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PubMed1976

The interaction of ligands with chemically modified phosphorylase b.

Phosphorylase b which had been inactivated with 5-diazo1H-tetrazole was specifically labelled with 4-iodoacetamidosalicylic acid (a fluorescent probe) or with N-(1-oxyl-2,2,6,6,-tetramethyl-4-piperidinyl)iodoacetamide (a spin label probe) so that the binding of ligands and accompanying conformational changes could be determined by fluorescence or electron spin resonance changes, respectively. The allosteric effector, AMP, causes conformational changes similar to those caused in the native enzyme. The affinity of binding of phosphate or AMP to the inhibited protein is the same as for the unmodified protein. The heterotropic interactions between glucose-1-phosphate or glycogen and AMP are much less in the inactivated enzyme than in unmodified phosphorylase. Using a light scattering assay, it is shown that the modified enzyme binds to glycogen less strongly than the native protein. Phosphorylase b which had been inactivated by carbodimide in the presence of glycine ethyl ester, resulting in the modification of one or more carboxyl groups, was labelled with the spin label probe described above. The modified enzyme has an affinity for AMP similar to that of the native enzyme. AMP binding to the modified enzyme is tightened by glycogen, weakened by glucose-6-phosphate and is unaffected by glucose-1-phosphate. The actions of 5-diazo-1H-tetrazole and carbodimide on phosphorylase are discussed in the light of the above observation.

Adenosine Monophosphate↗
PubMed1982

Liver glycogen synthase and phosphorylase changes in vivo with hypoxia and anesthetics.

Methods for obtaining and processing rat liver for determination of glycogen phosphorylase a and synthase I activity were studied. An extremely rapid and profound increase in phosphorylase was induced by hypoxia. The effect on synthase I was slower and less striking. Using alpha- and beta-adrenergic antagonists, a catecholamine-depleting agent, and a ganglionic blocking agent, it was determined that adrenergic stimulation secondary to the surgical procedure required to obtain the liver was not a significant factor. The anesthetic agent used also had a significant effect on the proportion of phosphorylase in the a form. Seconal anesthesia resulted in lower phosphorylase a levels than did ether or urethan anesthesia.

Anesthesia↗
PubMed2003

Cardioprotective effects of ingliforib, a novel glycogen phosphorylase inhibitor.

Interventions such as glycogen depletion, which limit myocardial anaerobic glycolysis and the associated proton production, can reduce myocardial ischemic injury; thus it follows that inhibition of glycogenolysis should also be cardioprotective. Therefore, we examined whether the novel glycogen phosphorylase inhibitor 5-Chloro-N-[(1S,2R)-3-[(3R,4S)-3,4-dihydroxy-1-pyrrolidinyl)]-2-hydroxy-3-oxo-1-(phenylmethyl)propyl]-1H-indole-2-carboxamide (ingliforib; CP-368,296) could reduce infarct size in both in vitro and in vivo rabbit models of ischemia-reperfusion injury (30 min of regional ischemia, followed by 120 min of reperfusion). In Langendorff-perfused hearts, constant perfusion of ingliforib started 30 min before regional ischemia and elicited a concentration-dependent reduction in infarct size; infarct size was reduced by 69% with 10 microM ingliforib. No significant drug-induced changes were observed in either cardiac function (heart rate, left ventricular developed pressure) or coronary flow. In open-chest anesthetized rabbits, a dose of ingliforib (15 mg/kg loading dose; 23 mg.kg(-1).h(-1) infusion) selected to achieve a free plasma concentration equivalent to an estimated EC(50) in the isolated hearts (1.2 microM, 0.55 microg/ml) significantly reduced infarct size by 52%, and reduced plasma glucose and lactate concentrations. Furthermore, myocardial glycogen phosphorylase a and total glycogen phosphorylase activity were reduced by 65% and 40%, respectively, and glycogen stores were preserved in ingliforib-treated hearts. No significant change was observed in mean arterial pressure or rate-pressure product in the ingliforib group, although heart rate was modestly decreased postischemia. In conclusion, glycogen phosphorylase inhibition with ingliforib markedly reduces myocardial ischemic injury in vitro and in vivo; this may represent a viable approach for both achieving clinical cardioprotection and treating diabetic patients at increased risk of cardiovascular disease.

