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PubMed1990

Phosphorylase-cross-reactive antibodies evoked by streptococcal M protein.

Rabbit antisera evoked by type 5 streptococcal M protein (M5) were screened by enzyme-linked immunosorbent assay (ELISA) for immunological cross-reactivity with purified rabbit muscle phosphorylases a and b. Of 10 pep M5 antisera tested, 3 showed significant cross-reactivity with both forms of the enzyme. ELISA inhibition studies using one of the pep M5 antisera showed that all of the phosphorylase b antibodies were inhibited by pep M5, the immunogen, and phosphorylase b, the ELISA antigen. All of the antibodies were also inhibited by pep M6 and pep M19, but not by pep M24, indicating that the cross-reactive epitopes were shared by multiple serotypes of M protein. Western blot (immunoblot) analyses showed that pep M5 antisera reacted strongly with the subunit of phosphorylase b. In addition, purified phosphorylase partially inhibited the binding of pep M5 antibodies to a 95-kilodalton protein of human myocardium. One of the three cross-reactive pep M5 antisera inhibited the enzymatic activity of phosphorylase a in a dose-related fashion, reaching a maximum inhibition of 75%. The enzymatic activity in the presence of antibody was totally restored when the antiserum was first incubated with pep M5.

Animals↗

PubMed1987

Distinction between substrate- and enzyme-directed effects of modifiers of rabbit liver phosphorylase a phosphatases.

The natural substrate (phosphorylase a) and two alternative ones (phosphorylated histone and a tetradecapeptide consisting of residues 5-18 of rabbit skeletal muscle phosphorylase a) were used to distinguish the modes of action of some physiologically important effectors of four different molecular forms of rabbit liver phosphorlase a phosphatases. In general, glucose, caffeine, AMP, ADP, Pi, and glucose-1-P showed substrate-directed effects for the holophosphatase forms, since they usually did not affect the activity on histone phosphate and, with one slight exception (Pi), never affected the activity on the tetradecapeptide phosphate. ADP, Pi, and glucose-1-P did affect directly the relative mass (Mr) 35,000 phosphatase, in addition to an inhibition mediated via phosphorylase a. ATP exerted both substrate- and enzyme-directed effects for the Mr 35,000 phosphatase and phosphatases 1 and 2A2, but only a substrate-directed effect for phosphatase 2A1, suggesting that the gamma-subunit of the type 2 phosphatases may prevent ATP binding to the phosphatase. Mg2+ showed substrate-directed effects for phosphatases 1, 2A1, and 2A2, and an additional enzyme-directed effect for the Mr 35,000 phosphatase form. Furthermore, Mg2+ could not abolish ATP inhibition of the tetradecapeptide phosphatase activity, but significantly overcame ATP inhibition of the phosphorylase a phosphatase activity, thus suggesting that its ability to reverse the ATP effect is by a substrate-directed mechanism. The substrate-directed effects seen for the different ligands on the different phosphatase forms strongly indicate the significance of this form of control in the regulation of phosphorylase a phosphatase activities and may serve to narrow the otherwise broad substrate specificities of the major phosphorylase a phosphatase activities in mammalian tissues: phosphatases 1 and 2A.

Animals↗

PubMed1984

Phosphorylase activation hypersensitivity in hearts of diabetic rats.

A hypersensitivity of glycogen phosphorylase activation by epinephrine and glucagon has been demonstrated in isolated perfused working and non-working hearts from diabetic rats. Accumulation of tissue cAMP and activation of cAMP-dependent protein kinase in response to epinephrine and glucagon were no greater and usually less in hearts of diabetic than of normal rats. Insulin deficiency was not associated with greater changes in epinephrine-induced activation of glycogen phosphorylase kinase than that observed in normal hearts. Perfusion of hearts with subphysiological concentrations of calcium (0.83 mM) partially reversed the diabetes-related hypersensitivity of phosphorylase activation by epinephrine. The phosphorylase activation hypersensitivity to epinephrine was completely reversed by adrenalectomizing diabetic rats 5 days before heart perfusion, an effect potentially caused by steroid-induced changes in cardiac calcium metabolism. These data are consistent with the hypothesis that phosphorylase activation by phosphorylase kinase is allosterically increased in the diabetic due to a diabetes-related increase in free intracellular calcium concentrations.

