Rabbit Skeletal Muscle Phosphorylase Kinase Research Articles

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.

Phosphorylation and activation of rabbit skeletal muscle phosphorylase kinase by a cyclic nucleotide- and Ca2+-independent protein kinase.

Phosphorylase kinase from rabbit skeletal muscle can be phosphorylated and activated by a cyclic nucleotide- and Ca2+-independent protein kinase previously identified as a glycogen synthase kinase (Itarte, E., and Huang, K.-P. (1979) J. Biol. Chem. 254, 4052-4057). This independent kinase phosphorylates the beta subunit of phosphorylase kinase approximately 15 times faster than it does the alpha subunit. The cAMP-dependent and -independent kinases separately catalyze the incorporation of 1 mol of phosphate into the beta subunit. Analyses of the tryptic peptides from the beta subunit phosphorylated with either kinase by isoelectric focusing and peptide mapping indicate that both kinases phosphorylate the same site on the beta subunit. Activation of phosphorylase kinase catalyzed by the independent kinase is only 60% of that observed with cAMP-dependent kinase. If phosphorylase kinase is first incubated with the independent kinase to phosphorylate the beta subunit, subsequent addition of cAMP-dependent kinase results in a predominant phosphorylation of the alpha subunit. This additional phosphorylation of the alpha subunit is accompanied by a further activation of the alpha subunit is accompanied by a further activation of phosphorylase kinase to the same extent as that achieved by cAMP-dependent kinase alone. Hence, the phosphorylation of the alpha subunit is clearly required for full activation of phosphorylase kinase, even at low [Mg2+].

The regulatory role of Mg 2+ on rabbit skeletal muscle phosphorylase kinase

The stimulation of phosphorylase kinase by Mg 2+ was studied. Both the nonactivated and activated kinases are stimulated by Mg 2+ at concentrations that are 100- to 200-fold greater than ATP. This stimulation is observed at both pH 6.8 and 8.2 and results in a 10-fold increase in the activity of the nonactivated kinase. Mg 2+ stimulation is additive with that observed by calmodulin. Both the Ca 2+-dependent and -independent activities of the kinase are stimulated by high [Mg 2+]. Kinetically this stimulation can be explained by a decrease in the K m for both phosphorylase b and ATP or an increase in V. The pH 6.8 8.2 ratio (0.06) is unaffected by [Mg 2+] between 5 and 20 m m, but increases when [Mg 2+] is less than 5 m m or greater than 20 m m. The stimulation by high [Mg 2+] is explained by a direct effect of this cation on the kinase molecule rather than on its protein substrate, phosphorylase. This activating effect of high [Mg 2+] does not result in any permanent change in the kinase molecule and can be readily reversed by diluting [Mg 2+] to a low value.

Rabbit skeletal muscle phosphorylase kinase. Interactions between subunits and influence of calmodulin on different complexes.

Aspects of the molecular interaction and subunit structure of rabbit skeletal muscle phosphorylase kinase, (alpha beta gamma delta)4, were investigated. Exogenous addition of the delta subunit (calmodulin) stimulated the activities of nonactivated phosphorylase kinase and the alpha gamma delta complex, but not the gamma delta complex. This stimulatory effect does not seem to affect the activity-pH profile of the native kinase and is expressed at least partly through an interaction of the calmodulin with the alpha subunit, resulting in an increase in the apparent Vm parameters. Low concentrations of trifluoperazine had only slight effect on the activities of the three forms of kinase, whereas high concentrations caused nonspecific inactivation. The effects of trifluoperazine and EGTA on the activities of the alpha gamma delta and gamma delta complexes were additive; together, they inactivated the two complexes to about 10% of their original activities. The delta subunit remained tightly bound to phosphorylase kinase and the alpha gamma delta complex, even in the presence of 8 M urea, but less so with the gamma delta complex. The nonactivated kinase is more stable towards heat inactivation at 37 degrees C than the alpha gamma delta complex, whereas the gamma delta complex is least stable. Similar to the holoenzyme, limited trypsin digestion activated the alpha gamma delta complex. The pH 6.8/8.2 activity ratio of this complex increased from 0.5 to 0.9, with concomitant degradation of the alpha subunit. In contrast, the gamma delta complex is fairly inert to limited proteolysis. By using sucrose density gradient ultracentrifugation and pretreatment of the gamma delta complex with EGTA, results indicated that the gamma subunit may be a Ca2+-independent kinase, which has about 30% of the original phosphorylase kinase activity.

