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PubMed1994

A historical view of protein kinase CK2.

The enzyme termed nowadays protein kinase CK2 was first described in liver extracts (as a mixture with protein kinase CK1), using casein as artificial substrate, by Burnett and Kennedy (1954). In 1960 it was shown that such casein/phosvitin phosphorylating activity was ubiquitous and distinct from phosphorylase kinase, i.e., the only other protein kinase known at that time. CK1 and CK2 were distinguished from each other at the end of the sixties, and during the seventies CK2 was purified to homogeneity in several laboratories and thoroughly characterized as far as its subunit structure (alpha 2 beta 2), site specificity, and in vitro responsiveness to various effectors were concerned. The first endogenous substrate for CK2 (eIF-3) was described in 1976, but it was during the eighties that it became clear that CK2 is a pleiotropic protein kinase committed with the phosphorylation of a myriad of cellular targets. More than 100 CK2 substrates are known, sharing typical phosphoacceptor sites specified by multiple acidic residues on the C terminal side of Ser/Thr. The definition of the primary structure of CK2 catalytic subunit, in 1987, definitely included CK2 in the big family of eukariotic protein kinases. The growing interest for CK2 is accounted for by its unusual properties, by the increasing number of its substrates, and by several coincidental arguments suggesting that this pleiotropic protein kinase plays a fundamental role in cellular regulation. A major and intriguing problem concerning CK2 is its apparent lack of regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

Activation and inactivation of glycogen phosphorylase isoenzymes purified from diabetic rat heart.

1. Hearts of diabetic rats gradually accumulate glycogen, although the activities of glycogen synthase and glycogen phosphorylase are altered in favor of a depletion of glycogen. 2. Phosphorylase in diabetic hearts has been reported to be even more activated in response to adrenaline than controls. 3. The situation is further complicated by the fact that in rat heart two isoenzymes of phosphorylase are present. Therefore we have studied the properties of phosphorylases purified from diabetic rat heart in more detail. 4. This investigation revealed that compared to controls: (A) the amount of enzyme protein which could be isolated from diabetic animals is drastically lower; (B) the affinities towards glycogen and inorganic phosphate are decreased; (C) the activation by phosphorylase kinase is delayed; and (D) the inactivation by protein phosphatase-1 is accelerated. 5. We conclude that all of the reported changes in diabetes might contribute to a phosphorylase system less able to catalyze glycogen breakdown effectively.

Animals↗
PubMed1979

Glycogen synthesis by rat hepatocytes.

1. Hepatocytes from starved rats or fed rats whose glycogen content was previously depleted by phlorrhizin or by glucagon injections, form glycogen at rapid rates when incubated with 10mM-glucose, gluconeogenic precursors (lactate, glycerol, fructose etc.) and glutamine. There is a net synthesis of glucose and glycogen. 14C from all three types of substrate is incorporated into glycogen, but the incorporation from glucose represents exchange of carbon atoms, rather than net incorporation. 14C incorporation does not serve to measure net glycogen synthesis from any one substrate. 2. With glucose as sole substrate net glucose uptake and glycogen deposition commences at concentrations of about 12--15mM. Glycogen synthesis increases with glucose concentrations attaining maximal values at 50--60mM, when it is similar to that obtained in the presence of 10mM glucose and lactate plus glutamine. 3. The activities of the active (a) and total (a+b) forms of glycogen synthase and phosphorylase were monitored concomitant with glycogen synthesis. Total synthase was not constant during a 1 h incubation period. Total and active synthase activity increased in parallel with glycogen synthesis. 4. Glycogen phosphorylase was assayed in two directions, by conversion of glycose 1-phosphate into glycogen and by the phosphorylation of glycogen. Total phosphorylase was assyed in the presence of AMP or after conversion into the phosphorylated form by phosphorylase kinase. Results obtained by the various methods were compared. Although the rates measured by the procedures differ, the pattern of change during incubation was much the same. Total phosphorylase was not constant. 5. The amounts of active and total phosphorylase were highest in the washed cell pellet. Incubation in an oxygenated medium, with or without substrates, caused a prompt and pronounced decline in the assayed amounts of active and total enzyme. There was no correlation between phosphorylase activity and glycogen synthesis from gluconeogenic substrates. With fructose, active and total phosphorylase activities increased during glycogen syntheses. 6. In glycogen synthesis from glucose as sole substrate there was a decline in phosphorylase activities with increased glucose concentration and increased rates of glycogen deposition. The decrease was marked in cells from fed rats. 7. To determine whether phosphorolysis and glycogen synthesis occur concurrently, glycogen was prelabelled with [2-3H,1-14C]-galactose. During subsequent glycogen deposition there was no loss of activity from glycogen in spite of high amounts of assayable active phosphorylase.

