SEARCH · PubMed Health

Results for “PHOSPHORYLASE KINASE”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 163 records · Page 9Linked to original sources

PubMed1984

[Activation of phosphorylase kinase from rabbit muscle by actin and calmodulin].

The activation of different forms of muscle phosphorylase kinase by actin has been studied. F-actin which is polymerized by 2 mM MgCl2 is a more effective activator of phosphorylase kinase than F-actin polymerized by 50 mM KCl. There is evidence suggesting that the activation of phosphorylase kinase b by actin is not due to the presence of trace amounts of calmodulin in actin preparations: (1) Troponin I and trifluoperazine inhibit the activation of phosphorylase kinase by calmodulin but do not inhibit the activation by actin. (2) The activation induced by saturating concentrations of calmodulin and actin is additive. (3) The activation of phosphorylase kinase by calmodulin and actin has different pH profiles. An addition of F-actin does not affect the apparent Km value for ATP but increases the sensitivity to phosphorylase b and the value of V. F-actin has no stimulating effect on the phosphorylated form (a) of phosphorylase kinase or on the form a previously activated by proteolysis.

Actins↗

PubMed1982

Regulation of phosphorylase kinase in rat ventricular myocardium. Role of calmodulin.

Conversion of phosphorylase b to a which is catalyzed by the enzyme phosphorylase kinase is known to require Ca++. Trifluoperazine, an inhibitor of calmodulin-dependent enzymes, was utilized in the present study to clarify the role in vivo of calcium-calmodulin regulation of phosphorylase kinase. Twenty-minute preperfusion of isolated rat ventricles with 10(-5) M trifluoperazine had no effect on basal levels of phosphorylase a but significantly attenuated phosphorylase activation induced by either calcium (3.75 mM) or isoproterenol (3 x 10(-9) M, 3 x 10(-8) M). The positive inotropic effect of both agents and cyclic adenosine 3',5'-monophosphate (cAMP) levels were not altered by trifluoperazine in the perfused hearts. In addition, no effects of 10(-5) M trifluoperazine were noted on beta-adrenergic receptor binding of [3H](+/-)carazolol or on adenylate cyclase activity. In vitro studies with partially purified rat cardiac phosphorylase kinase demonstrated 1.5- to 3-fold stimulation by exogenous calmodulin. The addition of 10(-5) M trifluoperazine prevented calmodulin stimulation but had little effect on activity in the absence of exogenous calmodulin. The present results suggest that reversible binding of calcium-calmodulin may represent a physiological means for regulating phosphorylase kinase activity in rat cardiac muscle.

Animals↗

PubMed1997

Antipeptide antibodies as probes of subunit-dependent structural changes in the regulatory domain of the gamma-subunit of phosphorylase kinase.

The gamma-subunit of phosphorylase kinase contains a protein kinase catalytic domain (residues 20-276) and a regulatory domain (residues 276-386). The purpose of the present investigation was to develop monospecific antibodies against four synthetic gamma-subunit regulatory domain peptides (PhK1: 362-386; PhK5: 342-366; PhK9: 322-346; PhK13: 302-326) to use as probes to study the structure of the regulatory domain. Each affinity-purified antibody was characterized with regard to its ability to bind three different structural forms of the gamma-subunit: the isolated gamma-subunit, the gamma-delta complex, and the holoenzyme complex (alpha beta delta gamma)4. Of the four antibodies, binding of affinity-purified anti-PhK13 was most affected by alterations in gamma-subunit interactions. Taken together, the data from this investigation indicate that the regulatory domain of the gamma-subunit can assume different immunochemically distinguishable conformations as the result of interactions among the alpha-, beta-, gamma-, and delta-subunits of phosphorylase kinase.

Amino Acid Sequence↗

PubMed1991

Identification and primary structure of calmodulin binding domains in the phosphorylase kinase holoenzyme.

Fast twitch skeletal muscle phosphorylase kinase was isolated and incubated with a radioactive, bifunctional, photoactivable, and cleavable cross-linker conjugated to calmodulin. Incubation of the holoenzyme only resulted in the labeling of the alpha-subunit in the presence of Ca2+. After cleavage with CNBr (and subdigestion with Asp-N protease), a sequence was identified (residues 1069-1087) in the alpha-subunit which had the predominant basic character and the propensity to form an amphiphilic helix like other calmodulin binding domains. If cross-linked calmodulin was incubated with the isolated subunits of phosphorylase kinase, radioactivity was recovered in seven CNBr peptides: three came from the alpha-subunits, one of them corresponding to the sequence labeled in the holoenzyme. Three came from the beta-subunit, and one came from the gamma-subunit. The latter contained the two adjacent calmodulin binding domains recently identified in the gamma-subunit (Dasgupta, M., Honeycutt, T., and Blumenthal, D. K. (1988) J. Biol. Chem. 264, 17156-17163).

