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PubMed1978

Purine nucleoside phosphorylase in lymphocytes of various mammalian species.

The distribution of purine nucleoside phosphorylase has been assessed by light and electron microscopy in peripheral lymphocytes of man, the rabbit, rat, mouse, guinea-pig, pig and dog. The enzyme activity was detected in the cytosol of the majority of lymphocytes in all species. The amount of reaction product was high in the rabbit, man, guinea-pig and dog, mpderate in the rat and very low in the pig and mouse. Other blood cell types are reactive as well, although there is a variation between species. A possible relationship of purine nucleoside phosphorylase with particular subpopulations of lymphocytes is suggested.

Animals↗
PubMed1981

Alkalinization of phosphorylase kinase-deficient muscle during tetanic contraction.

The intracellular pH of resting and stimulated muscle was monitored by two independent methods: measurement of pH in iodoacetate-treated homogenates of freeze-clamped tissue and the absorbance at 550-443 nm of intracellular neutral red dye in vivo. During tetanic stimulation, muscle of phosphorylase kinase-deficient mice shows a transient alkalinization whereas muscle in normal mice becomes more acid under similar conditions. The alkalinization appears to be caused by abnormally rapid AMP deamination associated with adaptation to phosphorylase kinase deficiency.

Animals↗
PubMed1977

Protein inhibitors of phosphorylase phosphatase and cyclic AMP-dependent protein kinase from rabbit skeleta muscle.

A heat-and acid-stable protein inhibitor of phosphorylase phosphatase is present in a highly purified preparation of protein inhibitor of cyclic AMP-dependent protein kinase from rabbit skeletal muscle. Although these two inhibitors have strikingly similar properties to each other, such as sensitivity to trypsin and behavior on gel permeation chromatography, they can be separated by polyacrylamide disc gel electrophoresis. This indicates that the phosphatase-inhibitory and kinase-inhibitory activities reside with different protein species. The inhibition of both the enzymes is not altered by incubating the inhibitor preparation with a general phosphoprotein phosphatase, with phosvitin kinase, or with cyclic AMP-dependent protein kinase. Inhibition of phosphorylase phosphatase is of a non-competitive type supporting the idea that the phosphatase inhibitor is not an alternative substrate for the enzyme. Inhibition of phosphatase activity is selective in that it does no occur when phosphorylated histone or phosphorylated protamine are used as substrates.

Animals↗
PubMed1977

Inhibition of rabbit skeletal muscle phosphorylase phosphatase by spermine.

The effect of three naturally occurring polyamines (putrescine, spermidine, and spermine) on the activity of rabbit skeletal muscle phosphorylase phosphatase was investigated. Only spermine significantly inhibited the enzyme. The mode of inhibition (ki value of 0.3 mM) of the phosphatase by spermine appears to be different from that caused by divalent metal ions or by other organic cations, such as arginine and lysine esters, since it is noncompetitive with respect to the substrate, phosphorylase a.

Animals↗
PubMed1986

Regional pattern and heat-resistance of brain 5'-deoxy-5'-methylthioadenosine phosphorylase.

The distribution of 5'-deoxy-5'-methylthioadenosine (MTA) phosphorylase in 10 pig brain areas was determined. The observed regional differences of the enzymatic activity seem to reflect more the pattern of brain spermine distribution rather than that of spermidine. Moreover, comparative studies on the heat-resistance of MTA phosphorylase extracted from the whole brain of various species suggest structural differences in the enzyme molecules occurring in the brains of different animals.

Animals↗
PubMed1994

Uncooked cornstarch treatment for hepatic phosphorylase kinase deficiency.

UNLABELLED: A 5-year-old boy with short stature, hepatomegaly and motor weakness due to hepatic phosphorylase kinase deficiency is described. Laboratory data showed mild hypoglycaemia and metabolic acidosis, hepatic dysfunction, and a low insulin-like growth factor-I level. Mild hypoglycaemia, marked ketosis and insufficient growth hormone secretion were revealed at night. Serum total and free carnitine levels were low and the acyl/total carnitine ratio was high. Urinary acylcarnitine profile using fast atom bombardment and tandem mass spectrometry showed increased excretion of acetylcarnitine and dicarboxylylcarnitines. These endocrinological and metabolic abnormalities and clinical symptoms were improved with uncooked cornstarch treatment. CONCLUSION: Uncooked cornstarch treatment may be helpful in hepatic phosphorylase deficiency.

Child↗
PubMed1987

Expression and regulation of glycogen phosphorylase in preneoplastic and neoplastic hepatic lesions in rats.

