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PubMed1992

Comparative studies on the properties of glycogen phosphorylases from the foot muscles of two benthic bivalves, Sunetta scripta and Villorita cyprenoides, having different habitats.

Glycogen phosphorylase (1,4-alpha-D-glucan:orthophosphate-alpha-D-glucosyl transferase, EC 2.4.1.1) was partially purified from two bivalves found in different habitats, viz. Villorita cyprenoides, an estuarine bivalve, and Sunetta scripta, a marine bivalve, and their properties compared with other animal phosphorylases. While the kinetic mechanism was same as that of phosphorylases from other animal sources, it differed in the control mechanism from other phosphorylases. The observed differences support the earlier finding that the control mechanism adopted by different animals is dependent on the evolutionary status and energy needs.

Animals↗
PubMed1991

Identification of a tissue-specific regulatory element within the human muscle glycogen phosphorylase gene.

Northern blot analysis showed that human muscle glycogen phosphorylase is developmentally regulated in human adult and fetal skeletal muscle. Furthermore, muscle phosphorylase mRNA expression is temporally regulated in the C2C12 mouse muscle cell line. To define regulatory elements that control expression of the human muscle glycogen phosphorylase gene, the structure of the 5' end of the gene was determined, and 1,129 base pairs of the 5'-flanking region were subcloned and sequenced. Primer extension, RNase protection, and S1 nuclease protection experiments mapped the transcription start site to 76 base pairs upstream from the starting methionine. Sequential deletions of the 5'-flanking region were tested for the ability to activate chloramphenicol acetyltransferase (CAT) expression in fused or proliferating C2C12 cells. Inclusion of the 43 base pairs between -612 and -570 led to a 9-fold increase in CAT activity in fused myotubes. No increase was observed in proliferating myoblasts. This region contains a 10-base pair sequence, CTCCAAAAGG, at -592, which is also repeated at -252. Mutation of the sequence at -592 results in a 50% decrease in CAT activity compared with the amount of CAT activity observed with the normal or a control mutation. These results indicate that a regulatory element is found within -612 to -570 of the 5'-flanking DNA of the human muscle glycogen phosphorylase gene which activates transcription only in differentiated muscle cells.

Animals↗
PubMed1990

[Studies on 5-FU concentration and thymidine phosphorylase activity in tissues of patients with colorectal cancer after SF-SP administration].

5-fluorouracil (5-FU) concentrations in peripheral blood, portal blood, normal and cancer tissues were evaluated in 26 patients with colorectal cancer after SF-SP administration (800 mg/day for 10 days). Thymidine phosphorylase activity in cancer tissues was also evaluated. 5-FU concentration in cancer tissues was significantly higher than that in other three specimens, and much higher than 0.05 microgram/g which was reported to be the minimum effective concentration. 5-FU concentration in portal blood was lower than MEC (0.05 microgram/ml). As for the relationship with the pathological features of cancer, the protruding lesions showed a higher 5-FU concentration than the ulcerative ones, and the lesions invaded only to submucosa or proper muscle showed a higher concentration than others. 5-FU concentration ratio in cancer tissues per in peripheral blood (T/B ratio) was related to thymidine phosphorylase activity. The higher was the thymidine phosphorylase activity, the greater T/B ratio. The results suggest that the tumor with higher thymidine phosphorylase activity might have a more pronounced anticancer efficacy of 5-FU.

Colorectal Neoplasms↗
PubMed1989

Skeletal muscle glycogen content, structure, and metabolism are normal in rats with hepatic glycogen phosphorylase kinase deficiency.

Skeletal muscle glycogen content and structure, and the activities of several enzymes of glycogen metabolism are reported for the hepatic glycogen phosphorylase b kinase deficient (gsd/gsd) rat. The skeletal muscle glycogen content of the fed gsd/gsd rat is 0.50 +/- 0.11% tissue wet weight, and after 40 hours of starvation this value is lowered 40% to 0.30 +/- 0.05% tissue wet weight. In contrast the gsd/gsd rat liver has an elevated glycogen content which remains high after starvation. The skeletal muscle phosphorylase b kinase, glycogen phosphorylase, glycogen synthase and acid alpha-glucosidase activities are 17.2 +/- 2.9 units/g tissue, 119.9 +/- 6.4 units/g tissue, 12.2 +/- 0.4 units/g tissue and 1.4 +/- 0.4 milliunits/g tissue, respectively, with approx. 20% of phosphorylase and approx. 24% of synthase in the active form (at rest). These enzyme activities resemble those of Wistar skeletal muscle, and again this contrasts with the situation in the liver where there are marked differences between the Wistar and the gsd/gsd rat. Fine structural analysis of the purified glycogen showed resemblance to other glycogens in branching pattern. Analysis of the molecular weight distribution of the purified glycogen indicated polydispersity with approx. 66% of the glycogen having a molecular weight of less than 250 X 10(6) daltons and approx. 25% greater than 500 X 10(6) daltons. This molecular weight distribution resembles those of purified Wistar liver and skeletal muscle glycogens and differs from that of the gsd/gsd liver glycogen which has an increased proportion of the low molecular weight material.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