Animals↗
PubMed1985

Creatine kinase and phosphorylase in cardiac lymph: coronary occlusion and reperfusion.

Cardiac lymph, collected from conscious dogs, was monitored for glycogen phosphorylase and creatine kinase (CK) enzymatic activity during control state, circumflex coronary artery (CFX) occlusion, and reperfusion. CFX occlusions, lasting for intervals as short as 10 min, initiated a release of phosphorylase and CK into the cardiac lymph, which was immediately observed during reperfusion of the ischemic tissue. Blood plasma levels did not appear for several hours. In the absence of reperfusion, the appearance of enzymes in cardiac lymph was delayed and peaked later. Glycogen phosphorylase and CK entered the lymph in greater quantities with reperfusion as the length of occlusion was increased. Histological examination of multiple sections of the reperfused hearts showed infarcts in hearts where CFX occlusions lasted 20 min or longer; occlusions of 10-15 min showed evidence of cell injury and death in two hearts and no definable infarct in the majority. Ischemic intervals of short duration release functionally active glycogen phosphorylase and CK, which reflect changes in myocardial cell egress of macromolecules and/or cell death.

Animals↗
PubMed1992

Adrenergic activation of glycogen phosphorylase in primary culture diabetic cardiomyocytes.

The basis of catecholamine-induced activation of glycogen phosphorylase was investigated in adult rat cardiomyocytes isolated from normal and alloxan-diabetic animals. Cells derived from diabetic animals exhibited a hypersensitive response to epinephrine stimulation that was apparent 3 h after cell isolation and was further enhanced on maintenance of the myocytes in culture for 24 h. Normal cells initially lacked the hypersensitive response to epinephrine stimulation, although on maintenance of these cells in culture for 24 h, the hypersensitive response was acquired in vitro. To assess alpha- and beta-adrenergic mediation of the response, normal and diabetic cardiomyocytes were incubated with propranolol, a beta-blocker, before direct alpha 1-receptor stimulation with phenylephrine. Both normal and diabetic myocytes failed to undergo activation of phosphorylase in 3- or 24-h cell cultures. In addition, the effects of epinephrine on phosphorylase activation were completely inhibited by propranolol, whereas prazosin, an alpha-blocker, was unsuccessful. The present data suggest that the hypersensitive response of glycogen phosphorylase in normal and diabetic cardiomyocytes is solely mediated through beta-adrenergic receptor activation.

Animals↗
PubMed1986

Rat liver free cytosolic Ca2+ and glycogen phosphorylase in endotoxicosis and sepsis.

Rats were treated with Escherichia coli endotoxin (ET) either acutely or chronically or rendered septic by cecal ligation and puncture. At 6 h after ET injection, at various intervals of continuous ET infusion, and at 17-18 h after the onset of peritonitis, animals were killed and hepatocytes were isolated. Cytosolic [Ca2+] ([Ca2+]c) was measured by quin 2 during the resting state and after stimulation with epinephrine and vasopressin. Basal and epinephrine-, vasopressin- and glucagon-stimulated glycogen phosphorylase activity were also determined. In hepatocytes from acutely ET-treated rats, resting levels of [Ca2+]c were decreased 46% from 245.8 +/- 11.0 to 131.0 +/- 8.5 nM (n = 4-6, P less than 0.05). In septic rats a 39.5% decrease was noted [i.e., from 154.0 +/- 17.7 (n = 4, sham) to 93.3 +/- 91 nM (n = 5, septic, P less than 0.05)]. These decreased [Ca2+]c levels were associated with changes of glycogen phosphorylase activity in a manner suggesting a cause and effect relationship; e.g., acute ET treatment resulted in greater than 80% depression of phosphorylase a activity, whereas sepsis induced a 58% decrease in the activity of this enzyme. In ET-infused rats the resting level of [Ca2+]c and its response to hormonal stimulation were not different from hepatocytes of saline-infused rats, although glycogen phosphorylase activity was less responsive to these hormones. The effect on the enzyme's response to Ca2+-mobilizing hormones was more marked than to glucagon. This is consistent with the concept that information flow in the Ca2+-messenger system is a site of metabolic lesions produced by endotoxicosis and sepsis.