Rabbit skeletal muscle phosphorylase kinase. Catalytic and regulatory properties of the active alpha gamma delta and gamma delta complexes.

Rabbit skeletal muscle phosphorylase kinase. Catalytic and regulatory properties of the active alpha gamma delta and gamma delta complexes.

The activation of rabbit skeletal muscle phosphorylase kinase requires the binding of 3 Ca 2+ per δ subunit

Rabbit skeletal muscle phosphorylase kinase (EC 2.7.1.38) binds 12 Ca 2+, i.e. 3 per δ subunit; among these three Ca-binding sites, one has a high affinity (K diss = 0.31 μM) and two have a lower affinity (K diss = 2.15 μM). Thus, the binding of calcium to the enzyme-bound calmodulin, called δ subunit, is different from the binding to free calmodulin. The activation of phosphorylase kinase occurs when three Ca 2+ are bound to the δ subunit.

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.

Dephosphorylation of rabbit skeletal muscle phosphorylase kinase. Evidence against the operation of the “second-site phosphorylation” mechanism of regulation.

The dephosphorylation of rabbit skeletal muscle phosphorylase kinase was studied using two purified rabbit skeletal muscle protein phosphatases. The first enzyme (Mr = 32,000) corresponds to the form we have previously termed protein phosphatase C. Phosphorylase kinase was found to be rapidly dephosphorylated by this enzyme. The site of dephosphorylation was examined, and it was shown that this enzyme was relatively specific for the dephosphorylation of the beta-subunit phosphate, as compared to the alpha-subunit phosphate, of phosphorylase kinase. Phosphate release from the beta-subunit was approximately 100-fold faster than from the alpha-subunit. More importantly, dephosphorylation of the beta-subunit phosphate was not significantly affected by phosphorylation of the alpha-subunit. The dephosphorylation of phosphorylase kinase by a second low molecular weight protein phosphatase, Mr = 33,500, was also studied. The specific activity of this enzyme toward phosphorylase kinase was only a fraction of that exhibited by the Mr = 32,000 phosphatase. This enzyme removed phosphate from both the alpha- and beta-subunits but more rapidly (about 4-fold) from the alpha-subunit. With neither of these enzyme preparations was there any evidence for the regulation of beta-subunit dephosphorylation by phosphorylation of the alpha-subunit as proposed by Cohen and Antoniw ((1973) FEBS Lett. 34, 43-47).

Differential interaction of rabbit skeletal muscle phosphorylase kinase isozymes with calmodulin.

Rabbit skeletal muscle contains two phosphorylase kinase isozymes arising from the two different muscle types, the white and the red muscle (Jennissen, H. P., and Heilmeyer, L. M. G. (1974) FEBS Lett. 42, 77-80). The two phosphorylase kinase isozymes could be separated by affinity chromatography on a calmodulin-Sepharose 4B column. In media containing high concentrations of Ca2+, about 2 mM, both isozymes were bound to the affinity column. When the column was eluted with a buffer containing 0.2 mM Ca2+, the red muscle isozyme was eluted, whereas white muscle isozyme was eluted from the column by an ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid. The purified white muscle isozyme can be distinguished from the red muscle isozyme by its ability to inhibit calmodulin-dependent cyclic nucleotide phosphodiesterase. The two isozymes are also regulated differently by calmodulin. Both isozymes contain tightly bound calmodulin as a subunit (Cohen, P., Burchell, A., Foulkes, J. G., Cohen, P. T. W., Vanaman, T. C., and Nairn, A. C. (1978) FEBS Lett. 92, 287-293), which renders the enzyme sensitive to Ca2+. Only the white muscle isozyme can be activated by exogenous calmodulin.