On the release of glycogen phosphorylase from heart muscle: effect of substrate depletion, ischemia and of imipramine.

The release of glycogen phosphorylase from isolated, perfused rabbit heart was studied after substrate depletion and after global ischemia. The assayed efflux of the enzyme was found to be not only related to alterations in membrane integrity but also to the amount of glycogen in the injured heart tissue. The differential efflux profile of phosphorylase as a constituent of the sarcoplasmic reticulum-glycogenolysis complex in cardiac cells in comparison to cytosolic creatine kinase was found to be more pronounced employing K+-arrested, hypothermically perfused hearts exposed to imipramine (0.4 to 0.6 mM), known for altering specifically membrane integrity. Under these conditions only a rise in the release of creatine kinase occurs, whereas both glycogen content and the efflux of phosphorylase remains uneffected. It is suggested that the metabolical dependence of phosphorylase efflux from the injured heart muscle is of importance for its high sensitivity being a marker of acute heart infarction.

Animals↗
PubMed1989

[Regulation of the activity of rabbit skeletal muscle phosphorylase kinase by glycogen].

The effects of glycogen on the non-activated and activated forms of phosphorylase kinase were studied. It was found that in the presence of glycogen the activity of non-activated kinase at pH 6.8 and 8.2 and that of the activated (in the course of phosphorylation) form are enhanced. The degree of activation depends on glycogen concentration. At saturating concentrations, this enzyme activity increases 2-3-fold; the enzyme affinity for the protein substrate, phosphorylase b, also shows an increase. The polysaccharide has no effect on the activity of phosphorylase kinase stimulated by limited proteolysis. In the presence of glycogen, the rate of autocatalytic phosphorylation of the enzyme is increased. Glycogen stabilizes the enzyme activity upon dilution. The experimental results suggest that the polysaccharide directly affects the phosphorylase kinase molecule. The maximal binding was shown to occur at the enzyme/polysaccharide ratio of 1:10 (w/w) in the presence of Ca2+ and Mg2+.

Animals↗
PubMed1987

Two types of phosphorylase from etiolated soybean cotyledons.

Two types of phosphorylase [EC 2.4.1.1] from the etiolated soybean (Glycine max) cotyledons were separated by column chromatography on DEAE-Sephacel and further purified to apparent homogeneities. Molecular weights of the subunits were 100,000 and 113,000 for phosphorylases I and II, respectively. The native enzymes I and II were a dimer (200,000) and tetramer (450,000), respectively. Electrophoretic analysis by the Hedrick and Smith method indicated that the two phosphorylases were distinct proteins with no correlation. The apparent Km values for glucose 1-phosphate, glycogen, and maltoheptaose of enzyme I were 4.00 mM, 0.18 mg/ml, and 10.3 mM, respectively, while those values of enzyme II were 5.43 mM, 23.8 mg/ml, and 0.30 mM, respectively. The relative activity of enzyme I increased with increasing chain length of the substrate glucans, and waxy maize amylopectin was the best substrate among the 11 saccharides examined. For enzyme II, maltooligosaccharides with degree of polymerization (DP) 5-7 were the better substrates than amylose (DP 38) and glycogen. These results indicated that the soybean phosphorylases I and II have similar properties to a cytoplasmic and chloroplastic type of plant leaf enzyme, respectively.

Chromatography, DEAE-Cellulose↗
PubMed1985

Asymptomatic decreased activities of hepatic glucose-6-phosphatase and glycogen phosphorylase in a number of children with chronic liver disease.

