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PubMed1984

Effects of acyclovir and its metabolites on purine nucleoside phosphorylase.

Acyclovir (9-(2-hydroxyethoxymethyl)guanine), the clinically useful antiherpetic agent, is an "acyclic" analogue of 2'-deoxyguanosine. Purine nucleoside phosphorylase partially purified from human erythrocytes did not catalyze detectable phosphorolysis of this drug or any of its metabolites (less than 0.07% of the rate with Guo). However, these compounds were competitive inhibitors of this enzyme with Ino as the variable substrate. Acyclovir per se was a relatively weak inhibitor. Its Ki value (91 microM) was much greater than that for its 8-hydroxy metabolite (Ki = 4.7 microM) but less than that for its carboxylic acid metabolite (9-carboxymethoxy-methylguanine) (K'i = 960 microM). The phosphorylated metabolites of acyclovir were more potent inhibitors than were their guanine nucleotide counterparts. At a phosphate concentration of 50 mM, the apparent Ki values for the mono- (120 microM), di- (0.51 microM), and tri (43 microM)-phosphate esters of acyclovir were 1/2, 1/1200, and 1/26 those for dGMP, dGDP, and dGTP, respectively. The concentration of phosphate did not markedly affect the Ki value of acyclovir but dramatically affected those of its phosphorylated metabolites and their nucleotide counterparts. Decreasing phosphate to a physiological concentration (1 mM) decreased the apparent Ki values for the mono-, di-, and triphosphate esters of acyclovir to 6.6, 0.0087, and 0.31 microM, respectively. Inhibition of the enzyme by acyclovir diphosphate was also influenced by pH. This metabolite of acyclovir is the most potent inhibitor of purine nucleoside phosphorylase reported to date. It has some features of a "multisubstrate" analogue inhibitor.

Acyclovir↗
PubMed1978

Inactivation of phosphorylase b by potassium ferrate. Identification of a tyrosine residue involved in the binding of adenosine 5'-monophosphate.

The site of reaction of potassium ferrate (K2FeO4) with rabbit muscle phosphorylase b has been further characterized in an extension of previously published studies (Lee, Y. M., and Benisek, W. F. (1976) J. Biol, Chem. 251, 1553-1560) reporting inactivation of the enzyme by this reagent. The tryptic peptide composed of residues 70 to 80 of the enzyme's polypeptide chain was shown to contain a tyrosine residue which is chemically modified by ferrate and which is protected by 5'-AMP. The sequence of this peptide obtained from both untreated and ferrate-treated phosphorylase b was determined, and the results showed that tyrosine-75 was the residue with which ferrate reacts.

Adenosine Monophosphate↗
PubMed1980

Differential interaction of rabbit skeletal muscle phosphorylase kinase isozymes with calmodulin.

Rabbit skeletal muscle contains two phosphorylase kinase isozymes arising from the two different muscle types, the white and the red muscle (Jennissen, H. P., and Heilmeyer, L. M. G. (1974) FEBS Lett. 42, 77-80). The two phosphorylase kinase isozymes could be separated by affinity chromatography on a calmodulin-Sepharose 4B column. In media containing high concentrations of Ca2+, about 2 mM, both isozymes were bound to the affinity column. When the column was eluted with a buffer containing 0.2 mM Ca2+, the red muscle isozyme was eluted, whereas white muscle isozyme was eluted from the column by an ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid. The purified white muscle isozyme can be distinguished from the red muscle isozyme by its ability to inhibit calmodulin-dependent cyclic nucleotide phosphodiesterase. The two isozymes are also regulated differently by calmodulin. Both isozymes contain tightly bound calmodulin as a subunit (Cohen, P., Burchell, A., Foulkes, J. G., Cohen, P. T. W., Vanaman, T. C., and Nairn, A. C. (1978) FEBS Lett. 92, 287-293), which renders the enzyme sensitive to Ca2+. Only the white muscle isozyme can be activated by exogenous calmodulin.

Animals↗
PubMed1981

Kinetic alpha-deuterium isotope effects for Escherichia coli purine nucleoside phosphorylase-catalyzed phosphorolysis of adenosine and inosine.