Animals↗

PubMed1976

[Glycogen phosphorylase activity of full term immature human placentas].

Human placenta contains glycogen phosphorylase activity (E.C. 2.4.1.1) in two interconvertible forms, one is already active and the other is actibatable by 5' AMP. The human placenta contains also a sufficient amount of endogenous phosphorylase b kinase and phosphorylase a phosphatase, to allow this interconversion. The phosphorylase activity is higher in immature placentas than in full-term organs, as in the latter the inactive phosphorylase is only partially revealed by 5' AMP as in other steroidogenic organs. By contrast, in immature placentas the inactive glycogen phosphorylase is completely revealed by this nucleotide.

Cyclic AMP↗

PubMedDate not supplied

[Phosphorylase activity and glycogen content in cerebral and myocardium tissues in rats of different age].

The phosphorylase activity (EC 2.4.1.1) and the glycogen content were determined in the brain and myocardium of adult and old rats. In the cortex and while substance of great cerebral hemispheres, the phosphorylase a activity decreases and the phosphorylase b activity increases, while the total enzyme activity remains unchanged with aging. In the myocardium, the activity of phosphorylase a increases, while that of phophorylase b decreases against a background of an insignificant decrease in the total phosphorylase activity. During aging, the glycogen content decreases sharply in the myocardium, whereas in the cerebral tissue it remains unchanged. Age peculiarities of the adrenaline effect upon myocardial phosphorylase activity are presented.

Aging↗

PubMed2005

Localization of thymidine phosphorylase in breast cancer tissue.

Thymidine phosphorylase levels are higher in some human cancer tissues than in adjacent normal tissue. However, the ultrastructural localization of thymidine phosphorylase in cancer tissue has been demonstrated only in advanced gastric and colorectal cancer. We investigated the localization of thymidine phosphorylase in breast cancer tissue by immunohistochemistry and its ultrastructural localization by immunoelectron microscopy. Surgically resected specimens from 30 cases of breast cancer were analyzed. Immunohistochemical analysis revealed that cancer cells were positive in 13 cases. However, there were 21 cases that showed thymidine phosphorylase-positive inflammatory cells in cancer tissue. Thymidine phosphorylase-positive staining was detected among both cancer cells and inflammatory cells in 11 cases. Thymidine phosphorylase was diffusely positive in the cytoplasm of cancer cells and specifically positive in mitochondria of neutrophils and specific cytoplasmic granules of macrophages in cancer tissue by immunoelectron microscopy. These findings suggest that thymidine phosphorylase is produced by macrophages and is present in mitochondria of neutrophils and cytoplasmic granules of cancer cells.

5'-Nucleotidase↗

PubMed1997

Efficient downstream processing of maltodextrin phosphorylase from Escherichia coli and stabilization of the enzyme by immobilization onto hydroxyapatite.

Downstream processing by biospecific chromatography of maltodextrin phosphorylase from Escherichia coli, overexpressed in E. coli, was substantially improved by a novel approach using ceramic hydroxyapatite. Wild-type and a less active mutant enzyme were purified from crude bacterial cell extracts in one efficient separation step that yielded phosphorylase in purity > 95% in at least 90% recoveries. At pH 6.9 and 25 degrees C, wild-type and mutant phosphorylases eluted from the hydroxyapatite column at a phosphate concentration of 0.4 M whereas calcium ions failed to displace the enzymes. The dynamic capacity for phosphorylase binding in the presence of bulk proteins was approximately 3 mg enzyme ml-1 matrix. The interaction of E. coli phosphorylase with hydroxyapatite seems to be mediated by surface amino groups, so that the bound enzyme retained almost full catalytic activity. Compared to the soluble enzyme, immobilization onto hydroxyapatite resulted in a more than 30-fold stabilization of wild-type phosphorylase against thermal and proteolytic inactivation and thus could improve the operational stability of phosphorylase during conversion of polysaccharide to glucose 1-phosphate.

