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At least 1,639 records · Page 91Linked to original sources
PubMed1989

Genetic deficiency of purine nucleoside phosphorylase in the mouse. Characterization of partially and severely enzyme deficient mutants.

Two independent mutations of purine nucleoside phosphorylase were identified in the first-generation progeny of male mice that had been treated with the mutagen N-ethylnitrosourea and mated to untreated females. The common allele in inbred strains is Np-1a and the mutants are assigned the gene symbols Np-1e and Np-1f. Heterozygotes had approximately half normal purine nucleoside phosphorylase activity in erythrocytes and activity of homozygotes was 17 and 5% of NP-1A for NP-1E and NP-1F, respectively. The following properties are consistent with both Np-1e and Np-1f being point mutations: the expression of residual but markedly reduced activity with normal Michaelis constants for inosine and phosphate, altered isoelectric points, and increased thermal lability. The reduction in erythrocyte activity was also evident in other tissues. A metabolic consequence of the mutations was increased purine nucleoside excretion. Inosine and guanosine, total 150 +/- 84 microM, and inosine, deoxyinosine, guanosine, and deoxyguanosine, total 1490 +/- 190 microM, were present in urine of Np-1e/Np-1e and Np-1f/Np-1f mice, respectively, but not in normal urine, less than 10 microM.

Animals↗
PubMed1982

In situ enzymatic removal of orthophosphate by the nucleoside phosphorylase catalyzed phosphorolysis of nicotinamide riboside.

An enzymatic orthophosphate removal system is described which can be effectively used to continuously remove orthophosphate from biochemical samples. The phosphorolysis of nicotinamide riboside is catalyzed by calf spleen nucleoside phosphorylase to give ribose-1-PO4 and nicotinamide along with a proton. At pH 8 the production of ribose-1-PO4 from orthophosphate is essentially quantitative. This reaction can be monitored optically or by 31P nuclear magnetic resonance (NMR). Equations are given for determining the time required to remove a given amount of phosphate from a typical NMR sample with a known amount of nucleoside phosphorylase. The effects of a competing orthophosphate-producing reaction are considered.

Animals↗
PubMed1985

Rabbit liver phosphorylase a phosphatase: regulation by glucose and caffeine.

The modulation of three molecular forms of liver phosphorylase a phosphatases, the "catalytic" subunit phosphatase (35 000 relative mass (Mr) and phosphatases 2A1 and 2A2 by glucose and caffeine and some physiologically important compounds were studied, using 32P-labelled phosphorylase a obtained from rabbit liver as substrate. Glucose and caffeine showed independent and additive activations. The caffeine effect was seen at micromolar to millimolar concentrations and glucose caused activation even at concentrations below the normal blood glucose level. The nucleotides ATP and AMP, at their presumed physiological concentrations in the liver, were strongly inhibitory. Inhibition by these nucleotides and other inhibitors tested showed varied responses to the presence of the activators glucose and caffeine, depending on the phosphatase form. Thus, significant relief of ATP inhibition was afforded by glucose and caffeine acting independently for the 35 000 Mr phosphatase, whereas relief of inhibition for phosphatases 2A1 and 2A2 required a combination of glucose, caffeine, and Mg2+. The Km of the liver 35 000 Mr phosphatase was about 50 microM for the liver substrate as compared with 4 microM for the muscle substrate. The Km of phosphatase 2A2 was about 16 microM and for phosphatase 2A1 it was about 20 microM, using liver substrate in the absence of any stimulators. Mg2+ inhibited the 35 000 Mr phosphatase, but became stimulatory for phosphatase 2A2 and was an almost obligatory requirement for phosphatase 2A1.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗
PubMed1991

Purification and characterization of purine nucleoside phosphorylase from developing embryos of Hyalomma dromedarii.

Purine nucleoside phosphorylase from Hyalomma dromedarii, the camel tick, was purified to apparent homogeneity. A molecular weight of 56,000 - 58,000 was estimated for both the native and denatured enzyme, suggesting that the enzyme is monomeric. Unlike purine nucleoside phosphorylase preparations from other tissues, the H. dromedarii enzyme was unstable in the presence of beta-mercaptoethanol. The enzyme had a sharp pH optimum at pH 6.5. It catalyzed the phosphorolysis and arsenolysis of ribo- and deoxyribo-nucleosides of hypoxanthine and guanine, but not of adenine or pyrimidine nucleosides. The Km values of the enzyme at the optimal pH for inosine, deoxyinosine, guanosine, and deoxyguanosine were 0.31, 0.67, 0.55, and 0.33 mM, respectively. Inactivation and kinetic studies suggested that histidine and cysteine residues were essential for activity. The pKa values determined for catalytic ionizable groups were 6-7 and 8-9. The enzyme was completely inactivated by thiol reagents and reactivated by excess beta-mercaptoethanol. The enzyme was also susceptible to pH-dependent photooxidation in the presence of methylene blue, implicating histidine. Initial velocity studies showed an intersecting pattern of double-reciprocal plots of the data, consistent with a sequential mechanism.

