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PubMed1987

Trypanosoma cruzi adenine nucleoside phosphorylase. Purification and substrate specificity.

An adenine nucleoside phosphorylase has been partially purified from extracts of epimastigotes of the Peru strain of Trypanosoma cruzi, the causative agent of Chagas' disease. The purification procedure separated this enzyme from the three other nucleoside-cleaving enzymes found in extracts. The adenine nucleoside phosphorylase, which efficiently cleaved 5'-deoxy-5'-methylthioadenosine (MTA), had a particle weight of 68,000 and exhibited a broad pH optimum between pH 6 and 8. In addition to MTA, the purified enzyme cleaved and synthesized adenosine and 2'-deoxyadenosine with high efficiency. This contrasts to the enzyme from S-180 cells which has been reported to cleave adenosine poorly and not to cleave 2'-deoxyadenosine. Several observations suggested that the three substrates, MTA, adenosine and 2'-deoxyadenosine, use a common catalytic site: (a) all served as alternate-substrate inhibitors exhibiting mutually competitive inhibition with Ki values equivalent to their respective Km values, (b) 5'-chloroformycin A exhibited a competitive Ki value of 4 microM with each nucleoside substrate, and (c) the Km value of phosphate derived from initial velocity studies (180 +/- 20 microM) was independent of the nucleoside substrate. Substrate specificity studies in both the synthesis and cleavage direction indicated that the enzyme had a broad specificity for bases and nucleosides. For the synthesis of nucleosides, the enzyme demonstrated a preference for an amino group in the position equivalent to the 6 position of purine. Compounds containing a hydroxyl group in this position were not substrates. Although a hydrogen or methyl group could substitute for a 6-amino group, a marked decrease in substrate efficiency was observed with these compounds. Alterations in the purine ring led to decreases in the maximal velocity values as evidenced by the substrate or nonsubstrate properties of 1-, 3-, and 7-deazaadenine and 4-aminopyrazolo[3,4-d]pyrimidine. The Km values for 5-methylthioribose 1-phosphate, ribose 1-phosphate and 2'-deoxyribose 1-phosphate with adenine serving as acceptor were 21, 150 and 370 microM. For nucleoside cleavage, the T. cruzi enzyme catalyzed the phosphorolysis of a variety of 5'-substituted adenine-containing nucleosides including those possessing 5'-hydrogen-, hydroxyl-, halogeno-, alkylthio-, amino- and azido-moieties. Inclusion of an ionized group in the 5'-position, such as 5'-carboxy-5'-deoxyadenosine or AMP, precluded substrate activity. 3'-Deoxyadenosine, arabinosyladenine and alpha-adenosine did not serve as substrates.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenine↗
PubMed1989

Inhibition of uridine phosphorylase from Giardia lamblia by pyrimidine analogs.

Fifty-six pyrimidine analogs were tested as possible inhibitors of uridine phosphorylase from Giardia lamblia. Values of Ki were determined for eight of these which demonstrated an inhibition greater than 60% under the standard conditions of uridine at 1 mM (approximately 1.5 times the Km) and inhibitor at 1 mM. All were competitive with respect to uridine. The most effective inhibitors were uracil analogs substituted at the C-5 position with electron withdrawing groups (nitro groups or halogens). The inhibitory effect at the 5-position appeared to be further enhanced by substitution at the C-6 position with electron releasing groups. The order of effectiveness as inhibitors was 6-methyl-5-nitrouracil greater than 6-amino-5-nitrouracil greater than 5-benzylacyclouridine greater than 5-nitrouracil greater than 5-fluorouracil greater than 5-bromouracil greater than 6-benzyl-2-thiouracil greater than 1,3-dimethyluracil with Ki values of 10, 12, 44, 56, 119, 230, 190 and greater than 1000 microM, respectively. The compounds were also effective inhibitors of the thymidine phosphorylase activity of the enzyme. The effect of the more potent compounds on G. lamblia in in vitro culture are currently under investigation.

Animals↗
PubMed1993

Circadian rhythm of orotate phosphoribosyltransferase, pyrimidine nucleoside phosphorylases and dihydrouracil dehydrogenase in mouse liver. Possible relevance to chemotherapy with 5-fluoropyrimidines.