Amino Acid Sequence↗

PubMed1984

Comparison of the phosphorylation of rabbit skeletal muscle phosphorylase kinase by cAMP-dependent protein kinase and cAMP-independent glycogen synthase (casein) kinase-1.

We have previously reported that rabbit skeletal muscle phosphorylase kinase is phosphorylated by glycogen synthase (casein) kinase-1 (CK-1) primarily on the beta subunit (beta = 1 mol of PO4; alpha = 0.2 mol of PO4) when the reaction was carried out in beta-glycerophosphate. The resultant enzyme activation was 16-fold (Singh, T. J., Akatsuka, A., and Huang, K.-P. (1982) J. Biol. Chem. 257, 13379-13384). In the present study we found that in Tris-Cl buffer CK-1 catalyzes the incorporation of greater than 2 mol of PO4/monomer into each of the alpha and beta subunits. Phosphorylase kinase activation resulting from the higher level of phosphorylation remained 16-fold. 32P-Labeled tryptic peptides from the alpha and beta subunits were analyzed by isoelectric focusing. Cyclic AMP-dependent protein kinase (A-kinase) phosphorylates a single major site in each of the alpha and beta subunits at 1.5 mM Mg2+. In addition to these two sites, A-kinase phosphorylates at least three other sites in the alpha subunit at 10 mM Mg2+. CK-1 also catalyzes the phosphorylation of multiple sites in both the alpha and beta subunits. Of the two major sites phosphorylated by CK-1 in the beta subunit, one of these sites is also recognized by A-kinase. At least three sites are phosphorylated by CK-1 in the alpha subunit. One of these sites is recognized by CK-1 only after a prior phosphorylation of phosphorylase kinase by A-kinase at a single site in each of the alpha and beta subunits at 1.5 mM Mg2+. The roles of the different phosphorylation sites in phosphorylase kinase activation are discussed.

Animals↗

PubMed1977

Studies on the specificity of phosphorylase kinase using peptide substrates.

The action of phosphorylase kinase on synthetic peptides is reported. These peptides are variants of the amino acid sequence. Ser-Asp-Gln-Glu-Lys-Arg-Lys-Gln-Ile-Ser-Val-Arg-Gly-Leu, found in the natural substrate, phosphorylase b. The effects of size, the cluster of basic groups at the NH2-terminal side, the phosphorylatable seryl residue, the hydrophobic groups surrounding serine, and the arginyl function at the COOH-terminal side were tested and analyzed by evaluation of the kinetic parameters, Km and Vmax. The first 6 residues were found to be nonessential, but substitution of residues in the sequence. Lys-Gln-Ile-Ser-Val-Arg, had a large effect on phosphorylation. A comparison was made between the action of nonactivated and activated phosphorylase kinase on selected peptides and phosphorylase b. Various forms of phosphorylase b were tested as substrates for cyclic AMP-dependent protein kinase in the presence of effectors and salts. Although phosphorylase would not serve as a substrate for protein kinase, the aforementioned synthetic peptide of the phosphorylase b sequence would do so, indicating that the primary sequence surrounding the phosphorylatable serine did not block phosphorylation, which suggests that higher order structural features prohibit the phosphorylation.

Amino Acids↗

PubMed1992

Coordinated expression of phosphorylase kinase subunits in regenerating skeletal muscle.

The developmental expression of the alpha, beta, and gamma subunits of skeletal muscle phosphorylase kinase has been examined in regenerating muscle. Rat extensor digitorum longus (EDL) muscles, treated with bupivacaine, promptly undergo a rapid degeneration of the muscle, followed by regeneration and recovery of essentially normal morphology and physiology by 3-4 weeks post-treatment (Hall-Craggs, E. C. B., and Seyan, H. S. (1975) Exp. Neurol. 46, 345-354). Phosphorylase kinase activity dropped to approximately 10% of control within 3 days of bupivacaine treatment and remained at this low level for several days but had attained at least 60% of normal levels by day 21. The pH 6.8/8.2 activity ratio was unusually high during the period of low activity, suggesting that the catalytic activity was not under normal regulation at this time. The subunit mRNAs were readily detected in control EDL but were undetectable at day 3 post-bupivacaine treatment. Very small amounts of message for all three subunits were evident by day 6 and began to approach normal levels by day 12-15. The mRNA for both the alpha and alpha' subunits of phosphorylase kinase exhibited a similar pattern of recovery, as did also the mRNA for phosphorylase. In contrast to both phosphorylase kinase and phosphorylase, actin mRNA exhibited a quite a different pattern, with a nearly full recovery of message levels by day 6 post-bupivacaine. These data indicate that synthesis of phosphorylase and the alpha, beta, and gamma subunits of phosphorylase kinase appears to be coordinately regulated at the level of message accumulation and that the expression of phosphorylase kinase activity is likely to be also regulated post-transcriptionally.

