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PubMed1988

The ATP-binding site in gamma subunit of phosphorylase kinase.

To reveal the structure of the ATP-binding site(s) in rabbit muscle phosphorylase kinase, we modified the enzyme with adenosine polyphosphopyridoxals. Adenosine tri- and tetraphosphopyridoxals at micromolar concentrations effectively inactivated the enzyme in a time-dependent manner. Inactivation of the enzyme was accelerated by the addition of Ca2+ and Mg2+. Protection from inactivation was afforded by adenylyl beta,gamma-imidodiphosphate and ADP. In reversible inhibition kinetics, adenosine polyphosphopyridoxals as well as their reduced compounds (adenosine polyphosphopyridoxines) competed with ATP. These results suggest that adenosine polyphosphopyridoxals bind to the ATP-binding site(s) in phosphorylase kinase. When phosphorylase kinase was incubated with adenosine triphosphopyridoxal in the presence of Ca2+ and Mg2+, incorporation of the label into alpha, beta, and gamma subunits was observed. In the absence of both cations, larger amounts of the label were incorporated into all the subunits. Structural study on adenosine triphosphopyridoxal-modified sites in the gamma subunit (having a catalytic site) revealed that Lys-151 is mainly labeled. Based on the results of the present and other studies, it is suggested that the site around Lys-151 is involved in recognition of the substrate protein.

Adenosine Triphosphate↗
PubMed1987

Properties of the gamma subunit of phosphorylase kinase.

Enzymatic properties of the isolated, active gamma subunit of phosphorylase kinase were characterized. Kinetic parameters indicated that the gamma subunit binds the substrates MgATP and phosphorylase b as well as the holoenzyme with a Km (MgATP) of 98 microM and a Km (phosphorylase b) of 80 microM at pH 8.2, but maximal velocities are significantly lower than the holoenzyme's. Unlike the gamma-calmodulin complex, the gamma subunit activity is dependent on pH in the range of pH 6.2-9.0, with a ratio of activity at pH 6.8 to activity at pH 8.2 of 0.5-0.6. Calmodulin activates the gamma subunit more at low pH than at high pH. ADP inhibits the gamma subunit in a competitive manner with a Ki of 60 microM. Free Mg2+ stimulates gamma subunit activity 3.5-fold at both pH 6.8 and 8.2. MnATP is equivalent to MgATP as a substrate for the enzyme, but free Mn2+ inhibits gamma subunit activity. Several protein substrates of holophosphorylase kinase were found also to be phosphorylated by the gamma subunit. These included kappa-casein, myelin basic protein, the troponin complex, and troponin T alone. In the troponin complex, the proportion of 32P incorporated by the gamma subunit into troponin I compared with troponin T was not Ca2+ dependent, but with the holoenzyme, this proportion was changed greatly by Ca2+ concentration.

Animals↗
PubMed1986

The complete amino acid sequence of potato alpha-glucan phosphorylase.

The complete amino acid sequence of potato alpha-glucan phosphorylase has been determined. The monomer contains 916 amino acids with a molecular weight of 103,916. About one-fourth of the amino-terminal threonine is blocked by an acetyl group. Sequence comparison among phosphorylases from potato tuber, rabbit muscle, and Escherichia coli reveals the presence of a characteristic 78-residue insertion in the middle of the polypeptide chain of the potato enzyme. Except for the large inserted portion, 51 and 40% of the amino acids in the potato enzyme are identical with the rabbit muscle and E. coli enzymes, respectively. The regions relevant to the regulation of activity are completely different among the three enzymes, whereas those involved in the catalytic reaction are well conserved. The potato enzyme sequence is consistent with the tertiary structure of the rabbit muscle enzyme. The 78-residue insertion is located at the junction of the amino- and carboxyl-terminal domains on the molecular surface near the glycogen storage site. This insertion could account for the substrate discrimination of the potato enzyme. The molecular evolution of phosphorylase is discussed based on the presence of the large insertion of the potato enzyme.

Amino Acid Sequence↗
PubMedDate not supplied

[Separation of glycogen phosphorylase isoenzymes using electrophoresis in acetate cellulose].

A new electrophoretic procedure is developed for estimation of glycogen phosphorylase isoforms using acetate cellulose films. In the course of incubation of a mixture containing substrate the reaction was directed towards formation of glucose-I-phosphate, concentration of which was estimated in the coupled reactions based on equimolar increase in content of NADH. Content of the reduced nucleotide was evaluated by means of fluorescent densitometry. Three fractions of glycogen phosphorylase I, II and III were detected after electrophoretic separation of an extract from heart muscle homogenate. Only the isoenzyme I was found in blood serum of patients with myocardium infarction. In subsequent studies clinical validity of the procedure should be evaluated in order to prove the suitability of glycogen phosphorylase isoenzymes estimations for diagnostics of myocardial infarction.

