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At least 613 records · Page 34Linked to original sources
PubMed1988

Characteristics of acetylcholine-induced phosphorylase a activity in uterine segments as a substitute for contractile response to acetylcholine.

Studies were made on whether the ACh-induced phosphorylase a activity in isolated rat uterine muscle segments could be used as a substitute for the contractile response to ACh. This ACh-induced phosphorylase a activity was dependent upon the concentration of ACh and was inhibited by atropine, suggesting that it was linked to muscarinic ACh receptors. Both extracellular calcium and an increase of the intracellular calcium concentration were needed for its activation by ACh. Ca2+-antagonists such as Co2+, diltiazem, nitrendipine and verapamil inhibited the ACh-induced activity, suggesting that the activation by ACh required the influx of calcium ions into the uterine muscle through Ca2+-antagonist sensitive Ca2+ channels. The IC50 values of CoCl2, diltiazem, nitrendipine and verapamil on the ACh-induced phosphorylase a activity were 3.4 x 10(-3) M, 2.5 x 10(-4) M, 2.5 x 10(-5) M and 1.1 x 10(-4) M, respectively. These values were comparable with the IC50 values of these Ca2+-antagonists on the contractile response of isolated rat uterine muscle segments to 3 x 10(-4) M ACh. The inhibitory effects of Co2+, nitrendipine and verapamil, but not diltiazem, on ACh-induced phosphorylase a activity were attenuated by higher concentrations of CaCl2 (0.36 to 2 mM). These findings suggested that the ACh-induced phosphorylase a activity in isolated rat uterine muscle segments could be used as a substitute for the contractile response to ACh.

Acetylcholine↗
PubMed1975

Inactivation of glycogen phosphorylase of human polymorphonuclear leukocytes.

Glycogen phosphorylase of human polymorphonuclear leukocytes is dephosphorylated during incubation of a gel-filtered cell extract. The dephosphorylated enzyme (b form) retains 25 per cent of the activity of the phosphoenzyme (a form) when measured without AMP but a high glucose-1-phosphate concentrations. The ratio of activity -AMP/+AMP for the a enzyme is 0.8-1.0 and for the b enzyme 0.2. Leukocyte phosphorylase is not activated by -SH groups, but the b enzyme is stimulated by 0.4 mol/1 Na2SO4. The phosphatase which catalyzes the conversion of phosphorylase a to b is inhibited by glucose-1-phosphate and AMP both a 14 degrees C and 25 degrees C. Glucose counteracts the AMP inhibition but not the glucose-1-phosphate inhibition at both temperatures. Glucose alone had no effect at 25 degrees C, but it accelerated the phosphatase reaction at 14 degrees C. Glucose-6-phosphate or glycogen alone or in the presence of AMP or glucose-1-phosphate did not affect the phosphatase reaction. From previous and present experiments it is concluded that the phosphorylase of human polymorphonuclear leukocytes is closely related to liver phosphorylase and that the inactivation of the enzyme is mainly controlled by AMP and glucose.

Adenosine Monophosphate↗
PubMedDate not supplied

[Glycogen disease caused by disorders in the liver phosphorylase system].

Concentration of glycogen and activity of enzymes, participating in its metabolism, were studied in punctates of liver tissue obtained from three patients with clinical manifestations of hepatic forms of glycogenosis. Accumulation of glycogen in liver tissue of the patients was caused by distinct decrease in the phosphorylase activity. In two patients low activity of phosphorylase A was due to impairment of one of the enzymes, activating the phosphorylase in liver tissue, and in one patient phosphorylase B itself exhibited low activity. The in vitro data on glycogen metabolism were correlated with those, obtained in study of glycogen turnover in vivo, after loading with adrenaline. The data obtained enable to develop methods for treatment of the patients with impairments in activity of liver phosphorylase.

Child, Preschool↗
PubMed1980

[Interaction of phosphorylase B with SH-reagents and properties of the modified enzyme].

