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At least 1,009 records · Page 56Linked to original sources
PubMed1987

[Interaction of muscle glycogen phosphorylase b with nicotinic acid, nicotinamide, N-nicotinyl-gamma-aminobutyric acid and nicotinamide coenzymes].

The inhibitory action of nicotinic acid, nicotinamide, N-nicotinoyl-gamma-aminobutyric acid, NAD, NADH, NADP, and NADPH on the rabbit skeletal muscle glycogen phosphorylase b has been studied. The inhibition is reversible and positively cooperative (the value of Hill coefficients were determined for the following compounds: nicotinic acid (28 mM; 1.4), nicotinamide (4.4 mM; 1.2), N-nicotinoyl-gamma-aminobutyric acid (9.5 mM; 1.4), NAD (4.4 mM; 1.2), NADH (0.93 mM; 1.2). NADH-binding site of glycogen phosphorylase b subunit was characterized by the sedimentation velocity method. Microscopic dissociation constant was found to be 86 +/- 9 microM (pH 6.8; 20 degrees C). AMP-induced association of glycogen phosphorylase b is hindered by NADH.

Animals↗
PubMed1986

A spectrophotometric study of vitamin B2 and its coenzyme forms binding to muscle glycogen phosphorylase b.

The vitamin B2 and its coenzyme forms binding to glycogen phosphorylase b from rabbit skeletal muscle has been studied by the spectrophotometric method. The spectral properties of riboflavin, FMN and FAD bound to muscle glycogen phosphorylase b were found to be identical at the wavelengths of 300 to 500 nm. According to data on spectrophotometric titration of muscle glycogen phosphorylase b by FMN, each subunit of the enzyme contains one flavin-binding site.

Animals↗
PubMed1988

[Interaction of muscle glycogen phosphorylase b with riboflavin, its tetraacetyl derivative and their analogs].

The interaction of rabbit skeletal muscle glycogen phosphorylase b with riboflavin, 2',3',4',5'-tetraacetylriboflavin and their analogues, containing different substituents in the positions 6, 8 and 8 alpha, has been studied. Dissociation constant for the complex of the enzyme and riboflavin was determined to be 12.5 microM (pH 6.8; 20 degrees C) by sedimentation velocity method. Riboflavin and its analogues have been found to inhibit glycogen phosphorylase b. The inhibitor half-saturation concentration values increase in the following order: riboflavin (18 microM), 8-methoxy(nor)rifoblavin (23 microM), 8 alpha-bromo-2',3',4',5'-tetraacetylriboflavin (40 microM), 6-bromoriboflavin (40 microM), 8 alpha-hydroxyriboflavin (60 microM), 8-hydroxy(nor)riboflavin (90 microM), 8 alpha-(gamma-carboxypropylamino-2',3',4',5'-tetraacetylriboflav in (90 microM), 8 alpha-[p-(5-ethyl-1,3,4-thiodiazol-2-ylsulfamido)phenylamino ]- 2',3',4',5'-tetraacetylriboflavin (100 microM), 8 alpha-(L-methionyno)-2',3',4',5'-tetraacetylriboflavin (120 microM), 8 alpha-[p-(thiazol-2-ylsulfamido)phenylamino]- 2',3',4',5'-tetraacetylriboflavin (140 microM), 8 alpha-(p-sulfamidophenylamino)-2',3',4',5'-tetraacetylriboflavi n (180 microM), 8 alpha-(p-carboxyphenylamino)-2',3',4',5'-tetraacetylriboflavin+ ++ (210 microM), 2',3',4',5'-tetraacetylriboflavin (250 microM), 8 alpha-(L-homoserino)-2',3',4',5'-tetraacetylriboflavin (340 microM), 8 alpha-(L-glutamo)-2',3',4',5'-tetraacetylriboflavin (360 microM). The existence of glycogen phosphorylase b complexes with riboflavin and its analogues has been proved by methods of absolute and difference spectrophotometry.

Animals↗
PubMed1988

[Inhibition of muscle glycogen phosphorylase b by 5-methyltetrahydrofolic acid, 3'-chloro- and 3',6'-dichloromethotrexates].

