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PubMed1976

Stimulation of glycogenolysis by beta adrenergic agonists in skeletal muscle of mice with the phosphorylase kinase deficiency mutation (I strain).

The mechanism by which beta adrenergic agonist stimulate glycogenolysis in intact skeletal muscle was investigated in mice with the phosphorylase kinase deficiency mutation (I strain). Although extracts of I strain diaphragm muscle had only 3.7% of the phosphorylase kinase activity found in extracts of the control strain (C57BL), incubation of I strain hemidiaphragms in Krebs-Ringer bicarbonate buffer with either isoproterenol or epinephrine resulted in a stimulation of the rate of glycogenolysis. In C57BL diaphragms, the EC50 values for isoproterenol and epinephrine were 2 and 14 nM, respectively. With I strain diaphragms, dl-isoproterenol or l-epinephrine stimulated glycogenolysis as a linear function of the log of the drug concentration with no apparent plateau of response up to concentrations of 30 to 40 mugM. For each 10-fold increase in drug concentration, isoproterenol and epinephrine stimulated glycogenolysis in I strain muscles an additional 0.37 to 0.42 mg/g/hr, a slope in the concentration-response relationship of 0.17 and 0.37, respectively, of that measured in C57BL diaphragms at concentrations around the EC50. The highest glycogenolytic response measured in I strain hemidiaphragms (at 40 mugM isoproterenol) was 80% of the maximal catecholamine-stimulated glycogenolysis in C57BL diaphragms. Both 4 nM and 4 mugM isoproterenol, in a concentration-dependent manner, stimulated phosphorylase b to a conversion in I and C57BL diaphragms and increased cyclic adenosine 3':5'-monophosphate (cyclic AMP) concentrations. The glycogenolytic response to 10.1 nM dl-isoproterenol in both I and C57BL diaphragms was blocked by 34 nM l-propranolol but not by 34 nM d-propranolol. The response to 4 mugM isoproterenol was enhanced by the cyclic nucleotide phosphodiesterase inhibitors papaverine (27 mugM) or dl-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724, 3 mugM). From the results of these studies, we conclude: 1) Catecholamines stimulate glycogenolysis in skeletal muscle of I mice, as in C57BL mice, by interacting with the beta adrenergic receptor, thereby increasing tissue cyclic AMP concentrations and stimulating phosphorylase b to a conversion. 2) alternative hypotheses for the mechanism of the catecholamine-stimulated decrease in glycogen concentration in I skeletal muscle-inhibition of glycogen synthesis, hyposia and 5'-AMP stimulation of phosphorylase b activity-have been ruled out. 3) the activity of the mutant phosphorylase kinase, although it is only 3.7% of that in extracts of C57BL muscle, is sufficient to produce phosphorylase b to a conversion and thereby account for the glycogenolytic response of I strain muscle to catecholamines.

4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone↗

PubMed1972

Role of the sarcoplasmic reticulum in glycogen metabolism. Binding of phosphorylase, phosphorylase kinase, and primer complexes to the sarcovesicles of rabbit skeletal muscle.

Sarcoplasmic vesicles and beta-glycogen particles 30-40 mmicro in diameter were isolated from perfused rabbit skeletal muscle by the differential precipitation-centrifugation method. This microsomal fraction was subjected to zonal centrifugation on buffered sucrose gradients, in a B XIV Anderson type rotor, for 15 hr at 45,000 rpm in order to separate the two cytoplasmic organelles. Zonal profiles of absorbance at 280 mmicro, proteins, glycogen, and enzymatic activities (phosphorylase b kinase, phosphorylase b, and glycogen synthetase) were performed. Whereas the entire synthetase activity was found combined with the glycogen particles, 39% of phosphorylase and 53% of phosphorylase b kinase activities, present in the microsomal fraction, were recovered in the purified vesicular fraction (d = 1.175). This latter fraction consists of vesicles, derived from the sarcoplasmic reticulum, and of small particles 10-20 mmicro in diameter attached to the outer surface of the membranes. These particles disappear after alpha-amylase treatment. Incubation of the sarcovesicular fraction with (14)C-labeled glucose-1-phosphate confirms the localization of a polysaccharide synthesis at the level of the membranes. "Flash activation" of phosphorylase b, i.e. Ca "activation" of phosphorylase kinase followed by a conversion of phosphorylase b into a, was demonstrated in the purified sarcovesicular fraction. Moreover, the active enzymatic sites were detected on the membranes by electron microscopy. The presence of binding sites between the membranes of the sarcoplasmic vesicles and a glycogen-enzyme complex suggests that this association plays a role in the glycogenolysis during muscle contraction.

