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PubMed1986

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

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

Animals↗
PubMed1981

Rat liver phosphorylase kinase. Stimulation by heparin.

Rat liver phosphorylase kinase activity was increased up to 7-fold by low concentrations of heparin, but other glycosaminoglycans were relatively ineffective. A half-maximal effect was seen with 0.5 units of heparin/ml, corresponding to 150 nM. The stimulation was rapid, i.e. occurring within 15 s, and was prevented or reversed by addition of the heparin-binding protein antithrombin III. The action of heparin was also sharply reduced by prior activation of the enzyme by MgATP. In the absence of heparin, the time course of the phosphorylase kinase reaction was concave upward, whereas in the presence of heparin, a constant rate was seen. The stimulatory effect of heparin was inversely proportional to the concentration of phosphorylase b. The data suggest that heparin reversibly stimulates a low activity form of liver phosphorylase kinase to a degree similar to that found with activation by phosphorylation.

Animals↗
PubMed1985

Phosphorylase kinase from chicken gizzard. Partial purification and characterization.

Phosphorylase kinase was partially purified (530-970-fold) from chicken gizzard smooth muscle by a procedure involving ammonium sulfate fractionation, chromatography on 8-(6-aminohexyl)adenosine-5'-phosphate--Sepharose 4B and glycerol density gradient ultracentrifugation. The final and most efficient purification step takes advantage of the relatively high molecular mass of gizzard phosphorylase kinase, which was found to be similar to that of rabbit skeletal muscle enzyme. The gizzard kinase, further purified to near homogeneity by calmodulin-Sepharose 4 B affinity chromatography, showed one main protein band of 61 kDa, upon dodecyl sulfate acrylamide gel electrophoresis. Four minor protein bands of higher molecular mass were also present but no protein stain was seen at the position of the gamma subunit. The gizzard phosphorylase kinase showed a high pH 6.8/8.2 activity ratio of 0.53, it was stimulated by Ca2+, inhibited up to 80% by EGTA and it was activated about 1.9-fold by calmodulin. The km value for ATP was 0.45 mM, while the K0.5 for rabbit muscle phosphorylase b was extremely low, more than 200-fold lower than the Km of nonactivated skeletal muscle phosphorylase kinase for its protein substrate. High concentrations of phosphorylase b were found to be inhibitory. At 10 mg/ml phosphorylase b, the maximum activity of the kinase was inhibited fivefold. No evidence has been obtained indicating autophosphorylation or the existence of active and inactive forms of gizzard phosphorylase kinase. Limited proteolysis of the smooth muscle kinase with trypsin was accompanied by a twofold activation at pH 6.8.

Animals↗
PubMed1984

Phosphorylation of rat liver glycogen synthase by phosphorylase kinase.

Phosphorylation of rat liver glycogen synthase by rabbit skeletal muscle phosphorylase kinase results in the incorporation of approximately 0.8-1.2 mol of PO4/subunit. Analyses of the tryptic peptides by isoelectric focusing and thin layer chromatography reveal the presence of two major 32P-labeled peptides. Similar results were obtained when the synthase was phosphorylated by rat liver phosphorylase kinase. This extent of phosphorylation does not result in a significant change in the synthase activity ratio. In contrast, rabbit muscle glycogen synthase is readily inactivated by rabbit muscle phosphorylase kinase; this inactivation is further augmented by the addition of rabbit muscle cAMP-dependent protein kinase or cAMP-independent synthase (casein) kinase-1. Addition of cAMP-dependent protein kinase after initial phosphorylation of liver synthase with phosphorylase kinase, however, does not result in an inactivation or additional phosphorylation. The lack of additive phosphorylation under this condition appears to result from the phosphorylation of a common site by these two kinases. Partial inactivation of liver synthase can be achieved by sequential phosphorylation with phosphorylase kinase followed by synthase (casein) kinase-1. Under this assay condition, the phosphate incorporation into the synthase is additively increased and the synthase activity ratio (-glucose-6-P/+glucose-6-P) is reduced from 0.95 to 0.6. Nevertheless, if the order of the addition of these two kinases is reversed, neither additive phosphorylation nor inactivation of the synthase is observed. Prior phosphorylation of the synthase by phosphorylase kinase transforms the synthase such that it becomes a better substrate for synthase (casein) kinase-1 as evidenced by a 2- to 4-fold increase in the rate of phosphorylation. This increased rate of phosphorylation of the synthase appears to result from the rapid phosphorylation of a site neighboring that previously phosphorylated by phosphorylase kinase.