Inhibition of rabbit skeletal muscle glycogen phosphorylase b by 5-methyl-5,6,7,8-tetrahydrofolic acid, 3'-chloro- and 3',5'-dichloromethotrexates has been studied. The inhibition is reversible and characterized by positive kinetic cooperativity (Hill coefficient exceeds 1). The values of pterin concentration causing two-fold diminishing of the enzymatic reaction rate increased in the order: 3',5'-dichloromethotrexate, 3'-chloromethotrexate, 5-methyl-5,6,7,8-tetrahydrofolic acid (0.24, 0.40 and 1.87 mM, respectively). Comparison of "half-saturation" concentrations for the above compounds and for methotrexate and folinic acid shows that pterin affinity to glycogen phosphorylase b is affected by substituents both in pteridine and in p-aminobenzoic moieties of the pterin molecule. The antagonism between 5-methyl-5,6,7,8-tetrahydrofolic acid, 3'-chloro- and 3',5'-dichloromethotrexates, on the one hand, and AMP and FMN, on the other, is revealed for combined action of modifiers on glycogen phosphorylase b.

Adenosine Monophosphate↗
PubMed1988

Ischaemia-induced changes in the phosphorylase activity in different parts of the cardiac conduction system.

The phosphorylase specific activity was lowered in total ischaemia in all examined parts of the myocardium and the conduction system of the heart. Sixty-minute ischaemia had a considerable effect on glycogen degradation in right bundle branch and in the working myocardium, however, its glycogenolytic effect on the AV-node and penetrating bundle of His was insignificant. Phosphorylase "a" evaluated as a percentage of the total phosphorylase activity decreased in the working myocardium and increased in the AV-node during ischaemia. The data obtained support the hypothesis according to which conductive tissue is more resistant to ischaemia than the working myocardium. The results serve evidence that the right bundle branch is more susceptible to metabolic damage caused by ischaemia than the AV-node and penetrating bundle of His.

Animals↗
PubMed1985

Ultraviolet radiation-induced decreases in tension and phosphorylase a formation in rat aorta.

Helical strips of rat aorta contracted with norepinephrine (10 nM) respond to ultraviolet (UV) radiation (340-360 nm) with a transient decrease in tension. The UV radiation-dependent relaxation is completely reversible and endothelial cell-independent. Although decreased tension is associated with a rise in tissue cGMP content, the cAMP level is unchanged after UV radiation. A significant inhibition of phosphorylase a formation which occurs coincidently with the rise in cGMP and decline in tension is observed with UV radiation. The effects of UV radiation on cGMP, phosphorylase a formation and tension were blocked by methylene blue. Relaxation and inhibition of phosphorylase a formation in response to UV radiation were also partially reversed with higher concentrations of norepinephrine (100 nM). Our results suggest that cGMP may mediate UV radiation-dependent reactions by reducing cytoplasmic Ca2+.

Animals↗
PubMed1983

Continuous production of maltooligosaccharides from glucose 1-phosphate by amylase-phosphorylase reactor.

Using an ultrafiltration cell, the continuous production of maltose and a mixture of other oligosaccharides was achieved by the combined action of alpha- or beta-amylase and phosphorylase. For the beta-amylase-phosphorylase system, a steady production of maltose was obtained until the beta-amylase was inactivated. For the alpha-amylase-phosphorylase system, it was difficult to maintain a stable production of oligosaccharides, probably due to the destruction of substrate glycogen by random cleavage by alpha-amylase.

Amylases↗
PubMed1984

[Interaction of muscle glycogen phosphorylase B with flavin mononucleotide and its analogs].