Animals↗

PubMed1988

The alpha and beta subunits of phosphorylase kinase are homologous: cDNA cloning and primary structure of the beta subunit.

We have cloned cDNA molecules encoding the beta subunit of phosphorylase kinase (ATP:phosphorylase-b phosphotransferase; EC 2.7.1.38) from rabbit fast-twitch skeletal muscle and have determined the complete primary structure of the polypeptide by a combination of peptide and DNA sequencing. In the mature beta subunit, the initial methionine is replaced by an acetyl group. The subunit is composed of 1092 amino acids and has a calculated molecular mass of 125,205 Da. Alignment of its sequence with the alpha subunit of phosphorylase kinase reveals extensive regions of homology, but each molecule also possesses unique sequences. Two of the three phosphorylation sites known for the beta subunit and all seven phosphorylation sites known for the alpha subunit are located in these unique domains.

Amino Acid Sequence↗

PubMed1990

Localisation of the gene encoding the catalytic gamma subunit of phosphorylase kinase to human chromosome bands 7p12-q21.

Skeletal muscle phosphorylase kinase has the structure (alpha beta gamma delta)4 where the alpha and beta subunits are regulatory components, the gamma subunit possesses catalytic activity and the delta subunit is identical to the calcium binding protein calmodulin. A rabbit skeletal muscle cDNA for the gamma subunit has been used to map the human gene (PYKG1) to 7p12-q21, by analysis of somatic cell hybrids and in situ hybridisation. The data suggest that the skeletal muscle gamma subunit gene is located just above the centromere of chromosome 7, with further cross-hybridising sequences at 7q21 and 11p11-14. The liver gamma subunit is distinct and its mRNA does not cross-hybridize with the skeletal muscle gamma subunit cDNA. These results indicate that autosomal human phosphorylase kinase deficiencies affecting both liver and muscle are likely to be caused by a defect in the autosomally determined beta subunit, rather than the gamma subunit.

Blotting, Northern↗

PubMed1997

The structural effects of endogenous and exogenous Ca2+/calmodulin on phosphorylase kinase.

The activity of phosphorylase b kinase (PbK) is stimulated by Ca2+ ions, which act through its endogenous calmodulin subunit (delta), and further stimulated by the Ca2+-dependent binding of exogenous calmodulin (delta'). In contrast to their highly characterized effects on activity, little is known regarding the structural effects on the (alphabetagammadelta)4 PbK holoenzyme induced by Ca2+ and delta'/Ca2+. We have used mono- and bifunctional chemical modifiers as conformational probes to compare how the two effectors influence the structure of the catalytic gamma subunit and the interactions among all of the subunits. As determined by reductive methylation and carboxymethylation, Ca2+ increased the accessibility of the gamma subunit; it also increased the formation by phenylenedimaleimide of an alphagammagamma conjugate that is characteristic of activated conformations of PbK (Nadeau, O. W., Sacks, D. M., and Carlson, G. M. (1997) J. Biol. Chem. 272, 26196-26201); however, Ca2+ also had structural effects that were clearly distinct from other activators. Moreover, similar structural effects of Ca2+ were observed with PbK that had been activated by phosphorylation, consistent with the fact that such activation does not eliminate the catalytic dependence of the enzyme on Ca2+. Our results suggest tiers of conformational transitions in the activation of PbK, with the most fundamental being induced by Ca2+. Analysis of the various cross-linked conjugates formed in the presence of Ca2+ by o-phenylenedimaleimide or m-maleimidobenzoyl-N-hydroxysuccinimide ester showed that the binding of Ca2+ to the delta subunit triggers changes in the interactions among all subunits, including between protomers, indicating an extensive communication network throughout the PbK complex. Most of the structural effects of delta'/Ca2+ were qualitatively similar to, but quantitatively greater than, the effects of Ca2+ alone; but delta'/Ca2+ also had distinct effects, especially involving cross-linking of the delta subunit.