Animals↗
PubMed1975

The phosphorylase kinase deficiency (Phk) locus in the mouse: evidence that the mutant allele codes for an enzyme with an abnormal structure.

Female (I/St X C57BL/St) F1 mice heterozygous at the sex-linked phosphorylase kinase deficiency locus (Phk) have phosphorylase kinase activities averaging 86% that of mice homozygous for the wild-type allele (C57BL/St), i.e., 72% greater than the sum of one-half the activities of the parental strains. Approximately one-half the phosphorylase kinase activity in the (I X C57BL) F1 muscle extracts had a stability at 42.5 C similar to that of the activity in C57BL extracts (t1/2 = 13.2 min); the other half of the activity in the F1 extracts was more labile (t1/2 = 3.9 min). Two species of phosphorylase kinase activity in F1 muscle extracts were also differentiated with an antiserum prepared in guinea pigs against purified rabbit skeletal muscle phosphorylase kinase. This anti-serum cross-reacted with phosphorylase kinase in C57BL muscle extracts but did not cross-react with skeletal muscle extracts of mice hemi- or homozygous for the mutant allele (I/LnJ). The guinea pig antiserum precipitated 52% as much protein from (I X C57BL)F1 muscle extracts compared to those of C57BL. However, an antiserum prepared against purified rabbit skeletal muscle phosphorylase kinase in the goat cross-reacted with the mutant phosphorylase kinase. The ratio C57BL:(I X C57BL)F1:I of immunoprecipitated protein from skeletal muscle extracts with this antiserum was 1:0.97:1.08. Polyacrylamide gel electrophoresis of the immunoprecipitates in the presence of 0.1% sodium dodecylsulfate showed three subunits for mouse phosphorylase kinase with molecular weights of 139,000, 118,000, and 41,000; these values are similar to the ones obtained with purified rabbit skeletal muscle phosphorylase kinase. These three subunits were also observed in immunoprecipitates from I/LnJ muscle extracts. These results offer substantial evidence (1) that in skeletal muscle extracts of mice heterozygous at the Phk locus the mutant phosphorylase kinase is active, (2) that the gene product of the mutant allele is an enzyme with an abnormal structure, and (3) that the phosphorylase kinase deficiency in I/LnJ skeletal muscle extracts is not the result of the absence of phosphorylase kinase or one of its subunits.

Alleles↗
PubMed1990

Purification and characterization of native and proteolytic forms of rabbit liver phosphorylase kinase.

1. Two forms of phosphorylase kinase having mol. wt of 1,260,000 (form I) and 205,000 (form II) have been identified by gel filtration chromatography of rabbit liver crude extracts. 2. Form I was the majority when the homogenization buffer was supplemented with a mixture of proteinase inhibitors. This form has been purified through a protocol including ultracentrifugation, gel filtration and affinity chromatography on Sepharose-heparin. 3. Form II was purified by a combination of chromatographic procedures including ion exchange, gel filtration and affinity chromatography on Sepharose-Blue Dextran and Sepharose-histone. 4. Upon electrophoresis in the presence of sodium dodecyl sulfate two subunits of 69,000 and 44,000 were identified for this low molecular weight enzyme. Thus, a tetrameric structure comprising two subunits of each kind can be proposed. 5. Treatment of form I with either trypsin or chymotrypsin gave an active fragment having a molecular weight similar to that of form II. On the contrary, other dissociating treatments with salts, thiols and detergents failed in producing forms of lower molecular weight. 6. The similarities between proteolyzed forms I and II were stressed by their behavior in front of antibodies raised against the muscle isoenzyme of phosphorylase kinase. 7. The study of the effect of magnesium and fluoride ions on the activity of both forms showed an inhibitory effect of magnesium when its concentration exceeded that of ATP. 8. The inhibition could nevertheless be reverted by including 50 mM NaF in the reaction mixture. 9. Form I and form II could be distinguished by their pH dependence in the presence of an excess of magnesium ions over ATP, whereas the affinity for both substrates was not significantly different.

