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PubMed1991

Kinetic characterization of the calmodulin-activated catalytic subunit of phosphorylase kinase.

The phosphorylase kinase holoenzyme from skeletal muscle is composed of a catalytic and three different regulatory subunits. Analysis of the kinetic mechanism of the holoenzyme is complicated because both the natural substrate phosphorylase b and also phosphorylase kinase itself have allosteric binding sites for adenine nucleotides. In the case of the kinase, these allosteric sites are not on the catalytic subunit. We have investigated the kinetic mechanism of phosphorylase kinase by using its isolated catalytic gamma-subunit (activated by calmodulin) and an alternative peptide substrate (SDQEKRKQISVRGL) corresponding to the convertible region of phosphorylase b, thus eliminating from our system all known allosteric binding sites for nucleotides. This peptide has been previously employed to study the kinetic mechanism of the kinase holoenzyme before the existence of the allosteric sites on the regulatory subunits was suspected [Tabatabai, L. B., & Graves, D. J. (1978) J. Biol. Chem. 253, 2196-2202]. This peptide was determined to be as good an alternative substrate for the isolated catalytic subunit as it was for the holoenzyme. Initial velocity data indicated a sequential kinetic mechanism with apparent Km's for MgATP and peptide of 0.07 and 0.47 mM, respectively. MgADP used as product inhibitor showed competitive inhibition against MgATP and noncompetitive inhibition against peptide, whereas with phosphopeptide as product inhibitor, the inhibition was competitive against both MgATP and peptide. The initial velocity and product inhibition studies were consistent with a rapid equilibrium random mechanism with one abortive complex, enzyme-MgADP-peptide. The substrate-directed, dead-end inhibitors 5'-adenylyl imidodiphosphate and Asp-peptide, in which the convertible Ser of the alternative peptide substrate was replaced with Asp, were competitive inhibitors toward their like substrates and noncompetitive inhibitors toward their unlike substrates, further supporting a random mechanism, which was also the conclusion from the report cited above that used the holoenzyme.

Amino Acid Sequence↗
PubMed1990

[The effect of media pH and autocatalytic phosphorylation on the interaction of kinase phosphorylase with calmodulin].

Using calmodulin covalently labeled with dansyl, the Ca2(+)-dependent interaction of phosphorylase kinase with calmodulin has been studied. It has been shown that at pH 6.8 the (alpha beta gamma delta) protomer of the enzyme binds 2.1 +/- 0.8 mol of calmodulin with Kd = (6.67 +/- 1.77).10(-8) M. The enzyme activation induced by the pH increase up to 8.2 does not affect the enzyme interaction with calmodulin [2.14 +/- 0.58 mol calmodulin per mol of (alpha beta gamma delta)]; Kd = (4.14 +/- 1.22).10(-8) M. However, the enzyme activation during its autocatalytic phosphorylation eliminates this effect practically completely.

Animals↗
PubMed1983

Regulation of yeast phosphorylase by phosphorylase kinase and cAMP-dependent protein kinase.

Yeast phosphorylase is phosphorylated and activated by a cyclic AMP-independent protein kinase (called phosphorylase kinase) and a cyclic AMP-dependent protein kinase. Only in the presence of both kinases is phosphorylase fully activated and phosphorylated. No evidence was found for the presence of two phosphorylation sites as an identical phosphopeptide pattern of phosphorylase is obtained after phosphorylation by either one or both kinases. The kinases probably phosphorylate identical sites but recognize different subunits of phosphorylase. Phosphorylase kinase phosphorylates the high-Mr subunit while cAMP-dependent protein kinase phosphorylates the low-Mr subunit.

Enzyme Activation↗
PubMed2005

Fatal congenital heart glycogenosis caused by a recurrent activating R531Q mutation in the gamma 2-subunit of AMP-activated protein kinase (PRKAG2), not by phosphorylase kinase deficiency.