Electrophoresis, Cellulose Acetate↗
PubMed1986

Cooperative behavior in the thiol oxidation of rabbit muscle glycogen phosphorylase in cysteamine/cystamine redox buffers.

Glycogen phosphorylase a and b are irreversibly inactivated by oxidation with the disulfide cystamine. The mechanism is complex and involves oxidation of at least two classes of sulfhydryl groups. The oxidation of one or more of the first class of 4 +/- 1 sulfhydryl groups is reversible, but the equilibrium constant for the oxidation is so unfavorable (1 X 10(-4)) that the micromolar concentrations of cysteamine released stoichiometrically with enzyme oxidation are sufficient to prevent complete oxidation even in the presence of 100 mM cystamine. The rapid phase of inactivation of phosphorylase b, which is first order in cystamine (k = 2.9 +/- 0.3 M-1 min-1), is followed by the oxidation of 5 +/- 1 groups in an irreversible process that is second order in cystamine concentration (k = 3.9 +/- M-2 min-1). Similar behavior is observed for phosphorylase a, although the behavior is complicated by association/dissociation equilibrium. The second-order dependence of the rate of irreversible inactivation on cystamine concentration is interpreted in terms of a "cooperative" model in which a rapidly reversible thermodynamically unfavorable equilibrium oxidation of one or more sulfhydryl groups must precede the irreversible oxidation of one or more additional sulfhydryl groups. The thiol/disulfide oxidation equilibrium constant for the initial reversible reaction is estimated to be at least 10(4) less favorable than that for the reversible oxidation of phosphofructokinase.

Adenosine Monophosphate↗
PubMed1987

The three-dimensional structure of acarbose bound to glycogen phosphorylase.

Acarbose, a pseudotetrasaccharide with a conduritol ring at the nonreducing terminus, is a naturally occurring inhibitor of amylases. It is shown here to be an inhibitor of glycogen phosphorylase and to bind more tightly to the enzyme than the equivalent malto-oligosaccharide substrate. X-ray crystallographic studies of the acarbose-phosphorylase a complex in the presence of glucose and caffeine reveal the structure of acarbose as bound to the storage site of phosphorylase. The acarbose binds in an orientation such that the conduritol ring makes no protein contacts. As with malto-oligosaccharides bound at this site, the observed conformation of acarbose is stabilized by O-2-O-3' hydrogen bonding and is similar to, but not identical with, that predicted by hard-sphere exo-anomeric effect calculations and justified by 1H nuclear magnetic resonance studies (Bock, K., and Pedersen, H. (1984) Carbohydr. Res. 132, 142-149). Intramolecular O2-O3' hydrogen bonds appear to play an important role in stabilizing the conformation observed in these studies, even for those residues closely associated with the protein.

Acarbose↗
PubMed1986

Stimulation of glycogen phosphorylase kinase by phospholipids.

The acidic phospholipids phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-biphosphate (PIP2) and the neutral phospholipid lysophosphatidylcholine (LPC) were found to stimulate (3 to 8-fold) the activity of nonactivated rabbit skeletal muscle phosphorylase kinase at pH 6.8, without significantly affecting the activity at pH 8.2. In this respect, phosphatidylcholine and phosphatidylethanolamine were ineffective, while the anionic detergent sodium dodecyl sulfate (SDS) and the anionic steroid dehydroisoandrosterone sulfate (DIAS) were able to mimic the action of phospholipids. SDS was also found to be a very efficient activator of the autophosphorylation of phosphorylase kinase (20-fold activation at 200 microM). The activating effect of phospholipids largely depends on the size of lipid vesicles, which is connected with the procedure of their preparation. These results suggest that phosphorylase kinase belongs to the class of Ca2+-dependent enzymes, which are sensitive to stimulation by calmodulin, limited proteolysis and anionic amphiphiles.

Animals↗
PubMed1985

Intravenous thiobarbital anaesthesia for determination of liver glycogen phosphorylase activity in rats subjected to various forms of stress.