Adenosine Triphosphate↗
PubMed1991

Phosphorylase kinase from bovine stomach smooth muscle: a Ca2(+)-dependent protein kinase associated with an actin-like molecule.

Phosphorylase kinase was purified (110-fold) from bovine stomach smooth muscle by a procedure involving DEAE-cellulose chromatography, ammonium sulfate fractionation and glycerol density ultracentrifugation. On sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) the final enzyme preparation shows a single protein band of 43 kDa. The purified protein exhibits a close similarity with bovine aortic actin, as revealed by amino acid analysis and sequencing of a tryptic decapeptide fragment, although it differs widely from actin in several respects. In our effort to separate phosphorylase kinase activity from the 43 kDa protein we used a variety of chromatographic procedures, but in all cases the catalytic activity (when eluted) was accompanied by the 43 kDa protein band. Bovine stomach phosphorylase kinase exhibits an apparent molecular mass of 950 kDa, it shows a low Vmax value for phosphorylase b (85 nmol.min-1.mg-1), a pH 6.8/8.2 activity ratio of 0.23, it has an absolute requirement for Ca2+ and it is activated 1.8-fold by Ca2+/calmodulin. Furthermore, the protein kinase activity is neither inhibited by antibodies against rabbit skeletal muscle phosphorylase kinase nor activated by protein phosphorylation. These results suggest that bovine stomach phosphorylase kinase is tightly bound to an aggregate of actin-like molecules.

Actins↗
PubMed1993

Phosphorylase kinase phosphorylates the calmodulin-binding regulatory regions of neuronal tissue-specific proteins B-50 (GAP-43) and neurogranin.

Neuronal tissue-specific proteins B-50 (GAP-43, neuromodulin) and neurogranin are phosphorylated by phosphorylase kinase with stoichiometries of 0.4 and 0.5 mol of phosphate/mol of protein, respectively. The apparent Km and kcat values determined at pH 8.2 for neurogranin phosphorylation are 28.4 microM and 139.3 min-1, respectively, and for B-50 phosphorylation are 22.8 microM and 33.2 min-1, respectively. As a substrate of phosphorylase kinase, phosphorylase is approximately 44 and approximately 13 times better than B-50 and neurogranin, respectively. Both proteins are better substrates of protein kinase C than of phosphorylase kinase and are phosphorylated on a single site by phosphorylase kinase. The sequence analyses of tryptic phosphopeptides isolated from neurogranin and B-50 phosphorylated by phosphorylase kinase revealed the same amino acid sequence, IQASF, indicating that phosphorylase kinase phosphorylates the calmodulin-binding regulatory regions of B-50 and neurogranin previously known to be phosphorylated by protein kinase C (Coggins, P. J., and Zwiers, H. (1989) J. Neurochem. 53, 1895-1901; Baudier, J., Deloulme, J. C., Dorsselaer, A. V., Black, D., and Matthes, W. D. (1991) J. Biol. Chem. 266, 229-237). In rat brain synaptosomes, a relatively high phosphorylase kinase specific activity is detected, and approximately 32% activity is associated with synaptic membranes where B-50 is localized. In rat brain homogenate and synaptosomal membranes, phosphorylation of a protein that co-migrates with B-50 on SDS-polyacrylamide gel electrophoresis is enhanced in the presence of exogenous phosphorylase kinase.

Amino Acid Sequence↗
PubMed1980

Multiplicity of phosphate acceptor proteins for muscle glycogen phosphorylase kinase.