Kinetic alpha-deuterium isotope effects have been measured for the purine nucleoside phosphorylase-catalyzed phosphorolysis of adenosine and inosine by a competitive double label technique at saturating concentrations of the second substrate, phosphate. Under these conditions the observed isotope effect, kH/kD, is on the second order rate constant, Vmax/Km, for reaction of nucleoside with the Michaelis complex of enzyme and phosphate. For adenosine, at neutral pH, the isotope effect is unity. For inosine, kH/kD was determined as a function of pH (the numbers in parentheses are the ratios of Vmax at that pH to Vmax at pH 7.3): 1.10 at pH 5.0 (0.19); 1.10 at pH 6.1 (0.72); 1.01 at pH 7.3 (1.00); 1.16 at pH 8.4 (0.22); and 1.18 at pH 9.4 (0.04). These values suggest a mechanism for purine nucleoside phosphorylase involving a change in rate-limiting step from one at pH values near neutrality for which cleavage of the nucleoside C-N bond is not rate limiting to a step at extremes of pH with a transition state having considerable oxocarbonium ion-like character.

Adenosine↗
PubMed1980

Phosphorylation of deoxyguanosine by B and T lymphocytes: evidence against selective trapping of deoxyguanosine by T lymphocytes in purine nucleoside phosphorylase deficiency.

T and B lymphocytes from blood and tonsils, as well as T and B lymphoid cell lines, phosphorylate deoxyguanosine which is probably the major toxic metabolite in purine nucleoside phosphorylase (PNP) deficiency. T and B cells also have similar activities of purine nucleoside phosphorylase. These findings indicate that the selective T cell defect in patients with PNP deficiency is unlikely to be due to a selective 'trapping' of deoxyguanosine by T lymphocytes.

Adenosine Deaminase↗
PubMedDate not supplied

[Glycogen content and phosphorylase activity in various muscles of rats and gophers during hypothermia, metabolic heating and hibernation].

Hypothermia (30 and 20 degrees C) of rats and gophers did not decrease the level of glycogen and did not change the activity of phosphorylase in musculus masseter, musculus gastrocnemius, musculus soleus. In musculus trapezius of rats the content of glycogen decreased by 34.5% under cooling down to 30 degrees and by 59.5% under cooling down to 20 degrees. A fall in muscular glycogen was observed under self-heating of rats and gophers after 20 degrees hypothermia and especially in awakening from hibernation. The portion of phosphorylase a increased in muscular tissues of gophers during a three-month hibernation (10-12 degrees) and awakening.

Animals↗
PubMed1983

Isozymes of glycogen phosphorylase in human cartilage and cartilage tumors.

Isozyme patterns of glycogen phosphorylase in normal fetal and adult human cartilage and benign and malignant cartilage tumors were investigated by means of polyacrylamide gel disc electrophoresis. In both normal cartilage and cartilage tumors four distinct bands were observable: these were of the liver, liver-like and fetal types and of a hybrid type between the muscle and fetal types judging by their mobilities in the small-pore gels containing 0.01% and 0.1% glycogen. The isozyme pattern of benign cartilage tumors was almost identical with that of normal cartilage. In normal fetal and adult cartilage and benign cartilage tumors, the liver and liver-like types were predominant, whereas in malignant cartilage tumors they were minor and the fetal type was predominant. These results indicate fetal deviation of phosphorylase isozymes in malignant transformation of cartilage cells.

Adult↗
PubMed1980

[Effect of the ts 15 mutation of transcription termination factor rho on expression of the uridine phosphorylase gene of Escherichia coli].

The expression of nucleoside-catabolizing genes was studied in two isogenic strains, SA1030(rho+) and AD1600(rho ts 15). The deo-enzymes activities do not differ markedly in these two strains, though when cells were grown on glycerol medium the deo-enzymes level appeared to be higher than on glucose medium. The synthesis of uridine phosphorylase in AD1600 strain was found to be constitutive. It was found also that the cytR regulation system was not altered in AD1600 strain and a wild-type allele of udp gene was present in the genome of AD1600 strain. A conclusion is made that constitutive synthesis of uridine phosphorylase is due to the presence of rho ts 15 mutation in the genome of the strain AD1600.

Escherichia coli↗
PubMed1980

[Glycogen phosphorylase isoenzymatic makeup changes in the liver of birds and mammals in ontogeny].

It was established by means of immunochemical titration that phosphorylase of the chickliver, beginning from the 8th day of embryogenesis, was represented by an isozyme, immunochemically identical or closely related to mammalian phosphorylase L. At the early stage of development (4th day) the chick embryo contained both isozyme L (ca. 60%) and isozyme, closely related to mammalian isozyme I. In the liver of 15 days old rat embryos the ratio of isozymes I and L equaled 1 : 2. It is suggested that the ratio of L and IL is close to 1 : 1. The isozyme IL is replaced for the isozyme L completely during the second ten day period of postnatal development.

