. Purified liver phosphorylase a was used. Activity was measured in the direction of glycogenolysis, at 37 degrees C, pH 7.0, and under initial rate conditions. Both a Km and a near-saturating concentration of inorganic phosphate (substrate) were used in the assays. A physiological concentration of AMP was saturating. It decreased the Km for Pi by approximately 50% and stimulated activity. ADP, ATP, and glucose inhibited activity. Fructose-1-P inhibited activity only at a high and nonphysiological concentration. Glucose-6-P and UDP-glucose were not significant inhibitors. Inhibition of activity by ADP was little affected by the addition of AMP. However, AMP partially abolished the inhibitory effect of ATP and completely abolished the inhibitory effect of glucose. When AMP, ADP, ATP, glucose-6-P, UDP-glucose, glucose, and fructose-1-P were added together, the net effect was no change in phosphorylase a activity compared to the activity without any effectors. In addition, changes in glucose concentration did not affect activity. K glutamine modestly stimulated activity. Numerous other metabolites were tested and were without effect. The present data indicate that the known endogenous allosteric effectors cannot explain the smaller than expected in vivo phosphorylase a activity or the regulation of phosphorylase a activity.

Adenine Nucleotides↗

PubMed1991

A comparative study of the microsomal S6 phosphatase and phosphorylase phosphatase activities in rat liver.

Rat liver microsomes contain type-1 S6 phosphatase (acting on the serine residues phosphorylated by protein kinase A) and type-1 phosphorylase phosphatase activities. The main aim of this study has been to characterize the microsomal S6 phosphatase activity and to compare its properties with those of the phosphorylase phosphatase activity in the same microsomal preparation. The specific activities of both microsomal S6 phosphatase and phosphorylase phosphatase were 1.6- to 1.7-fold higher in the smooth endoplasmic reticulum than in the rough sarcoplasmic reticulum. Both phosphatase activities were inhibited to a similar extent by MgCl2 (10 mM) and NaF (22 mM), were completely suppressed by glycerophosphate (80 mM) and ZnCl2(10 mM), and were stimulated by MnCl2(1 mM). When analyzed by gel filtration on Sephadex G-100 superfine, both phosphatase activities eluted as broad peaks, stretching from the void volume to 45-60 kDa. The microsomal S6 phosphatase and phosphorylase phosphatase activities also displayed the following distinct characteristics: (a) Mn2+ stimulated the S6 phosphatase activity 2.9-fold more than the phosphorylase phosphatase activity, (b) limited trypsin digestion of microsomal preparations increased the phosphorylase phosphatase activity by 1.5- to 2-fold, but decreased the S6 phosphatase activity by 50%, (c) a synthetic peptide analog of S6 (S6229-239) (200 microM), which did not act as a substrate for the microsomal S6 phosphatase and did not affect its activity, inhibited the microsomal phosphorylase phosphatase activity by about 50%, and (d) the elution profile of the phosphorylase phosphatase activity was markedly broader than that of the S6 phosphatase activity. A series of in vivo studies showed that streptozotocin-diabetes and insulin replacement therapy as well as ip injection of insulin or vanadate, which modified the microsomal S6 phosphatase activity, had no statistically significant effects on the microsomal phosphorylase phosphatase activity. Taken together, these results suggest that the microsomal S6 phosphatase and phosphorylase phosphatase activities are due to two distinct enzyme populations.

Animals↗

PubMed1978

Evidence for the non-identity of proteins having synthase phosphatase, phosphorylase phosphatase and histone phosphatase activity in rat liver.