Chromatography↗

PubMed1991

Primary structure of sweet potato starch phosphorylase deduced from its cDNA sequence.

Sweet potato (Ipomoea batatas) starch phosphorylase cDNA clones were isolated by screening an expression library prepared from the young root poly(A)(+) RNA successively with an antiserum, a monoclonal antibody, and a specific oligonucleotide probe. One cDNA clone had 3292 nucleotide residues in which was contained an open reading frame coding for 955 amino acids. This sequence was compared with those of potato (916 residues plus 50-residue putative transit peptide) and rabbit muscle (841 residues) phosphorylases. The sweet potato phosphorylase has an overall structural feature highly homologous to that reported for potato phosphorylase, in conformity with the finding that they belong to the same class of plant phosphorylase. High divergencies of the two enzymes are found in the about 70 residue N-termini each including a putative transit peptide, and the midchain 78 residue insert typical of type I plant phosphorylase. We consider that the very high dissimilarity found in the midchain inserts is related to the difference in proteolytic lability of the two plant phosphorylases. Some structural features of the cDNA clone were also discussed.

Journal Article↗

PubMed1967

Thymidine and thymine incorporation into deoxyribonucleic acid: inhibition and repression by uridine of thymidine phosphorylase of Escherichia coli.

Thymidine is poorly incorporated into deoxyribonucleic acid (DNA) of Escherichia coli. Its incorporation is greatly increased by uridine, which acts in two ways. Primarily, uridine competitively inhibits thymidine phosphorylase (E.C.2.4.4), and thereby prevents the degradation of thymidine to thymine which is not incorporated into normally growing E. coli. Uridine also inhibits induction of the enzyme by thymidine. It prevents the actual inducer, probably a deoxyribose phosphate, from being formed rather than competing for a site on the repressor. The inhibition of thymidine phosphorylase by uridine also accounts for inhibition by uracil compounds of thymine incorporation into thymine-requiring mutants. Deoxyadenosine also increases the incorporation of thymidine, by competitively inhibiting thymidine phosphorylase. Deoxyadenosine induces the enzyme, in contrast to uridine. But this is offset by a transfer of deoxyribose from deoxyadenosine to thymine. Thus, deoxyadenosine permits incorporation of thymine into DNA, even in cells induced for thymidine phosphorylase. This incorporation of thymine in the presence of deoxyadenosine did not occur in a thymidine phosphorylase-negative mutant; thus, the utilization of thymine seems to proceed by way of thymidine phosphorylase, followed by thymidine kinase. These results are consistent with the data of others in suggesting that wild-type E. coli cells fail to utilize thymine because they lack a pool of deoxyribose phosphates, the latter being necessary for conversion of thymine to thymidine by thymidine phosphorylase.

DNA, Bacterial↗

PubMed1988

Glycogen phosphorylase 'b' in Dictyostelium: stability and endogenous phosphorylation.

The slime mold Dictyostelium discoideum has two forms of the enzyme glycogen phosphorylase. The inactive phosphorylase 'b' form requires 5' AMP for activity and is present in early development. The active phosphorylase 'a' form is 5' AMP independent and occurs during later development. We here show that the 92 kd 'b' enzyme subunit exists either as a singlet or a doublet upon SDS-PAGE, depending on the method of sample extraction. In the presence of exogenously added Mn2+ and ATP, the phosphorylase 'b' shows apparent conversion into a 5' AMP independent form as measured by enzyme activity. In addition, Mn2+ and ATP also support an in vitro phosphorylation of the 92 kd phosphorylase 'b' subunit. We also demonstrate phosphorylation of the 'b' enzyme subunit in vivo by 32-P incorporation into the enzyme protein. A protein kinase responsible for the observed in vitro phosphorylation of the phosphorylase 'b' subunit is characterized.