In female mice (30-35 g) maintained in standardized conditions of 12 hr light (0600-1800 hr) alternating with 12 hr darkness (1800-0600 hr), food and water ad lib., there was a 24-hr cycle change (P < 0.0001, Cosinor analysis) in the activity of hepatic orotate phosphoribosyltransferase (OPRTase; EC 2.4.2.10), uridine phosphorylase (UrdPase; EC 2.4.2.3), and dihydrouracil dehydrogenase (DHUDase; 1.3.1.2) but not thymidine phosphorylase (EC 2.4.2.4). The peaks of both OPRTase and UrdPase activities occurred in the activity span at around 18 and 15 hours after light onset (HALO) and the trough at 6 and 3 HALO, respectively, when samples were taken every 4 hr. Conversely, the peak of DHUDase occurred in the rest span at around 3 HALO and the trough at 15 HALO. The maximal enzyme activities (3146 +/- 172, 561 +/- 25, and 6.7 +/- 0.7 pmol/min/mg protein) was 210, 400 and 560% higher than the minimal activities (1507 +/- 172, 139 +/- 25, and 1.2 +/- 0.7 pmol/min/mg protein), for OPRTase UrdPase, and DHUDase, respectively. A circadian rhythm was also observed when the light-dark cycle was shifted (reverse cycle) so that the lights went on at 2200 hr and off at 1000 hr. Under the reverse cycle condition there was a corresponding shift in UrdPase and DHUDase activities with a period of 1 hr difference in the time of maximum and minimum enzyme activities. OPRTase, on the other hand, showed little change after 4 weeks of adaptation under the reverse light cycle. The circadian rhythm of these key enzymes of pyrimidine metabolism, the interrelationship of their activities, and their role in the regulation of uridine bioavailability could be of particular significance in modulating the therapeutic regimens with 5-fluorinated pyrimidines.

Animals↗
PubMed1993

Structure-activity relationship of nucleobase ligands of uridine phosphorylase from Toxoplasma gondii.

Seventy-nine nucleobase analogs were evaluated as potential inhibitors of Toxoplasma gondii uridine phosphorylase (UrdPase), and the apparent Ki (appKi) values for these compounds were determined. Based on the inhibition data, a structure-activity relationship for the binding of nucleobase analogs to the enzyme was formulated, using uracil as a reference compound. Two compounds were identified as very potent inhibitors of T. gondii UrdPase, 5-benzyloxybenzylbarbituric acid and 5-benzyloxybenzyluracil, which had appKi values of 0.32 and 2.5 microM, respectively. A comparison of the results from the present study, with similar studies on mammalian UrdPase and thymidine phosphorylase (dThdPase) (Niedzwicki et al., Biochem Pharmacol 32: 399-415, 1993) revealed that there are both similarities and differences between the catalytic site of T. gondii UrdPase and the catalytic sites of the mammalian enzymes with respect to binding of uracil analogs. One compound, 6-benzyl-2-thiouracil, was identified as a potent, specific inhibitor (appKi = 14 microM) of T. gondii UrdPase, relative to mammalian UrdPase and dThdPase.

Animals↗
PubMed1983

Organization of the mouse cerebral cortex: a histochemical study using glycogen phosphorylase.

The postmortem phosphorylase a activity in cryostat sections taken from the brains of mice killed without any prior period of maintained anaesthesia ('unanaesthetized' mice) was compared histochemically with the activity in sections taken from mice anaesthetized with pentobarbitone prior to decapitation ('anaesthetized' mice). This study provides evidence for the existence of two separate overlapping modular subdivisions of the somatosensory cortex. The modules observed in the 'unanaesthetized' mice were restricted to layer IV and corresponded to the hollows of the whisker barrels where the thalamic afferents terminate. In contrast, the modules observed in the 'anaesthetized' mice extended from layer I to layer V, and formed a mosaic of cylinders that was out of register with the whisker barrels. These cylinders may correspond to the terminal fields of corticocortical afferents. In a few of the 'unanaesthetized' mice bands of high phosphorylase a activity are evident in the visual areas 17 and 18. This suggests that in the mouse, as in higher mammals, the thalamic input to the visual cortex gives rise to a columnar organization. In the 'anaesthetized' mice the mosaic of modules observed in the somatosensory cortex is also present in the auditory and motor areas. The modules in both groups of mice have similar diameters of between 200 and 350 microns.