The interaction of phosphorylase B with the SH-reagents, i.e. 2-chloromercuri-4-nitrophenol and ethylmercurichloride was studied. It was shown that phosphorylase B inhibition obeys the pseudo-first-order kinetics, the inactivation rate constants being equal to 11 M-1 s-1 and 17,5 M-1 s-1, respectively. Data from the SH-group titration with 2-chloromercuri-4-nitrophenol and p-chloromercuri benzoate suggest that the number of modified cysteine residues and the amount of bound 2-chloromercuri-4-nitrophenol in the phosphorylase B dimer is equal to 2. In the modified phosphorylase B the absorption maximum of pyridoxal phosphate is decreased at 330 nm and is increased at 410 nm. The binding of 2-chloromercuri-4-nitrophenol is accompanied by quenching of the protein and coenzyme fluorescence. Upon interaction with ethylmercurichloride only the pyridoxalphosphate fluorescence is quenched. The increase of the spin label mobility in the modified enzyme calculated from the EPR spectra of the spin-labelled preparations is indicative of the changes in the protein conformation coupled with the blocking of one SH-group in the enzyme monomer. The rate of enzyme inactivation under effects of the SH-reagents is a function of pH and is considerably increased within the pH range of 5.7-6.7. The pH-optimum of activity of partly modified enzyme remains practically unchanged; however, at the pH shift towards the acidic values the activity is drastically decreased as compared to that of the native enzyme. The data obtained suggest that the enzyme inactivation is due to modification of one SH-group in the phosphorylase B monomer vicinal to the pyridoxal phosphate binding site and probably involved in the enzymatic reaction.

Animals↗
PubMed1978

[Reaction between adenosine-5'-chloromethylphosphonate and the allosteric center of phosphorylase B].

The interaction between phosphorylase B and an AMP analog, adenosine-5'-chloromethylphosphonate, is found to be irreversible. Their binding stechiometry is calculated from the differential absorption spectrum. Maximal inhibition is reached when 1,3--1,5 moles of the analogue is bound per mole of monomer phosphorylase B. The enzyme-inhibitory complex exhibited 50% activity is characterized by a sigmoid curve of the reaction rate dependency on the substrate concentration, by a decrease of the affinity to glucose-1-phosphate and the maximal rate, and by an increase of Hill's coefficient. Similar SH-groups titration curves were obtained for modified and native phosphorylase in the presence of AMP. Apophosphorylase was incapable of the complete reactivation by pyridoxalphosphate in the presence of adenosine-5'-chloromethylphosphonate. The complex of phosphorylase B and the AMP analogue is found to be inhibited by glucose-6-phosphate, like the native enzyme in the presence of AMP. The results of ultracentrifugation and disc electrophoresis show that the AMP analogue contributes the formation of the tetrameric form of the enzyme. The data obtained indicate the tight binding of adenosine-5'-chloromethylphosphonate in the active site of phosphorylase B. The properties of the complex confirm the importance of the phosphate group for the AMP binding in the allosteric site and for the enzyme activation.

Adenosine Monophosphate↗
PubMed1985

Coupled diminished energy turnover and phosphorylase a formation in contracting hypothyroid rat muscle.

Hypothyroidism leads to a diminished phosphorylase a formation and a reduction in both glycogen breakdown and lactate production during tetanic stimulation of fast-twitch skeletal muscle (mixed type). Phosphorylase kinase activity is almost 50% lower in the hypothyroid (Tx) group, and the possibility that this might explain the reduction in phosphorylase a formation is discussed. In both the Tx group and the euthyroid (C) group the reconversion of phosphorylase a to b correlates well with a decrease in the energy cost for contraction.

Animals↗
PubMed1999

Angiogenesis and expression of thymidine phosphorylase by inflammatory and carcinoma cells in ductal carcinoma in situ of the breast.