Amino Acid Sequence↗

PubMed1989

[Molecular mechanisms of the regulation of phosphorylase kinase from skeletal muscles of mammals and birds].

Red and white avian skeletal muscles (chicken and pigeon) contain the same alpha'-isoenzyme of phosphorylase kinase. According to data from gradient polyacrylamide slab electrophoresis in the presence of SDS, the molecular masses of beta- and gamma-subunits of phosphorylase kinase from rabbit, chicken and pigeon muscles are not identical. Electron microscopy data suggest that the quaternary structure of chicken and pigeon phosphorylase kinase is of the same type. The alpha'-isozyme of chicken and pigeon phosphorylase kinase is strongly activated by calmodulin and troponin C. Avian phosphorylase kinase is activated 2--3-fold by phosphorylation with cAMP-dependent protein kinase and by autophosphorylation. This activation is associated with the phosphorylation of both alpha'- and beta-subunits. The affinity of pigeon phosphorylase kinase a for Ca2+ is 20 times as high as that of phosphorylase kinase b.

Animals↗

PubMed1975

The hormonal control of activity of skeletal muscle phosphorylase kinase. Phosphorylation of the enzyme at two sites in vivo in response to adrenalin.

Phosphorylase kinase was activated 5--10-fold in vivo by an intravenous injection of adrenalin. Sodium fluoride an inhibitor of phosphorylase kinase phosphatase, was required to prevent the reversal of this process; the activated and non-activated forms of the enzyme were indistinguishable by dodecylsulphate gel electrophoresis. This suggested that the activation had resulted from a phosphorylation of the enzyme, and that it was not a consequence of the well known activation by proteolytic cleavage that can be demonstrated in vitro. Phosphorylase kinase activated in vivo was purified and digested with trypsin, and the two tryptic peptides which contain the serine residues which are phosphorylated in vitro by the action of cyclic-AMP (adenosine 3':5'-monophosphate) dependent protein kinase, were isolated. It was found that the same nine-amino-acid segment of the beta chain and the same seven-amino-acid segment of the alpha chain had become phosphorylated in vivo in response to adrenalin, as were phosphorylated in vitro. The degree of phosphorylation of each of the two sites was at least 50%. The data provide direct proof that the activation of phosphorylase kinase which occurs in vivo in response to adrenalin results from a phosphorylation of the enzyme. They also indicate that the novel form of regulation associated with the phosphorylation of the alpha subunit, the stimulation of protein dephosphorylation by "second site phosphorylation", can now be regarded as a new form of enzyme control mechanism which operates in vivo. The regulation of phosphorylase kinase activity was studied in the protein - glycogen complex from skeletal muscle. The enzyme could be rapidly converted to a phosphorylated form in a cyclic-AMP-stimulated reaction upon addition of magnesium ions and ATP, but the conversion of phosphorylase b to phosphorylase a in the complex still showed an absolute requirement for calcium ions. The implications of these findings and major problems in the hormonal control of skeletal muscle glycogenolysis which are not yet resolved, are discussed.

Adenosine Triphosphate↗

PubMed1990

Localization of phosphoserine residues in the alpha subunit of rabbit skeletal muscle phosphorylase kinase.

The alpha subunit of skeletal muscle phosphorylase kinase, as isolated, carries phosphate at the serine residues 1018, 1020 and 1023. Employing the S-ethyl-cysteine method, these residues are found to be phosphorylated partially, i.e. differently phosphorylated species exist in muscle. Serine 1018 is a site which can be phosphorylated by the cyclic-AMP-dependent protein kinase. The serine residues 972, 985 and 1007 are phosphorylated by phosphorylase kinase itself when its activity is stimulated by micromolar concentrations of Ca2+. These phosphorylation sites are not identical to those found to be phosphorylated already in the enzyme as prepared from freshly excised muscle. A 'multiphosphorylation loop' uniquely present in this but not in the homologous beta subunit contains all the phosphoserine residues so far identified in the alpha subunit.