Phosphorylation of rabbit skeletal muscle phosphorylase kinase by cyclic GMP-dependent protein kinase

Phosphorylation of rabbit skeletal muscle phosphorylase kinase by cyclic GMP-dependent protein kinase

Rabbit skeletal muscle phosphorylase kinase. Comparison of glycogen synthase and phosphorylase as substrates.

Rabbit skeletal muscle phosphorylase kinase. Comparison of glycogen synthase and phosphorylase as substrates.

Identification of the Ca 2+-dependent modulator protein as the fourth subunit of rabbit skeletal muscle phosphorylase kinase

Kakuichi et al. [l] and Cheung [2,3] were the first to demonstrate the presence of a factor in brain homogenates, which in the presence of Ca”‘, stimulated the activity of one of the cyclic nucleotide phosphodiesterases of this tissue. This factor was subsequently shown to be a small heat stable calcium binding protein, which was present in high concentrations in a wide variety of animal tissues [4-61. Following its purification to apparent homogeneity from bovine brain [7] and bovine heart [8] , it was noted that its physico-chemical properties were very similar to the calcium-binding subunit of rabbit skeletal muscle troponin, troponln-C, the protein which confers calcium sensitivity to actomyosin ATPase [9,10] . This idea was substantiated by the determination of the amino acid sequence of the ‘calcium-dependent modulator’ from bovine brain [ 1 l] and rat testis [ 121, which showed extensive homology with troponin-C, and by the finding that the ‘modulator’ could substitute for troponin-C in restoring calcium sensitivity to actomyosin ATPase in reconstituted systems [ 131. Troponin-C can also substitute for the ‘modulator’ in the activation of cyclic nucleotide

Postnatal development of phosphorylase kinase in mouse skeletal muscle

The postnatal development of phosphorylase kinase activity was studied in mouse (strain Ha ICR ) skeletal muscle, and the properties of the enzymes from skeletal muscle of 6- to 10-day-old and adult mice were compared. Phosphorylase kinase activity in skeletal muscle was found to increase more than 12-fold during postnatal development with over 80% of the increase occurring after the second week of life. Comparison of the phosphorylase kinase activity in muscle from 6- to 10-day-old and adult mice showed the two enzymes to have similar kinetic properties, but three lines of evidence indicated that they have different structures: (i) They differed in their cross-reactivity in quantitative immunotitrations using antisera prepared to purified rabbit skeletal muscle phosphorylase kinase. (ii) They differed in their heat lability at 42.5 °C. (iii) They differed in their subunit structure as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The phosphorylase kinase from muscle of 6- to 10-day-old mice had only the α′,β, and γ subunits, while the enzyme from adults showed α,α′,β, and γ subunits. These results indicate that skeletal muscle of adult mice has a different phosphorylase kinase isozyme(s) from that present in muscle of newborn mice.

Purification and properties of debranching enzyme from dogfish muscle.

Glycogen debranching enzyme (4-alpha-glucanotransferase amylo-1,6-glucosidase, EC 2.4.1.25 + 3.2.1.33) was purified 140-fold from dogfish muscle in a rapid, high-yield procedure that takes advantage of a strong binding of the enzyme to glycogen, and its quantitative adsorption to concanavalin A-Sepharose only when the polysaccharide is present. The final product was hrophoresis in the presence and absence of dodecyl sulfate. A molecular weight of 162,000 +/- 5000 was determined by sedimentation equilibrium analysis in good agreement with the value of 160,000 estimated by gel electrophoresis, but a low-sedimentation constant of 6.5 S suggests that the enzyme is asymmetric. The molecule appears to be made up of a single polypeptide chain with no evidence for multiple repeating sequences: it could not be dissociated into smaller fragments by dodecyl sulfate even after complete carboxymethylation; tryptic cleavage of the native protein yielded only two fragments of molecular weight 20,000 and 140,000 without loss of enzymatic activity. The amino acid composition of the enzyme is reported; no covalently bound phosphate or carbohydrate could be detected. All 32 sulfhydryl groups present were titrated with 5,5'-dithiobis(2-nitrobenzoic acid) under denaturing conditions; eight reacted readily in the native enzyme without loss of catalytic activity, while substitution of eight additional ones lowered the activity by 50%. Inactivation was greatly reduced by glycogen; the polysaccharide also influenced markedly the electrophoretic behavior of the enzyme and large filamentous aggregates were formed when solutions of both were mixed. Purified debranching enzyme releases 3 mumol of glucose min-1 mg-1 at 19 degrees C, pH 6.0, from a glycogen limit dextrin and one-tenth this amount when the native polysaccharide is used as substrate; glycogen is quantitatively degraded in the presence of phosphorylase. None of the usual sugar phosphates or nucleotide effectors of glycolysis affected enzymatic activity. No phosphorylation by either dogfish or rabbit skeletal muscle protein kinase or phosphorylase kinase could be demonstrated, nor any direct interaction with phosphorylase as measured by SH-group reactivity, enzymatic activity, or rate of phosphorylase b to a conversion. Purification of the 160,000 molecular weight M-line protein of skeletal muscle resulted in the quantitative removal of debranching enzyme, indicating that the two proteins are different.