The evaluation of hepatic degradation of glycogen in patients with different chronic liver diseases was carried out on the basis of: a) specific activities of hepatic enzymes involved in catabolism of glycogen; b) level of glycogen in liver biopsies; c) concentration of glucose and cAMP in serum after the intravenous administration of glucagon. In 13 out of 35 patients investigated the activity of glucose-6-phosphatase was decreased to 14-50% of the control value. In the livers of 3 patients glycogen phosphorylase activity was decreased to 10% of the control value. In patients with the significantly low activities of hepatic glucose-6-phosphatase and phosphorylase a, however, normal catabolism of glycogen in the liver was observed, neither hypoglycemia nor abnormal glycogen storage in liver biopsies nor abnormal response to glucagon being found. In the group of patients with decreased and normal activities of glucose-6-phosphatase and phosphorylase a, biochemical parameters in the serum (i.e. markers of liver damage) were not detectable. Possible causes of the selective and asymptomatic decrease in the activities of glucose-6-phosphatase and phosphorylase a are discussed.

Adolescent↗
PubMed1987

Purification and properties of polycation-stimulated phosphorylase phosphatases from rabbit skeletal muscle.

Four types of polycation-stimulated (PCS) phosphorylase phosphatases have been isolated from rabbit skeletal muscle. They are called PCSH (390 kDa), PCSM (250 kDa), and PCSL (200 kDa) phosphatase according to the apparent molecular weight of the native enzymes in gel filtration. Two forms of PCSH phosphatase could be separated by Mono Q fast protein liquid chromatography: PCSH1 and PCSH2. In the absence of polycations, the specific activities of the PCSH1, PCSH2, PCSM, and PCSL phosphatase were 400, 680, 600, and 3000 units/mg, respectively, using phosphorylase a as a substrate. They all contain a 62-65- and a 35-kDa subunit, the latter being the catalytic subunit. In addition PCSH1 phosphatase contains a 55-kDa subunit and the PCSM phosphatase a 72-75-kDa subunit in a substoichiometric ratio. All the PCS phosphatases are insensitive to Ca2+ calmodulin, inhibitor-1, and modulator protein. They display a high specificity for the alpha-subunit of phosphorylase kinase and a broad substrate specificity. The PCSH1 and PCSH2 phosphatases, but not the catalytic subunit (PCSC phosphatase), show a high degree of specificity for the deinhibitor protein. During the purification the phosphorylase to inhibitor-1 phosphatase activity ratio (10:1) remained constant for the PCSH and PCSL enzymes but decreased for the PCSM phosphatase. The stimulation observed with low concentrations of polycations is enzyme directed. The different enzyme forms show a characteristic concentration optimum and degree of stimulation. At higher concentrations, polycations become inhibitory and a time-dependent deactivation of the phosphatases is observed.

Animals↗
PubMed1986

[The presence of glycogen synthase in phosphorylase kinase preparations isolated from rabbit skeletal muscles].

Phosphorylase kinase isolated from rabbit skeletal muscle contains a protein whose molecular mass as determined by polyacrylamide gel electrophoresis is 571 000 Da. The protein was found to possess a higher affinity for glycogen as compared to phosphorylase kinase and phosphorylase. The protein separated from kinase by chromatography on a DEAE-cellulose column produced during SDS electrophoresis one protein band corresponding to Mr of 95 200 Da. The above properties of the protein and the glycogen synthetase activity revealed in the presence of glucose-6-phosphate suggest that phosphorylase kinase preparations contain a hexameric form of glycogen synthetase.

Animals↗
PubMed1987

Synergistic inhibition of polyamine synthesis and growth by difluoromethylornithine plus methylthioadenosine in methylthioadenosine phosphorylase-deficient murine lymphoma cells.