Animals↗
PubMed1982

[Properties of the catalytically active fragment obtained by limited proteolysis of phosphorylase kinase].

The activation of phosphorylase kinase by limited proteolysis with subtilisin results in a formation of new enzyme forms differing in their molecular weights. Using gradient electrophoresis in polyacrylamide gel, it was shown that the high molecular weight fraction is made up of active fragments having different molecular weights. The low molecular weight fraction was found to contain only one active fragment with molecular weight of 80 000. Disc electrophoresis in polyacrylamide gel demonstrated that the active fragments of the high and low molecular weight fractions are not homogenous. The kinetic properties of the low molecular weight fragment were investigated. It was found that at pH 8.2 the native non-activated kinase and the catalytically active fragment have identical Km values for the substrates (phosphorylase B and MgATP); however, unlike the non-activated kinase, this fragment possesses a decreased sensitivity to Ca2+ and effectors (glycogen and glucose 6-phosphate) and has no optimum of activity within the pH range of 6.0-9.0.

Animals↗
PubMed1980

Changes in the glycogen phosphorylase isozyme pattern of AH130 during cell growth.

Changes in the glycogen phosphorylase isozyme patterns of AH130 and AH66F during cell growth were investigated immunochemically and electrophoretically. The content of the liver-like type increased as the cell growth in the ascites decreased, while the level of the fetal type (or prototype) remained approximately constant. The isozyme shift from the fetal type to liver-like type was more marked in subcutaneous solid AH130. In AH130 cells cultured in vitro under growth-inhibited conditions, the content of the hybrid between the above two types was increased, with increased amount of liver-like subunit. These results indicate that the phosphorylase isozyme patterns of Yoshida ascites hepatomas vary during cell growth under different conditions.

Animals↗
PubMed1981

[Changes in the glycogen phosphorylase isoenzyme spectrum in the rat liver and skeletal muscles in ontogeny].

Changes in the isozyme patterns of glycogen phosphorylase in the rat liver and skeletal muscles were studied during ontogenesis. The presence of a minor component both in the muscles and the liver during embryogenesis was shown by means of immunochemical titration and disc-electrophoresis. The possible nature of this component, its identity with the "embryonic" phosphorylase isozyme [1] and the hybrid form of IL enzyme [2,3].

Animals↗
PubMed1980

The role of pyridoxal 5'-phosphate in plant phosphorylase.

The removal of pyridoxal 5'-phosphate from potato phosphorylase [EC 2.4.1.1] was achieved by incubation in an acidic ammonium sulfate solution containing hydroxylamine. Potato apophosphorylase is catalytically inactive, but reactivated by incubation with pyridoxal phosphate. Upon titration, the degree of recovery of activity agreed well with the degree of incorporation of pyridoxal phosphate. Therefore, it can be concluded that pyridoxal phosphate in the plant enzyme is the cofactor required for the enzyme activity, as it has been shown to be in the animal enzyme. The reconstitution of the apoenzyme using six pyridoxal phosphate analogues modified at the 5' position indicates that the structural properties of the cofactor binding site is similar in potato and rabbit muscle phosphorylases. This suggests that the plant enzyme has also a substrate binding site neighboring with the 5'-phosphate moiety of the bound cofactor similar to that shown to exist in the animal enzyme. On the other hand, the analysis of the far-ultraviolet circular dichroism spectra of these two enzymes shows that the secondary structures are rather different from each other; the potato enzyme is estimated to have 16% alpha-helix and 37% beta-structure, and the muscle enzyme b 41% alpha-helix and 21% beta-structure. This indicates that the cofactor binding locus must have been more strictly conserved than other regions.

Ammonium Sulfate↗
PubMed1978

[Determination of glycogen phosphorylase B--a contribution to the enzymatic diagnosis of acute myocardial infarct].

On the basis of a small number of patients with the tentative diagnosis acute myocardial infarction the diagnostic valency of the glycogen phosphorylase was tested. A separation between severe stenocardia and myocardial infarction is possible. After simplification of the methodology the glycogen phosphorylase might substitute in higher specifity the creatinine kinase.

Aged↗
PubMed1995

Slow step in activation of muscle glycogen phosphorylase b by adenosine 5'-monophosphate.