Angiogenesis is essential for tumour growth and important in metastasis and for prognosis in invasive carcinoma of the breast. Two patterns of increased vascularity have been shown in mammary ductal carcinoma in situ (DCIS): a cuff of vessels close to the involved ducts, and vessels in the interductal stroma. Inflammation may potentially promote angiogenesis by release of angiogenic factors and digestive enzymes. A correlation has previously been found between the intensity of perivascular inflammation and stromal vascularity in DCIS, but no strong relationship has been observed between inflammation and angiogenesis in invasive carcinoma. Tumour angiogenesis is regulated by a number of angiogenic factors, including thymidine phosphorylase (platelet-derived endothelial cell growth factor), which is expressed at high levels in macrophages. Using immunohistochemical methods, thymidine phosphorylase expression and vascularity have been studied in DCIS (n = 34) and invasive carcinoma (n = 32). Stromal vascularity in DCIS was associated with thymidine phosphorylase expression in the perivascular inflammatory cells and in the cytoplasm of carcinoma cells. In invasive carcinoma, no relationship was found between vascularity and thymidine phosphorylase expression in either the carcinoma or the inflammatory cells. This study suggests that thymidine phosphorylase expression in both inflammatory and carcinoma cells may contribute to one of the patterns of vascularity in DCIS, but not in invasive disease.

Breast Neoplasms↗
PubMed1997

Association of thymidine phosphorylase concentration with ultrasound-derived indices of blood flow in ovarian masses.

BACKGROUND: The aim of this study was to determine the relationship between the concentration of thymidine phosphorylase (a known angiogenic factor) and indices of blood flow in physiologic ovarian tissues and overt (benign and malignant) tumors. METHODS: The ovaries of all patients were examined by transvaginal ultrasonography, with color Doppler imaging and pulsed Doppler spectral analysis, within the 24 hours preceding laparotomy. Ovaries removed at surgery were dissected into their main components (follicles, corpus luteum, and tumor) and, where possible, into areas of high blood velocity according to the results of color Doppler imaging. The concentration of thymidine phosphorylase was measured by an enzyme-linked immunosorbent assay. RESULTS: Thirty-eight tissue aliquots (16 from normal ovaries and 22 from ovarian tumors) were obtained from 33 patients. Twenty-nine tissue samples (76%) came from areas of measurable (high) blood velocity. The concentration of thymidine phosphorylase was significantly higher in tissue associated with high blood velocity (median 17.9, range 1.8-78.3 units per mg of protein vs. median 6.8, range 1.3-24.7 units per mg of protein, respectively; P < 0.05, Mann-Whitney U test). All of 8 corpora lutea, 12 of 14 benign tumors, and 7 of 7 malignant tumors had measurable blood velocity. There was a significant correlation between the concentration of thymidine phosphorylase and the peak systolic velocity in benign tumors (correlation coefficient [r] = 0.79, P < 0.01) and malignant tumors (r = 0.87, P < 0.05). CONCLUSIONS: High intratumoral peak systolic velocity as determined by transvaginal color Doppler imaging and spectral analysis reflects high production of thymidine phosphorylase. This finding may aid the development of antivascular therapy for patients with ovarian carcinoma.

Adolescent↗
PubMed1978

McArdle disease: the mystery of reappearing phosphorylase activity in muscle culture--a fetal isoenzyme.

To understand the apparently paradoxical appearance of phosphorylase in muscle cultured from patients with McArdle disease, the enzyme in muscle culture was studied immunologically and electrophoretically. Antibody against normal human muscle phosphorylase completely inhibited the enzyme of adult muscle, but it had no effect on phosphorylase activity of muscle cultures from normal individuals or patients with McArdle disease. Also, amounts of antibody that would completely inhibit phosphorylase in mature muscle left about 30% of the activity in muscle obtained from human fetus at four months' gestation. Acrylamide-disc and slab-gel electrophoresis showed a single band of phorphorylase activity in adult muscle and two bands in fetal muscle. This suggested that at four months' gestation, both fetal and mature forms are present but that only the mature isoenzyme is inhibited by the antibody. The enzyme from cultured muscle gave only a single band, with the electrophoretic mobility of the fetal isoenzyme. These data suggest that phosphorylase activity in muscle cultured from patients with McArdle disease is due to a fetal isoenzyme whose genetic control is different from that of the mature enzyme.