Synthase phosphatase, phosphorylase phosphatase and histone phosphatase in rat liver were measured using as substrates purified liver synthase D, phosphorylase alpha and 32P-labelled phosphorylated f1 histone, respectively. The three phosphatase enzymes had different sedimentation characteristics. Both synthase phosphatase and phosphorylase phosphatase were found to sediment with the microsomal fraction under our experimental conditions. Only 10% of histone phosphatase was in this fraction; the majority was in the cytosol. No change in histone phosphatase was observed in the adrenalectomized fasted rat whereas synthase phosphatase and phosphorylase phosphatase activities were decreased 5-10 fold. Fractionation of liver extract with ethanol produced a dissociation of the three phosphatase activities. When a partially purified fraction was put on a DEAE-cellulose column, synthase phosphatase and phosphorylase phosphatase both exhibited broad elution profiles but their activity peaks did not coincide. Histone phosphatase eluted as a single discrete peak. When the supernatant of CaCl2-treated microsomal fraction was put on a Sepharose 4B column, the majority of synthase phosphatase was found to elute with the larger molecular weight proteins whereas the majority of phosphorylase phosphatase eluted with the smaller species. Histone phosphatase migrated as a single peak and was of intermediate size. Synthase phosphorylase phosphatase by synthase D (Ki approximately 2 units/ml). The inhibition of synthase phosphatase by phosphorylase alpha was kinetically non-competitive with substrate. Histone phosphatase activity was not inhibited by synthase D or by phosphorylase alpha. The above results suggest that different proteins are involved in the dephosphorylation of synthase D, phosphorylase alpha and histone in the cell.

Animals↗

PubMedDate not supplied

The role of blood platelets in nucleoside metabolism: assay, cellular location and significance of thymidine phosphorylase in human blood.

The enzyme thymidine phosphorylase (thymidine: orthophosphate deoxyribosyltransferase, EC 2.4.2.4), which plays a crucial role in nucleic acid metabolism in both prokaryotic and eukaryotic cells by regulating the availability of thymidine, is present in mammalian blood. Here we describe a simple, rapid HPLC-based micromethod for the assay of blood thymidine phosphorylase. We have arbitrarily defined 1 unit of blood thymidine phosphorylase activity as the activity required to produce a 1-nM increment in the plasma concentration of thymine after incubation for 1 h at 37 degrees C with a saturating concentration of exogenous thymidine. In normal adults, whole (peripheral venous) blood thymidine phosphorylase activity with blood cells intact was 64 +/- 11 units (mean +/- S.D., n = 20, range 45-89). The apparent Michaelis constant for thymidine was of the order of 10(-4) M but varied nearly 5-fold between different individuals. Activity increased when blood cells were permeabilised or lysed with non-ionic detergents, implying that thymidine phosphorylase is an intracellular enzyme which may be influenced by exogenous as well as intracellular factors. When blood from normal donors was fractionated, thymidine phosphorylase activity consistently co-isolated with platelets. Whole-blood thymidine phosphorylase activity correlated well with platelet parameters. Although thymidine phosphorylase activity was also detected in plasma and serum, the small size and notorious fragility of platelets suggest its platelet origin. Blood from leukaemic donors showed significantly increased thymidine phosphorylase activity compared to normal controls (mean activity +/- S.D. was 96 +/- 27 units; range 58-140, n = 8). Thymine formation from thymidine was temperature- and pH-dependent in whole blood. 2'-Deoxyuridine and 3 of its 5-halogenated analogues (but not 3'-azido-3'-deoxythymidine (AZT), were catabolised by blood thymidine phosphorylase, even during blood clotting at room temperature. Assumptions about in vivo concentrations of these compounds should therefore be interpreted cautiously. In the presence of high concentrations of thymine and suitable deoxyribose donors, small amounts of thymidine were formed in some blood samples, so it is conceivable that thymidine catabolism may be reversible in vivo under some circumstances.

Blood Platelets↗

PubMed1982

Some comparative aspects of regulation of muscle and liver phosphorylase kinase.