Glycogen phosphorylase activity was determined in rat livers obtained by laparotomy in thiobarbital (Inactin, PROMONTA) anaesthesia induced by injection of the drug via a polyethylene catheter into a jugular vein as described previously [Németh et al. 1983a]. Intact rats and animals exposed to open field stress without or after i.p. injection of the alpha blocker phentolamine (Regitine, CIBA; 20 mg kg-1, 90 min before stress) or the beta blocker propranolol (Inderal, ICI; 2 mg kg-1, 30 min before stress) were studied. In stressed animals a net increase of phosphorylase activity was observed. This response was abolished after alpha blockade, while after beta blockade it was potentiated. In a further series on animals subjected to the same type of anaesthesia, the activating effect of immobilization stress on phosphorylase activity was confirmed in both fed and fasted animals, the response of the latter issuing from a lower initial level but being of the same extent as in the fed rats.

Animals↗
PubMed1985

[Enhancement of expression of Escherichia coli uridine phosphorylase gene as a result of duplication].

The thymine-requiring (thy) Escherichia coli strains normally convert thymine to thymidine through the action of thymidine phosphorylase, an enzyme, whose synthesis is directed by tpp gene of the deo operon. Selection for efficient thymine utilization in the thy mutants carrying a deletion in the tpp gene allows to obtain clones with constitutive synthesis of an alternative enzyme, uridine phosphorylase coded by the udp gene. Such clones are usually formed, due to "promoter-up" constitutive udpP mutants. Further selection for increased expression of the udp gene was performed on the background of udpP1 and udpP18 "promoter-up" constitutive mutants isolated previously. Several lines of evidence obtained by using transposon insertions suggest that the increased synthesis of uridine phosphorylase in some newly isolated mutants is due to duplications of the udp gene. In two mutants the adjacent metE gene is involved in duplications simultaneously with the udp gene.

Cloning, Molecular↗
PubMed1986

Analyses of phosphorylase kinase by transmission and scanning transmission electron microscopy.

Under conventional electron microscopy negatively stained phosphorylase kinase exhibits a bilobal structure resembling two bridged opposing parentheses. In this predominant particle orientation, usually only one bridge is observed; however, in many particles two bridges can be seen. Scanning transmission electron microscopy of unstained phosphorylase kinase shows very similar structures, with a particle mass equivalent to that of the hexadecameric holoenzyme. Partial digestion of the enzyme with chymotrypsin, which preferentially hydrolyzes the alpha-subunits, causes no significant changes in the structure; however, when both the alpha and beta subunits are degraded by trypsin, single lobed particles appear, i.e. the connecting bridges are missing. Mass analysis of scanning transmission electron microscopy images of trypsinized enzyme indicates that the protease does, in fact, split the particle into halves. Transmission electron microscopy of an alpha gamma delta complex isolated after incubation of the holoenzyme with LiBr shows only small particles approximately one-fourth the size of the holoenzyme. Thus, integrity of the beta subunit may be necessary in order for the two lobes of phosphorylase kinase to be bridged. These data also indicate that the subunits are arranged as a bridged dimer of octamers 2 (alpha 2 beta 2 gamma 2 delta 2).

Animals↗
PubMed1985

Adenosine 5'-diphosphate as an allosteric effector of phosphorylase kinase from rabbit skeletal muscle.

Equilibrium binding and activity studies indicate that adenosine 5'-diphosphate binds to phosphorylase kinase with high affinity at a site, or sites, distinct from the catalytic site. Equilibrium dialysis at pH 6.8 and 8.2, with and without Mg2+, and with phosphorylated and nonphosphorylated enzyme preparations revealed approximately 8 ADP binding sites per alpha 4 beta 4 gamma 4 delta 4 hexadecamer, with Kd values ranging from 0.26 to 17 microM. Decreasing the pH from 8.2 to 6.8 or removing the Mg2+ enhanced the affinity for ADP. At pH 6.8, ADP stimulated the phosphorylase conversion and autophosphorylation activities of the nonactivated enzyme. Analogs of ADP with modifications at the 2'-, 3'-, and 5'-positions allowed determination of structural requirements for the stimulation of activity. ADP seems to alter the conformation of the beta subunit because addition of the nucleotide inhibits its dephosphorylation by phosphoprotein phosphatase and its chemical cross-linking by 1,5-difluoro-2,4-dinitrobenzene. The binding affinities and effects of ADP suggest that it may function physiologically as an allosteric effector of phosphorylase kinase.

Adenosine Diphosphate↗
PubMed1984

Evidence for the role of phosphorylase kinase, protein kinase C, and other Ca2+-sensitive protein kinases in the response of hepatocytes to angiotensin II and vasopressin.