The inhibition of rabbit skeletal muscle glycogen phosphorylase B by FMN and its analogues with substituents in the positions 6 and 8 has been studied. Inhibiting action of FMN is manifested in reducing the limiting rate of enzymic reaction and in increasing the half-saturation concentration of AMP. The inhibitor half-saturation values (in microM) increase in the following order: FMN (13,5), 6-bromo-FMN (27), 8 alpha-hydroxy-FMN (30), 8-dimethylamino(nor)-FMN (33), 6-(N-acetyl-L-cysteine-S-yl)-FMN (44), 6-amino-FMN (96), 8-hydroxy(nor)-FMN (109), 6-nitro-FMN (170), 8 alpha-(N-acetyl-L-cysteine-S-yl)-FMN (260). The existence of the glycogen phosphorylase B complexes with FMN or its analogues has been proved by spectrophotometry and sedimentation in analytical ultracentrifuge. FMN has been shown to hinder AMP-induced transition of dimeric form of the enzyme to tetrameric one. AMP at high concentrations has been found to inhibit glycogen phosphorylase B.

Animals↗
PubMed1981

Rose bengal as a tool in studying the ligand binding of phosphorylase b.

The interaction of rose bengal (RB) with rabbit skeletal muscle phosphorylase b (1,4-alpha-D glucan: orthophosphate alpha-glucosyl-transferase, E.C. 2.4.1.1.) was studied by kinetic and absorption photometric methods. RB inhibited the phosphorylase b activity. Inhibition was strictly competitive with respect to substrate G-1-P and activator AMP with inhibition constants 2 x 10(-6) M and 2.2. x 10(-7) M, respectively. The association of the dye with the enzyme elicited a red shift in the spectrum of RB indicating an apolar binding site. According to difference absorption measurements, the enzyme binds two dye molecules per dimer in the presence and absence of both G-1-P and AMP. Binding constants determined from photometric titrations are consistent with those obtained from kinetic measurements. The present findings allow to carry out detailed kinetic investigations on the activator AMP and substrate G-1-P binding of phosphorylase b.

Adenosine Monophosphate↗
PubMed1978

Role of SH groups in the activity of pig phosphorylase b isoenzymes.

The reaction of phosphorylases from rabbit skeletal muscle, pig skeletal muscle and pig heart with DTNB and the effects of AMP and glucose-6-phosphate on the above reaction were examined and compared. The SH groups of pig heart-specific phosphorylase b were found to be the same as those of rabbit skeletal muscle. Pig skeletal muscle phosphorylase b differed from the foregoing enzymes in that its slowly reacting SH groups played a minor role in the catalytic activity and AMP and glucose-6-phosphate affected neither the DTNB reaction nor the small decrease of activity during the reaction.

Animals↗
PubMed1993

Phosphorylase kinase, a metal ion-dependent dual specificity kinase.

Phosphorylase kinase is shown to be a dual specificity kinase. The specificity of phosphorylation is determined by divalent cation. Mg2+ causes seryl phosphorylation of phosphorylase b, but Mn2+ activates tyrosine phosphorylation of angiotensin II. In contrast to seryl phosphorylation, the tyrosine kinase activity of holoenzyme is not regulated by Ca2+. Preincubation of the holoenzyme with Ca2+, Mg2+ and ATP that causes autophosphorylation activates tyrosine kinase activity. The tyrosyl kinase activity is a property of the gamma subunit. Addition of varying amounts of Mn2+ to a truncated form of the gamma subunit of phosphorylase kinase containing MgATP inhibits serine kinase but activates tyrosine kinase activity. This result along with an oxidative reaction caused by Cu2+ and site-directed mutagenesis of the putative catalytic base inhibiting both serine and tyrosine kinase activity suggest that one active site is involved in both activities. Kinetic studies with Mn2+ and ATP show that Km for nucleotide is not changed with a seryl or tyrosyl substrate. The Vm values are different, and the value for tyrosyl phosphorylation is similar to other tyrosyl kinases. We propose two conformations for the active site; one favors seryl phosphorylation, and the second tyrosyl phosphorylation is caused by the binding of divalent cation at a second metal ion binding site.

Adenosine Triphosphate↗
PubMed1999

Induction of thymidine phosphorylase expression and enhancement of efficacy of capecitabine or 5'-deoxy-5-fluorouridine by cyclophosphamide in mammary tumor models.