Although muscle glycogen phosphorylase kinase reacts preferentially with an inactive form of phosphorylase, the enzyme is able to phosphorylate in vitro multiple species of unidentified endogenous proteins in mammalian tissues such as liver. The reactions absolutely require Ca2+. Phosphate acceptor proteins are most abundant in the soluble and microsomal fractions. Sodium lauryl sulfate-slab gel electrophoresis analysis has revealed that the spectrum of phosphate acceptor proteins entirely differs from that for cyclic AMP-dependent protein kinase, although the biological significance of these reactions is unclear. Nevertheless, it is suggested that the enzyme is potentially multifunctional and plays roles in controlling some of the Ca2+-dependent processes. In contrast, myosin light chain kinase which is another species of calmodulin-dependent protein kinase seems to be strictly specific for this particular protein, and does not utilize any other endogenous protein so far tested.

Animals↗
PubMed1985

ADP-ribosylation of phosphorylase kinase and block of phosphate incorporation into the enzyme.

Phosphorylase kinase purified from rabbit skeletal muscle was ADP-ribosylated by hen liver nuclear ADP-ribosyltransferase. This modification, as was seen in cAMP-dependent phosphorylation, was observed only in alpha and beta subunits of the phosphorylase kinase and the latter was more rapidly modified. Analysis of the ADP-ribosylated amino acid residue sequenced in alpha and beta subunits showed that both subunits were modified at the area of the arginine residue. The Km for NAD was 0.10 mM and the pH optimum was 9.0. When the ADP-ribosylated phosphorylase kinase was phosphorylated by cAMP-dependent protein kinase, a reduction in phosphate incorporation occurred with increase in the ADP-ribosylation. ADP-ribosylation also suppressed autophosphorylation, to a lesser degree than observed with cAMP-dependent phosphorylation. The ADP-ribosylation-dependent reduction of phosphorylation resulted in a suppression of the phosphorylation-dependent activation of the phosphorylase kinase. These results together with findings of ADP-ribosyltransferase activity in the rabbit skeletal muscle [Soman, G. et al. (1984) Biochem. Biophys. Res. Commun. 120, 973-980] suggest that ADP-ribosylation participates in the regulation of the phosphorylase kinase activity through changes in the rate of phosphorylation.

Adenosine Diphosphate Ribose↗
PubMed1982

[Properties of the catalytically active fragment obtained by limited proteolysis of phosphorylase kinase].

The activation of phosphorylase kinase by limited proteolysis with subtilisin results in a formation of new enzyme forms differing in their molecular weights. Using gradient electrophoresis in polyacrylamide gel, it was shown that the high molecular weight fraction is made up of active fragments having different molecular weights. The low molecular weight fraction was found to contain only one active fragment with molecular weight of 80 000. Disc electrophoresis in polyacrylamide gel demonstrated that the active fragments of the high and low molecular weight fractions are not homogenous. The kinetic properties of the low molecular weight fragment were investigated. It was found that at pH 8.2 the native non-activated kinase and the catalytically active fragment have identical Km values for the substrates (phosphorylase B and MgATP); however, unlike the non-activated kinase, this fragment possesses a decreased sensitivity to Ca2+ and effectors (glycogen and glucose 6-phosphate) and has no optimum of activity within the pH range of 6.0-9.0.

Animals↗
PubMed1983

Characterization of initial autophosphorylation events in rabbit skeletal muscle phosphorylase kinase.

Initial autophosphorylation of nonactivated rabbit skeletal muscle phosphorylase kinase at pH 8.0 caused an increase in enzymatic activity that closely paralleled phosphorylation of the beta subunit. Peptide maps revealed that the first phosphate incorporated into the beta subunit during autophosphorylation was on the same tryptic peptide previously isolated from phosphorylase kinase that had been phosphorylated by cAMP-dependent protein kinase (Cohen P., Watson, D.C., and Dixon, G.H. (1975) Eur. J. Biochem. 51, 79-92). When preincubated with phosphorylase kinase for one min, Ca2+ and Mg2+ synergistically stimulated subsequent autophosphorylation at pH 6.8. After this treatment phosphorylation of both the alpha and beta subunits became linear, and the first site phosphorylated on the beta subunit at pH 6.8 corresponded to the first site phosphorylated at pH 8.0. Removal of the lag as a consequence of the synergistic action of the metal ions allowed determination of a Km for MgATP of approximately 20 microM during initial autophosphorylation at either pH 6.8 or 8.2. With phosphorylase b as the substrate the Km values for MgATP under identical conditions were determined to be approximately 30 and 60 microM at pH 6.8 and 8.2, respectively. Initial rates of autophosphorylation over a 30-fold range of phosphorylase kinase concentrations suggest that incorporation of the first 1 to 2 mol of phosphate per alpha beta gamma delta tetramer occurs through an intramolecular mechanism.