Angiotensin II, catecholamines, and vasopressin can stimulate the phosphorylation of 10 hepatic cytosolic proteins via a Ca2+-linked, cyclic AMP-independent mechanism. To explore the role of known Ca2+-sensitive protein kinases in this response, [32P]PO4(3-)-labeled hepatocytes were stimulated with various agonists, the cytoplasmic proteins were separated on two-dimensional gels, and the resulting autoradiographs were computer analyzed. The role of phosphorylase kinase was examined using hepatocytes from gsd/gsd rats which are deficient in this enzyme. The phosphorylation state of phosphorylase was not increased by glucagon, angiotensin II, or vasopressin in hepatocytes from the gsd/gsd animals. The phosphorylation state of all other substrates was changed by glucagon or the Ca2+-linked hormones to the same extent in gsd/gsd hepatocytes as in normal Wistar controls, suggesting that phosphorylase kinase plays a restricted role in the hormone response. The role of the Ca2+- and phospholipid-sensitive protein kinase (protein kinase C) was examined by stimulating hepatocytes with phorbol esters which are thought to activate protein kinase C by substituting for diacylglycerol. Phorbol esters increased the phosphorylation state of 3 of the 10 substrates affected by angiotensin II or vasopressin, but did not stimulate Ca2+ fluxes in hepatocytes. Treatment of hepatocytes with the Ca2+ ionophore A23187 mimicked the effect of the Ca2+-linked hormones on the phosphorylation of the other 7 substrates. The results demonstrate that at least three Ca2+-sensitive protein kinases are involved in the response of hepatocytes to Ca2+-linked hormones. Since these kinases can be activated independently by phorbol esters or A23187, the results imply that hormones such as vasopressin generate two intracellular messengers, diacylglycerol and Ca2+ ion.

Angiotensin II↗
PubMed1984

Potentiation of 5-fluoro-2'-deoxyuridine antineoplastic activity by the uridine phosphorylase inhibitors benzylacyclouridine and benzyloxybenzylacyclouridine.

At a nontoxic dose (50 microM), the two potent uridine phosphorylase inhibitors, benzylacyclouridine and benzyloxybenzylacyclouridine (BBAU), potentiated 5-fluoro-2'-deoxyuridine (FdUrd) growth inhibition of human pancreatic carcinoma (DAN) and, to a lesser extent, human lung carcinoma (LX-1) cells in culture. BBAU was more effective than benzylacyclouridine. BBAU (50 microM) enhanced the cytocidal effect of FdUrd (1 microM, 3 hr) on DAN grown on soft agar from 75 to 88%. In antithymocyte serum-immunosuppressed mice bearing DAN, the mean tumor weight in animals treated with FdUrd (50 mg/kg/day for 2 days) was 11% less than that of untreated controls. When BBAU (10 mg/kg/day for 2 days) was coadministered, the mean tumor weight at Day 10 was 78% less than untreated controls, with no apparent host toxicity, clearly demonstrating the potentiation of the antitumor effects of FdUrd by BBAU. The fact that DAN responded better than LX-1 to benzylacyclouridine and BBAU could be due, in part, to the lower relative activity of thymidine phosphorylase to uridine phosphorylase in DAN compared to LX-1. The activities of other enzymes involved in FdUrd metabolism, thymidine kinase, uridine kinase, orotate phosphoribosyltransferase, 5'-nucleotidase, and dihydrouracil dehydrogenase, did not differ between the two cell lines.

Animals↗
PubMed1980

Interaction between alpha and beta adrenergic receptors and cholinergic receptors in isolated perfused rat heart: effects on cAMP-protein kinase and phosphorylase.

The ability of acetylcholine to antagonize catecholamine-induced activation of myocardial cyclic AMP dependent protein kinase and glycogen phosphorylase activity was assessed using isolated perfused rat hearts. Perfused hearts were treated with either saline, epinephrine, epinephrine plus phentolamine or isoproterenol. After 1 minute of infusion of the indicated drug a second infusion containing acetylcholine was started. After an additional minute hearts were frozen and analyzed for cyclic nucleotide content and enzyme activity. In the presence of the alpha receptor blocking agent, phentolamine, epinephrine is a more effective activator of protein kinase than in its absence. Under these conditions the antagonistic action of acetylcholine on protein kinase activation is more pronounced. In the presence of epinephrine plus phentolamine or in the presence of isoproterenol the antagonistic action of acetylcholine on phosphorylase activity can be accounted for by a reduction in cyclic AMP-protein kinase. This same action of acetylcholine on epinephrine-stimulated phosphorylase in the aabsence of phentolamine, however, cannot be totally accounted for by a reduction in cyclic AMP content or in protein kinase activity.

Acetylcholine↗
PubMed1983

Adenosine deaminase and purine nucleoside phosphorylase activities in peripheral blood cells of patients with neoplastic diseases. I. Bronchogenic carcinoma.