Effect of Mg2+ concentration on the cAMP-dependent protein kinase-catalyzed activation of rabbit skeletal muscle phosphorylase kinase.

Phosphorylase kinase was found to be activated and phosphorylated at 10mM Mg2+ by the cAMP-dependent protein kinase-catalyzed reaction ot much higher levels than observed previously when reactions were carried out in 1 to 2 mM Mg2+ (Cohen, P. (1973) Eur. J. Biochem. 34, 1; Hayakawa, T., Perkin, J.P., and Krebs, E.G. (1973) Biochemistry 12, 574). That the reaction at 10 mM Mg2+ is protein kinase-catalyzed is supported by several observations: (a) the reaction is facilitated by the addition of protein kinase; (b) the reaction depends on cAMP when protein kinase holoenzyme is uded; (c) the reaction is not inhibited by 1 mM ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetate which is known to inhibit autoactivation and autophosphorylation of phosphorylase kinase; and (d) the protein inhibitor of protein kinase inhibits this reaction. The phosphorylation and activation of phosphorylase kinase seem to occur in two phases. At low Mg2+ only the first phase is manifested and involves the incorporation of 2 mol of phosphate, 1 mol into each of Subunits A and B. At high Mg2+ additional sites are phosphorylated almost exclusively on Subunit A, with phosphate incorporation approaching the final level of 7 to 9 mol. Enzyme activity at high Mg2+ is 2 to 3 times higher than that observed when activation is studied at low Mg2+. The observation that both casein and type II histone are phosphorylated to the same extent at 1 mM and 10 mM Mg2+ suggested that high Mg2+ may be altering the conformation of phosphorylase kinase thus rendering more phosphorylation sites accessible to protein kinase. Since the phosphorylation of phosphorylase kinase by either the protein kinase-catalyzed or autocatalytic reaction can result in the incorporation of 7 to 9 mol of phosphate, the finding that only about seven sites become phosphorylated by both mechanisms acting together suggest that activation by these two mechanisms may involve common phosphorylation sites.

Demonstration of the Control of Rabbit Skeletal-Muscle Phosphorylase Kinase Activity in vivo by Adenosine 3′5′-Cyclic Monophosphate-Dependent Protein Kinase