Animals↗
PubMed1995

Activation of glycogen phosphorylase by serotonin and 3,4-methylenedioxymethamphetamine in astroglial-rich primary cultures: involvement of the 5-HT2A receptor.

Neurotransmitters, neuropeptides, and ions regulate glycogen levels in the brain by modulating the activity of glycogen synthase (GSase) and glycogen phosphorylase (GPase). GPase is co-localized with glial fibrillary acidic protein (GFAP), an astroglia-specific marker, suggesting that glycogen is localized in astroglial cells. Additionally, functional serotonin (5-HT) receptors are found in both neurons and glia, and 5-HT is known to stimulate glycogenolysis. It is reported that 3,4-methylenedioxymethamphetamine (MDMA), a drug of abuse, stimulates the release and inhibits the reuptake of 5-HT, and selectively inhibits the activity of MAO-A. These biochemical consequences of MDMA lead to increased extra-cellular 5-HT levels. This study investigates the effects of MDMA(+) and serotonin (5-HT) on glycogen metabolism in the rat brain. A histochemical method was designed to visualize active glycogen phosphorylase (GPase) in an astroglial-rich primary culture. Serotonin activated GPase in a concentration-dependent manner (100 nM-100 microM). Maximal activation by 5-HT was achieved by 50 microM and resulted in a 167% increase in the number of reactive sites (P < 0.001). MDMA(+) (500 nM-50 microM) directly stimulated GPase activity with maximal activation induced by 5 microM, which caused a 70% increase in the number of reactive sites (P < 0.001). The 5-HT2 receptor agonist, 1-(2,5-dimethoxy-4-bromophenyl)-2-aminopropane (DOB), also displayed a concentration-dependent increase in the number of GPase reactive sites. Maximal stimulation by DOB occurred at 100 nM which increased the number of reactive sites by 166% (P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

3,4-Methylenedioxyamphetamine↗
PubMed1978

A micromethod for determining adenosine deaminase and purine nucleoside phosphorylase activity in cells from human peripheral blood.

A radiochromatographic method is described for measuring adenosine deaminase and purine nucleoside phosphorylase activity in cells from human peripheral blood. The respective substrates, [8-14C]adenosine or [8-14C]inosine, are converted either to inosine and hypoxanthine or hypoxanthine, respectively. A single simple and rapid chromatographic procedure is used to isolate the products of both reactions. The mean normal activity (nmol h-1mg-1) of ADA for erythrocytes is 63 +/- 24 (+/- 1 S.D.) for leukocytes, 750 +/- 280 and for lymphocytes, 2105 +/- 1170. Corresponding activities for purine nucleoside phosphorylase are 1850 +/- 490, 3665 +/- 1170 and 5890 +/- 2030. With the described methods a further patient with severe combined immuno-deficiency and adenosine deaminase deficiency has been identified.

Adenosine Deaminase↗
PubMed1985

The degradation of 5'-deoxy-5-fluorouridine by pyrimidine nucleoside phosphorylase in normal and cancer tissues.

We describe an assay procedure for the quantification of pyrimidine nucleoside phosphorylase, the enzyme thought to degrade the pro-drug 5'-deoxy-5-fluorouridine to 5-fluorouracil. The method is based on the known differences in the ultraviolet absorption spectra in alkaline medium between pyrimidines and their nucleosides. Analogous to the cleavage of uridine by uridine phosphorylase, the enzymatic degradation of 5'-deoxy-5-fluorouridine in the presence of inorganic phosphate yields 5-fluorouracil and ribose-1-phosphate. We have shown that the rate of phosphorolysis of this pyrimidine nucleoside could be readily measured by spectrophotometry. The method described is simple, specific for the 'pro-drug' as well as reproducible. We have also applied this method to define tissue enzyme activities in extracts of human tumours, normal tissues of the same organ and tissues of mice.