By means of sensitive and specific radiochromatographic methods the activities of adenosine deaminase and purine nucleoside phosphorylase in peripheral blood cells of patients with bronchogenic carcinoma were estimated. It has been shown that purine nucleoside phosphorylase activity was two- to three-fold higher in lymphocytes of patients with epidermoid as well as other types of bronchogenic carcinoma in comparison with a group of healthy individuals. Adenosine deaminase activity in lymphocytes was elevated only in the group of patient with nonepidermoid carcinoma. The authors suggest that especially the estimation of purine phosphorylase activity in lymphocytes would be a useful laboratory test in the study of immune responsiveness of patients with bronchogenic carcinoma.

Adenosine Deaminase↗
PubMed1978

[Inhibition of the phosphorylase kinase activity by ATP analogs and their binding to the enzyme subunits].

The interaction between phosphorylase kinase (EC 2.7.1.38), isolated from rabbit skeletal muscles, and the ATP analogs with the modified triphosphate fragment: adenosine-5'-chloromethane pyrophosphonate (1), adenosine-5'-chloroethyl phosphate (2), adenosine-5'-bromethane pyrophosphonate (3), adenosine-5'-bromoethane phosphonate (4), adenosine-5'-chloroacetylaminomethane phosphonate (5), adenosine-5'-chloroacetylaminomethane pyrophosphonate (6) and adenosine-5'-chloromethane phosphonate (7), was studied. The compounds 1, 2 and 3 irreversibly inhibit the enzyme activity. In the presence of ATP the rate of inactivation is decreased. The radioactive compounds 1, 2 and 3 are stoicheometrically incorporated into the beta- and gamma-subunits of phosphorylase kinase. A correlation is shown to exist between the degree of the beta-subunit modification by compound 1 and the enzyme inactivation. The compounds 4, 5 and 6 inhibit the enzyme reversibly: in the presence of ATP complete protection of the enzyme activity is observed. The compound 7 does not affect the kinase activity; however, it binds itself to the beta-subunit of the enzyme. The binding of analogs 1 and 7 to the beta-subunit occurs at different sites. The data obtained are indicative of the catalytic role of the beta-subunit of phosphorylase kinase.

Adenosine Triphosphate↗
PubMed1983

Histochemical investigations on phosphorylase, branching enzyme and glycogen in guinea pig livers in experimental anaphylactic and histaminic shock.

The investigations have been carried out on 116 guinea pigs divided in three groups: the control (first group), the experimental group with animals after acute anaphylactic shock (second group), the animals after histaminic shock (third group). The animals of the experimental (second) group were sensitized with 25% egg white suspension in 0.9% NaCl applied subcutaneously. The same animals were exposed to the action of the antigen in aerosol (second group). The healthy animals were exposed to the action of 1% solution of dihydrochloride histamine (third group). In acute anaphylactic shock a decrease of histoenzymatic activity of phosphorylase A and branching enzyme in liver parenchyma was observed. It has been concluded that in anaphylactic shock there occurred disturbances in the function of the phosphorylase A--branching enzyme system. In histaminic shock the phosphorylase reaction becomes intensified in numerous liver cells. This is possible because the exogenic histamine may lead to the activation of the enzymatic system under studies.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1980

Characterization of purine nucleoside phosphorylase from human granulocytes and its metabolism of deoxyribonucleosides.

Purine nucleoside phosphorylase (EC 2.4.2.1) has been purified 715-fold from human granulocytes by formycin B affinity chromatography. The purified enzyme has a subunit molecular weight of 32,800 +/- 1,300 and an estimated native molecular weight of 102,000, which indicates a trimeric subunit structure. The purified enzyme migrates as a single band upon denaturing and nondenaturing polyacrylamide gel electrophoresis. Its amino acid composition has been determined. Isoelectric focusing of the purified enzyme produces three protein bands with isoelectric pH values of 5.8 to 6.1. The purified enzyme catalyzes the phosphorolysis of inosine, deoxyinosine, guanosine, and deoxyguanosine with apparent Km values of 0.21 mM, 0.32 mM, 0.21 mM, and 0.24 mM, respectively. The ribonucleosides and deoxyribonucleosides of adenine and the pyrimidines are not substrates. There are no major differences in metabolism of ribonucleosides and deoxyribonucleosides. Purine nucleoside phosphorylase activity is inhibited by formycin B in a competitive manner and by adenine in a mixed noncompetitive manner. Antiserum to the purified protein has been prepared and is useful in the immunochemical assay of phosphorylase over the range 10 to 100 ng of protein.

Amino Acids↗