Acute Disease↗
PubMed1992

Defective insulin response of phosphorylase phosphatase in insulin-resistant humans.

Insulin-stimulated glycogen synthase activity in human muscle is reduced in insulin-resistant subjects. Insulin regulation of human muscle glycogen synthase may require activation of a type-1 protein phosphatase (PP-1). We investigated the change of phosphorylase phosphatase and glycogen synthase activities in muscle biopsies obtained during a 2-h hyperinsulinemic euglycemic clamp in 12 insulin-sensitive (group S) and 8 insulin-resistant (group R) subjects. Fasting phosphorylase phosphatase activity was lower in group R than in group S, and did not increase significantly with insulin infusion in group R until 20 min. In group S, phosphorylase phosphatase was significantly stimulated by 10 min, remaining significantly higher than in group R at all time points. The insulin-mediated changes in phosphatase activities were not decreased by 3 nM okadaic acid but were completely inhibited by 1 microM okadaic acid, thereby verifying that insulin-stimulated phosphorylase phosphatase is accounted for by a PP-1. Subcellular fractionation demonstrated reduced fasting PP-1 activities in both the glycogen and cytosolic fractions of muscle obtained from subjects in group R compared to those in group S. These results suggest that insulin activation of PP-1 could contribute to the stimulation of glycogen synthase by this hormone in human muscle. Lower fasting PP-1 activity in cytosol and glycogen fractions plus lower insulin-stimulated PP-1 activity could explain, in part, reduced insulin-stimulated glycogen synthase in skeletal muscle of insulin-resistant subjects.

Adult↗
PubMed2003

Severe phenotype of phosphorylase kinase-deficient liver glycogenosis with mutations in the PHKG2 gene.

Phosphorylase kinase-deficient liver glycogenosis manifests in infancy with hepatomegaly, growth retardation, and elevated plasma aminotransferases and lipids. It can be caused by mutations in three different genes of phosphorylase kinase subunits: PHKA2, PHKB, and PHKG2. It is usually a benign condition, often with complete resolution of symptoms during puberty. A minority of patients displays a more severe phenotype with symptomatic fasting hypoglycemia and abnormal liver histology that may progress to cirrhosis. Three patients with liver cirrhosis in childhood analyzed previously all had PHKG2 mutations. This suggested that this genotype may generally cause a more severe clinical manifestation, but to date PHKG2 mutations have been identified in only seven patients. Here, we report mutation analysis in three new patients with liver phosphorylase kinase deficiency and recurrent hypoglycemia, liver fibrosis, and lack of glucagon response but no overt cirrhosis. In all three patients, PHKG2 mutations were found (H89fs[insC], E157K, D215N, W300X). Three of these mutations are novel, bringing the total number of distinct human PHKG2 mutations to 11, found in 10 patients. We conclude that liver phosphorylase kinase deficiency with a severe phenotype, with or without cirrhosis, is indeed often caused by PHKG2 mutations. These patients require active measures to maintain normoglycemia (raw cornstarch, nocturnal tube feeding), which may also alleviate growth retardation and the development of abnormal liver histology.

Female↗
PubMed1981

Effects of perhexiline on myocardial phosphorylase activity, myocardial catecholamine content and heart rate.

A study was conducted on the effects of perhexiline on myocardial phosphorylase activity, myocardial catecholamine content and heart rate. Phosphorylase a activity and heart rate were investigated as an indicator of sympathetic nerve tone in order to clarify the characteristic of perhexiline with regard to the effects on myocardial metabolism in hyperthyroid rats and catecholamine deficient rats. Myocardial catecholamine and phosphorylase a activity were measured by von Euler's method and Cori's method respectively. Conclusion of this study are summarized as follows: 1) Perhexiline reduces the heart rate, and its effect is not always dependent upon the changes in myocardial norepinephrine content. 2) Perhexiline reduces myocardial phosphorylase a activity. It cannot be always said that the reduction is dependent upon the changes in myocardial norepinephrine. 3) Though slightly different from propranolol and dichloroisoproterenol, perhexiline possesses cardiac effects resembling beta-blockers.