The interaction of muscle and liver phosphorylase kinase with some proteins has been studied. It was shown that muscle G-actin has a visible stimulating effect on the dephosphorylated form of muscle phosphorylase kinase. The effect of F-actin on this enzyme is very low. The interaction of phosphorylase kinase with G-actin probably is one of the additional links between glycogenolysis and muscle contraction. To answer the question what subunit(s) of phosphorylase kinase is involved in the interaction with G-actin we studied the influence of actin on the kinase preparations previously activated to a different degree by partial proteolysis with endogenous protease(s) or with trypsin. G-actin has almost no stimulating effect on the preparations of phosphorylase kinase deeply activated by proteolysis (pH 6.8-8.2 activity ratio more than 0.2). The experiments with partial proteolysis allow us to suppose that alpha-subunit is involved in the interaction of phosphorylase kinase with G-actin. Skeletal muscle G-actin activates purified preparations of liver phosphorylase kinase but to a lower degree than muscle enzyme. Brain and liver calmodulin has a low activating effect on liver phosphorylase kinase in the presence of calcium. Calcium-independent action of calmodulin on the preparations of liver phosphorylase kinase is stronger; probably it is connected with a nonspecific effect of this small acidic protein on the liver enzyme. The basic protein protamine has a strong inhibitory effect on liver phosphorylase kinase.

Actins↗

PubMed1983

The comparative effects of 5'-methylthioadenosine and some of its analogs on cells containing, and deficient in, 5'-methylthioadenosine phosphorylase.

The antiproliferative effects of 5'-methylthioadenosine and the 5'-methylthioadenosine analogs, 5'-isobutylthioadenosine, 5'-deoxyadenosine and 5'-methylthiotubercidin were examined using two mouse cell lines, one 5'-methylthioadenosine phosphorylase-deficient the other containing 5'-methylthioadenosine phosphorylase. All of the compounds were found to be growth inhibitory to both cell lines, demonstrating that these compounds need not be degraded to exert their inhibitory effects. A correlation was observed between the potency of the growth inhibitory effect and the ability of the cells to degrade these compounds. 5'-Methylthioadenosine, 5'-deoxyadenosine and 5'-isobutylthioadenosine, all of which are substrates for the 5'-methylthioadenosine phosphorylase in vitro, were more growth inhibitory to the 5'-methylthioadenosine phosphorylase-deficient cells than to the 5'-methylthioadenosine phosphorylase-containing cells, whereas, the 7-deaza analog, 5'-methylthiotubercidin, a nondegradable inhibitor of the 5'-methylthioadenosine phosphorylase, was a more potent inhibitor of the 5'-methylthioadenosine phosphorylase-containing cell line. Due to the inhibition by 5'-methylthiotubercidin on 5'-methylthioadenosine phosphorylase in vitro the disposition of cellularly-synthesized 5'-methylthioadenosine was explored using both cell types. 5'-Methylthiotubercidin inhibited the accumulation of exogenous 5'-methylthioadenosine from 5'-methylthioadenosine phosphorylase-deficient cells with no effect on intracellular 5'-methylthioadenosine. In contrast, 5'-methylthiotubercidin caused a large accumulation of extracellular 5'-methylthioadenosine with a concomitant smaller increase intracellularly in 5'-methylthioadenosine phosphorylase-containing cells. That cellularly-synthesized 5'-methylthioadenosine as well as the cellular excretion of this nucleoside are altered in response to treatment with 5'-methylthiotubercidin suggests two possible sites at which 5'-methylthiotubercidin may exert its effect.

Adenosine↗

PubMed1987

Metabolism of pyrimidine bases and nucleosides by pyrimidine-nucleoside phosphorylases in cultured human lymphoid cells.