The antiproliferative effects of the ornithine decarboxylase inhibitor alpha-difluoromethylornithine (DFMO) are limited by the inability of the compound to deplete completely cellular polyamine pools. 5'-Deoxy-5'-methylthioadenosine (MeSAdo), the purine end product of the polyamine biosynthetic pathway, is an inhibitor of spermine and spermidine synthesis. Furthermore, a substantial number of human tumors are deficient in MeSAdo phosphorylase, and cannot degrade MeSAdo. It therefore seemed possible that DFMO and MeSAdo could interact synergistically to inhibit polyamine synthesis in MeSAdo phosphorylase-deficient malignant cells. To test this hypothesis, we have analyzed the effects of DFMO, in combination with MeSAdo, on polyamine synthesis and growth in a MeSAdo phosphorylase-deficient murine lymphoma cell line (R1.1-H), and a MeSAdo resistant mutant (R1.1-H3). Cultivation of the R1.1-H3 cells in medium containing 250 microM DFMO and 500 microM MeSAdo caused profound depletion of putrescine, spermidine, and spermine, and the accumulation of both decarboxylated S-adenosylmethionine and its acetylated derivative to levels that exceeded by nearly 3-fold the total cellular content of S-adenosylmethionine. Similarly, DFMO sensitized the lymphoma cells to the growth inhibitory effects of MeSAdo. Supplementation of the medium with putrescine, spermidine, or spermine partially protected R1.1-H3 cells from the DFMO-MeSAdo drug combination. It is conceivable that MeSAdo, or related nucleosides, may potentiate the cytostatic effects of DFMO toward MeSAdo phosphorylase-deficient tumors.

Adenosine↗
PubMed1987

McArdle's & Hers' diseases: glycogen phosphorylase transcriptional expression in human tissues.

We have cloned the cDNA encoding human liver glycogen phosphorylase (glycogenosis type VI) from a fetal brain cDNA library. Liver(L) and muscle(M) phosphorylase cDNA probes were used to determine the relative abundance of mRNA encoding the L- and M-isozymes of phosphorylase in human fetal and adult tissues. The transcript encoding the M-isozyme is 3.4 kb; the L-isozyme transcript is 3.3 kb. Transcriptional expression of the L-isozyme in human and primate tissues was found to differ from the isozyme's reported tissue specificity in non-primate mammals. Furthermore, using degenerate oligonucleotide probes to two different coding regions of M-phosphorylase, a novel 4.1-kb transcript was demonstrated to be present in human fetal and adult brain.

Amino Acid Sequence↗
PubMed1986

[Glycogen phosphorylase of skeletal muscles].

A review is given on the affinity modification of pyridoxal phosphate and AMP-binding sites as well as on the chemical modification of essential amino acid residues of phosphorylase (histidine residue of the substrate-binding site and cysteine residue of the coenzyme-binding site). The role of allosteric effectors (AMP and glucose-6-phosphate) and functionally important centers of the protein in conformational transitions of rabbit muscle phosphorylase b is discussed. The kinetic properties of rabbit and bovine muscle phosphorylase are compared. Bovine muscle phosphorylase is shown to be a partly phosphorylated form of the enzyme. Some peculiarities of the pH-dependence of kinetic behaviour of the hybrid form of the bovine muscle enzyme are discussed.

Animals↗
PubMed1987

Phosphorylase kinase conformers. Detection by proteases.

A variety of proteases have been evaluated as potential structural and conformational probes of nonphosphorylated and phosphorylated phosphorylase kinase. In general, the enzyme's alpha subunit is rapidly degraded, followed in most cases by hydrolysis of the beta subunit; the gamma subunit is resistant to most proteases. Trypsin clearly distinguishes between the nonactivated and activated conformers of phosphorylase kinase, in that the beta subunit in phosphorylated enzyme, as opposed to nonphosphorylated enzyme, is markedly protected from tryptic attack. In contrast, only a small difference in the rates of proteolysis of the alpha subunit in phosphorylated and nonphosphorylated enzyme is seen, even when a protease is used that is highly selective for the alpha subunit, such as chymotrypsin or endoproteinase Arg C. Incubation of nonphosphorylated phosphorylase kinase with either Mg2+ or Ca2+, which are activating cations, also protects the beta subunit from tryptic hydrolysis, whereas Mn2+, which inhibits the kinase activity, has little effect on proteolysis. The allosteric activator ADP also causes the beta subunit to become refractory to trypsin and mimics the effects of phosphorylation. Similar effector-induced conformational changes in the beta subunit are also observed with enzyme in which the alpha subunit has previously been selectively destroyed. These data indicate that activation of phosphorylase kinase by dissimilar mechanisms is associated with a conformational change in the enzyme's beta subunit that is detectable by trypsin and confirm earlier studies from this laboratory employing a chemical cross-linker as a conformational probe for activated and nonactivated conformers of the enzyme (Fitzgerald, T. J., and Carlson, G. M. (1984) J. Biol. Chem. 259, 3266-3274).

Adenosine Diphosphate↗
PubMed1987

Diurnal variation in glycogen phosphorylase activity in rat liver. A quantitative histochemical study.