The incubation of glycogen phosphorylase b from rabbit skeletal muscles with the allosteric activator (AMP) for 10-15 min has been found to result in an additional increase in the initial rate of the enzymatic reaction (v) as compared with the corresponding value of v measured by the initiation of the enzymatic reaction by the addition of a mixture of glucose 1-phosphate and AMP (0.02 M HEPES, pH 6.8; 37 degrees C). Glycogen with molecular mass of 270.10(6) daltons was used in the kinetic experiments. It is assumed that the enhancement of the rate of the phosphorylase b reaction is due to association of the enzyme molecules adsorbed to a glycogen particle resulting in an increase in the affinity of the enzyme for glycogen.

Adenosine Monophosphate↗
PubMed1995

Adrenaline stimulated glycogen breakdown in rat epitrochlearis muscles: fibre type specificity and relation to phosphorylase transformation.

The effect of adrenaline on glycogen breakdown in different skeletal muscle fibre types was investigated in the epitrochlearis muscle in vitro. Histochemical studies showed that adrenaline stimulated glycogen breakdown in all three major fibre types, with a higher absolute glycogen breakdown in type IIB fibres compared to type IIA and type I fibres. In biochemical studies we found that the glycogenolytic rate decreased during prolonged incubation with adrenaline, although the percentage phosphorylase in the a form and the concentration of glucose-6-phosphate (G-6-P) remained high. In the dose response studies we found an EC50 of 2.2 x 10(-8) M adrenaline for adrenaline stimulated glycogen breakdown, with an EC50 of 2.0 x 10(-7) M for adrenaline stimulated accumulation of G-6-P, excluding G-6-P as the key inhibitor of phosphorylase a activity.

Animals↗
PubMed1995

Enhancement of 5-fluoro-2'-deoxyuridine antitumor efficacy by the uridine phosphorylase inhibitor 5-(benzyloxybenzyl)barbituric acid acyclonucleoside.

5-(Benzyloxybenzyl)barbituric acid acyclonucleoside (BBBA) was recently synthesized as a potent and specific inhibitor of uridine phosphorylase (EC 2.4.2.3), the enzyme responsible for the catabolism of 5-fluoro-2'-deoxyuridine (FdUrd) in many types of tumors that are deficient or have little thymidine phosphorylase (EC 2.4.2.4) activity. The effect of BBBA on modulating the antitumor efficacy of FdUrd was evaluated in vitro, against the human colon carcinomas DLD-1 and HCT-15 grown in culture, and in vivo, against DLD-1 grown as xenografts in anti-thymocyte serum immunosuppressed mice. The concentrations of FdUrd that produced 50% growth inhibition after a 3-h exposure were 88 and 340 nM for HCT-15 and DLD-1, respectively. BBBA alone, at all concentrations tested, had no significant effect on the growth of DLD-1 and HCT-15 in culture. However, BBBA at 5, 10, 20, and 40 nM potentiated (P < 0.05) the cytotoxicity of FdUrd (340 nM; 3 h) against DLD-1 in culture by 20, 33, 55, and 63%, respectively. Similarly, BBBA at 10 and 20 nM potentiated the cytotoxicity of FdUrd (88 nM; 3 h) against HCT-15 in culture by 37 and 45%, respectively. In soft agar, BBBA (10 nM) also enhanced the cytocidal effect of FdUrd (10 and 32 nM) against DLD-1 by 41 and 55%, respectively, and against HCT-15 by 6 and 31%, respectively. Increasing BBBA dose to 20 nM enhanced further the FdUrd (10 and 32 nM) cytotoxicity against DLD-1 by 76 and 77%, respectively, and HCT-15 by 31 and 48%, respectively. BBBA also potentiated the chemotherapeutic efficacy of FdUrd in anti-thymocyte serum immunosuppressed mice bearing DLD-1 xenografts with no apparent host toxicity. At a low tumor burden (2.5 x 10(6) cells/mouse), 2 days treatment with FdUrd alone (50 mg/kg/day x 2) did not result in significant reduction in tumor volume. Coadministration of BBBA at 5 and 10 mg/kg/day x 2 did not potentiate the efficacy of FdUrd over that achieved by FdUrd alone, but it significantly reduced the tumor volume by 27 and 32%, respectively, when compared with untreated controls. FdUrd alone at 150 mg/kg/day x 2 reduced the tumor volume by 29%. This reduction in tumor volume was enhanced 1.8-fold by coadministration of BBBA (10 mg/kg/day x 2). At a higher tumor burden (5 x 10(6) cells/mouse) and 4 days treatment, BBBA at 10 and 30 mg/kg/day x 4 reduced further the tumor volume produced by FdUrd alone (200 mg/kg/day x 4) by 1.2- and 1.4-fold, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