Animals↗
PubMed1986

Developmental alteration in adrenergic regulation of hepatic glycogen phosphorylase.

The effects of development upon adrenergic regulation of glycogenolysis were characterized using isolated hepatocytes from 3 different age groups of male rats (6 week-old, 8 week-old and 30 week-old). The phosphorylase a response in isolated hepatocytes to alpha-adrenergic stimulation decreased moderately with advancing age; whereas, that to beta-adrenergic stimulation declined more rapidly and almost disappeared at the age of 30 weeks. This developmental alteration in relative contribution of alpha- and beta-adrenergic regulation of phosphorylase was further confirmed by the experiments with specific antagonists. Also, the dramatic decrease of beta-adrenergic response on glycogen phosphorylase activity was found to be closely associated with a similar change of cAMP generation. In addition, the glucagon effect on cAMP production was found to be declined with advancing animal age. These results demonstrate that the glycogenolytic response of isolated rat hepatocytes to catecholamines can be mediated by different pathways according to the age of the animal; thus, juvenile male rats exhibit both the alpha- and beta-adrenergic mechanism for activation of phosphorylase and the maturation is associated with a modest decline of alpha receptor-mediated effect and a dramatic attenuation of a beta-adrenergic/cAMP response.

Aging↗
PubMed1978

[Studies on the activities of glycogen synthetase and glycogen phosphorylase in the human endometrium (author's transl)].

Glycogen content, glycogen synthetase and glycogen phosphorylase were studied in the endometrial tissues of 28 women with normal menstrual cycles and in the decidual tissues of 24 women with normal early pregnancies. The endometrial glycogen synthetase enzyme increased gradually from the proliferative phase to the secretory phase and reached maximal activity during the sixteenth to twenty-third days of the cycle, a time coincident with maximal glycogen content, while the glycogen phosphorylase reached its maximal activity on and after the twenty-fourth day of the cycle. In addition, glycogen phosphorylase activity in the decidual tissue during the early period of gestation (4--10 weeks) was lower than that in the endometrial tissue during the late secretory phase of the cycle, and in particular, the active form (-AMP) was significantly (p less than 0.005) low, with the result that the glycogen content in the decidua was significantly (p less than 0.05) higher than that in the endometrium. On the other hand, in 20 normal pregnant women during the 6--10th weeks of gestation, the glycogen contents in the decidua and in the placental villi were 599 +/- 44 mg/100 g wet weight (mean +/- standard error of the mean) and 731 +/- 55 mg/100 g, respectively, but the difference between them was statistically not significant. The levels of glycogen synthetase and glycogen phosphorylase enzyme in the decidua were significantly (p less than 0.005) higher than those in the placental villi.

Chorionic Villi↗
PubMed1988

Multiple ubiquitination of vertebrate calmodulin by reticulocyte lysate and inhibition of calmodulin conjugation by phosphorylase kinase.

Mammalian calmodulin containing trimethyllysine 115 can be covalently coupled to ubiquitin in a Ca2+-dependent manner in the presence of ATP/Mg2+ by reticulocyte lysate. This conjugation reaction can be quantitated in a novel test employing fluphenazine-Sepharose. It is shown that at least 3 ubiquitin molecules can be coupled to calmodulin indicating that more than one lysine residue is involved in the ubiquitination reaction. In addition only the free form of calmodulin can be ubiquitinated. Neither calmodulin bound to phosphorylase kinase as an integral subunit (delta-subunit) nor that bound as a peripheral subunit (delta'-subunit) is ubiquitinated. A total binding of equimolar calmodulin to phosphorylase kinase occurs since the affinity of binding of calmodulin to phosphorylase kinase as integral (KCaMm unknown) or peripheral subunit (KCaMm ca. 30-50nM) is several order of magnitude higher than the corresponding affinity of calmodulin for the ubiquitin-conjugating enzyme (KCaMm ca. 8 microM). We conclude that the "protective" effect of phosphorylase kinase towards calmodulin conjugation is due to a changed conformation of bound calmodulin and/or inacessibility of the ubiquitination sites (e.g. at subunit-subunit interface). Thus Ca2+-dependent ubiquitination only of free calmodulin may provide an efficient scavanging mechanism (with subsequent breakdown) for all free calmodulin in excess of that amount which can be bound by the calmodulin-binding proteins in the cell.

