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PubMed1981

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).

Animals↗
PubMed1981

Phosphorylation and activation of the cardiac isoenzyme of phosphorylase kinase by the cAMP-dependent protein kinase.

The cAMP-dependent protein kinase catalyzes the phosphorylation of the alpha- and beta-subunits of the cardiac isozyme of phosphorylase kinase. beta-Subunit phosphorylation achieves a maximum level of between 1 to 2 mol of phosphate/mol of phosphorylase kinase, a value less than the stoichiometric content of beta-subunits in the enzyme. This, less than stoichiometric incorporation, is not a result of the presence of endogenous phosphate in equivalent sites in the remaining beta-subunit moieties. Pretreatment of phosphorylase kinase with phosphoprotein phosphatase, under conditions proven to dephosphorylate such sites, does not modify the observed extent of beta-subunit phosphorylation. alpha'-Subunit phosphorylation is initiated at a slower rate than beta but achieves a higher maximum level of incorporation. alpha'-Subunit phosphorylation, but not the extent of beta-subunit phosphorylation, is stimulated by MnCl2 and partially inhibited by NaF; neither is effected by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The activation of cardiac phosphorylase kinase that occurs concomitantly with phosphorylation appears to be dependent upon phosphate incorporation into both the alpha- and beta-subunits. At low levels of activation a close correlation is observed between activation and either alpha-subunit phosphorylation, beta-subunit phosphorylation, or total phosphorylation. However, the cAMP-dependent catalyzed phosphorylation of alpha, at a time after which beta-subunit phosphorylation is already maximal, also results in activation of cardiac phosphorylase kinase.

Animals↗
PubMed1984

Histochemical study of phosphorylase in proliferating cells of intestinal metaplasia and carcinoma of the human stomach.

A morphologic histochemical study of phosphorylase was carried out to investigate the relationship between gastric carcinoma and intestinal metaplasia. Intense phosphorylase activity was observed in the carcinoma cells, especially in well-differentiated adenocarcinoma, and in the proliferating cells of some intestinal metaplasias. Metaplastic epithelium other than the proliferating cells occasionally showed a positive reaction. Phosphorylase was negative in normal gastric epithelium, even in its proliferating cells. There was an apparent coincidence between the location of well-differentiated adenocarcinoma and the distribution of intestinal metaplasia, with the proliferating cells showing positive reaction for phosphorylase. These data suggest that the relationship between the proliferating cells of intestinal metaplasia showing phosphorylase activity and well-differentiated adenocarcinoma is apparently closer than the much-debated relationship between the epithelium of intestinal metaplasia and gastric carcinoma.

Adenocarcinoma↗
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↗
PubMed1983

Free energy coupling in the interactions between Ca2+, calmodulin, and phosphorylase kinase.

Interactions between Ca2+, exogenous calmodulin, and white skeletal muscle phosphorylase kinase have been quantitatively studied by equilibrium gel filtrations and analyzed by means of the so-called "linked functions" theory (Weber, G. (1975) Adv. Protein Chem. 29, 1-83). Four moles of calmodulin, each saturated with at least 3 Ca2+ ions, bind to 1 mol of phosphorylase kinase with a Kdiss of 2.3 nM. The activation of the enzyme as a function of free [Ca2+] shows that the intrinsic Ca-binding properties of phosphorylase kinase do not change upon binding of exogenous calmodulin, and confirms that alpha beta gamma delta X Ca3 is the functional catalytic unit through which activation occurs. Direct binding studies reveal that the intrinsic Ca-binding properties of the enzyme remain the same in the presence of either 0.5 or 8 mM Mg2+, indicating that phosphorylase kinase is endowed with Ca-specific sites. Upon interaction with the enzyme, calmodulin acquires strong positive cooperativity in Ca2+-binding: whereas its first two stoichiometric Ca-binding constants are not significantly different from those of free calmodulin, the third Ca2+ ion binds with an affinity at least 10(5)-fold higher than the corresponding one in free calmodulin. Calmodulin liganded with 1 or 2 Ca2+ displays the same low affinity for the enzyme as calmodulin depleted of Ca2+ (approximate Kdiss = 10(-4)-10(-3) M). The alpha beta gamma delta X calmodulin X Ca3 complex is strengthened by a free energy coupling of -8 kcal/mol upon complexation. The quantitative analysis of our results predicts that in spite of this high free energy barrier the dissociation of the complex (i.e. the inactivation of phosphorylase kinase) occurs rapidly upon lowering free [Ca2+].