The anabolism of pyrimidine ribo- and deoxyribonucleosides from uracil and thymine was investigated in phytohemagglutinin-stimulated human peripheral blood lymphocytes and in a Burkitt's lymphoma-derived cell line (Raji). We studied the ability of these cells to synthesize pyrimidine nucleosides by ribo- and deoxyribosyl transfer between pyrimidine bases or nucleosides and the purine nucleosides inosine and deoxyinosine as donors of ribose 1-phosphate and deoxyribose 1-phosphate, respectively: these reactions involve the activities of purine-nucleoside phosphorylase, and of the two pyrimidine-nucleoside phosphorylases (uridine phosphorylase and thymidine phosphorylase). The ability of the cells to synthesize uridine was estimated from their ability to grow on uridine precursors in the presence of an inhibitor of pyrimidine de novo synthesis (pyrazofurin). Their ability to synthesize thymidine and deoxyuridine was estimated from the inhibition of the incorporation of radiolabelled thymidine in cells cultured in the presence of unlabelled precursors. In addition to these studies on intact cells, we determined the activities of purine- and pyrimidine-nucleoside phosphorylases in cell extracts. Our results show that Raji cells efficiently metabolize preformed uridine, deoxyuridine and thymidine, are unable to salvage pyrimidine bases, and possess a low uridine phosphorylase activity and markedly decreased (about 1% of peripheral blood lymphocytes) thymidine phosphorylase activity. Lymphocytes have higher pyrimidine-nucleoside phosphorylases activities, they can synthesize deoxyuridine and thymidine from bases, but at high an non-physiological concentrations of precursors. Neither type of cell is able to salvage uracil into uridine. These results suggest that pyrimidine-nucleoside phosphorylases have a catabolic, rather than an anabolic, role in human lymphoid cells. The facts that, compared to peripheral blood lymphocytes, lymphoblasts possess decreased pyrimidine-nucleoside phosphorylases activities, and, on the other hand, more efficiently salvage pyrimidine nucleosides, are consistent with a greater need of these rapidly proliferating cells for pyrimidine nucleotides.

Cells, Cultured↗

PubMed1987

Liver glycogen metabolism in endotoxin shock. II. Endotoxin administration increases glycogen phosphorylase activities in dog livers.

The effects of E. coli endotoxin administration on hepatic glycogen phosphorylase activities in dogs were investigated. Hepatic glycogen phosphorylase activities in both control and endotoxic dogs were inactivated spontaneously by preincubation of enzyme preparations at 25 degrees C. Total glycogen phosphorylase activity was not significantly altered during preincubation. The activity of glycogen phosphorylase a was increased by 83 and 80% at 1 and 2 hr postendotoxin, respectively, without preincubation; and by 203 and 133% at 1 and 2 hr postendotoxin, respectively, after 30 min preincubation. Without preincubation, the glycogen phosphorylase percentage a activity was increased from the control value of 37 to 58% at 1 hr postendotoxin and to 53% at 2 hr postendotoxin. After 30 min preincubation, the glycogen phosphorylase percentage a activity was increased from the control value of 10 to 28% at 1 hr postendotoxin and to 20% at 2 hr postendotoxin. The time required for half maximum inactivation of percentage a activity was 16.5, 33, and 24 min for control, 1 and 2 hr postendotoxin, respectively. Although the Vmax and Km for glucose-1-P for total glycogen phosphorylase were not affected by endotoxin administration, the Vmax for glucose-1-P for glycogen phosphorylase a was increased by 57.3 and 42.7% at 1 and 2 hr postendotoxin, respectively, with no change in the Km values. Glucose inhibited glycogen phosphorylase a activity both in control and endotoxin-injected dogs, but the I50 value was increased by 35% in endotoxin-injected (2 hr) dogs. AMP activated glycogen phosphorylase b activity both in control and endotoxin-injected dogs with no change in A0.5 values between the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Monophosphate↗

PubMed1998

Immunocytochemical localization of glycogen phosphorylase kinase in rat brain sections and in glial and neuronal primary cultures.