Adenosine Triphosphate↗
PubMedDate not supplied

The activity of thymidine phosphorylase obtained from human uterine leiomyomas and studied in the presence of pyrimidine derivatives.

Partially purified samples of thymidine phosphorylase were obtained from four preparations of human uterine leiomyomas and uteri using the method of Yoshimura et al. (1990), Biochim. Biophys. Acta 1034, 107-113. Among the studied twelve pyrimidine derivatives, 5-bromouracil, 5-nitrouracil, 5-fluorouracil, 6-aminouracil, 4, 6-dihydroxy-5-nitropyrimidine are competitive inhibitors, while allyloxymethylthymine is an uncompetitive inhibitor of thymidine phosphorylase activity, 6-benzyl-2-thiouracil inhibits the activity of the enzyme in a mixed way. The most potent inhibitor of the thymidine phosphorylase activity is 5-bromouracil and uracil the weakest one. Stronger inhibition of these compounds on the activity of thymidine phosphorylase was found in uterine leiomyomas than in uteri.

Adult↗
PubMed1999

Leptin enhances glycogen storage in hepatocytes by inhibition of phosphorylase and exerts an additive effect with insulin.

The effects of the adipocyte-derived hormone leptin on glucose metabolism in hepatocytes were investigated. Incubation of hepatocytes from Wistar rats with leptin for 16 h caused a dose-dependent increase in incorporation of [14C]glucose into glycogen, with a maximal effect at 30 nmol/l leptin. This effect of leptin was observed over a range of glucose concentrations (10-25 mmol/l) and was associated with stimulation of net glycogen deposition. It was not counteracted by mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase, indicating that it is not due to increased gluconeogenic flux. Leptin also enhanced the short-term stimulation of glycogen synthesis by insulin. These effects of leptin were associated with inhibition of phosphorylase a, which occurred after 4 h of exposure to leptin. Culture with leptin for 16 h did not affect the activities of glucose-6-phosphatase or glucokinase or the activation state of glycogen synthase. Leptin did not affect glycolysis determined from the detritiation of [3-(3)H]glucose. The inhibitory effects of leptin on phosphorylase a were counteracted by short-term incubation with glucagon but were additive with the inhibitory effects of insulin and also with the inhibition caused by resorcinol (25 pmol/l), which inhibits phosphorylase kinase by a mechanism analogous to the antidiabetic drug proglycosyn. These results show that leptin has additive effects with insulin in inhibiting phosphorylase and stimulating glycogen storage in hepatocytes, indicating that these hormones act by separate but convergent mechanisms. It is concluded that the primary action of leptin in hepatocytes is to enhance glycogen storage. This may be an important compensatory mechanism for the inhibition of insulin secretion.

Animals↗
PubMed1980

Formycin B, purine nucleoside phosphorylase and lymphocyte function.

A genetic deficiency of purine nucleoside phosphorylase (NP) is associated with immune dysfunctin that specifically affects T cells. An absence of adenosine deaminase (ADA), the preceding enzyme in the pathway of purine recycling, is associated with failure of both T and B lymphocyte function. Formycin B, an analogue of inosine and an inhibitor of purine nucleosidase phosphorylase, was used in cell culture systems to stimulate deficiency of this enzyme. The cells used were normal circulating lymphocytes and lymphoblastoid cell lines (LCL) with T and B cell characteristics. Formycin B inhibited the growth of these cells, but its primary effect was found to be due to a mechanism other than purine nucleoside phosphorylase inhibition. The possibility that formycin B was inhibiting adenosine deaminase, for which it is a product analogue, was studied by the analysis of reaction progress curves using the integrated rate equation. The molecular structure of formycin B, whilst preventing its phosphorylytic cleavage by purine nucleoside phosphorylase, could enable this compound to function as a substrate for adenosine kinase. The resultant formation of formycin B nucleotide probably causes the observed inhibition of growth in cultured cells.