Animals↗
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↗
PubMedDate not supplied

High uridine phosphorylase activity in human melanoma tumor.

Uridine (Urd) phosphorylase and Urd kinase activities were examined in 4 human tumor types including melanoma and human and mouse melanoma cell lines. Urd phosphorylase activity in melanoma tumor specimens was higher than in specimens of colon, ovarian, and breast tumors. Urd kinase activity levels were similar in the 4 tumor types. In 3 human melanoma cell lines examined, Urd phosphorylase activity was markedly greater than in mouse B16 melanoma cells, while Urd kinase activity did not differ appreciably in the human and mouse cell lines. Urd phosphorylase activity in crude extract preparations from different melanoma cell lines showed similar substrate affinity and sensitivity to 1-(2'-deoxy-beta-D-glucopyranosyl)-thymine, a specific inhibitor. The high Urd phosphorylase activity found in melanoma tumor tissue may be exploited in the treatment of malignant melanoma with antipyrimidine agents. Cultured human melanoma cells retain this biochemical characteristic and may serve as appropriate in vitro models for the human tumor in studies concerning pyrimidine metabolism.

Animals↗
PubMed1981

[Properties of phosphorylase kinase activated by subtilisin].

The activation of phosphorylase kinase during limited proteolysis by subtilisin was studied. It was shown that phosphorylase kinase undergoes rapid activation and its activity remains unchanged throughout a prolonged incubation. Electrophoresis in the presence of Na-SDS revealed a rapid decomposition of the alpha-subunit and a gradual disappearance of the beta-subunit; the protein molecule was shown to be composed of the degradation products of alpha- and beta-subunits with different molecular weights and unchanged proteolysis of the gamma-subunit. The phosphorylase kinase hydrolysate was separated using chromatography on a cellulose phosphate column. The active protein fraction contains a new form of phosphorylase kinase with a low molecular weight (approximately 80 000) which is insensitive to Ca2+. The subtilisin-activated phosphorylase kinase does not affect the activity of phosphodiesterase from cyclic nucleotides.

Animals↗
PubMedDate not supplied

[Corticosteroids and the adrenaline activation of glycogen phosphorylase in heart tissue].

Study of changes in the glycogen phosphorylase activity under the effect of adrenaline in the hearts of intact, adrenalectomized and sham-adrenalectomized rats demonstrated that stress increased the phosphorylase A level considerably. Against the background of these changes perfusion of the heart with 0.5.10(-7)M adrenaline produced no further elevation of the enzyme activity. Following restoration of the basal phosphorylase A to the normal level adrenaline again acquired the capacity to Phosphorylase activation. Stable absence of response of the heart tissue to adrenaline by increase of phosphorylase A activity was noted on the 5th postadrenalectomy day; this apparently followed disturbances in the potassium ions accumulation in the cells.

Adrenal Cortex Hormones↗
PubMed1995

[Delayed activation of muscle glycogen phosphorylase b on incubation of the enzyme with adenosine-5'-monophosphate].

When studying the enzyme activity of glycogen phosphorylase b from rabbit skeletal muscles by the turbidimetric method or by the method based on the determination of inorganic phosphate (the product of the enzymatic reaction) in the direction of glycogen synthesis we observed that 10-15 min preincubation of the enzyme with the allosteric activator (AMP) results in an increase in the initial rate of the enzymatic reaction (nu) in relation to the corresponding value of nu measured by the initiation of the enzymatic reaction by the addition of the mixture of glucose 1-phosphate and AMP (0.02 M Hepes, pH 6.8; 37 degrees C). Glycogen with molecular mass of (264-276).10(6) dalton was used in the kinetic experiments. For 1 mM AMP the time-dependent process of phosphorylase b activation under the action of AMP follows the exponential law with the apparent rate constant of the first order equal to 0.43 min-1 (turbidimetric method of measurement of enzyme activity). When AMP concentration increases, the degree of activation of phosphorylase b reaches a limiting value equal to 1.75 (6 mM glucose 1-phosphate, 0.2 mg/ml glycogen). The activation effect decreases with increasing glycogen concentration and disappears at saturating concentrations of high-molecular weight substrate. Incubation of phosphorylase b with AMP causes the lowering of Michaelis constant for glucose 1-phosphate. It is assumed that enhancement of the rate of the enzymatic reaction catalyzed by phosphorylase b during incubation with AMP is due to association of the enzyme molecules adsorbed to a glycogen particle resulting in an increase in the affinity of the enzyme for glycogen.