Animals↗
PubMed1988

A sensitive non-radioactive assay for pyrimidine nucleoside phosphorylase using reversed-phase high performance liquid chromatography.

A new sensitive assay for pyrimidine nucleoside phosphorylase using non-radioactive substrates is described. With the natural substrate uridine (UR) and the analog, 5'-deoxy-5-fluorouridine (5'dFUR) conditions have been optimized to measure the product formation with reversed-phase high-performance liquid chromatography. Using automated injection large series of samples may be analyzed. The assay for UR phosphorylase appeared to be comparable to existing methods with radiolabeled UR as substrate regarding sensitivity and linearity. The assay has been used to measure kinetic parameters for 5'dFUR and UR in two cell lines from intestinal origin.

Cell Line, Transformed↗
PubMed1978

Reserpine-induced supersensitivity to the inotropic and phosphorylase activating effects of glucagon in the perfused rat heart.

Glucagon (0.125--2.0 microgram) produced a dose-dependent increase in cyclic AMP, % phosphorylase a and force of contraction in the isolated perfused rat heart. Pretreatment of animals with reserpine (2.5 mg/kg, 24 h) resulted in an enhancement of the inotropic and phosphorylase activating effects of glucagon but the effect on cyclic AMP was not altered. It is suggested that reserpine-induced supersensitivity in the rat heart occurs at a point beyond the cyclic AMP step.

Animals↗
PubMed1982

Identification of a calmodulin-dependent glycogen synthase kinase in rabbit skeletal muscle, distinct from phosphorylase kinase.

A glycogen synthase kinase that is completely dependent on Ca2+ and calmodulin has been identified in mammalian skeletal muscle, and purified approximately 3000-fold by chromatography on phosphocellulose and calmodulin--Sepharose. The presence of 50 mM NaCl in the homogenisation buffer was critical for extraction of the enzyme. The calmodulin-dependent glycogen synthase kinase (app. Mr 850 000) is distinct from myosin light-chain kinase and phosphorylase kinase, but phosphorylates the same serine residue on glycogen synthase as phosphorylase kinase. The physiological role of the enzyme is discussed.

Animals↗
PubMed1987

Isolation and sequence analysis of a cDNA clone encoding the entire catalytic subunit of phosphorylase kinase.

Synthetic oligonucleotides have been used to isolate a 1.85 kb clone containing the full length coding sequence for the catalytic subunit of rabbit skeletal muscle phosphorylase kinase from a cDNA library constructed in lambda gt10. Sequence analysis of the clone predicted an amino acid sequence in agreement with a published primary structure. Inspection of the codon usage revealed a strong preference for G or C nucleotides at the third codon position as found for several other skeletal muscle proteins. This cDNA clone should facilitate identification of functional domains, including the calmodulin-binding site, and investigation of the molecular basis of X-linked phosphorylase kinase deficiencies.

Amino Acid Sequence↗
PubMed1989

Molecular cloning and sequencing of the glycogen phosphorylase gene from Escherichia coli.

The glgP gene, which codes for glycogen phosphorylase, was cloned from a genomic library of Escherichia coli. The nucleotide sequence of the glgP gene contained a single open reading frame encoding a protein consisting of 790 amino acid residues. The glgP gene product, a polypeptide of Mr 87,000, was confirmed by SDS-polyacrylamide gel electrophoresis. The deduced amino acid sequence showed that homology between glgP of E. coli and rabbit glgP, human glgP, potato glgP, and E. coli malP was 48.6, 48.6, 42.3, and 46.1%, respectively. Within this homologous region, the active site, glycogen storage site, and pyridoxal-5'-phosphate binding site are well conserved. The enzyme activity of glycogen phosphorylase increased after introduction on a multicopy of the glgP gene.

Amino Acid Sequence↗
PubMed1994

Curcumin is a non-competitive and selective inhibitor of phosphorylase kinase.