Amino Acid Sequence↗

PubMed1993

Features of calmodulin that are important in the activation of the catalytic subunit of phosphorylase kinase.

Calmodulin (CaM) is an integral subunit, called delta, of the phosphorylase kinase hexadecamer, and the activity of the isolated catalytic gamma-subunit of the kinase is stimulated by CaM. We report here the first analysis of functionally important features of CaM for activation of the gamma-subunit. A set of genetically engineered CaMs, in which acidic residues in each of the four E-helices of the "EF-hands" were changed to basic lysine residues, was used to probe the relative importance of charge features in each domain of CaM. The maximal activation of the isolated gamma-subunit was diminished by all of the charge reversal mutations. The gamma-subunit was especially sensitive to reversals in the second and third E-helix of CaM (residues 45-47 and 82-84), the latter being present in the central helix. The results suggest the functional importance of electrostatics in the interactions between the delta-subunit (CaM) and the catalytic gamma-subunit of phosphorylase kinase, which is similar to results obtained with CaM-dependent myosin light chain kinase (MLCK) from chicken gizzard and CaM-dependent protein kinase II (CaMPK-II). However, novel features of the interaction between CaM and the gamma-subunit of phosphorylase kinase are the significant contribution of electrostatics throughout the CaM molecule, including residues in both halves and on more than one face of CaM, and the lack of a major effect of the CaM mutations on substrate kinetic parameters, unlike the effects observed with MLCK and CaMPK-II. These results are consistent with a model in which the delta-subunit (CaM) of phosphorylase kinase interacts with an extended region or multiple regions of the gamma-subunit and suggest that the mechanism of CaM activation of the gamma-subunit may have features that are distinct from those of MLCK and CaMPK-II.

Amino Acid Sequence↗

PubMed1992

[Isolation and properties of three forms of phosphorylase and phosphorylase kinase from human skeletal muscle].

Three forms of phosphorylase (I, II and III), two of which (I and II) were active in the presence of AMP and one (III) was active without AMP, were isolated from human skeletal muscles. The pI values for phosphorylases b(I) and b(II) were found to be identical (5.8-5.9). During chromatofocusing a low molecular weight protein (M(r) = 20-21 kDa, pI 4.8) was separated from phosphorylase b(II). This process was accompanied by an increase of the enzyme specific activity followed by its decline. During reconstitution of the complex the activity of phosphorylase b(II) returned to the initial level. Upon phosphorylation the amount of 32P incorporated into phosphorylase b(II) was 2 times as low as compared with rabbit phosphorylase b and human phosphorylase b(I). It may be supposed that in the human phosphorylase b(II) molecule one of the two subunits undergoes phosphorylation in vivo. This form of the enzyme is characterized by a greater affinity for glycogen and a lower sensitivity to allosteric effectors (AMP, glucose-6-phosphate, caffeine) compared with phosphorylase b(I). Thus, among the three phosphorylase forms obtained in this study, form b(II) is the most unusual one, since it is partly phosphorylated by phosphorylase kinase to form a complex with a low molecular weight protein which stabilizes its activity. A partially purified preparation of phosphorylase kinase was isolated from human skeletal muscles. The enzyme activity necessitates Ca2+ (c0.5 = 0.63 microM). At pH 6.8 the enzyme is activated by calmodulin (c0.5 = 15 microM). The enzyme activity ratio at pH 6.8/8.2 is equal to 0.18.

Animals↗

PubMed1992

A kinetic re-interpretation of the regulation of rabbit skeletal-muscle phosphorylase kinase activity by Ca2+ and phosphorylation.

The regulation of phosphorylase kinase has been proposed to occur physiologically under conditions of zero-order ultrasensitivity [Meinke & Edstrom (1991) J. Biol. Chem. 266, 2259-2266]. This is also one of the conditions that recent theoretical approaches have indicated to be essential in order for an interconvertible enzyme cascade to generate a sensitive response to an effector [Cardenas & Cornish-Bowden (1989) Biochem. J. 257, 339-345]. In contrast, all published kinetic data to date have strongly suggested that activation of phosphorylase kinase by Ca2+ or phosphorylation is attributable solely to a change in affinity for phosphorylase, with no effect on the Vmax. of the reaction. In this study an attempt is made to resolve this conflict. Findings suggest that changes in Vmax. can fully account for the activation of phosphorylase kinase by the physiological mechanisms of cyclic AMP-dependent phosphorylation and increase in Ca2+ concentration.