Adenosine Monophosphate↗
PubMed1995

[Interaction between glycogen phosphorylase b and creatinine kinase from rabbit skeletal muscle].

Phosphorylase b association with creatine kinase has been studied by frontal elution affinity chromatography, using CNBr-Sepharose 4B immobilized creatine kinase as the affinity matrix. The quantitative parameters of this interaction were estimated from the volumes of phosphorylase b elution at various concentrations of the enzyme. The dissociation constants for phosphorylase b complexes with immobilized creatine kinase and of the phosphorylase b complex with free creatine kinase were found to be equal to 0.49 and 0.191 microM, respectively. In the presence of AMP the interaction between the proteins became weaker. With a rise in AMP concentration from 0.02 to 0.15 mM the value of the dissociation constants increased from 1.59 up to 9.66 microM. One molecule of AMP was shown to bind on the phosphorylase b-immobilized creatine kinase complex.

Adenosine Monophosphate↗
PubMed1996

[Effect of specific ligands on heat inactivation of muscle glycogen phosphorylase b].

It has been shown that the rate constant, k, for thermal inactivation of rabbit skeletal muscle glycogen phosphorylase b decreases as the enzyme concentration increases. This effect is interpreted within the framework of a kinetic model which includes two parallelly occurring processes, namely: phosphorylase b denaturation in solution and denaturation of the enzyme absorbed on test-tube walls. The contribution of the latter process increases with a decrease in the enzyme concentration. The protective effect of the allosteric activator (AMP), allosteric inhibitors (glucose 6-phosphate and FMN) and the competitive inhibitor (glucose) against heat denaturation of glycogen phosphorylase b has been demonstrated. Quantitative analysis of the dependence of the rate constant, k, on concentration of AMP, glucose 6-phosphate and FMN allows the calculation of microscopic constants for dissociation of phosphorylase b complexes with these ligands for the given experimental conditions as equal to 0.34, 0.50 and 0.30 mM, respectively. The S-shaped dependence of the rate constant of thermal inactivation on glucose concentration points to the existence of positive cooperative interactions between glucose-binding sites in the dimeric molecule of phosphorylase b (nH = 1.8).

Allosteric Regulation↗
PubMed1977

Activation of glycogen phosphorylase kinase by a calcium-activated, cyclic nucleotide-independent protein kinase system.

A protein kinase, which was produced from its proenzyme occurring in rat brain upon limited proteolysis by a Ca2+-dependent protease from the same tissue (Inoue, M., Kishimoto, A., Takai, Y., and Nishizlka, Y. (1977) J. Biol. Chem. 252, 7610-7616, was capable of phosphorylating alpha and beta subunits of rabbit skeletal muscle glycogen phosphorylase kinase, resulting in a marked enhancement of the enzymatic activity. This protein kinase was entirely independent of cyclic nucleotides and differed from the catalytic subunit of cyclic AMP-dependent protein kinase. The activation of phosphorylase kinase by this active protein kinase was not inhibited by a protein inhibitor of cyclic AMP-dependent protein kinase, nor by ethylene glycol bis(beta-aminoethyl ether)N',N'-tetraacetic acid, which prevented autophosphorylation of phosphorylase kinase. The proenzyme was distinguishable from cyclic nucleotide-dependent protein kinases, since it did not bind cyclic AMP and cyclic GMP, and was inactive in the phosphorylation and activation of phosphorylase kinase both in the presence and absence of these cyclic nucleotides. Neither the protein kinase nor its proenzyme showed phosphorylase kinase activity. Available evidence indicates that the Ca2+-activated, cyclic nucleotide-independent protein kinase system as well as cyclic AMP-dependent protein kinase shows an ability to stimulate glycogen breakdown as far as tested in vitro.

Animals↗
PubMed1976

Glycogen phosphorylase in Dictyostelium discoideum. I. Purification and properties of the enzyme.