Fatal congenital nonlysosomal cardiac glycogenosis has been attributed to a subtype of phosphorylase kinase deficiency, but the underlying genes and mutations have not been identified. Analyzing four sporadic, unrelated patients, we found no mutations either in the eight genes encoding phosphorylase kinase subunits or in the two genes encoding the muscle and brain isoforms of glycogen phosphorylase. However, in three of five patients, we identified identical heterozygous R531Q missense mutations of the PRKAG2 gene, which encodes the gamma 2-subunit of AMP-activated protein kinase, a key regulator of energy balance. Biochemical characterization of the recombinant R531Q mutant protein showed >100-fold reduction of binding affinities for the regulatory nucleotides AMP and ATP but an enhanced basal activity and increased phosphorylation of the alpha -subunit. Other PRKAG2 missense mutations were previously identified in patients with autosomal dominant hypertrophic cardiomyopathy with Wolff-Parkinson-White syndrome, characterized by juvenile-to-adult clinical onset, moderate cardiac glycogenosis, disturbed excitation conduction, risk of sudden cardiac death in midlife, and molecular perturbations that are similar to--but less severe than--those observed for the R531Q mutation. Thus, recurrent heterozygous R531Q missense mutations in PRKAG2 give rise to a massive nonlysosomal cardiac glycogenosis of fetal symptomatic onset and rapidly fatal course, constituting a genotypically and clinically distinct variant of hypertrophic cardiomyopathy with Wolff-Parkinson-White syndrome. R531Q and other PRKAG2 mutations enhance the basal activity and alpha -subunit phosphorylation of AMP-activated protein kinase, explaining the dominant nature of PRKAG2 disease mutations. Since not all cases displayed PRKAG2 mutations, fatal congenital nonlysosomal cardiac glycogenosis seems to be genetically heterogeneous. However, the existence of a heart-specific primary phosphorylase kinase deficiency is questionable, because no phosphorylase kinase mutations were found.

AMP-Activated Protein Kinases↗
PubMed1983

The phosphorylase kinase activity of hearts from phosphorylase kinase-deficient mice.

In an assay measuring radioactive incorporation from gamma--P32P]ATP into phosphorylase b, cardiac muscle extracts from mice with the phosphorylase kinase deficiency mutation showed significant, calcium-dependent phosphorylase kinase activity that was 10 to 15% of that of Swiss mice, the control strain. Isoproterenol stimulated significant phosphorylase a accumulation in both isolated atria and right ventricular strips of phosphorylase kinase-deficient mice, and the drug-stimulated increases in phosphorylase a activity the the contractile responses of right ventricular strips were similar in Swiss and phosphorylase kinase/deficient mice.

Animals↗
PubMed1996

Hysteretic properties of rabbit skeletal muscle phosphorylase kinase: synergistic activation by phosphorylase b, Ca2+, and Mg2+.

To study the hysteretic properties of rabbit skeletal muscle phosphorylase kinase the method of continuous registration of the kinetics of the kinase reaction developed by us earlier has been used. It was shown that duration of the lag period on the kinetic curves is independent of the phosphorylase kinase concentration and the simultaneous presence of phosphorylase b, Ca2+, and Mg2+ is required for the complete transition of the enzyme into the activated state.

Adenosine Triphosphate↗
PubMed1991

The ATPase activity of phosphorylase kinase is regulated in parallel with its protein kinase activity.

Phosphorylase kinase from rabbit skeletal muscle has been found to have an intrinsic ATPase activity that occurs at a rate approximately 0.2% of that of its phosphorylase conversion activity and about three times that of its autophosphorylation activity. The characteristics of this ATPase activity were in all aspects tested essentially the same as the kinase's phosphorylase conversion activity. The ATPase requires Mg2+ and is dramatically stimulated by Ca2+ ions. At neutral pH there is a pronounced lag in the rate of product formation that is not present at alkaline pH, a condition that greatly stimulates both the phosphorylase conversion and ATPase activities. ATP is preferentially hydrolyzed over GTP and the Km for MgATP determined in the ATPase assay is 0.14 mM. ADP, an allosteric activator of phosphorylase conversion, also stimulates the ATPase activity, whereas beta-glycerophosphate, an inhibitor of phosphorylase conversion, is an inhibitor of the ATPase activity. Phosphorylation or partial proteolysis of the kinase, which are known to activate phosphorylase conversion, also activate the ATPase activity. Because the phosphorylase conversion and ATPase activities are regulated in parallel, we conclude that activation of the two catalytic activities must share a common underlying basis, namely an enhanced phosphotransferase activity that is independent of the phosphoryl acceptor.

Adenosine Diphosphate↗
PubMed1995

X-linked liver phosphorylase kinase deficiency is associated with mutations in the human liver phosphorylase kinase alpha subunit.