Adenosine Triphosphate↗
PubMed1982

Purification of rat liver phosphorylase kinase.

A rapid method for the purification of rat liver phosphorylase kinase 30,000-fold over homogenate values is described. The method allows the isolation of a near homogeneous preparation of phosphorylase kinase initially associated with the glycogen pellet to be accomplished within 24 h. The enzyme has Mr (apparent) = 1.3 million by gel filtration and is composed of subunits similar in size to those of skeletal muscle phosphorylase kinase. The enzyme is phosphorylated by the cAMP-dependent protein kinase: phosphate is incorporated into two of the subunits (Mr = 140,000 and Mr = 116,000) and is closely paralleled by activation of the enzyme. The enzyme is partially inhibited by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and is stimulated by 10(-8)-10(-6) M Ca2+. The pH optimum of the nonactivated enzyme is 7.0. Activation by cAMP-dependent protein kinase does not appear to alter the Ca2+ sensitivity of the enzyme. However, it results in a large increase in activity at pH 7 through 8, but not at pH below 6.5. Purified rat liver phosphorylase kinase thus shows many similarities to purified skeletal muscle phosphorylase kinase, but differs in respect to its incomplete inhibition by ethylene glycol bis(beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid and to the effects of phosphorylation by cAMP-dependent protein kinase on its pH activity profile and Ca2+ sensitivity.

Animals↗
PubMed1980

Phosphorylase kinase from rabbit skeletal muscle: identification of the calmodulin-binding subunits.

Phosphorylase kinase has the structure (alpha beta gamma delta)4 where the delta-subunit is identical to the calcium-binding protein termed calmodulin [Shenolikar et al. (1979) Eur. J. Biochem. 100, 329--337]. The delta-subunit was tightly bound to phosphorylase kinase in the absence of calcium ions, and its rate of exchange with [14C]calmodulin was only 15% per week. The delta-subunit remained associated with phophorylase kinase in the presence of 8 M urea provided that calcium ions were present and this property enabled electrophoretic techniques to be used which demonstrated that the delta-subunit was associated with the gamma-subunit. This finding was confirmed by cross-linking experiments with dimethylsuberimidate which resulted in the formation of a gamma delta complex. Phosphorylase kinase was shown to bind one additional molecule of calmodulin per alpha beta gamma delta unit, termed the delta'-subunit. Glycerol gradient centrifugation in the presence of [14C]calmodulin indicated that the interaction of the delta'-subunit with phosphorylase kinase only occurred in the presence of calcium ions, and that the Kd value was near 0.01 microM. This was similar to the concentration of delta'-subunit which produced half-maximal activation. The delta'-subunit did not remain associated with phosphorylase kinase in the presence of 8 M urea, either in the presence or absence of calcium ions. The very slow exchange between the delta-subunit and [14C]calmodulin, and the calcium-dependent binding of the delta'-subunit allowed cross-linking experiments to be used which demonstrated that the delta'-subunit was bound to both the alpha and beta subunits. This result was supported by the finding that selective proteolysis of either the alpha-subunit, or the alpha and beta subunits, decreased or abolished the ability of phosphorylase kinase to bind to calmodulin-Sepharose. The roles of the different subunits in the regulation of phosphorylase kinase activity are discussed.

Animals↗
PubMed1986

Interaction of flavonoids with rabbit muscle phosphorylase kinase.