Animals↗

PubMed1990

Mapping of the phosphorylase kinase alpha subunit gene on the mouse X chromosome.

Phosphorylase kinase is a glycogenolytic enzyme in several animal tissues. Within the last few years all four subunits of the enzyme have been cloned. The beta, gamma, and delta subunits are known to be autosomal. We have mapped the alpha subunit of phosphorylase kinase, recently cloned by Zander et al. (1988), in an interspecific mouse pedigree and localized it on the X chromosome, where it maps between the X-linked zinc finger protein and phosphoglycerate kinase genes, close to the latter. In man and mouse several X-linked disorders of this enzyme have been described. Although the X-linked phosphorylase kinase deficiency in mice may be caused by a mutation in the structural gene for the alpha subunit, mapped here, the existence of a separate regulatory locus, important in the normal expression or function of the enzyme in muscle, still remains a possibility.

Animals↗

PubMed1984

Ca2+-dependent stimulation of muscle and liver phosphorylase kinase by heparin.

Heparin stimulates the activity of nonactivated and activated skeletal muscle phosphorylase kinase in a Ca2+-dependent manner. The stimulatory effect of heparin on the activity of nonactivated phosphorylase kinase is also expressed in the presence of calmodulin and glycogen. Heparin acted in synergism with glycogen. Heparin increases the affinity of phosphorylase kinase to Ca2+ 5-12 fold depending upon the activation conditions. Ca2+ influences the stimulation of liver phosphorylase kinase by heparin in a similar way.

Animals↗

PubMed1991

[Interaction of phosphorylase kinase with thin filament proteins of rabbit skeletal muscles].

The binding of phosphorylase kinase to thin filaments and their effects on the enzyme activity as well as the contribution of the enzyme to contractile protein phosphorylation have been studied. The data obtained suggest that the kinase binding to thin filaments is controlled by the regulatory proteins, troponin and tropomyosin. The bulk of the enzyme is bound to the F-actin-tropomyosin-troponin complex which activates the enzyme in a far greater degree than each of its constituent components. Ca2+ and ATP control the kinase binding to F-actin. ATP increases the enzyme binding 6-fold; Ca2+ decrease the S0.5 value for F-actin 5-fold. In acetone powder extracts phosphorylase kinase phosphorylates thin filament-bound phosphorylase b, troponin T and troponin I as well as 51-58 kDa and 114 kDa proteins. These results suggest that phosphorylase kinase plays a role in the mechanism of synchronization of glycogenolysis and muscle contraction rates.

Actins↗

PubMed1986

Phosphorylase kinase isozymes in normal and electrically stimulated skeletal muscles.

Phosphorylase kinase was quantitatively immunoprecipitated from extracts of different rabbit skeletal muscles, subjected to electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate, and stained with silver. Amounts of the two isozymes, enzymes r and w. were determined by the staining intensities of the alpha'- and alpha -subunits, respectively. The kinase in muscles composed primarily of slow oxidative fibers was almost entirely enzyme r, but several times more of this isozyme was found in muscles with a high proportion of fast oxidative-glycolytic fibers. Enzyme w predominated in muscles composed mostly of fast glycolytic fibers. To investigate the possible role of muscle activity in controlling isozyme levels, tibialis anterior muscles were activated by chronic electrical stimulation of the peroneal nerves. After 10 wk of continuous stimulation, the amount of phosphorylase kinase was decreased by approximately 80%, and the ratio of enzyme r to enzyme w was doubled. These results demonstrate that increased muscle activity can decrease levels of phosphorylase kinase and suggest that activity may regulate the expression of the isozymes in different muscle fiber types.

Animals↗

PubMed1985

Interaction of phosphorylase kinase with polymyxins.

Nonactivated rabbit skeletal muscle phosphorylase kinase is inhibited by the polymyxins A, B, D and E when assayed at pH 8.6. Polymyxin B is the most effective inhibitor, causing 50% inhibition at 0.3 mM. Following the effect of polymyxin B on the kinase activity toward troponin, no inhibition was observed. In contrast, polymyxin B was found to greatly stimulate the autophosphorylation of phosphorylase kinase. About 10 mol of phosphate per tetramer (alpha beta nu delta) were incorporated in presence of polymyxin B (full autophosphorylation). This incorporation was about 6-fold higher than that observed without polymyxin. The stimulation of autophosphorylation by polymyxin B was accompanied with enhancement of the rate of autoactivation at pH 6.8.

Animals↗