Animals↗

PubMedDate not supplied

[Phosphorylase kinase: mathematic modeling].

A mathematical model of the dynamic behavior of phosphorylase kinase was devised. Based on the results obtained, the function of this protein is discussed. It is suggested that phosphorylase kinase doses in a cAMP-dependent manner additional portions of glucoso-l-phosphate, which the muscle cell receives in response to contraction.

Calcium↗

PubMed1999

A recombinant form of the catalytic subunit of phosphorylase kinase that is soluble, monomeric, and includes key C-terminal residues.

Residues 302-326 of the catalytic (gamma) subunit of phosphorylase kinase (PhK) may comprise an autoinhibitory, pseudosubstrate domain that binds calmodulin. To study this, the cDNA corresponding to rabbit muscle PhKgamma was expressed using Escherichia coli. This yielded two stable, high-activity PhKgamma forms (35 and 42 kDa by SDS-PAGE) that were smaller than an authentic sample of rabbit muscle PhKgamma (45 kDa by SDS-PAGE). Each recombinant form was purified to homogeneity. The N-terminal sequence of the larger, 42-kDa form (pk42) matched that of the rabbit muscle enzyme. This suggested that pk42 consisted of PhKgamma residues 1-362, including the putative calmodulin-binding, autoinhibitory domain. Kinetic parameters obtained for pk42 were like those previously reported for the intact gamma subunit. This implied that the lack of 25 PhKgamma C-terminal residues did not affect phosphorylase kinase activity, but greatly improved enzyme stability. An additional 60 residues were removed from the C-terminus of pk42 using the protease m-calpain. This increased the kinase activity 1.5-fold. Consistent with this, the activity of a mutant PhKgamma that consisted of residues 1-300, denoted gamma1-300, was like that of the m-calpain-treated enzyme. Therefore, although the effect was small, some influence by the C-terminus of pk42 was noted. Moreover, when pk42 was incubated with ATP alone, a C-terminal threonine residue became phosphorylated. Although the influence of this autophosphorylation cannot be inferred from this data, it was evidence that the C-terminus accessed the enzyme's active site. Taken together, these data imply that pk42 will be useful to study phosphorylase kinase structure/activity relationships.

Adenosine Triphosphate↗

PubMed1991

Phosphorylase kinase activity in I/strain neonatal skeletal muscle with a deficiency in alpha/alpha' subunit mRNAs.

In the adult I/LnJ mouse skeletal muscle, phosphorylase kinase activity is 0.2% of that in normal. This deficiency results from a paucity of mRNA's for the phosphorylase kinase regulatory subunit- alpha and its isoform alpha'. However, in the I/LnJ neonatal skeletal muscle phosphorylase kinase activity is 20-25% of that in normal. During the first two months of development this activity decreases while in normal tissue it increases. The developmental differences in the magnitude of the I/LnJ deficiency indicate the possibility of stage specific mechanisms regulating the accumulation of alpha/alpha' mRNAs. To investigate this possibility, the abundance of alpha/alpha' mRNAs and of the catalytic subunit, gamma, mRNAs were compared by Northern Blot analysis. The results demonstrate that neonatal and adult I/LnJ skeletal muscle have a similar paucity of alpha/alpha' mRNAs whereas accumulation of gamma mRNAs is not significantly different from normal.

Aging↗

PubMed1981

The regulation of muscle phosphorylase kinase by calcium ions, calmodulin and troponin-C.