We have examined the effect of several flavonoids on the activity of phosphorylase kinase from rabbit skeletal muscle. From 14 flavonoids tested, the flavones quercetin and fisetin were found to be efficient inhibitors of nonactivated phosphorylase kinase when assayed at pH 8.2, causing 50% inhibition at a concentration of about 50 microM, while the flavanone hesperetin stimulated phosphorylase kinase activity about 2-fold when tested at 250 microM. The efficiency of quercetin in inhibiting the kinase is higher when the enzyme is stimulated either by ethanol or by alkaline pH. Both casein and troponin phosphorylation by phosphorylase kinase and the autophosphorylation of the kinase were inhibited by quercetin. In addition, quercetin was found to be a competitive inhibitor of ATP for the phosphorylation of phosphorylase b at pH 8.2. These observations suggest that the inhibitory effect of the flavone is directly on the phosphorylase kinase molecule. Trypsin-activated phosphorylase kinase was inhibited by quercetin and stimulated by hesperetin, as for the native enzyme.

Adenosine Monophosphate↗
PubMed2002

The regulatory alpha subunit of phosphorylase kinase may directly participate in the binding of glycogen phosphorylase.

The yeast two-hybrid screen has been used to identify potential regions of interaction of the largest regulatory subunit, alpha, of phosphorylase kinase (PhK) with two fragments of its protein substrate, glycogen phosphorylase b (Phb). One fragment, corresponding to residues 17-484 (PhbN'), contained the regulatory domain of the protein, but in missing the first 16 residues was devoid of the sole phosphorylation site of Phb, Ser14; the second fragment corresponded to residues 485-843 (PhbC) and contained the catalytic domain of Phb. Truncation fragments of the alpha subunit were screened for interactions against these two substrate fragments. PhbC was not found to interact with any alpha constructs; however, PhbN' interacted with a region of alpha (residues 864-1014) that is near the phosphorylatable region of that subunit. PhbN' was also screened for interactions against a variety of fragments of the catalytic gamma subunit of PhK; however, no interactions were detected, even with full-length gamma. Our results support the idea that amino acid residues proximal to the convertible serine of Phb are important for its specific interaction with the catalytic subunit of PhK, but that regions distinct from the convertible serine residue of Phb and from the catalytic domain of PhK may also be involved in the interaction of these two proteins.

Catalytic Domain↗
PubMed1997

Autosomal recessive phosphorylase kinase deficiency in liver, caused by mutations in the gene encoding the beta subunit (PHKB).

The association of autosomal recessive phosphorylase kinase deficiency in liver of a 3 1/2-year-old female child with mutations in the gene encoding the common part of the beta subunit of phosphorylase kinase is reported. The proband had a severe deficiency of phosphorylase kinase in liver, while the phosphorylase kinase activity in erythrocytes was only slightly diminished. She had no symptoms of muscle involvement. The complete coding sequences of the liver gamma subunit and of the beta subunit of phosphorylase kinase of the proband were analyzed for the presence of mutations, by either reverse-transcribed PCR or SSCP analysis. Three deviations from the normal sequence were found in the region encoding the common part of the beta subunit of phosphorylase kinase-namely, a 1827G-->A (W609X) transition, a 2309A-->G (Y770C) transition, and a deletion of nucleotides 2896-2911-whereas no mutations were detected in the sequence encoding the liver gamma subunit of phosphorylase kinase. The 1827G-->A mutation and the deletion both result in the formation of early stop codons. Investigation of DNA showed that the deletion is caused by a splice-acceptor site mutation (IVS30(-1),g-->t). Family analysis revealed that the 1827G-->A and IVS30(-1),g-->t substitutions are located on different parental chromosomes and that compound heterozygosity for these mutations segregates with the disease. The 2309A-->G mutation was detected in 2%-3% of the normal population. Thus, it is concluded that the deficiency of phosphorylase kinase in this proband is caused by compound heterozygosity for the 1827G-->A and the IVS30(-1),g-->t mutations and that the 2309A-->G mutation is a polymorphism. This implies that a defect in the sequence encoding the common part of the beta subunit of phosphorylase kinase may present as liver phosphorylase kinase deficiency.

Carbohydrate Metabolism, Inborn Errors↗