Amino Acids↗
PubMed1977

Reserpine-induced supersensitivity to the activation of cardiac glycogen phosphorylase by calcium.

Rats receiving reserpine (2.5 mg/kg, 24 h before sacrifice) developed a supersensitivity to the phosphorylase activating effect of both noradrenaline and Ca2+. Supersensitivity to the inotropic and chronotropic effects of the agonists did not develop with this dose of reserpine. It is concluded that reserpine induced supersensitivity, as well as being dose and species dependent, is also dependent on the response measured. It is concluded further that the supersensitivity developed to noradrenaline may be due to an alteration in the responding system (in this case the phosphorylase activating system) to calcium.

Animals↗
PubMed1997

Accelerated glycogenolysis in uremia and under sucrose feeding: role of phosphorylase alpha regulators.

To understand the mechanism of hepatic glycogen depletion found in uremia and under sucrose feeding, we examined net hepatic glycogenolysis-associated active enzymes and metabolites during fasting. Liver was taken 2, 7, and 18 h after food removal in uremic and pair-fed control rats fed either a sucrose or cornstarch diet for 21 days. Other uremic and control rats fasted for 18 h were refed a sucrose meal to measure glycogen increment. Glycogen storage in uremia was normal, suggesting effective glycogen synthesis. During a short fast, sucrose feeding and uremia enhanced net glycogenolysis through different but additive mechanisms. Under sucrose feeding, there were high phosphorylase alpha levels associated with hepatic insulin resistance. In uremia, phosphorylase alpha levels were low, but the enzyme was probably activated in vivo by a fall of inhibitors (ATP, alpha-glycerophosphate, fructose-1,6-diphosphate, and glucose) and a rise of Pi, as verified in vitro. Enhanced gluconeogenesis was also suggested, but excessive hepatic glucose production was unlikely in uremia. During fasting, hypoglycemia occurred in uremia due to reduced glycogenolysis, inefficient hepatic gluconeogenesis, and impaired renal gluconeogenesis. This may be relevant to poor fasting tolerance in uremia, which could be aggravated under excessive sucrose intake.

Animals↗
PubMed2000

Regulation of glycogen phosphorylase and PDH during exercise in human skeletal muscle during hypoxia.

The present study examined the acute effects of hypoxia on the regulation of skeletal muscle metabolism at rest and during 15 min of submaximal exercise. Subjects exercised on two occasions for 15 min at 55% of their normoxic maximal oxygen uptake while breathing 11% O(2) (hypoxia) or room air (normoxia). Muscle biopsies were taken at rest and after 1 and 15 min of exercise. At rest, no effects on muscle metabolism were observed in response to hypoxia. In the 1st min of exercise, glycogenolysis was significantly greater in hypoxia compared with normoxia. This small difference in glycogenolysis was associated with a tendency toward a greater concentration of substrate, free P(i), in hypoxia compared with normoxia. Pyruvate dehydrogenase activity (PDH(a)) was lower in hypoxia at 1 min compared with normoxia, resulting in a reduced rate of pyruvate oxidation and a greater lactate accumulation. During the last 14 min of exercise, glycogenolysis was greater in hypoxia despite a lower mole fraction of phosphorylase a. The greater glycogenolytic rate was maintained posttransformationally through significantly higher free [AMP] and [P(i)]. At the end of exercise, PDH(a) was greater in hypoxia compared with normoxia, contributing to a greater rate of pyruvate oxidation. Because of the higher glycogenolytic rate in hypoxia, the rate of pyruvate production continued to exceed the rate of pyruvate oxidation, resulting in significant lactate accumulation in hypoxia compared with no further lactate accumulation in normoxia. Hence, the elevated lactate production associated with hypoxia at the same absolute workload could in part be explained by the effects of hypoxia on the activities of the rate-limiting enzymes, phosphorylase and PDH, which regulate the rates of pyruvate production and pyruvate oxidation, respectively.

Adenosine Triphosphate↗
PubMed1980

Uridine phosphorylase activity during postnatal development of the rat heart.