Although it has been believed for several years that calcium ions are the means by which glycogenolysis and muscle contraction are synchronized, it is only over the past three years that this concept has started to be placed on a firm molecular basis. It appears that the regulation of phosphorylase kinase in vivo is achieved through the interaction of the enzyme with the two calcium binding proteins, calmodulin and troponin-C, and that the relative importance of these proteins depends on the degree of phosphorylation of the enzyme (figure 3). In the dephosphorylated form of the enzyme, troponin-C rather than calmodulin is the dominant calcium dependent regulator providing an attractive mechanism of coupling glycogenolysis and muscle contraction, since the same calcium binding protein activates both processes. On the other hand, the phosphorylated form of the enzyme can hardly be activated at all by troponin-C, although it is still completely dependent on calcium ions. Calmodulin (the delta-subunit) is therefore the dominant calcium dependent regulator of phosphorylase kinase in its hormonally activated state. Recent work has demonstrated that phosphorylase kinase not only activates phosphorylase, but also phosphorylates glycogen synthase thereby decreasing its activity (45-49). The regulation of phosphorylase kinase by calcium ions may therefore also provide a mechanism for co-ordinating the rates of glycogenolysis and glycogen synthesis during muscle contraction.

Animals↗

PubMed1981

Interaction of phosphorylase kinase with the 2',3'-dialdehyde derivative of adenosine triphosphate. 1. Kinetics of inactivation.

The 2',3'-dialdehyde derivative of ATP (oATP) was found to be a valid affinity label for rabbit skeletal muscle phosphorylase kinase. Inactivation by oATP at pH 6.8 followed pseudo-first-order and saturation kinetics. An apparent Ki of approximately 6.7 microM was obtained in the presence of 0.6 mM Ca2+ plus 10 mM Mg2+. Protection against the rate of inactivation was provided by the natural substrate ATP. In addition, at pH 8.2, oATP could be used as a substrate to phosphorylate phosphorylase b, thus providing evidence that oATP can bind to the active site of phosphorylase kinase. Inactivation of phosphorylase kinase by oATP was sensitive to various effectors of the enzyme such as Ca2+, Mg2+, and pH. Ca2+ plus Mg2+ synergistically enhanced the rate of inactivation severalfold; each metal ion by itself had little effect on the rate of inactivation. This synergism was seen both at pH 6.8 and at pH 8.2; however, the rates of inactivation were much greater at pH 6.8. The enhancement of inactivation by Ca2+ plus Mg2+ was also more pronounced with activated than with nonactivated kinase.

Adenosine Triphosphate↗

PubMed1985

[Effect of ATP, ADP and magnesium ions on the activity of phosphorylase kinase from rabbit skeletal muscles].

The dependence of enzymatic activity of phosphorylase kinase on ATP and magnesium concentrations has been studied. The enzyme activity has been shown to be inhibited by the substrate surplus (Mg-ATP) but free Mg2+ stimulates the enzyme. At saturating concentrations of ATP the activating effect of Mg2+ is maximum at the Mg/ATP ratio of 6-10. The ADP inhibition action is characterized by an incompetitive type towards ATP. The apparent Ki value is equal to 0.2 mM. It is suggested that the specific ADP-binding site spatially removed from the active site has an importance for the phosphorylase kinase activity regulation.

Adenosine Diphosphate↗

PubMed1990

The quaternary structure of phosphorylase kinase as influenced by low concentrations of urea. Evidence suggesting a structural role for calmodulin.