The physiological function of brain glycogen and the role of phosphorylase kinase as a regulatory enzyme in the cascade of reactions associated with glycogenolysis in the brain have not been fully elucidated. As a first step toward elucidating such a function, we studied the localization of phosphorylase kinase in glial and neuronal primary cell cultures, and in adult rat brain slices, using a rabbit polyclonal antibody against skeletal muscle glycogen phosphorylase kinase. Immunocytochemical examination of rat astroglia-rich primary cultures revealed that a large number of cells were positive for glycogen phosphorylase kinase immunoreactivity. These cells were also positive for vimentin, a marker for immature glia, while they were negative for glial fibrillary acidic protein, a marker for mature astroglia, and for galactocerebroside, an oligodendroglial marker. Neurons in rat neuron-rich primary cultures did not show any kinase-positive staining. In paraformaldehyde-fixed adult rat brain sections, phosphorylase kinase immunoreactivity was detected in glial-like cells throughout the brain, with relatively high staining found in the cerebral cortex, the cerebellum, and the medulla oblongata. Phosphorylase kinase immunoreactivity could not be detected in neurons, with the exception of a group of large neurons in the brain stem, most likely belonging to the mesencephalic trigeminal nucleus. Phosphorylase kinase was also localized in the choroid plexus and to a lesser degree in the ependymal cells lining the ventricles. Phosphorylase kinase thus appears to have the same cellular distribution in nervous tissue as its substrates, i.e. glycogen phosphorylase and glycogen, which suggests that the physiological role of brain phosphorylase kinase is the mobilization of glycogen stores to fuel the increased metabolic demands of neurons and astrocytes.

Animals↗

PubMedDate not supplied

Evolution of catalytic and regulatory sites in phosphorylases.

Glycogen phosphorylase (E.C.2.4.1.1) was the first enzyme shown to be regulated by allosteric effectors and by protein phosphorylation. Transcriptional control of bacterial phosphorylases further extends the range of regulatory mechanisms by which phosphorylases contribute to the control of carbohydrate metabolism. Despite their regulatory differences, all known phosphorylases share catalytic and structural properties and a strongly conserved pyridoxal-5'-phosphate binding site; this makes phosphorylases highly attractive for investigations into the evolution of regulatory mechanisms. The primary and tertiary structure of rabbit muscle phosphorylase has been determined completely. Recently, comparable amino acid sequences from plants and bacteria have been resolved. Here we report the sequence of 687 amino acids of Escherichia coli maltodextrin phosphorylase, deduced from a cloned malP gene sequence. Alignment of animal and bacterial phosphorylase sequences shows strong homology (48%) throughout 91% of the polypeptide chain enclosing the extrinsic catalytic region. Within this region, structural homology identifies a presumed phosphate-binding site from which the allosteric 5' AMP binding site of rabbit muscle phosphorylase might have developed. From the decreased alignment at the N-terminus and the presence of additional residues compared with bacterial phosphorylases, we conclude that the regulatory sequences that also carry the phosphorylation site in the muscle enzyme were joined to a presumed ancestral precursor gene by gene fusion after separation of the eukaryotic and prokaryotic lines of descent.

Adenosine Monophosphate↗

PubMed2004

Glucose 6-phosphate causes translocation of phosphorylase in hepatocytes and inactivates the enzyme synergistically with glucose.

The role of glucose 6-P (glucose 6-phosphate) in regulating the activation state of glycogen synthase and its translocation is well documented. In the present study, we investigated the effects of glucose 6-P on the activation state and compartmentation of phosphorylase in hepatocytes. Glucose 6-P levels were modulated in hepatocytes by glucokinase overexpression or inhibition with 5-thioglucose and the effects of AMP were tested using AICAR (5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside), which is metabolized to an AMP analogue. Inhibition of glucokinase partially counteracted the effect of glucose both on the inactivation of phosphorylase and on the translocation of phosphorylase a from a soluble to a particulate fraction. The increase in glucose 6-P caused by glucokinase overexpression caused translocation of phosphorylase a to the pellet and had additive effects with glucose on inactivation of phosphorylase. It decreased the glucose concentration that caused half-maximal inactivation from 20 to 11 mM, indicating that it acts synergistically with glucose. AICAR activated phosphorylase and counteracted the effect of glucose 6-P on phosphorylase inactivation. However, it did not counteract translocation of phosphorylase by glucose 6-P. Glucose 6-P and AICAR had opposite effects on the activation state of glycogen synthase, but they had additive effects on translocation of the enzyme to the pellet. There was a direct correlation between the translocation of phosphorylase a and of glycogen synthase to the pellet, suggesting that these enzymes translocate in tandem. In conclusion, glucose 6-P causes both translocation of phosphorylase and inactivation, indicating a more complex role in the regulation of glycogen metabolism than can be explained from regulation of glycogen synthase alone.