Antibody Formation↗
PubMed1990

Adult phosphorylase b kinase deficiency.

Phosphorylase b kinase deficiency affecting muscle has been observed infrequently in children with weakness and hepatomegaly, and in 2 adults with cramps on exertion. We observed 2 additional adults with phosphorylase b kinase deficiency: Patient 1, aged 58, had progressive, predominantly distal weakness since age 46 but no cramps on exertion; Patient 2, aged 26, had cramps on exertion since age 6 but no weakness. Lactate production on ischemic exercise was impaired only in Patient 1. The serum creatine kinase level was elevated in both. Muscle specimens showed focal glycogen excess in both, and a necrotizing myopathy and mild denervation atrophy in Patient 1. Muscle phosphorylase b kinase activity was 0.5% and 8.9% of the lowest control value in Patients 1 and 2, respectively; erythrocyte phosphorylase b kinase activity was normal in both; liver phosphorylase b kinase activity, measured in Patient 1, was also normal. Other glycolytic enzymes in muscle were preserved in both.

Adult↗
PubMed1988

Glycogen phosphorylase in Dictyostelium discoideum: demonstration of two developmentally regulated forms, purification to homogeneity, immunochemical analysis, cAMP induction, in vitro translation, and molecular cloning.

A key step in the cellular differentiation of Dictyostelium is the degradation of glycogen to provide the precursors for synthesis of the structural end products of development. We have found that the enzyme that initiates this degradative pathway, glycogen phosphorylase (1,4-alpha-D-glucan:orthophosphate alpha-glucosyltransferase; EC 2.4.1.1), is developmentally regulated and exists as two forms. During the time course of development, a previously undescribed activity, the "b" form, decreases, while that of the "a" form increases. The "b" form is inactive unless 5'AMP is included in the reaction mixture. The two forms differ in their elution from DE52 cellulose, affinity constants, thermal stability, affinity for 5'AMP Sepharose, subunit molecular weight, and peptide maps. In crude extracts, anti-a antiserum stains a 104-kD protein that is associated with phosphorylase "a" activity and appears late in development, while anti-b antiserum stains a 92-kD protein that is associated with phosphorylase "b" activity and is present throughout development. We have also demonstrated in vitro phosphorylation of the "b" form by an endogenous protein kinase and a corresponding loss of 5'AMP dependence. If intact cells were exposed to exogenous cAMP, "b" activity decreased and was replaced by "a" activity, as well as the 104-kD protein band on SDS-PAGE. In order to determine if the two forms of the enzyme are different gene products, we screened lambda gt11 expression libraries with antibodies against the purified "a" and "b" forms. Three clones were found to be overlapping by Southern analysis. A yeast glycogen phosphorylase cDNA clone (gpy) and a human muscle glycogen phosphorylase clone (HM-11) cross-hybridized with the Dictyostelium inserts, and gpy shared a few common restriction fragments with the Dictyostelium clones on genomic blots. Northern analysis of Dictyostelium total RNA showed that the Dictyostelium inserts and gpy recognize an mRNA of 3.2 kb, while on poly A-enriched RNA, the yeast clone detects preferentially a 3.6-kb message.

Cloning, Molecular↗
PubMed1990

Purine nucleoside phosphorylase: a new marker for free oxygen radical injury to the endothelial cell.