Glycogen phosphorylase (PHO) was demonstrated immunocytochemically and enzyme histochemically in cryostat sections of liver from rats treated for 7 weeks with N-nitrosomorpholine (120 mg/l and 200 mg/l drinking water) and from untreated controls. The activity and distribution of PHO protein were studied in normal liver and correlated with morphologically defined stages of hepatic tumour development. In normal liver the amount of enzyme protein, as visualized by the immunoperoxidase method using antibodies against phosphorylase, showed some heterogeneity within the liver lobule. The intralobular and intracellular distribution of PHO protein was the same as that of glycogen, namely coarse and granular in periportal hepatocytes and very fine in perivenular cells. In glycogen storage foci the amount of PHO protein was increased. In contrast, PHO activity was generally decreased. In other preneoplastic and neoplastic lesions such as mixed cell foci, neoplastic nodules and hepatocellular carcinomas, PHO protein was increased in all glycogen-loaded cells while PHO activity was reduced. In all glycogen-poor and basophilic cells, both PHO protein and PHO activity were decreased or absent. It was concluded that the decrease in PHO activity in glycogen storage foci was not the direct consequence of genetic changes leading to a loss in enzyme protein but was due to a defect in the cascade of phosphorylation processes resulting in active PHO. Alteration in gene expression leading to a loss of PHO protein was a late event in the process of hepatocarcinogenesis.

Animals↗
PubMed1992

Glycogen phosphorylase hyperactive foci of altered hepatocytes in aged rats.

In untreated 12- to 24-month-old rats, the enzyme histochemical pattern of 45 focal hepatic lesions was investigated in serial sections. In addition to previously characterized glycogen storage foci, a new type of enzymatically altered hepatic focus was found. The outstanding feature of this was an increased glycogen phosphorylase activity. The frequent appearance of glycogen phosphorylase hyperactive foci simultaneously exhibiting excessive glycogen storage suggests a close relationship to the well known glycogen storage foci representing an early stage in the sequence of cellular changes which lead to hepatic tumors.

Aging↗
PubMed2001

Plastidic (Pho1-type) phosphorylase isoforms in potato (Solanum tuberosum L.) plants: expression analysis and immunochemical characterization.

Higher plants contain two types of phosphorylase (EC 2.4.1.1). One type is plastidic (Phol) and the other resides in the cytosol (Pho2). For Solanum tuberosum L., two highly homologous Pho1-type sequences (designated as Pho1a and Pho1b, respectively) have been described that occur both in a homodimeric, (Pho1a)2, and a heterodimeric, Pho1a-Pho1b, state [U. Sonnewald et al. (1995) Plant Mol Biol 27:567 576; T. Albrecht et al. (1998) Eur J Biochem 251:981-991]. We present a spatial and temporal analysis of the expression patterns of the Pho1-type phosphorylases in S. tuberosum. Expression was analyzed at transcript, protein and activity levels. The specificity of both the probes and the antibodies used was carefully determined to ensure selectivity of detection. For both the Pho1a and Pho1b probes the degree of cross-hybridization was estimated. Peptide scanning identified the epitopes of the anti-Pho 1a and anti-Pho 1b antibodies. Expression of the two Pho1-type genes was analyzed in various organs of the potato plant. In all organs studied the Pho1a transcript levels exceeded those of Pho1b. Furthermore, leaves of a given developmental stage were sampled during the light period and were analyzed for transcript and protein levels and for various carbohydrate pools as well. The data show that in leaves the Pho1a gene expression closely corresponds to starch accumulation, suggesting that the enzyme fulfils a metabolic function within the process of starch biosynthesis. In tubers, Pho1a is constitutively expressed in the parenchyma cells whereas expression of the Pho1b, gene is restricted to cells in close vicinity of the vascular tissue.

Amino Acid Sequence↗
PubMed2005

Prognostic significance of thymidine phosphorylase in superficial bladder carcinoma.

OBJECTIVE: We investigated the expression of thymidine phosphorylase (TP) in bladder carcinomas and assessed its prognostic significance in superficial bladder cancer samples. PATIENTS AND METHODS: We studied 142 primary bladder cancer samples immunohistochemically for nuclear thymidine phosphorylase (TPN), cytoplasmic (TPC) and stromal (TPSTR) expression. We correlated them with standard clinicopathological features (grade, stage, concurrent in situ, multiplicity, primary or recurrent status), as well with recurrence and progression. We examined also the relationship between TP and tumor microvessel density. RESULTS: The level of all types of TP correlated well with stage, while grade correlated well only with TPSTR and the presence of carcinoma in situ only with TPN. Patients with low levels of TPN had a longer tumor free interval, during a 38.6 months mean follow up time. Regarding the association between TP count and microvessel density we found the strongest association with TPSTR (p=0.003), a borderline statistical significance with TPC (p=0.049) and no relationship with TPN (p=0.072). CONCLUSIONS: We suggest that the assessment of TPN might be useful for predicting recurrence in superficial bladder cancer. We propose also that TP may stimulate angiogenesis.