Adenosine Monophosphate↗

PubMed1988

Renaturation of phosphorylase kinase activity from sodium dodecyl sulfate-polyacrylamide gels.

Phosphorylase kinase activity is renatured and detected in situ following electrophoresis of the denatured holoenzyme in a sodium dodecyl sulfate-polyacrylamide gel containing phosphorylase b that has been included in the gel polymerization according to the method of R. L. Geahlen et al. [(1986) Anal. Biochem. 153, 151-158]. Among the enzyme's four subunits, only gamma is catalytically active. When extract of rabbit muscle is electrophoresed and renatured in a similar manner, the phosphorylase-conversion activity is also associated only with a protein band that comigrates with the gamma subunit of phosphorylase kinase. This suggests that the gamma subunit of phosphorylase kinase may be the sole activity in rabbit muscle responsible for the phosphorylation of phosphorylase b. In an alternative method for the renaturation of activity from conventional sodium dodecyl sulfate-polyacrylamide gels, the subunits of the enzyme are visualized using 2.5 M KCl, excised from the gel, and eluted by diffusion into buffer containing sodium dodecyl sulfate, which is subsequently removed by acetone precipitation of the eluted subunits. Catalytic activity is recovered when the acetone precipitate of the extracted gamma subunit is dissolved in 6 M guanidine hydrochloride and diluted 50-fold into an activity assay. Inclusion of eluted alpha and beta subunits in the assay inhibits the activity of the gamma subunit, which supports our previous finding that the alpha and/or beta subunits suppress the activity of the catalytic gamma subunit [H. K. Paudel and G. M. Carlson (1987) J. Biol. Chem. 262, 11912-11915].

Adenosine Triphosphate↗

PubMed1980

Influence of fructose on the glycogen synthase and phosphorylase systems in rat liver.

Fructose and glucose, when administered as a single, large intravenous dose (500 mg/kg) produced opposite effects on key regulatory enzymes of glycogen metabolism in intact normal fed animals. Glucose rapidly stimulated glycogen synthase phosphatase activity and increased the proportion of glycogen synthase in the active (I) form as expected; fructose reduced synthase phosphatase activity and the proportion of synthase in the I form. Glucose also stimulated a reduction in the proportion of phosphorylase in the active (a) form, whereas fructose stimulated an increase in the proportion of phosphorylase in thea form. The effect of fructose was not mediated by an increase in cyclic adenylate (cAMP) concentration nor by a conversion of phosphorylase kinase b to phosphorylase kinase a. As expected, the concentration of ATP decreased significantly. The increase in proportion of phosphorylase in the a form may be due to stimulation of phosphorylase kinase b activity by a decrease in the intracellular ATP:Mg++ ratio or by increase in intracellular Ca++ concentration. The mechanism of the fructose-induced change in synthase phosphatase activity and in synthase I activity is unknown.

Animals↗

PubMed1995

Purification and properties of glycogen phosphorylase from the fat body of larval Manduca sexta.