Skeletal-muscle phosphorylase kinase is a hexadecameric oligomer composed of equivalent amounts of four different subunits, (alpha beta gamma delta)4. The delta-subunit, which is calmodulin, functions as an integral subunit of the oligomer, and the gamma-subunit is catalytic. To learn more about intersubunit contacts within the hexadecamer and about the roles of individual subunits, we induced partial dissociation of the holoenzyme with low concentrations of urea. In the absence of Ca2+ the quaternary structure of phosphorylase kinase is very sensitive to urea over a narrow concentration range. Gel-filtration chromatography in the presence of progressively increasing concentrations of urea indicates that between 1.15 M- and 1.35 M-urea the delta-subunit dissociates, allowing extensive formation of complexes larger than the native enzyme that contain equivalent amounts of alpha-, beta- and gamma-subunits. As the urea concentration is increased to 2 M and 3 M, nearly all of the enzyme aggregates to the heavy species devoid of delta-subunit. Addition of Ca2+, which is known to block dissociation of the delta-subunit [Shenolikar, Cohen, Cohen, Nairn & Perry (1979) Eur. J. Biochem. 100, 329-337], also blocks aggregation of the enzyme induced by the low concentrations of urea. These results suggest that in native phosphorylase kinase the delta-subunit, in addition to activating the catalytic subunit and conferring upon it Ca2(+)-sensitivity, may also serve a structural role in preventing aggregation of the alpha-, beta- and gamma-subunits, thus limiting to four the number of alpha beta gamma delta protomers that associate under standard conditions. In gel-filtration chromatography with urea a protein peak containing equivalent amounts of alpha- and gamma-subunits is also observed, as is a peak containing only beta-subunits. Increasing concentrations of urea have a biphasic effect on the activity of the holoenzyme, being stimulatory up to 1 M and then inhibitory. The concentration-dependence of urea in the inhibitory phase parallels its ability to induce dissociation of the delta-subunit.

Animals↗

PubMed1979

Mechanism of activation of glycogen phosphorylase by fructose in the liver. Stimulation of phosphorylase kinase related to the consumption of adenosine triphosphate.

1. A dose-dependent activation of phosphorylase and consumption of ATP was observed in isolated hepatocytes incubated in the presence of fructose; histone kinase and phosphorylase kinase activities were unchanged at doses of this sugar that were fully effective on phosphorylase. The activation of phosphorylase by fructose was also observed in cells incubated in a Ca2+-free medium as well as in the livers of rats in vivo. 2. In a liver high-speed supernatant, fructose, tagatose and sorbose stimulated the activity of phosphorylase kinase; this effect was dependent on the presence of K+ ions, which are required for the activity of fructokinase; it was accompanied by the transformation of ATP into ADP. In the presence of hexokinase, glucose also stimulated phosphorylase kinase, both in an Na+ or a K+ medium. 3. The activities of partially purified muscle or liver phosphorylase kinase were unchanged in the presence of fructose. 4. Some properties of liver phosphorylase kinase are described, including a high molecular weight and an inhibition at ATP/Mg ratios above 0.5, as well as an effect of ATP concentration on the hysteretic behaviour of this enzyme. 5. The effect of fructose on the activation of phosphorylase is discussed in relation to the comsumption of ATP.

Adenosine Triphosphate↗

PubMed1987

Localization of phosphorylase kinase subunits at the sarcoplasmic reticulum of rabbit skeletal muscle by monoclonal and polyclonal antibodies.

Molecular structures related to phosphorylase kinase have been localized by light and electron microscopy in tissue sections of rabbit skeletal muscle employing polyclonal antibodies directed against the holoenzyme as well as monoclonal antibodies specific for its alpha-, beta- or gamma-subunits. In frozen sections of prefixed muscle fibres both known major regions of glycogen deposition, the intermyofibrillar space and the perinuclear area, are stained predominantly. In sections of unfixed muscle in which cytosolic phosphorylase kinase was removed by extensive washes prior to immunostaining the immunolabel is mainly associated with the sarcoplasmic reticulum (SR). This membrane location is further confirmed by immunoblot analysis of proteins solubilized from isolated SR with Triton X-114. Employing monoclonal antibodies two membrane proteins are identified as the alpha- and beta-subunits of phosphorylase kinase by Western blots. Immunoprecipitates reveal also the gamma-subunit; the delta-subunit, i.e., calmodulin, is enriched with the solubilized enzyme. It proves that a SR membrane associated form of holophosphorylase kinase exists in muscle. Functionally, this kinase might be involved in phosphorylation of phosphatidylinositol present on the SR Ca2+ transport ATPase and thereby might play a role in regulation of Ca2+ transport.