The effect of ischemia and reperfusion on purine nucleoside phosphorylase was studied in an isolated perfused rat liver model. This enzyme is localized primarily in the cytoplasm of the endothelial and Kupffer cells; some activity is associated with the parenchymal cells. Levels of this enzyme accurately predicted the extent of ischemia and reperfusion damage to the microvascular endothelial cell of the liver. Livers from Lewis rats were subjected to 30, 45 and 60 min of warm (37 degrees C) no flow ischemia that was followed by a standard reperfusion period lasting 45 min. Purine nucleoside phosphorylase was measured at the end of the no flow ischemia and reperfusion periods as was superoxide generation (O2-). Bile production was monitored throughout the no flow ischemia and reperfusion periods. Control perfusions were carried out for 120 min. A significant rise in purine nucleoside phosphorylase levels as compared with controls was observed at the end of ischemia in all the three groups. The highest level, 203.5 +/- 29.2 mU/ml, was observed after 60 min of ischemia. After the reperfusion period, levels of purine nucleoside phosphorylase decreased in the 30- and 45-min groups 58.17 +/- 9.66 mU/ml and 67.5 +/- 17.1 mU/ml, respectively. These levels were equal to control perfusions. In contrast, after 60 min of ischemia, levels of purine nucleoside phosphorylase decreased early in the reperfusion period and then rose to 127.8 +/- 14.8 mU/ml by the end of reperfusion (p less than 0.0001). Superoxide generation at the beginning of reperfusion was higher than in controls with similar values observed at the end of 30, 45 and 60 min of ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

The binding of phosphorylase kinase to immobilized calmodulin.

The binding of phosphorylase kinase to calmodulin-Sepharose 4B was studied by column and batch methods. It was found that the Ca2+ dependence of the interaction strongly depended on the degree of substitution of agarose with calmodulin. Equilibrium adsorption isotherms (i.e., bulk ligand binding functions and lattice site binding functions) of phosphorylase kinase were measured on calmodulin-Sepharose. Sigmoidal bulk ligand binding functions (bulk adsorption coefficients: 1.5-5.8) were found which indicate intermolecular attraction during binding. Hyperbolic lattice site binding functions (lattice adsorption coefficients: 1.0) were obtained thus excluding the existence of a critical surface concentration of immobilized calmodulin and indicating single independent binding sites on the gel surface and on phosphorylase kinase. These findings were combined to optimize the adsorption of phosphorylase kinase on calmodulin-Sepharose, for purification procedures at low Ca2+ concentrations (5-10 microM) minimizing proteolysis by calpains. With this novel method phosphorylase kinase from rabbit and frog skeletal muscle could be purified ca 100- and 200-fold, respectively, in two steps.

Adsorption↗
PubMed1998

Purine nucleoside phosphorylase: its use in a spectroscopic assay for inorganic phosphate and for removing inorganic phosphate with the aid of phosphodeoxyribomutase.

The kinetics of the phosphorolysis of 7-methylated guanosine analogues catalyzed by purine nucleoside phosphorylase has been analyzed to understand the use of this system as a "Pi mop" to remove Pi from solutions and as a spectroscopic assay for Pi at micromolar concentrations. An expression system was developed for the phosphorylase from Escherichia coli: this protein (subunit molecular mass 26 kDa) and one from a commercial source (29 kDa) were used in this study. Rates of >50 s-1 were obtained for the phosphorolysis at 30 degrees C, so that when the phosphorylase is coupled to the phosphatase being studied, rates of Pi release from the phosphatase can be measured close to this rate. The kinetic mechanism appears to obey the Michaelis-Menten model in the steady state with the bond cleavage rate limiting. Slow hydrolysis of ribose-1-phosphate to Pi catalyzed by the phosphorylase limits the efficiency of the Pi mop. To overcome this, phosphodeoxyribomutase was used to catalyze the conversion of ribose-1-phosphate to ribose-5-phosphate, enabling the Pi mop to remove large amounts of Pi quantitatively. Acyclovir diphosphate provides a simple method to switch off the Pi mop as it is a tight inhibitor (Kd 12 nM) of purine nucleoside phosphorylase.