Aminoimidazole Carboxamide↗

PubMed2003

Inactivation of phosphorylase is a major component of the mechanism by which insulin stimulates hepatic glycogen synthesis.

Multiple signalling pathways are involved in the mechanism by which insulin stimulates hepatic glycogen synthesis. In this study we used selective inhibitors of glycogen synthase kinase-3 (GSK-3) and an allosteric inhibitor of phosphorylase (CP-91149) that causes dephosphorylation of phosphorylase a, to determine the relative contributions of inactivation of GSK-3 and dephosphorylation of phosphorylase a as alternative pathways in the stimulation of glycogen synthesis by insulin in hepatocytes. GSK-3 inhibitors (SB-216763 and Li+) caused a greater activation of glycogen synthase than insulin (90% vs. 40%) but a smaller stimulation of glycogen synthesis (30% vs. 150%). The contribution of GSK-3 inactivation to insulin stimulation of glycogen synthesis was estimated to be less than 20%. Dephosphorylation of phosphorylase a with CP-91149 caused activation of glycogen synthase and translocation of the protein from a soluble to a particulate fraction and mimicked the stimulation of glycogen synthesis by insulin. The stimulation of glycogen synthesis by phosphorylase inactivation cannot be explained by either inhibition of glycogen degradation or activation of glycogen synthase alone and suggests an additional role for translocation of synthase. Titrations with the phosphorylase inactivator showed that stimulation of glycogen synthesis by insulin can be largely accounted for by inactivation of phosphorylase over a wide range of activities of phosphorylase a. We conclude that a signalling pathway involving dephosphorylation of phosphorylase a leading to both activation and translocation of glycogen synthase is a critical component of the mechanism by which insulin stimulates hepatic glycogen synthesis. Selective inactivation of phosphorylase can mimic insulin stimulation of hepatic glycogen synthesis.

Amides↗

PubMed1998

Expression of thymidine phosphorylase in malignant ovarian tumors: correlation with microvessel density and an ultrasound-derived index of angiogenesis.

OBJECTIVE: The aim of this study was to determine whether the expression of thymidine phosphorylase by ovarian cancer cells correlates with the density of microvessels within the tumor, and with ultrasound-derived indices of blood flow. METHODS: Transvaginal ultrasonography with color Doppler imaging and pulsed Doppler spectral analysis was used to scan patients with an overt ovarian mass immediately before laparotomy. Sections of malignant tumors were analyzed for the cellular expression of thymidine phosphorylase and the intratumoral density of microvessels by immunohistochemistry using monoclonal antibodies to thymidine phosphorylase and factor VIII-related antigen, respectively. The main outcome measures were the histological classification of the tumor, the stage of the disease, whether or not the tumor cells were positive or negative for thymidine phosphorylase, the microvessel count and the peak systolic velocity (PSV). RESULTS: Forty-two tumors were studied (three of low malignant potential, 29 epithelial, four granulosa cell, two germ cell and four metastatic); 18 were stage I, six stage II, 11 stage III and three stage IV. Twenty-seven tumors (64%) were classified as thymidine phosphorylase-positive. The proportion of stage I tumors that was thymidine phosphorylase-positive (44%) was significantly lower (p = 0.022) than the corresponding value for stages II-IV (85%), but the values for microvessel count and PSV were similar. The microvessel count in thymidine phosphorylase-positive tumors was significantly higher than in thymidine phosphorylase-negative tumors (p = 0.005). Similarly, the PSV was significantly higher in thymidine phosphorylase-positive tumors (p = 0.009). There was a significant correlation between the microvessel count and the PSV (r = 0.354, p = 0.022). CONCLUSIONS: The expression of thymidine phosphorylase by malignant tumor cells is associated with an increase in microvessel density and PSV in patients with ovarian cancer.