Uridine phosphorylase activity was assayed in heart extracts from rats of different postnatal age. Activity was low at birth, increased to a maximum at 8--14 days of postnatal development and then decreased to a low adult level. The switch from cellular hyperplasia to hypertrophy determined by the ratio proteins/DNA occurred at day 18 of development. It is suggested that the activation of uridine phosphorylase activity during the early life of the rat may be a critical element of the onset of hypertrophy.

Animals↗
PubMed1980

A new spectrophotometric assay for enzymes of purine metabolism. III. Determination of purine nucleoside phosphorylases.

A spectrophotometric method especially suitable for biological materials is described for the determination of purine nucleoside phosphorylase activity. In combination with the enzymes xanthine oxidase, catalase and aldehyde dehydrogenase, and in the presence of ethanol and NAD(P), the purines formed by phosphorylysis of purine nucleosides are oxidized and the absorption of the NAD(P)H formed is taken for the calculation of nucleoside phosphorylase activity.

Animals↗
PubMed1984

Isolation and comparative studies of glycogen phosphorylase isoenzymes from lymphocytes and polymorphonuclear leucocytes.

Two glycogen phosphorylase isoenzymes have been identified in pig lymphocytes and polymorphonuclear leucocytes by DEAE cellulose chromatography. Both isophosphorylases have been further purified by affinity chromatography on Sepharose-AMP to almost homogeneity. The purified isophosphorylases were composed of subunits of molecular weight similar to the muscle and liver monomers. Isophosphorylase I was more related to the liver enzyme than isophosphorylase II based on immuno-inhibition experiments. Both isoenzymes were markedly different from liver and muscle phosphorylases in their activation by AMP, sodium sulfate and 1,2-dimethoxyethane.

Animals↗
PubMed2003

Subconjunctival doxifluridine administration suppresses rat choroidal neovascularization through activated thymidine phosphorylase.

PURPOSE: Doxifluridine (5'-deoxy 5-fluorouridine) is an oral anticancer drug with antiangiogenic effects, with vasoclastic action that is enhanced by a major member of the pyrimidine phosphorylases, thymidine phosphorylase (TP). Previous studies have demonstrated that TP is upregulated in the lesions where pathologic angiogenesis occurs and TP itself promotes angiogenesis. To investigate the possible role of TP and doxifluridine in choroidal neovascularization (CNV), the expression level of TP was measured and the effect of doxifluridine was investigated in rat eyes with experimental CNV. METHODS: CNV was induced in rat eyes by diode laser photocoagulation. The expression level of TP in the laser-treated and control eyes was examined with high-performance liquid chromatography (HPLC). For the evaluation of CNV activity, the intensity of fluorescein leakage from the photocoagulated lesions was scored, and the areas of CNV lesions were measured histologically in the control eyes and eyes treated with a subconjunctival injection of doxifluridine 14 days after photocoagulation. RESULTS: The expression level of TP was higher in the laser-treated eyes than in the control eyes. Fluorescein leakage from the CNV lesions significantly decreased in the eyes given a subconjunctival injection of doxifluridine compared with the control. Histologic analysis demonstrated that both the areas of CNV lesions and the degree of vascular formation in the subretinal membrane were reduced in the doxifluridine-treated eyes compared with the control eyes. CONCLUSIONS: TP may be involved in the formation of CNV. Subconjunctival injection of doxifluridine significantly reduced experimental CNV activity without apparent adverse effects. These results suggest the possibility that doxifluridine can be beneficial in treating CNV.

Administration, Oral↗
PubMed1980

Erythrocyte metabolism in purine nucleoside phosphorylase deficiency after enzyme replacement therapy by infusion of erythrocytes.

Purine nucleoside phosphorylase deficiency is associated with a severely defective T-cell immunity. A patient with purine nucleoside phosphorylase deficiency was treated with transfusions of irradiated erythrocytes and plasma. This resulted in a remarkable correction of the metabolic disturbances in the patient. The urinary excretion of inosine, deoxyinosine, guanosine, and deoxyguanosine decreased, whereas uric acid excretion as well as serum uric acid concentration increased. It could be shown that the enzyme activity of the circulating erythrocytes correlated inversely with the urinary excretion of nucleosides and directly with the excretion of uric acid. As a consequence of the therapy, several glycolytic intermediates of the erythrocytes were increased, especially 2,3-diphosphoglycerate. The high 2,3-diphosphoglycerate level caused a shift to the right of the oxygen dissociation curve (P50 = 32.9 mm Hg). The immunological status of the patient showed definite improvement after the enzyme replacement therapy.