The diurnal variations of the glycogen content and of glycogen phosphorylase activity in periportal and pericentral areas of rat liver parenchyma have been analyzed in periodic acid Schiff (PAS)-stained cryostat sections using quantitative microdensitometry. Glycogen content and phosphorylase activity were always higher in periportal areas than in pericentral areas throughout the daily cycle. The glycogen content was highest at the end of the active period during darkness and lowest at the end of the resting period. Phosphorylase activity appeared to be inversely correlated with the glycogen content in both areas. It is concluded that the glycogen content is regulated by phosphorylase activity, which may be due to local cAMP concentration.

Animals↗
PubMed1986

Amino acid sequence of cyanogen bromide fragments of potato phosphorylase.

Amino acid sequence analysis of the cyanogen bromide peptides of potato alpha-glucan phosphorylase was undertaken for comparison with rabbit muscle glycogen phosphorylase and for elucidation of the structural bases for the differences in the catalytic and regulatory properties between the animal and plant enzymes. The potato enzyme was carboxymethylated and cleaved with cyanogen bromide. The 17 distinct fragments produced were isolated by a combination of gel filtration, sulfopropyl ion exchange chromatography, and high performance liquid chromatography. The molecular weights of these fragments are distributed in a range of 300 to 30,000. Fragment CI has a blocked amino terminus, and has the same amino acid sequence as CII, which has been assigned as the amino-terminal fragment of potato phosphorylase. The blocking group was deduced to be an acetyl group from the results of fast atom bombardment mass spectrometry of an amino-terminal pentapeptide. This paper describes the sequence determination of all the cyanogen bromide fragments of potato phosphorylase. The complete structure is presented in the following paper (Nakano, K., and Fukui, T. (1986) J. Biol. Chem. 261, 8230-8236).

Animals↗
PubMed1977

[Escherichia coli K-12 mutants capable of catabolizing purine nucleosides in the absence of purine nucleoside phosphorylase].

Strains of Escherichia coli K-12 defective in purine nucleoside phosphorylase (pup gene) formed on the medium with inosine as the source of carbon and energy phenotypical reversions for the ability of utilizing inosine as source of carbon or purines. The phenotypical suppression of the purine nucleoside phosphorylase deficiency is the result of the mutations (called pnd), which are mapped on the chromosome of E. coli beyond the region of the structural pup-gene location and have phenotypic manifestation distinct from that of pup+ allele: a) pnd mutants divide into some groups for the ability of utilizing several purine nucleosides, including xantosine that cannot be metabolized by pnd+ strains of E. coli; b) pnd mutations do not restore the ability of purine auxotrophs (pur) defective in purine nucleoside phosphorylase (pup) and adenine phosphoribosyltransferase (apt) to grow on the medium with adenine as the sole source of purines. Cell-free extracts of pnd mutants fail to degrade the guanine nucleosides in the absence of phosphate or arsenate ions. These data (and also the ability of pnd mutants to utilize both purine ribonucleosides and deoxyribonucleosides) seem to indicate that the activities induced by pnd mutations are phosphorylase activities.

Chromosome Mapping↗
PubMed1984

Chronic maternal ethanol administration in the rat decreases the stimulation by (-) epinephrine of glycogen phosphorylase a in the livers of the progeny during development.

Previous results from this laboratory have shown that the progeny of alcoholic rats have diminished alpha 1-adrenergic receptors in the hepatic plasma membranes. Since these receptors mediate epinephrine action on glycogen metabolism, it was decided to determine whether this change might affect the activation of glycogen phosphorylase a in the livers of the alcoholic progeny. Pregnant female rats were divided into two groups of which one received a Metrecal-ethanol liquid diet throughout pregnancy and lactation. The pair-fed control group received a liquid sucrose-Metrecal diet over the same period. Phosphorylase a activity was determined in liver slices from the progeny during postnatal development. The basal hepatic phosphorylase a activity was identical between the control and experimental groups at 5, 15 and 25 days of age. Both epinephrine and phenylephrine were superior enzyme activators than was isoproterenol. Stimulation with epinephrine (10 microM) demonstrated a significantly diminished capacity of the enzyme in the alcoholic liver to be activated by the hormone. In every instance, the livers from 5, 15 and 25 day old pups from alcoholic mothers displayed diminished epinephrine-stimulated phosphorylase a activity of about 30%, compared with the controls.

Alcoholism↗