Adolescent↗

PubMed1997

Autosomal recessive phosphorylase kinase deficiency in liver, caused by mutations in the gene encoding the beta subunit (PHKB).

The association of autosomal recessive phosphorylase kinase deficiency in liver of a 3 1/2-year-old female child with mutations in the gene encoding the common part of the beta subunit of phosphorylase kinase is reported. The proband had a severe deficiency of phosphorylase kinase in liver, while the phosphorylase kinase activity in erythrocytes was only slightly diminished. She had no symptoms of muscle involvement. The complete coding sequences of the liver gamma subunit and of the beta subunit of phosphorylase kinase of the proband were analyzed for the presence of mutations, by either reverse-transcribed PCR or SSCP analysis. Three deviations from the normal sequence were found in the region encoding the common part of the beta subunit of phosphorylase kinase-namely, a 1827G-->A (W609X) transition, a 2309A-->G (Y770C) transition, and a deletion of nucleotides 2896-2911-whereas no mutations were detected in the sequence encoding the liver gamma subunit of phosphorylase kinase. The 1827G-->A mutation and the deletion both result in the formation of early stop codons. Investigation of DNA showed that the deletion is caused by a splice-acceptor site mutation (IVS30(-1),g-->t). Family analysis revealed that the 1827G-->A and IVS30(-1),g-->t substitutions are located on different parental chromosomes and that compound heterozygosity for these mutations segregates with the disease. The 2309A-->G mutation was detected in 2%-3% of the normal population. Thus, it is concluded that the deficiency of phosphorylase kinase in this proband is caused by compound heterozygosity for the 1827G-->A and the IVS30(-1),g-->t mutations and that the 2309A-->G mutation is a polymorphism. This implies that a defect in the sequence encoding the common part of the beta subunit of phosphorylase kinase may present as liver phosphorylase kinase deficiency.

Carbohydrate Metabolism, Inborn Errors↗

PubMed2004

The relation of starch phosphorylases to starch metabolism in wheat.

Tissues of wheat (Triticum aestivum L., var. Star) exhibit three starch phosphorylase activity forms resolved by non-denaturing polyacrylamide gel affinity electrophoresis (P1, P2 and P3). Compartmentation analysis of young leaf tissues showed that P3 is plastidic, whereas P1 and P2 are cytosolic. P1 exhibits a strong binding affinity to immobilized glycogen upon electrophoresis, whereas P2 and the chloroplastic P3 do not. Cytosolic leaf phosphorylase was purified to homogeneity by affinity chromatography. The single polypeptide product constituted both the P1 and P2 activity forms. Probes for the detection of phosphorylase transcripts were derived from cDNA sequences of cytosolic and plastidic phosphorylases, and these-together with activity assays and a cytosolic phosphorylase-specific antiserum-were used to monitor phosphorylase expression in leaves and seeds. Mature leaves contained only plastidic phosphorylase, which was also strongly evident in the endosperm of developing seeds at the onset of reserve starch accumulation. Germinating seeds contained only cytosolic phosphorylase, which was restricted to the embryo. Plastidic phosphorylase thus appears to be associated with transitory leaf starch metabolism and with the initiation of seed endosperm reserve starch accumulation, but it plays no role in the degradation of the reserve starch. Cytosolic phosphorylase may be involved in the processing of incoming carbohydrate during rapid tissue growth.

Amino Acid Sequence↗