Glycogen phosphorylase b has been purified to homogeneity from the fat body of larval Manduca sexta. The purification procedure involved ammonium sulfate precipitation, and chromatography of DEAE-cellulose, 5'-AMP-Sepharose and Q-Sepharose. The final product, which showed a single band on SDS-PAGE with a M(r) = 92,500, was purified 50-fold from the original homogenate in a yield of about 3%. The molecular mass of the native purified phosphorylase b was estimated to be 186,000 Da from gel filtration, suggesting that the native enzyme is a dimer. The apparent Km values for glycogen, phosphate and 5'-AMP were 1.4 mM, 82 mM and 1.1 mM, respectively. The enzyme had a pH optimum of 7.05, and was inhibited by ATP, ADP and glucose, but not by trehalose, even at high concentration. Conversion of phosphorylase b into the a form was achieved by incubation with rabbit phosphorylase kinase and Mg(2+)-ATP. The molecular mass of phosphorylase a was estimated to be 250,000 Da by gel filtration chromatography. The specific activity of the a form in the presence of 5'-AMP was 1.6-1.7-fold higher than the specific activity of the b form under the same conditions. Thus, 5'-AMP activates the a form by about 20%, whereas ATP has no effect on the phosphorylase a activity.

Animals↗

PubMed1984

A new type of glycogen storage disease caused by deficiency of cardiac phosphorylase kinase.

A five-month-old Japanese boy was found to have marked glycogen accumulation only in the heart. A survey of enzymes revealed normal activities of phosphorylase, cyclic AMP-dependent protein kinase, acid maltase and amylo-1,6-glucosidase. However, the heart had capacity of activating neither rabbit muscle phosphorylase b nor endogenous phosphorylase b, which was converted to active form only when supplemented rabbit muscle phosphorylase kinase. In contrast to the heart, activities of phosphorylase kinase were found within normal levels in other organ tissues so far tested. These findings indicate that the present case of the cardiac glycogenosis is caused by deficiency of cardiac phosphorylase kinase.

Animals↗

PubMed1992

Pitfalls in the histochemical demonstration of alpha-glucan phosphorylase activity in glycogen-depleted skeletal muscle fibres.

In the present communication, an investigation is described into the reliability of histochemical methods for the demonstration of alpha-glucan phosphorylase activity in glycogen-depleted skeletal muscle fibres. Human skeletal muscles with glycogen-depleted fibres from patients with diseases of the neuromuscular system and from subjects who had suffered from malignant hyperthermia were used for the study. The location of phosphorylase activity and glycogen was demonstrated with histochemical techniques. Biochemical techniques were used to assay the activity of phosphorylase and the content of glycogen. Biochemical determinations of phosphorylase activity did frequently not reveal significant differences between glycogen-depleted and non-depleted skeletal muscle fibres. In contrast, all histochemical methods investigated, showed little or no phosphorylase activity in the glycogen depleted fibres, indicating that none of the existing histochemical methods revealed reliable staining results in these fibres. Owing to the invalid staining results of the histochemical methods for glycogen-depleted muscle fibres, it is necessary that for metabolic studies a biochemical assay for phosphorylase activity is also to be performed.

Glycogen↗

PubMed1984

Does pyridoxal 5'-phosphate function in glycogen phosphorylase as an electrophilic or a general acid catalyst?

alpha-D-Glucose 1-diphosphate interacts with pyridoxal-reconstituted rabbit muscle phosphorylase b activated by AMP (AMP-S). Under these conditions, the glucose moiety of alpha-D-[14C]glucose 1-diphosphate is transferred to limit dextrin forming alpha(1----4) glycosidic bonds and simultaneously releasing pyrophosphate as shown by 31P NMR spectroscopy. Thus, specific structural requirements invoked to explain the reactions of pyridoxal(5')diphospho(1)-alpha-D-glucose need not to be assumed in the case of the reactions of alpha-D-glucose 1-diphosphate. Dianions isomorphous to phosphate activate pyridoxal phosphorylase regardless of their pK values while the same anions, when bound covalently to pyridoxal, are inactive. Thus, anions bound noncovalently to pyridoxal phosphorylase act differently than anions linked covalently to pyridoxal, such as the 5'-phosphate group of pyridoxal 5'-phosphate, which is postulated to be part of a proton donor-acceptor pathway. The reaction of 2,6-anhydro-1-deoxy-D-gluco-hept-1-enitol (heptenitol) with phosphorylase yields, in the presence of orthophosphate as a glycosyl acceptor, 1-deoxy-D-gluco-heptulose 2-phosphate (heptulose-2-P). This sugar phosphate is unreactive but a potent competitive inhibitor for rabbit muscle phosphorylase b and potato phosphorylase with respect to alpha-D-glucose 1-phosphate: Ki = 14 X 10(-6) M and 1.9 X 10(-6) M, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2003

Mutants of phosphorylase a altered in recognition by protein phosphatase-1.