Animals↗
PubMed1979

Mechanisms involved in alpha-adrenergic effects of catecholamines on liver metabolism.

Recent results have indicated that alpha-adrenergic receptors are the major mediators of catecholamine actions on liver metabolism in several species. It is well-established that cAMP and cAMP-dependent protein kinase are not involved in hepatic alpha-adrenergic effects. This review presents evidence that alpha-adrenergic stimulation of glycogenolysis in rat liver involves the mobilization of Ca2+ ions from mitochondria and stimulation of phosphorylase kinase by the resulting increase in cytosolic Ca2+ concentration. Possible mechanisms by which activation of alpha-adrenergic receptors causes release of mitochondrial Ca2+ and affects other cell processes are discussed.

Animals↗
PubMed1984

Isolation and partial characterization of two forms of rat heart glycogen phosphorylase.

Rat heart glycogen phosphorylase a has been purified to apparent homogeneity by a procedure involving precipitation with ammonium sulfate, DEAE-Sephacel chromatography, and AMP-Sepharose affinity chromatography. In contrast to the skeletal muscle enzyme which appeared as a single peak upon ion-exchange chromatography, heart phosphorylase was separated into two distinct peaks (I and II), both only active in the presence of AMP. The isoelectric points of skeletal muscle phosphorylase b (dephosphorylated form) and heart phosphorylase Ib were both at 6.2, that of heart phosphorylase IIb was 5.2. The Km of phosphorylase IIb for AMP was threefold lower (7 microM) than that of Ib (22 microM). The dissociation constant K8 of phosphorylase Ia (phosphorylated form) was 0.37 mM for glycogen and 5.6 mM for Pi. Increasing the levels of glycogen decreased the apparent Km for Pi and vice versa. No such interaction between substrate binding was observed with phosphorylase IIa, since the Ks values for glycogen (0.27 mM) and Pi (3.8 mM) were not significantly influenced by increasing concentrations of the other substrate. The specific activity of both isoenzymes was 46 units/mg of protein at pH 6.8 and 30 degrees C. The subunit Mr of both forms was 92,500. By incubation with purified phosphorylase kinase and [gamma-32P]ATP both forms were converted to their corresponding forms by 32P incorporation of 1 mol/mol of subunit. This suggests the existence of two native forms of phosphorylase b in rat heart.

Animals↗
PubMedDate not supplied

In situ 13C NMR quantification of hepatic glycogen.

We report on the 13C NMR visibility of the C-1 glycosidic carbon of alpha-particulate glycogen in perfused rat liver. We used rats fed ad libitum, animals refed after a 48 h fast with a sucrose supplement with or without glucocorticoid treatment, and gsd/gsd rats with a hepatic glycogen storage disease due to phosphorylase kinase deficiency. Thus we studied a wide range of glycogen levels (25-140 mg/g liver). All livers were perfused with 15 mM glucose, to maintain constant glycogen levels. Failure to activate glycogen phosphorylase ensures stable glycogen levels in gsd/gsd livers. Natural abundance 13C NMR signals were calibrated against a phantom containing a fixed amount of glycogen. Accumulated free induction decays were analysed after Fourier transformation by numerical integration, or by direct analysis of the signal in the time domain using a non-iterative method based on singular value decomposition. NMR quantification of the glycogen correlated well with the chemical determination over the whole concentration range. However, the precision (reproducibility) of glycogen determinations (be it by chemical methods or by NMR spectroscopy) may pose problems. Authors should be encouraged to report systematically on the precision of their methods.

Animals↗
PubMed1987

Purification and properties of polycation-stimulated phosphorylase phosphatases from rabbit skeletal muscle.