Thymidine phosphorylase (dThdPase) is an essential enzyme for the activation of the oral cytostatic drugs capecitabine (N(4)-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine, Xeloda(trade mark)) and its intermediate metabolite doxifluridine [5'-deoxy-5-fluorouridine (5'-dFUrd, Furtulon((R)))] to 5-fluorouracil (5-FUra) in tumors. In a previous study, we found that several cytostatics were able to up-regulate tumor levels of dThdPase in a human colon cancer xenograft model. In the present study, we confirmed that the administration of cytostatics used for breast cancer treatment, such as taxanes and cyclophosphamide (CPA), up-regulated the tumor level of dThdPase in mammary tumor models as well. Because the dThdPase up-regulation was observed even when CPA was given orally, we investigated further the usefulness of combination therapy with the 2 oral drugs, 5'-dFUrd/capecitabine and CPA in mammary tumor models. Daily oral administration of CPA up-regulated human dThdPase levels in the tumor tissue of mice bearing a human mammary tumor xenograft, MX-1, whereas in the small intestine and liver, it did not affect levels of pyrimidine nucleoside phosphorylases (PyNPase) including dThdPase and uridine phosphorylase. The preferential up-regulation of PyNPase activity in the tumor by CPA administration was also confirmed in mice bearing a syngeneic murine mammary adenocarcinoma, A755. In both models, combination therapy of 5'-dFUrd/capecitabine with CPA showed synergistic antitumor activity, without significant potentiation of toxicity. In contrast, treatment with CPA and either 5-FUra or UFT (a mixture of tegafur and uracil) in combination showed only additive activity. Our results suggest that CPA and capecitabine/5'-dFUrd, both available for oral administration, would be good partners, and that clinical trials with this drug combination against breast cancer are warranted.

Animals↗
PubMed1991

Towards time-resolved diffraction studies with glycogen phosphorylase.

Laue diffraction with high intensity, broad-spectrum synchrotron radiation sources allows three-dimensional data sets on protein crystals to be recorded in seconds or milliseconds and opens the way for time-resolved studies on dynamic events in crystals. This chapter briefly reviews the field and describes progress towards time-resolved studies with glycogen phosphorylase. Methods for the synchronization of the start of reaction with the start of data collection have been developed for the phosphorolytic reaction of glycogen phosphorylase. The compound 3,5-dinitrophenylphosphate is photolabile, yielding Pi and the by-product, 3,5-dinitrophenol, which is non-reactive with the enzyme. Spectroscopic studies show that the compound has good quantum yield and that photolysis is rapid (greater than 1000 s-1). Release of the dinitrophenylate anion, following a pulse of light from a xenon flash lamp, has been monitored with a diode array spectrophotometer specially adapted for measurements on crystals. In a laboratory X-ray experiment with crystals of glycogen phosphorylase b, release of Pi and formation of the enzyme-product complex have been demonstrated. The way is now open for Laue diffraction studies on the catalytic reaction in the crystal.

Crystallography↗
PubMed1979

Purine nucleoside phosphorylase deficiency: biochemical properties and heterogeneity in two families.

The biochemical features of two families with purine nucleoside phosphorylase deficiency are compared. Laboratory studies and an evaluation of kinetic and physical properties of erythrocyte purine nucleoside phosphorylase give evidence that a) the degree of abnormality in uric acid and nucleoside concentrations in plasma and urine reflect the severity of the enzymatic deficiency and b) structural alterations of the mutant enzymes result from structural gene mutations and demonstrate genetic heterogeneity in the disease purine nucleoside phosphorylase deficiency.

Child↗
PubMed1977

1,4-alpha-Glucan phosphorylase form Klebsiella pneumoniae covalently couple on porous glass.

A simplified procedure for the preparation of 1,4-alpha-glucan phosphorylase from Klebsiella pneumoniae is described. An 80-fold purification is achieved in two steps with an overall yield of about 50%. The specific activity of the homogeneous enzyme protein is 17.7 units/mg. Compared with glycogen phosphorylase from rabbit muscle the enzyme from K. pneumoniae shows a markedly higher stability against deforming and chaotropic agents. The 1,4-alpha-glucan phosphorylase was covalently bound to porous glass particles by three different methods. Coupling with glutaraldehyde gave the highest specific activity, i.e., 5.6 units/mg of bound protein or 133 units/g of glass with maltodextrin as substrate. This corresponds to about 30% of the specific activity of the soluble enzyme. With substrates of higher molecular weight, such as glycogen or amylopectin, lower relative activity was observed. The immobilized enzyme preparations showed pH activity profiles which were slightly displaced to higher values and exhibited an increased temperature stability.