Two Dutch patients with liver phosphorylase kinase (PhK) deficiency were studied for abnormalities in the PhK liver alpha (alpha L) subunit mRNA by reversed-transcribed-PCR (RT-PCR) and RNase protection assays. One patient, belonging to a large Dutch family that expresses X-linked liver PhK deficiency, had a C3614T mutation in the PhK alpha L coding sequence. The C3614T mutation leads to replacement of proline 1205 with leucine, which changes the composition of an amino acid region, containing amino acids 1195-1214 of the PhK alpha L subunit, that is highly conserved in different species. The patient showed normal levels of PhK alpha L mRNA. The second patient, from an unrelated family, was found to have a TCT (bp 419-421) deletion in the PhK alpha L coding sequence, resulting in a phenylalanine 141 deletion. The same deletion was found in the PhK alpha L coding sequence from lymphocytes of the patient's mother, together with a normal PhK alpha L coding sequence. The phenylalanine that is absent in the PhK alpha L coding sequence of the second patient is a highly conserved amino acid between species. Both the C3614T mutation and the TCT (bp 419-421) deletion were not found in a panel of 80 control X chromosomes. On the basis of these results, it is postulated that the mutations found are responsible for liver PhK deficiency in the two patients investigated.

Amino Acid Sequence↗
PubMed1985

Stimulation of the cyclic AMP-dependent protein kinase-catalyzed phosphorylation of phosphorylase kinase by micromolar concentrations of spermine.

The phosphorylation of phosphorylase kinase by cyclic AMP-dependent protein kinase (A-kinase) is stimulated approximately 2-fold by spermine and spermidine. Half maximal effects were observed at 10 microM and 150 microM of spermine and spermidine, respectively. The phosphorylations of other substrates of A-kinase such as glycogen synthase, histone, and casein are not stimulated by these two polyamines. The rates, but not the final extents, of phosphorylation of both the alpha and beta subunits of phosphorylase kinase by A-kinase are stimulated by spermine. The results indicate that spermine and spermidine may play an important role in the activation of glycogenolysis in skeletal muscle.

Animals↗
PubMed1995

[Kinetics of action of phosphorylase kinase in an enzymatic cascade system. II. Assessment of the initial rate of the enzymatic reaction catalyzed by phosphorylase kinase].

The turbidimetric method for determining the phosphorylase kinase activity has been developed. The reaction mixture contained, alongside with other components of the kinase reaction, also the substrates of glycogen phosphorylase a, the final product of the kinase reaction-glycogen and glucose 1-phosphate (or inorganic phosphate). The kinetics of the cascade enzymatic system were followed by the increment (decrement) of absorbance of the glycogen solution at 360 nm (delta A). The initial rate of the phosphorylase kinase-catalyzed enzymatic reaction, nu 0, can be calculated according to the formula: nu 0 = 2tg alpha/a2, where tg alpha is the initial slope of the kinetic curve in the coordinates: delta A-(time)2 and a2 is the specific enzymatic activity of phosphorylase a. The latter was estimated from the initial rates of the phosphorylase reaction measured by the addition of glycogen to the reaction mixture after the completion of the kinase reaction. The reliability of the turbidimetric method for determining the phosphorylase kinase activity was proved by comparison with a direct method based on the measurement of the amount of incorporated 32P.

Animals↗
PubMed1999

A mutation in GLUT2, not in phosphorylase kinase subunits, in hepato-renal glycogenosis with Fanconi syndrome and low phosphorylase kinase activity.

Fanconi-Bickel syndrome is characterized by hepato-renal glycogenosis with severe renal tubular dysfunction and rickets. It has recently been found to be associated with GLUT2 mutations in three families. In another family, low activities of liver phosphorylase kinase (Phk) have been observed, suggesting that Fanconi-Bickel syndrome might be genetically heterogeneous. We have analyzed this family for mutations in the GLUT2 gene and in the three Phk subunit genes that can cause liver glycogenosis (PHKA2, PHKB, and PHKG2). The coding sequences of all three Phk genes are normal but we have identified a homozygous missense mutation (Pro417Leu) in GLUT2. The affected proline residue is completely conserved in all mammalian glucose permease isoforms and even in bacterial sugar transporters and is believed to be critical for the passage of glucose through the permease. Seven affected individuals from different branches of the same large consanguineous sibship all are homozygous for this mutation. These findings indicate that there is no specific subtype of genetic Phk deficiency giving rise to hepato-renal glycogenosis. Rather, they provide further evidence that Fanconi-Bickel syndrome is caused by GLUT2 mutations. The low Phk activity is probably a secondary phenomenon that contributes to the deposition of glycogen in response to the intracellular glucose retention caused by GLUT2 deficiency.