Four types of polycation-stimulated (PCS) phosphorylase phosphatases have been isolated from rabbit skeletal muscle. They are called PCSH (390 kDa), PCSM (250 kDa), and PCSL (200 kDa) phosphatase according to the apparent molecular weight of the native enzymes in gel filtration. Two forms of PCSH phosphatase could be separated by Mono Q fast protein liquid chromatography: PCSH1 and PCSH2. In the absence of polycations, the specific activities of the PCSH1, PCSH2, PCSM, and PCSL phosphatase were 400, 680, 600, and 3000 units/mg, respectively, using phosphorylase a as a substrate. They all contain a 62-65- and a 35-kDa subunit, the latter being the catalytic subunit. In addition PCSH1 phosphatase contains a 55-kDa subunit and the PCSM phosphatase a 72-75-kDa subunit in a substoichiometric ratio. All the PCS phosphatases are insensitive to Ca2+ calmodulin, inhibitor-1, and modulator protein. They display a high specificity for the alpha-subunit of phosphorylase kinase and a broad substrate specificity. The PCSH1 and PCSH2 phosphatases, but not the catalytic subunit (PCSC phosphatase), show a high degree of specificity for the deinhibitor protein. During the purification the phosphorylase to inhibitor-1 phosphatase activity ratio (10:1) remained constant for the PCSH and PCSL enzymes but decreased for the PCSM phosphatase. The stimulation observed with low concentrations of polycations is enzyme directed. The different enzyme forms show a characteristic concentration optimum and degree of stimulation. At higher concentrations, polycations become inhibitory and a time-dependent deactivation of the phosphatases is observed.

Animals↗
PubMed1997

Intermolecular tuning of calmodulin by target peptides and proteins: differential effects on Ca2+ binding and implications for kinase activation.

Ca(2+)-activated calmodulin (CaM) regulates many target enzymes by docking to an amphiphilic target helix of variable sequence. This study compares the equilibrium Ca2+ binding and Ca2+ dissociation kinetics of CaM complexed to target peptides derived from five different CaM-regulated proteins: phosphorylase kinase. CaM-dependent protein kinase II, skeletal and smooth myosin light chain kinases, and the plasma membrane Ca(2+)-ATPase. The results reveal that different target peptides can tune the Ca2+ binding affinities and kinetics of the two CaM domains over a wide range of Ca2+ concentrations and time scales. The five peptides increase the Ca2+ affinity of the N-terminal regulatory domain from 14- to 350-fold and slow its Ca2+ dissociation kinetics from 60- to 140-fold. Smaller effects are observed for the C-terminal domain, where peptides increase the apparent Ca2+ affinity 8- to 100-fold and slow dissociation kinetics 13- to 132-fold. In full-length skeletal myosin light chain kinase the inter-molecular tuning provided by the isolated target peptide is further modulated by other tuning interactions, resulting in a CaM-protein complex that has a 10-fold lower Ca2+ affinity than the analogous CaM-peptide complex. Unlike the CaM-peptide complexes, Ca2+ dissociation from the protein complex follows monoexponential kinetics in which all four Ca2+ ions dissociate at a rate comparable to the slow rate observed in the peptide complex. The two Ca2+ ions bound to the CaM N-terminal domain are substantially occluded in the CaM-protein complex. Overall, the results indicate that the cellular activation of myosin light chain kinase is likely to be triggered by the binding of free Ca2(2+)-CaM or Ca4(2+)-CaM after a Ca2+ signal has begun and that inactivation of the complex is initiated by a single rate-limiting event, which is proposed to be either the direct dissociation of Ca2+ ions from the bound C-terminal domain or the dissociation of Ca2+ loaded C-terminal domain from skMLCK. The observed target-induced variations in Ca2+ affinities and dissociation rates could serve to tune CaM activation and inactivation for different cellular pathways, and also must counterbalance the variable energetic costs of driving the activating conformational change in different target enzymes.

Amino Acid Sequence↗
PubMed1991

Modulation of maximal glycogenolysis in perfused rat liver by adenosine and ATP.

Rat livers perfused at constant flow via the portal vein with dibutyryl cyclic AMP produced glucose equivalents at a steady maximal rate (6 mumol/min per g of liver). Addition of adenosine (150 microM) caused a biphasic effect. (i) First, the glycogenolytic rate rose transiently, to a mean peak of 150% of control levels after 2 min. This glycogenolytic burst was reproduced by two P1-receptor agonists, but not by ATP, and was blocked by a P1-antagonist (8-phenyltheophylline), as well as by inhibitors of eicosanoid synthesis (indomethacin, ibuprofen or aspirin). It did not occur in phosphorylase-kinase-deficient livers. The adenosine-induced glycogenolytic burst coincided with moderate and transient changes in portal pressure (+6 cmH2O) and O2 consumption (-20%), but it could not be explained by an increase in cytosolic Pi, since the n.m.r. signal fell precipitously. (ii) Subsequently, the rate of glycogenolysis decreased to one-third of the preadenosine value, in spite of persistent maximal activation of phosphorylase. The decrease could be linked to the decline in cytosolic Pi: both changes were prevented by the adenosine kinase inhibitor 5-iodotubercidin, whereas they were not affected by ibuprofen or 8-phenyltheophylline, and were not reproduced by non-metabolized adenosine analogues. In comparison with adenosine, ATP caused a slower decrease of Pi and of glycogenolysis. The fate of the cytosolic Pi was unclear, especially with administered ATP, which did not increase the n.m.r.-detectable intracellular ATP.