Drug Stability↗
PubMed1978

Procedure for the simultaneous large-scale isolation of pullulanase and 1,4-alpha-glucan phosphorylase from Klebsiella pneumoniae involving liquid-liquid separations.

A procedure for the simultaneous large-scale isolation of pullulanase and 1,4-alpha-glucan phosphorylase from Klebsiella pneumoniae is described. The pullulanase is solubilized from the cell wall by cholate treatment; cells and cell debris are removed by partition in a poly(ethylene glycol) (PEG)-dextran two-phase system and from the upper (PEG) phase of this system the pullulanase is isolated by ultrafiltration and precipitation with N-cetyl,N-,N-,N-trimethyl ammonium bromide to a purity of about 80% with a yield of 70%. The preparations are free of alpha-amylase activity. The cell containing dextran-rich phase is passed through a Manton-Gaulin homogenizer. Then the phosphorylase is separated from the cell debris by partition in a second PEG-dextran system. From the top phase of this system the phosphorylase is isolated by distribution in a PEG-salt two-phase system followed by batch adsorption on carboxymethyl-Sephadex in a yield of 55%, a purity of around 90%, and nearly free of glycosyltransferase activity. All steps in the isolation of the two enzymes can be performed easily in a large scale.

Cell Fractionation↗
PubMed1977

Incorporation of purine nucleosides in cultured fibroblasts from a patient with purine nucleoside phosphorylase deficiency and associated T-cell immunodeficiency.

Cultured skin fibroblasts from a patient with T-cell immune deficiency and an absence of purine nucleoside phosphorylase activity in red cells were assayed for their capacity to metabolize inosine and guanosine. The cultured fibroblasts were lacking activity of nucleoside phosphorylase and, compared to normal fibroblasts, could incorporate only 2% and 4% of 14C-inosine and 3H-guanosine, respectively, into acid precipitable material. Autoradiography visually confirmed the failure of the NP deficient cell line to incorporate the nucleosides into nuclear material. The physiological mechanism by which the deficiency of purine nucleoside phosphorylase causes T-cell dysfunction remains unclear.

Cell Line↗
PubMed1985

Uridine phosphorylase from Novikoff rat hepatoma cells: purification, kinetic properties, and its role in uracil anabolism.

Uridine phosphorylase activity was detected in sonic extracts of six different mammalian cell lines and, in conjunction with uridine kinase, provides a route for the conversion of uracil to UMP via uridine. Uracil phosphoribosyl transferase activity was not detected in any of eight different mammalian cell lines. Uridine phosphorylase was purified 5,330-fold from Novikoff rat hepatoma cells by ammonium sulfate precipitation, DEAE-Sephadex chromatography, hydroxyapatite chromatography, and Sephadex G-200 fractionation. The molecular weight of the enzyme by gel filtration was approximately 45,000. The kinetics of the purified enzyme were analyzed with respect to all four substrates at saturating cosubstrate concentration, yielding the parameters KmUra = 360 microM, KmRib-1-P = 88 microM, KmUrd = 16 micron, and KmPi = 130 microM. However, in intact cells the phosphorolysis of uridine proceeded with an apparent Km of 231 microM. Novikoff cells treated with 0.5 mM inosine exhibited an increase in uracil uptake rate which was proportional to an observed increase in intracellular ribose-1-phosphate. Nevertheless, in cells whose de novo synthesis of pyrimidines was blocked by pyrazofurin or N-(phosphonacetyl)-L-aspartate ("PALA"), the uptake of uracil was insufficient to support proliferation, even when enhanced by inosine. These observations are consistent with the kinetic characteristics of the enzyme and provide evidence that the intracellular level of ribose-1-phosphate plays a rate-limiting role in the uptake of uracil mediated by uridine phosphorylase.

Animals↗