Amino Acid Sequence↗
PubMed2002

Differentiation-dependent mechanisms of transcriptional regulation of the catalytic subunit of phosphorylase kinase.

The amount of phosphorylase kinase in skeletal muscle is exquisitely sensitive to developmental signals such as differentiation and innervation, and is clearly regulated in such a manner so as to always maintain the gamma catalytic subunit under the control of its regulatory alpha, beta and gamma subunits. To identify how the transcription of the gamma subunit is regulated, we have analysed 3.8 kb of the upstream regulatory region using a luciferase reporter system. A complex sequence of interdependent regulations is evident. The gamma catalytic subunit gene contains two inhibitory controls with very dominant features. Also evident are an array of multiple positive regulatory elements, prominent amongst which are four E-boxes, of which two are downstream, one is upstream and one is in the middle of the CAAT-TATA core promoter. Differentiation-dependent positive regulation arises as a consequence of both E-box regulation and the activation of at least one other regulatory element. The primary mode of transcriptional regulation of the gamma catalytic subunit gene appears to occur by the relief of regulation of an otherwise default inhibitory status. It is noteworthy that such a mode of regulation mirrors the regulation of the enzymic activity of many protein kinases, including phosphorylase kinase. With phosphorylase kinase, both its transcriptional regulation as well as the regulation of the protein itself, are primed to maintain the gamma catalytic subunit either unexpressed or inactivate respectively, until a positive signal occurs to override an otherwise dominant default inhibitory condition.

Animals↗
PubMed1986

Chemical modification of rabbit skeletal muscle phosphorylase kinase with phenylglyoxal.

Nonactivated phosphorylase kinase from rabbit skeletal muscle is inactivated by treatment with phenylglyoxal. Under mild reaction conditions, a derivative that retains 10-15% of the pH 8.2 catalytic activity is obtained. The kinetics of inactivation profile, differential effects of modification on pH 6.8 and 8.2 catalytic activities, and the insensitiveness of the modified enzyme to activation by ADP reveal that the 10-15% of catalytic activity remaining is very likely due to intrinsic catalytic activity of the derivative rather than to the presence of unmodified enzyme molecules. The kinetic results also suggest that the inactivation is correlatable with the reaction of one molecule of the reagent with the enzyme without any prior binding of phenylglyoxal. The phenylglyoxal modification reduces the autophosphorylation rate of the kinase. Autophosphorylated phosphorylase kinase is inactivated by phenylglyoxal at a much slower rate than the inactivation of nonactivated kinase. Thus, phenylglyoxal modification influences the phosphorylation and vice versa. The modified enzyme can be reactivated by treatment with trypsin or by dissociation using chatropic salts. The activity of the phenylglyoxal-modified enzyme after trypsin digestion or dissociation with LiBr reaches the same level as that of the native enzyme digested with trypsin or treated with LiBr under identical conditions. The results suggest that the effect of modification is overcome by dissociation of the subunits of phosphorylase kinase and that the catalytic site is not modified under conditions when 85% of the pH 8.2 catalytic activity is lost. Among various nucleotides and metal ions tested, only ADP, with or without Mg2+, afforded effective protection against inactivation with phenylglyoxal. At pH 6.8, 1 mM ADP afforded complete protection against inactivation. Experiments with 14C-labeled phenylglyoxal revealed that ADP seemingly protects one residue from modification. This result is in agreement with the kinetic result that the inactivation seemingly is due to reaction of one molecule of the reagent with the enzyme. The results confirm the existence of a high-affinity ADP binding site on nonactivated phosphorylase kinase and suggest the involvement of a functional arginyl residue at or near the ADP binding site in the regulation of of pH 8.2 catalytic activity of the enzyme.

Adenosine Diphosphate↗
PubMed1982

Synergistic effect of Ca2+ and Mg2+ upon the interaction of phosphorylase kinase with glycogen.