Animals↗

PubMed1987

Autophosphorylation of the alpha subunit of phosphorylase kinase from rabbit skeletal muscle.

The autophosphorylation of the alpha subunit of phosphorylase kinase occurs simultaneously at multiple sites during incorporation of the first mol of phosphate. The predominant and initial autophosphorylation site on this subunit is different than the major site phosphorylated by cAMP-dependent protein kinase, which also phosphorylates multiple sites, as evidenced by two-dimensional phosphopeptide maps. All of the sites on the alpha subunit phosphorylated by cAMP-dependent protein kinase comigrate on peptide maps with autophosphorylation phosphopeptides; however, several phosphopeptides observed after autophosphorylation are not evident following phosphorylation by cAMP-dependent protein kinase. The phosphopeptide maps of the alpha subunit are the same whether autophosphorylation is carried out at pH 6.8 or 8.2 or whether MnATP is used instead of MgATP; there is only a slight difference in the maps brought about by EGTA-insensitive autophosphorylation. The autophosphorylation is shown to be an intrinsic activity of the phosphorylase kinase molecule; this conclusion is based on the observed copurification of the autophosphorylation activity with activities toward phosphorylase b and kappa-casein and the unaltered influence of various effectors on these activities throughout different sequential adsorption chromatography purification steps. Additional support to that already in the literature that the initial autophosphorylation events are predominantly intramolecular is gained by showing that previously autophosphorylated enzyme has little ability to catalyze the phosphorylation of nonphosphorylated enzyme.

Adenosine Triphosphate↗

PubMed1992

cDNA cloning of a liver isoform of the phosphorylase kinase alpha subunit and mapping of the gene to Xp22.2-p22.1, the region of human X-linked liver glycogenosis.

We have cloned cDNA molecules encoding another isoform of the alpha subunit of phosphorylase kinase (ATP:phosphorylase-b phosphotransferase, EC 2.7.1.38). Sequence comparison with the previously characterized muscle isoform reveals a pattern of highly conserved and variable domains and demonstrates that the isoforms are the products of distinct genes. In contrast to the muscle isoform gene, PHKA1, the gene of this additional isoform, PHKA2, is predominantly expressed in liver and other nonmuscle tissues. It was mapped to the distal short arm of the human X chromosome (Xp22.2-p22.1), the same region to which human X-linked liver glycogenosis due to phosphorylase kinase deficiency has been mapped. Thus, X-linked liver glycogenosis is probably caused by mutations affecting PHKA2.

Amino Acid Sequence↗

PubMed1994

The gamma phosphorylase kinase gene, Phkg, maps to mouse chromosome 5 near Gus.

Phosphorylase kinase is a multimeric regulatory enzyme in the glycogenolytic pathway. Interest in various types of phosphorylase kinase enzyme deficiency has focused attention on cloning and mapping the enzyme subunits. We report the mapping of the catalytic gamma subunit gene, Phkg, to mouse Chromosome (Chr) 5 near beta-glucuronidase (Gus), between alpha fetoprotein (Afp) and erythropoietin (Epo). In addition, PCR-based polymorphism assays have been developed for the human (EPO) and mouse erythropoietin genes, and a unique recombinant inbred strain distribution pattern has been defined for Epo, a distal anchor marker on mouse Chr 5.

Alleles↗

PubMed1989

Expression and characterization of the tau subunit of phosphorylase kinase.

A cDNA encoding the entire tau subunit of rabbit skeletal muscle phosphorylase kinase was reconstructed and inserted into a plasmid containing the Escherichia coli ptac promoter and a constructed plasmid containing the ptac promoter and bacterial chloramphenicol acetyl transferase (CAT) gene, respectively. A significant phosphorylase kinase activity was found, in the first case. In the second case, a fused protein containing 73 amino acids from the CAT protein was obtained. After renaturation, the CAT-tau subunit protein shows enzymatic activity similar to the HPLC-purified and renatured tau subunit.

Animals↗