Acyclovir↗
PubMed2002

Inverse relationship of skeletal muscle glycogen from wild-type and genetically modified mice to their phosphorylase a activity.

Leg muscle was biopsied and frozen for storage at -70 degrees C. from 5 wild-type mice, two knocked out acid alpha-glucosidase (GAA) gene mice, and seven glycogen synthase plus glucose muscle transporter transgenic mice. All of the wild-type mice had very little muscle glycogen (3.58 +/- 1.67 micromols glucosyl subunits per g muscle), and 52% or more of its glycogen phosphorylase activity without AMP (69% +/- 17% glycogen phosphorylase a). In contrast the GAA knockout and transgenic mice had glycogen ranging from 63 to 297 micromols glucosyl subunits per g muscle, and very little or no glycogen phosphorylase activity without 1.00 mM AMP (4.8% and less glycogen phosphorylase a). This suggests that there is an inverse relationship between mouse muscle phosphorylase a and the muscle's glycogen content.

Animals↗
PubMed1989

The rate of degradation of liver glycogen phosphorylase is specifically decreased in the C57BL/KsJ-db/db mouse.

We have recently demonstrated that the activity of liver glycogen phosphorylase, the rate-limiting enzyme of glycogenolysis, is elevated in genetically diabetic (db/db) mouse and that it is primarily due to the presence of increased amounts of this enzyme. In the present study, we examined the turnover of glycogen phosphorylase in vivo in order to elucidate the mechanism for this specific increase. The rate of phosphorylase synthesis was slightly decreased in the diabetic mouse compared to controls. However, the relative rates of synthesis were similar in these two groups. The rate of degradation of this enzyme was decreased 20% (p less than 0.05) in the diabetic mouse compared to controls. More importantly, the relative rate of degradation of phosphorylase was found to be lower in the diabetic animals. This indicates that the elevated concentration of phosphorylase in the liver of the db/db mouse is likely due to a specific decrease in its rate of degradation.

Amino Acids, Branched-Chain↗
PubMed1992

Immunohistochemical co-localization of glycogen phosphorylase with the astroglial markers glial fibrillary acidic protein and S-100 protein in rat brain sections.

Immunofluorescence double-labelling and immunoenzyme double-staining methods were used to examine the location of glycogen phosphorylase brain isozyme with the astrocyte markers glial fibrillary acidic protein (GFAP) and S-100 protein in formaldehyde-fixed, paraffin-embedded slices from adult rat brain. Astrocytes in the cerebellum and the hippocampus, which express GFAP or S-100 protein immunoreactivity, show glycogen phosphorylase immunoreactivity. Regional intensity and intracellular distribution of the three antigens vary characteristically. In ependymal cells, glycogen phosphorylase immunoreactivity is co-localized with S-100 protein immunoreactivity, but not with GFAP immunoreactivity. These findings confirm that glycogen phosphorylase in the rat brain is exclusively localized in astrocytes and ependymal cells. All astrocytes, as far as they express GFAP or S-100 protein, do contain glycogen phosphorylase.

Animals↗
PubMed1978

Phosphorylase activity as a histochemical marker of specialized tissue of heart.

Phosphorylase activity has been investigated histochemically under controlled conditions in the specialized tissue and the general myocardium of goat heart using glucose-1-phosphate as the substrate. It has been observed that the phosphorylase content of the nodal and conducting tissue is very high as compared to the general myocardium. The phosphorylase content of the cardiac neural elements is also high. Since the connective tissue which surrounds the nodal and conducting tissue has almost no phosphorylase activity, the nodal and conducting tissue is sharply demarcated by the histochemical reaction for phosphorylase. This reaction can, therefore, be used as a specific and expedient method for the localization of specialized tissue as an exploratory and confirmatory test.

Animals↗