Phosphorylase kinase (ATP: phosphorylase-b phosphotransferase, EC 2.7.1.38) forms molecular complexes with glycogen over a range of conditions. The extent of interaction at pH 7.0, 20 degrees C, is greatly enhanced in the simultaneous presence of Ca2+ and Mg2+. If either cation is absent, the extent of complex formation is substantially reduced. Significant changes in the physical properties of phosphorylase kinase are observed in the presence of Ca2+ plus Mg2+, suggesting that a conformational change may occur under these conditions.

Animals↗
PubMed1996

Divalent cations but not other activators enhance phosphorylase kinase's affinity for glycogen phosphorylase.

To better understand the physical interaction between glycogen phosphorylase-b (P-b) and its only known kinase, phosphorylase kinase (PbK) and the relationship of this interaction to the activation of PbK, direct binding studies are necessary. By utilizing an enzyme-linked immunosorbent assay, a method was developed for measuring the binding of PbK to immobilized P-b under a variety of experimental conditions. A monoclonal antibody specific for the alpha subunit of PbK that had no effect on the phosphorylation of P-b by PbK or on the interaction of PbK with known effectors was used to detect PbK bound to plated P-b. Hyperbolic binding curves were obtained regardless of whether the concentration of Pbk or P-b was varied, and the assay detected changes in relative affinity caused by certain effectors of the kinase. The allosteric effector ADP, alkaline pH, and phosphorylation by cAMP-dependent protein kinase, all activators of PbK, did not cause significant changes in its relative affinity for P-b; however, Ca2+ and Mg2+ ions, which also stimulate PbK, increased its affinity for P-b, with Mg2+ being more effective. Mn2+, which inhibits the P-b conversion activity of PbK, was found to be the most potent enhancer of its affinity for P-b, although divalent cations may enhance binding. Inclusion of ATP analogs in the binding assay with Ca2+ and Mg2+ to stimulate catalytic assay conditions did not further affect the apparent affinity for P-b, which is consistent with the previously reported rapid equilibrium random bi-bi kinetic mechanism for P-b conversion.

Animals↗
PubMed1983

Purification and partial characterization of bovine heart phosphorylase kinase.

Bovine heart phosphorylase kinase has been isolated by a procedure involving precipitation with polyethylene glycol, DEAE-Sephacel chromatography and calmodulin-Sepharose affinity chromatography. The isolated enzyme had a specific activity of 8.3 IU/mg of protein at pH 8.2 at 30 degrees C in the presence of 1% glycogen. The native enzyme had a sedimentation coefficient of 23 S and the Mr of the alpha', beta, gamma, and delta subunits, were 140,000, 130,000, 46,000, and 18,000, respectively. Activation of the phosphorylase kinase by the catalytic subunit of bovine heart cAMP-dependent protein kinase increases the pH 6.8/8.2 activity ratio from 0.01 to 0.32-0.38. Glycogen (1%) decreased the Km of the activated phosphorylase kinase at pH 6.8 for phosphorylase b from 5.5 to 1.25 mg/ml. Trypsin treatment increased the pH 6.8 activity but decreased the pH 8.2 activity. During this process the alpha' subunit was converted to a Mr 110,000 polypeptide and the enzyme activity was converted essentially to a 5.9 S species having an apparent Mr of 100,000 as determined by gel filtration. On extended trypsin treatment only one major polypeptide corresponding to the beta subunit remained. The same polypeptide was present in the active fractions following gel filtration of the trypsinized kinase.

Animals↗
PubMed1995

Isolation of cDNA encoding the human liver phosphorylase kinase alpha subunit (PHKA2) and identification of a missense mutation of the PHKA2 gene in a family with liver phosphorylase kinase deficiency.

X-linked liver glycogenosis (XLG) due to liver phosphorylase kinase (PHK) deficiency is the most frequent liver glycogen storage disease. The affected patients present in early childhood with hepatomegaly and growth retardation. We isolated and determined the structure of human liver alpha subunit of PHK (PHKA2) cDNA. The 3705 base pair open reading frame encodes a polypeptide of 1235 amino acid residues, and the deduced amino acid sequence shows 93 and 68% homology to that of rabbit liver alpha subunit of PHK and human muscle alpha subunit of PHK, respectively. We identified a missense mutation, a valine substitution for glycine at amino acid 193, in the PHKA2 gene of a family with XLG.

Amino Acid Sequence↗