Recently, we reported that curcumin (diferuloylmethane) inhibits the growth of several different kinds of tumor cells. In order to investigate the mechanism of this inhibition, we examined the effects of curcumin on different protein kinases: highly purified protein kinase A (PkA), protein kinase C (PkC), protamine kinase (cPK), phosphorylase kinase (PhK), autophosphorylation-activated protein kinase (AK) and pp60c-src tyrosine kinase. While all kinases tested were inhibited by curcumin, only PhK was completely inhibited at relatively lower concentrations. At around 0.1 mM curcumin, PhK, pp60c-src, PkC, PkA, AK, and cPK were inhibited by 98%, 40%, 15%, 10%, 1%, and 0.5%, respectively. Lineweaver-Burk plot analysis indicated that curcumin is a non-competitive inhibitor of PhK with a Ki of 0.075 mM. Overall, our results indicate that curcumin is a potent and selective inhibitor of phosphorylase kinase, a key regulatory enzyme involved in the metabolism of glycogen. This has important implications for the anti-proliferative effects of curcumin.

Binding, Competitive↗
PubMed1996

Effects of temperature shifts on the activities of Neurospora crassa glycogen synthase, glycogen phosphorylase and trehalose-6-phosphate synthase.

Conidiospore germlings of Neurospora crassa submitted to a heat shock at 45 degrees C accumulate trehalose and degrade glycogen. The opposite occurs upon reincubation at a physiologic temperature (30 degrees C). These observations suggest a temperature-dependent mechanism for the preferential synthesis of one or the other sugar reserve. Here we show that concomitant with these shifts of temperature, occurred reversible changes in the activities of glycogen synthase and phosphorylase. Glycogen synthase was inactivated at 45 degrees C while phosphorylase was activated. The reverse was true when the cells were shifted back to 30 degrees C. Addition of cycloheximide did not prevent the reversible enzymatic changes, which remained stable after gel filtration. Apparently, the effects of temperature shifts occurred at the level of reversible covalent enzymatic modifications. Trehalose-6-phosphate synthase properties were also affected by temperature. For instance, the enzyme was less sensitive to in vitro inhibition by inorganic phosphate at 50 degrees C than at 30 degrees C. Fructose-6-phosphate partially relieved the inhibitory effect of phosphate at 30 degrees C but not at 50 degrees C. These effects of the assay temperature, inorganic phosphate, and fructose-6-phosphate, on trehalose-6-phosphate synthase activity, were more evident for crude extracts obtained from heat-shocked cells. Altogether, these results may contribute to explain the preferential accumulation of trehalose 45 degrees C, or that of glycogen at 30 degrees C.

Cycloheximide↗
PubMed1996

Activation of glycogen phosphorylase and glycogenolysis in rat skeletal muscle by AICAR--an activator of AMP-activated protein kinase.

We determined whether the cell permeable molecule AICAR, whose metabolite activates AMP-activated protein kinase (AMPK) in cells, affected glycogen metabolism in rat soleus muscle preparations in vitro. The basal and insulin-stimulated rates of radiochemical lactate formation, net lactate release and glycogen synthesis were determined. AICAR stimulated net lactate release (but not radiochemical lactate formation) only at a basal concentration of insulin. An increased rate of glycogenolysis was the likely cause of increased net lactate release as glycogen phosphorylase activity was significantly increased by AICAR. AICAR-stimulated net lactate release and phosphorylase activity were potently inhibited by insulin.

AMP-Activated Protein Kinases↗
PubMed1986

Epinephrine effect on glycogen phosphorylase activity in catfish liver and muscles.

In catfish, the percentage of the active (a) form of glycogen phosphorylase with respect to the total (a + b) form varied in control slices from about 90% to 65% and 20%, respectively for liver, red, and white muscles. Epinephrine added to the incubation medium of liver and white muscle slices caused a significant increase in the specific activity of phosphorylase a in liver and white muscle, but not in red muscle. In liver slices epinephrine showed its effect from the concentration of 3.5 X 10(-8) M. The increase of enzyme activity explains the lowering of the glycogen level in liver and white muscle induced by epinephrine.

Adenosine Monophosphate↗