Rabbit skeletal-muscle phosphorylase kinase has an activity at physiological pH (6.8) only 1-2% of that observed at the pH optimum (8.2), but may be activated @fold at pH6.8 after incubation with cyclic AMP-dependent protein kinase, cyclic AMP and ATP-MgZ+ in vitro (Walsh et al., 1971 ; Cohen, 1973). Two molecules of covalently bound phosphate are incorporated during the reaction. The phosphorylation at the primary site on the 8-subunit parallels the increase in activity (Hayakawa et al., 1973; Cohen, 1973), whereas the slower phosphorylation of a second site, on the a-subunit, controls the rate of dephosphorylation of the 8-subunit catalysed by phosphorylase kinase phosphatase (Cohen & Antoniw, 1973). The specificity of this reaction in vitro has been confirmed by the isolation of a nine-residue tryptic phosphopeptide from the 8-subunit (Cohen, 1974) and a 39-residue tryptic phosphopeptide from the a-subunit (P. Cohen, unpublished work). Although phosphorylase kinase activity previously has been shown to increase after an intravenous injection of adrenalin (Posner et al., 1965; Drummond et al., 1969), this need not necessarily be a consequence of the activation of cyclic AMP-dependent protein kinase. Phosphorylase kinase activity can also be stimulated by two further mechanisms in vitro, by a calcium-dependent phosphorylation of phosphorylase kinase by itself (Walsh et al., 1971) and by proteolysis (Huston & Krebs, 1968). In the present paper, phosphorylase kinase was activated by an intravenous injection of adrenalin, and purified through the (NH4)2S04 step by the procedure previously described for the non-activated enzyme (Cohen, 1973). The pH6.8/8.2 activity ratio in the muscleextract was0.22 incontrast with thevalueof0.03f0.01 observedin theabsenceof adrenalin. NaF (50m~) was included in the purification to inhibit phosphorylase kinase phosphatase, but there was nevertheless a slight decline of the activity ratio to 0.16 during the 8 h isolation. Phosphorylase kinase (1 .Oms), which had been phosphorylated in vitro at both the aand 8-subunit sites, was then added to phosphorylase kinase activated tn vivo (200mg), and the aand 8-subunit phosphopeptides were isolated by using the trace of radioactivemarker protein to locate the position of the peptides and to calculate the yield at each step.

On the hysteretic response of rabbit skeletal muscle phosphorylase kinase.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTHysteretic response of rabbit skeletal muscle phosphorylase kinaseGiho Kim and Donald J. GravesCite this: Biochemistry 1973, 12, 11, 2090–2095Publication Date (Print):May 1, 1973Publication History Published online1 May 2002Published inissue 1 May 1973https://doi.org/10.1021/bi00735a011RIGHTS & PERMISSIONSArticle Views40Altmetric-Citations25LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (584 KB) Get e-Alerts Get e-Alerts

Activation of Skeletal Muscle Phosphorylase Kinase by Adenosine Triphosphate and Adenosine 3′,5′-Monophosphate

Abstract Rabbit skeletal muscle phosphorylase kinase has been obtained as a nearly homogeneous protein showing a single peak on electrophoresis and in the ultracentrifuge. Incubation of the purified kinase with γ32P-ATP in the presence of Mg++ ions activates the enzyme; this process is accompanied by phosphorylation of the protein. Activation and phosphorylation of phosphorylase kinase occur more rapidly when cyclic 3',5'-AMP or glycogen is present in activation reaction mixtures, but these two substances apparently act by different mechanisms since their effects are additive. Cyclic 3',5'-AMP and glycogen stimulate activation of the kinase more than they affect phosphorylation. No evidence was obtained to indicate that cyclic 3',5'-AMP undergoes any modification when it exerts its effect on phosphorylase kinase activation. Activation of the kinase was shown to be autocatalytic, but the possibility that cyclic 3',5'-AMP acts through stimulation of a second enzyme involved in the system was suggested by the failure to find any evidence for a significant degree of binding of this nucleotide to purified phosphorylase kinase.

Reversal of Phosphorylase Kinase Activation

Abstract When purified rabbit skeletal muscle phosphorylase kinase is activated by preliminary incubation with γ-32P-ATP in the presence of Mg++ ions, it incorporates covalently bound 32P. All of the 32P is bound as alkali-labile phosphate, presumably on seryl residues. Serine phosphate has been isolated after partial acid hydrolysis of the labeled enzyme. Skeletal muscle and other tissues contain a phosphatase that catalyzes the dephosphorylation of activated phosphorylase kinase, and it has been shown that this reaction is accompanied by reversal of activation. The phosphatase, tentatively designated phosphorylase kinase phosphatase, has a different tissue distribution profile than the phosphatase which catalyzes dephosphorylation of p-nitrophenyl phosphate and casein. Phosphorylase kinase phosphatase is activated by metal ions and is inhibited by sodium fluoride. When the phosphatase acts on activated phosphorylase kinase, various phosphorylated sites appear to be attacked at different rates.