Adenosine↗
PubMed1986

The protein phosphatases involved in cellular regulation. Evidence that dephosphorylation of glycogen phosphorylase and glycogen synthase in the glycogen and microsomal fractions of rat liver are catalysed by the same enzyme: protein phosphatase-1.

Glycogen synthase (labelled in sites-3) and glycogen phosphorylase from rabbit skeletal muscle were used as substrates to investigate the nature of the protein phosphatases that act on these proteins in the glycogen and microsomal fractions of rat liver. Under the assay conditions employed, glycogen synthase phosphatase and phosphorylase phosphatase activities in both subcellular fractions could be inhibited 80-90% by inhibitor-1 or inhibitor-2, and the concentrations required for half-maximal inhibition were similar. Glycogen synthase phosphatase and phosphorylase phosphatase activities coeluted from Sephadex G-100 as broad peaks, stretching from the void volume to an apparent molecular mass of about 50 kDa. Incubation with trypsin decreased the apparent molecular mass of both activities to about 35 kDa, and decreased their I50 for inhibitors-1 and -2 in an identical manner. After tryptic digestion, the I50 values for inhibitors-1 and -2 were very similar to those of the catalytic subunit of protein phosphatase-1 from rabbit skeletal muscle. The glycogen and microsomal fractions of rat liver dephosphorylated the beta-subunit of phosphorylase kinase much faster than the alpha-subunit and dephosphorylation of the beta-subunit was prevented by the same concentrations of inhibitor-1 and inhibitor-2 that were required to inhibit the dephosphorylation of phosphorylase. The same experiments performed with the glycogen plus microsomal fraction from rabbit skeletal muscle revealed that the properties of glycogen synthase phosphatase and phosphorylase phosphatase were very similar to the corresponding activities in the hepatic glycogen fraction, except that the two activities coeluted as sharp peaks near the void volume of Sephadex G-100 (before tryptic digestion). Tryptic digestion of the hepatic glycogen and microsomal fractions increased phosphorylase phosphatase about threefold, but decreased glycogen synthase phosphatase activity. Similar results were obtained with the glycogen plus microsomal fraction from rabbit skeletal muscle or the glycogen-bound form of protein phosphatase-1 purified to homogeneity from the same tissue. Therefore the divergent effects of trypsin on glycogen synthase phosphatase and phosphorylase phosphatase activities are an intrinsic property of protein phosphatase-1. It is concluded that the major protein phosphatase in both the glycogen and microsomal fractions of rat liver is a form of protein phosphatase-1, and that this enzyme accounts for virtually all the glycogen synthase phosphatase and phosphorylase phosphatase activity associated with these subcellular fractions.

Animals↗
PubMed1993

Glycogen phosphorylase activity in the liver of the frog Rana esculenta.

1. Phosphorylase activity has been assayed in liver extracts of the frog, Rana esculenta, during the winter period. In native conditions, most of the phosphorylase is present as AMP-independent activity and shows properties similar to those of the a form of the liver enzyme from other vertebrates. 2. It is suggested that regulation of phosphorylase activity is through interconversion between a and b forms operated by endogenous phosphorylase kinase and phosphatase. 3. Kinetic studies show hyperbolic saturation curves for glycogen with apparent Km of 2.91 mM and 9.67 mM for a and b forms, respectively. 4. A hyperbolic saturation curve is also observed for glucose 1-P in the case of phosphorylase a, with an apparent Km of 3.95 mM, whereas a sigmoidal kinetic is shown by the b form for the same substrate; from Hill plots an S0.5 of 24.2 mM was derived. 5. Hyperbolic responses were observed in the case of AMP, and Ka of 70 microM and 0.31 mM were calculated for phosphorylase a and b, respectively.

Adenosine Monophosphate↗