To develop our knowledge of specificity determinants for protein phosphatase-1, mutants of phosphorylase b have been converted to phosphorylase a and examined for their efficacy as substrates for protein phosphatase-1. Mutants focused on the N-terminal primary sequence surrounding the phosphoserine (R16A, R16E, and I13G) and at a site that interacts with the phosphoserine in phosphorylase a, (R69K and R69E). The success achieved studying protein kinase substrate specificity with peptide substrates has not extended to protein phosphatases. Protein phosphatases are believed to recognize higher order structure in substrates in addition to the primary sequence surrounding the phosphoserine or threonine. Peptide studies with protein phosphatase-1 have revealed a preference for basic residues N-terminal to the phosphoserine. Arginine 16 in phosphorylase a may be a positive determinant. In this work, protein phosphatase-1 preferred the positive charge on arginine 16. R16A exhibited a similar K(m) but reduced V(max), and R16E had an increased K(m) and a decreased V(max) when compared to phosphorylase. I13G had a similar K(m) but an increased V(max). The R69 mutants were also dephosphorylated preferentially over phosphorylase a. The K(m) for R69K was unchanged but had a higher V(max). R69E exhibited the most changes, with a 4-fold increase in K(m) and a 10-fold increase in V(max). These results suggest that proper presentation of the phosphoserine can greatly affect the rate of dephosphorylation.

Amino Acid Substitution↗

PubMed1995

Identification of the substrate and pseudosubstrate binding sites of phosphorylase kinase gamma-subunit.

Using site-directed mutagenesis, we proposed that an autoinhibitory domain(s) is located at the C-terminal region (301-386) of the phosphorylase kinase gamma-subunit (Huang, C.-Y.F., Yuan C.-J., Livanova, N.B., and Graves, D.J. (1993) Mol. Cell. Biochem. 127/128, 7-18). Removal of the putative inhibitory domain(s) by truncation results in the generation of a constitutively active and calmodulin-independent form, gamma 1-300. To probe the structural basis of autoinhibition of gamma-subunit activity, two synthetic peptides, PhK13 (gamma 303-327) and PhK5 (gamma 343-367), corresponding to the two calmodulin-binding regions, were assayed for their ability to inhibit gamma 1-300. Competitive inhibition of gamma 1-300 by PhK13 was found versus phosphorylase b (Ki = 1.8 microM) and noncompetitive inhibition versus ATP. PhK5 showed noncompetitive inhibition with respect to both phosphorylase b and ATP. Calmodulin released the inhibition caused by both peptides. These results indicate that there are two distinct auto-inhibitory domains within the C terminus of the gamma-subunit and that these two domains overlap with the calmodulin-binding regions. Two mutant forms of gamma 1-300, E111K and E154R, were used to probe the enzyme-substrate-binding region using peptide substrate analogs corresponding to residues 9-18 of phosphorylase b (KRK11Q12ISVRGL). The data suggest that Glu111 interacts with the P-3 position of the substrate (Lys11) and Glu154 interacts with the P-2 site (Gln12). Both E111K and E154R were competitively inhibited with respect to phosphorylase b by PhK13, with 14- and 8-fold higher Ki values, respectively, than that observed with the wild-type enzyme. These data are consistent with a model for the regulation of the gamma-subunit of phosphorylase kinase in which PhK13 acts as a competitive pseudosubstrate that directly binds the substrate binding site of the gamma-subunit (Glu111 and Glu154).

Amino Acid Sequence↗