Aged↗
PubMed1979

Cellular immune deficiency with autoimmune hemolytic anemia in purine nucleoside phosphorylase deficiency.

Immunologic and metabolic abnormalities were studied in a five year old boy with 0.07 per cent of normal erythrocyte purine nucleoside phosphorylase activity. The clinical course is characterized by severe autoimmune hemolytic anemia, a transient neurologic disorder with tremor and ataxia, and minor infectious illnesses. There is severe lymphopenia with decreased absolute numbers of T and B lymphocytes. Mitogen-stimulated blastogenesis is reduced, but response to allogeneic lymphocytes is normal. A monoclonal IgG protein is present. There is hypouricemia, elevated plasma inosine level, hypouricosuria and an increase in the urinary concentration of inosine and guanosine. The pattern of heterozygote distribution in the patient's family is compatible with an autosomal recessive trait in which heterozygotes are identifiable. In addition, the unusual laboratory and clinical manifestations of this patient illustrate the heterogeneity of the clinical syndrome associated with purine nucleoside phosphorylase deficiency.

Anemia, Hemolytic, Autoimmune↗
PubMed1983

A bioluminescent assay for glycogen phosphorylase in cultured cells.

A new method for the determination of glycogen phosphorylase (1,4 alpha-D-glucose:orthophosphate alpha-glucosyltransferase, EC 2.4.1.1) in cultured cells is described. The assay utilizes bacterial luciferase (EC 2.7) in a liquid scintillation spectrometer to measure NAD(P)H formed in a coupled enzyme reaction comprising glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and phosphoglucomutase (EC 2.7.5.1). This assay is highly sensitive, easily detecting as little as 10 microU phosphorylase, fast and simple to perform. With modifications this procedure can be extended to measure other glycogenolytic enzymes and intermediates.

Animals↗
PubMed1989

A new isotopic assay for purine nucleoside phosphorylase.

We have developed a new assay for purine nucleoside phosphorylase which is based on the release of tritium when [2-3H]inosine is used as the substrate and the reaction is coupled with xanthine oxidase. After the reaction is terminated, residual [2-3H]inosine is adsorbed on charcoal and the supernatant solution is assayed for radioactivity by liquid scintillation spectrometry. The new method gave results indistinguishable from those obtained by spectrophotometric determination of uric acid produced by the phosphorylase-xanthine oxidase-coupled reaction or by radioassay of chromatographically isolated [8-14C]hypoxanthine when [8-14C]inosine was used as substrate. The new method is faster than those involving chromatographic isolation of products. In comparison with spectrophotometric methods, it not only requires less manual time, but it also has the advantage that it can be used to study inhibitors whose ultraviolet absorption might interfere with spectrophotometric determination of uric acid.

Animals↗
PubMed1983

Calmodulin-dependent glycogen synthase kinase: identification in liver of normal and phosphorylase kinase-deficient rats.

We have purified a calmodulin-dependent glycogen synthase kinase from livers of normal and phosphorylase kinase-deficient (gsd/gsd) rats. No differences between normal and gsd/gsd rats were apparent in either (a) the ability of liver extracts to phosphorylate exogenous glycogen synthase in a Ca2+- and calmodulin-dependent manner or (b) the purification of the calmodulin-dependent synthase kinase. Although extracts from rat liver, when compared to rabbit liver extracts, had a significantly reduced ability to phosphorylate exogenous synthase, the calmodulin-dependent synthase kinase could be purified from rat liver using a protocol identical to that described for rabbit liver. Moreover, the synthase kinase purified from rat liver had properties very similar to those of the rabbit liver enzyme. The enzyme was completely dependent on calmodulin for activity against glycogen synthase, was unable to phosphorylate phosphorylase b, catalyzed the rapid incorporation of 0.4 mol phosphate/mol of glycogen synthase subunit, selectively phosphorylated sites 1b and 2 in the glycogen synthase molecule, had a Stokes' radius of about 70 A, and appeared to be composed of subunits of Mr 56,000 and 57,000. These observations led us to conclude that (1) calmodulin-dependent glycogen synthase kinase is distinct from other kinases previously described and (2) the rat liver kinase and the rabbit liver kinase are very similar enzymes.

Animals↗
PubMed1984

Predominance of beta-adrenergic over alpha-adrenergic receptor functions involved in phosphorylase activation in liver cells of cholestatic rats.