Adenosine Deaminase↗
PubMed2000

Activation of human liver glycogen phosphorylase by alteration of the secondary structure and packing of the catalytic core.

Glycogen phosphorylases catalyze the breakdown of glycogen to glucose-1-phosphate, which enters glycolysis to fulfill the energetic requirements of the organism. Maintaining control of blood glucose levels is critical in minimizing the debilitating effects of diabetes, making liver glycogen phosphorylase a potential therapeutic target. To support inhibitor design, we determined the crystal structures of the active and inactive forms of human liver glycogen phosphorylase a. During activation, forty residues of the catalytic site undergo order/disorder transitions, changes in secondary structure, or packing to reorganize the catalytic site for substrate binding and catalysis. Knowing the inactive and active conformations of the liver enzyme and how each differs from its counterpart in muscle phosphorylase provides the basis for designing inhibitors that bind preferentially to the inactive conformation of the liver isozyme.

Adenosine Monophosphate↗
PubMed1979

[Mutations in the purine nucleoside phosphorylase (pup) gene of Escherichia coli K-12 characterized by leaky damage to enzymatic activity and a pleiotropic effect].

The phenotype of earlier obtained mutants AIR38 and AIR6 is caused by leaky mutations of the structural gene for purine nucleoside phosphorylase (pup). These mutants are unable to grow on the medium with inosine as the only carbon source in the presence of thymidine. In contrast to ordinary leaky mutations, AIR38 and AIR6 are dominant in heterozygotes. When the strain F' with mutations AIR38 or AIR6 on episomes was used for conjugational matings with F- recA (pup+), recombinants with unexpected phenotype were observed: about 20% of recombinants F' became pup+ and thus the convertion of AIR38 and AIR6 alleles to pup+ took place. AIR38 mutation, unlike AIR6, is mapped in the proximal region of the pup gene and is characterized by pleyotropic effect on deo-genes: the inosine-induction of purine nucleoside phosphorylase in AIR38 mutant is absent and the induction of thymidine phosphorylase by exogenous thymidine is decreased. A speculation was made that the mutation AIR38 altered the structure of purine nucleoside phosphorylase in the site responsible for its interaction with membrane.

Conjugation, Genetic↗
PubMed2003

[Thymidine phosphorylase activity and neo-angiogenesis in ovarian cancer].

Neo-angiogenesis seems to play an important role in the progression of ovarian cancer and in formation of distant metastases. Data from literature on role of phosphorylase in neoplasmatic disease and in neo-angiogenesis are controversial. In mammalian cytosole there are two different pirymidine nucleosyde phosphorylases: thymidine (PT) and uridine (PU). Both of them play important role in the metabolism of nucleosides as well as in the recycling of pyrimidine base. Recently thymidine phosphorylase is identified with platelet derived endothelial cell growth factor (PD-ECGF). It has been demonstrated, that PD-ECGF/PT influence on neo-angiogenesis and correlates with degree of neoplasmatic invasion. In literature the data about thymidine phosphorylase activity and its correlation with neoplasmatic angiogenesis in ovarian tumors are controversial. The aim of the study was to evaluate the activity of PT together with the intensity of angiogenesis in epithelial ovarian tumors. 42 patients with ovarian cancer were included into the study. The enzyme activity was measured in ovarian cancer tissue and in the serum in the spectrophotometer. Intratumoral microvessel density (IMD) was evaluated in tumor using immunohistochemical methods. 10 woman with normal ovaries, treated surgically due to non-oncological reasons served as a control. Activity of PT in ovarian tumor and in serum was compared to the control group. Correlation between the intensity of angiogenesis and PT activity in ovarian cancer was also investigated. Significantly higher PT activity was stated both in tumor and serum when compared to the control. Positive correlation between enzyme activity in the serum and neoplasmatic tissue was found. Surprisingly, the negative correlation between neo-angiogenesis and PT activity in ovarian cancer was observed. Neo-angiogenesis is higher in ovarian cancer, when compared to the group of borderline malignancy tumors. Positive correlation between PT activity and staging in ovarian cancer was observed. No correlation between grading and histopathological type of epithelial cancer was observed. PT activity and neo-angiogenesis evaluation might be useful in diagnostics of ovarian cancer.

Adult↗