Blood Transfusion↗
PubMed1993

Expression of muscle-type phosphorylase in innervated and aneural cultured muscle of patients with myophosphorylase deficiency.

Patients with McArdle's myopathy lack muscle glycogen phosphorylase (M-GP) activity. Regenerating and cultured muscle of patients with McArdle's myopathy presents a glycogen phosphorylase (GP) activity, but it is not firmly established whether M-GP or non-M-GP isoforms are expressed. We have cultured myoblasts from biopsy specimen of five patients with McArdle's myopathy. Skeletal muscle was cultured aneurally or was innervated by coculture with fetal rat spinal cord explants. In the patients' muscle biopsies and in their cultured innervated and aneural muscle we studied total GP activity, isoenzymatic pattern, reactivity with anti-M-GP antiserum, and presence of M-GP mRNA. There was no detectable enzymatic activity, no immunoreactivity with anti-M-GP antiserum, and no M-GP mRNA in the muscle biopsy of all patients. GP activity, M-GP isozyme, and anti-M-GP antiserum reactivity were present in patients' aneural cultures, increased after innervation, and were undistinguishable from control. M-GP mRNA was demonstrated in both aneural and innervated cultures of patients and control by primer extension and PCR amplification of total RNA. Our studies indicate that the M-GP gene is normally transcribed and translated in cultured muscle of patients with myophosphorylase deficiency.

Adult↗
PubMed1980

Thymidine phosphorylase: a possible marker of cell maturation in acute myeloid leukaemia.

Thymidine Phosphorylase activity has been measured in two populations of human Acute Myeloid Leukaemia cells (AML) and in normal human macrophages. During short-term culture in vitro of AML cells the activity of this enzyme increases progressively and approaches that of normal non-dividing macrophages at a time when the leukaemia cells in culture show evidence of maturation as assayed by functional, enzymatic and morphological criteria. It is suggested that the level of Thymidine Phosphorylase activity may be a marker of maturation in AML.

Cells, Cultured↗
PubMed2003

Familial spastic paraplegia as the presenting manifestation in patients with purine nucleoside phosphorylase deficiency.

We report two siblings with purine nucleoside phosphorylase deficiency revealed by isolated spastic paraplegia, whereas symptoms of immune deficiency did not become apparent until 3 years of age. As the concurrence of immunodeficiency and neurologic problems strongly suggests the diagnosis of purine nucleoside phosphorylase deficiency, special attention should be paid to counts of lymphocytes in any infant with spastic paraplegia.

Cerebral Cortex↗
PubMed1978

Fatal infantile form of muscle phosphorylase deficiency.

A girl had generalized, rapidly progressive weakness beginning at age 4 weeks, and causing severe respiratory insufficiency and death at age 13 weeks. Histochemical and biochemical investigations of a muscle biopsy showed increased glycogen concentration and complete lack of phosphorylase activity. The enzyme protein appeared to be absent by immunodiffusion, and the metabolic block was documented by studies of anaerobic glycolysis in vitro. The biochemical basis for the unusual clinical picture is obscure, but muscle phosphorylase deficiency has to be considered in the differential diagnosis of the "floppy baby syndrome".

Female↗
PubMed1984

Metabolic basis of improved exercise tolerance: muscle phosphorylase deficiency after glucagon administration.

A 26-year-old girl with muscle phosphorylase deficiency had exercise intolerance and experienced an occasional "second wind" phenomenon. Muscle glycogen concentration was about three times the normal level, whereas each glycolytic intermediate below the phosphorylase step was equivalent to only 10% of a normal level. Semi-ischemic forearm exercise tests disclosed no elevation of the venous lactate or pyruvate level, but they showed remarkable increases of serum creatine kinase and ammonia. Glucagon administration markedly augmented exercise tolerance. Forearm exercise after glucagon injection significantly increased venous lactate. Thus, the beneficial effect of glucagon is attributable to blood glucose utilization by muscle.

Adult↗
PubMed2001

Structure-activity relationships for a collection of structurally diverse inhibitors of purine nucleoside phosphorylase.

Values of inhibition constants, Ki, and concentrations required for 50% inhibition, IC50, for a collection of structurally diverse competitive inhibitors of calf spleen purine nucleoside phosphorylase have been determined employing inosine as substrate. These values have been employed to create predictive quantitative structure-activity relationships (QSAR) which link structure to values of Ki and IC50. These QSAR models have substantial power to predict values and the associated uncertainties for Ki and IC50 for unknown, structurally diverse inhibitors of purine nucleoside phosphorylase.

Enzyme Inhibitors↗