Hepatocytes isolated from normal and cholestatic rats responded to adrenergic agonists and antagonists in a quite different manner. Much greater activation of glycogen phosphorylase was caused by phenylephrine, an alpha-agonist, than by isoproterenol, a beta-agonist, in normal rat hepatocytes, and vice versa in the cholestatic rat cells. Epinephrine activation of phosphorylase was antagonized more efficiently by phenoxybenzamine, an alpha-antagonist, than by propranolol, a beta-antagonist, in normal rats, whereas it was antagonized totally by propranolol but only partially by phenoxybenzamine in cholestatic rat hepatocytes. The number of alpha-adrenergic receptors, measured by [3H]prazosin binding to membranes, as well as alpha-receptor-mediated increases in 32Pi incorporation into phosphatidylinositol and in 45Ca efflux, were reduced in hepatocytes after induction of cholestasis. The reduction of these parameters of alpha-receptor-linked functions was associated with the reciprocal increase in the number of beta-receptors and enhancement of beta-receptor-mediated accumulation of cyclic AMP in cholestatic rat hepatocytes. The affinity of epinephrine for beta-receptors was higher in cholestatic rat cells than in normal rat cells; this difference in affinity was abolished by the addition of guanylylimidodiphosphate, indicating that induction of cholestasis rendered hepatic beta-receptors more tightly coupled to the GTP-binding protein. Thus, the cascade reactions arising from beta-receptors are predominant over those from alpha-receptors, eventually leading to glycogen breakdown in cholestatic rat hepatocytes, principally because of not only the elevated beta to alpha ratio of the membrane receptor density but also the tight coupling of beta-receptors to the adenylate cyclase system via the guanine nucleotide regulatory protein.

Animals↗
PubMed1984

Phosphorylation and inactivation of rabbit skeletal muscle glycogen synthase: distinction between kinase Fa-, phosphorylase kinase-, and glycogen synthase (casein) kinase-1-catalyzed reactions.

Rabbit skeletal muscle glycogen synthase was phosphorylated by kinase Fa, phosphorylase kinase, and cAMP-independent synthase (casein) kinase-1 to determine the differences among these kinase-catalyzed reactions. The stoichiometry of phosphate incorporation, the extent of inactivation, and the sites of phosphorylation were compared. Synthase (casein) kinase-1 catalyzes the highest level of synthase phosphorylation (4 mol/subunit) and inactivation (reduction of the activity ratio to below 0.05). The sites, defined by characteristic tryptic peptides, phosphorylated by synthase (casein) kinase-1 are distinguishable from those by kinase Fa and phosphorylase kinase. In addition, synthase (casein) kinase-1, unlike kinase Fa, does not activate ATP X Mg2+-dependent protein phosphatase. These results demonstrate that synthase (casein) kinase-1 is a distinct glycogen synthase kinase.

Animals↗
PubMed1985

Identification and partial characterization of bovine heart cytosolic phosphorylase phosphatases.

The properties of phosphatases in bovine heart cytosol were studied. Two isozymic forms of protein phosphatase H (H-1 and H-2) were resolved by chromatography on DEAE-Sephacel. The two isoenzymes had identical physical properties (Mr 260,000, 7.9 S). Treatment with 80% ethanol activated both isozymes and converted H-1 to a Mr 35,500 form and H-2 to Mr 67,000 and Mr 35,500 forms. Both H-1 and H-2 and their lower Mr activated forms had essentially identical Km values for phosphorylase a. The heart cytosol also contained a latent phosphatase (Fc) which could be activated by preincubation with either ATP X Mg and an activating factor (FA), or by Mn/trypsin treatment. The latter procedure converted the latent Fc (Mr 200,000) to a Mn2+-independent Mr 34,500 form. Both activated forms of Fc had similar Km values which were fourfold lower than the affinity of the protein phosphatase H forms for the phosphorylase a substrate.

Adenosine Triphosphate↗
PubMed1987

Purification and properties of phosphorylase from Phymatotrichum omnivorum.

The glycogen phosphorylase (EC 2.4.1.1) from the mycelium of Phymatotrichum omnivorum was purified by ammonium sulfate fractionation, gel filtration on Sephacryl S-200, and DEAE-cellulose ion-exchange chromatography to more than 100-fold. The purified enzyme was homogeneous; this was confirmed by polyacrylamide gel electrophoresis. Sodium dodecyl sulfate-gel electrophoresis indicated the relative molecular size of the enzyme was around 145,000. The approximate molecular weight by gel filtration was 116,000. The optimum pH of the enzyme was 7.0 and the enzyme was more specific for glycogen, with a Km value of 0.36 mg/ml. Nucleotides AMP, ADP, and ATP and compounds containing an "SH" group inhibited the enzyme activity. Diethyldithiocarbamate, EDTA, ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid, and Cu2+ were the potent inhibitors of the glycogen phosphorylase activity, Ca2+, Cu2+, Co2+, and Fe2+ stimulated the enzyme activity. The enzyme preparation was stable at 4 degrees C during a period of 30 days.

Cations, Divalent↗