The glycogen phosphorylase of Dictyostelium discoideum has been purified over 200-fold from cells in the culmination stage of development. Analytical gel electrophoresis of the purified enzyme indicates one major protein band with a molecular weight of approximately 210,000. Gel elution verified the presence of phosphorylase activity associated with the protein band. Electrophoresis of partially purified extracts prepared from amoebae cells revealed the absence of phosphorylase protein. Sodium dodecyl sulfate electrophoresis on 6% gels indicated that the purified phosphorylase is composed of subunits, 95,000 in molecular weight. The purified enzyme exhibited normal Michaelis-Menten kinetics and activity was not stimulated by added nucleotides such as 5'-AMP. Nucleotide sugars (GDP-glucose, UDP-glucose, ADP-glucose) were competitive inhibitors of the phosphorylase reaction.

Dictyostelium↗
PubMed1976

Glycogen phosphorylase in Dictyostelium discoideum. II. Synthesis and degradation during differentiation.

A purified preparation of glycogen phosphorylase from Dictyostelium discoideum was used to elicit specific antisera in rabbits. The antisera were used to quantitate the amount of precipitable phosphorylase protein from cell extracts prepared at various stages of the developmental cycle. Following isotope incorporation studies in differentiating cells, the specific radioactivity of enzyme isolated by antibody precipitation was compared to that of acid-insoluble protein. Prior to 5 hours of development, glycogen phosphorylase could not be detected enzymatically or immunologically. Between aggregation and culmination, the rate of enzyme synthesis increased about 6-fold, then decreased to an insignificant value in young sorocarps. The rate of enzyme degradation was negligible during the period of maximal enzyme accumulation, then increased to a peak value of 40% after culmination, coincident with a rapid drop in phosphorylase activity. The data indicated that the increase in glycogen phosphorylase activity during development results from an increase in the rate of enzyme synthesis.

Dictyostelium↗
PubMed1983

Ligand effects on the dephosphorylation of heart and skeletal muscle specific phosphorylases.

Pseudo first order rate constants were determined for the dephosphorylation of heart and skeletal muscle specific phosphorylase a isoenzymes isolated from rabbit and pig using rabbit muscle phosphorylase phosphatase (mol. wt 34,000). The rate constants determined in the absence of ligands, were 4-5 fold lower for heart specific phosphorylases than for skeletal muscle specific ones. Glucose 6-phosphate (0.5-1 mM) enhances the rate of dephosphorylation of heart specific isophosphorylases 3-fold and suspends inhibition by 10(-5) M AMP, however, it has no significant effect on the dephosphorylation of skeletal muscle specific enzymes under the same conditions. Our data support characteristic functional differences between heart and skeletal muscle specific phosphorylases both in rabbit and pig.

Adenosine Monophosphate↗
PubMed1990

The interaction of phosphorylase a with D-glucose displays alpha-stereospecificity.

Half-maximal inhibition of phosphorylase a required a much lower concentration of alpha-D-glucose (4 mM) than of the beta-anomer (14 mM) and of 1-deoxyglucose (about 25 mM). beta-D-Glucose was almost ineffective at concentrations of 1-2.5 mM, but at 50 mM the two anomers were equipotent. A similar picture emerged when the stimulatory effects of the glucose anomers and of 1-deoxyglucose were investigated on the inactivation of phosphorylase by phosphorylase phosphatase. However, upon addition of either glucose anomer (5-20 mM) to a suspension of isolated hepatocytes, the inactivation of phosphorylase occurred at the same rate. It is shown that, in the latter conditions, the rate of intracellular mutarotation considerably exceeds the rate of glucose transport. This results presumably in a rapid anomeric equilibrium in the liver cells.

Animals↗
PubMed1995

Modification of kinetic parameters of glycogen phosphorylase from mantle tissue of Mytilus galloprovincialis by a phosphorylation mechanism.

Initial rate and affinity studies on mantle Mytilus phosphorylase a were carried out in order to find possible differences in its kinetic properties with respect to phosphorylase b. Phosphorylase a was not stimulated for any AMP concentrations. Michaelis constants (Km) are 0.05 mg/ml glycogen, 1.15 mM inorganic phosphate and 1.50 mM glucose-1-phosphate. The Kms for the substrates, in the direction of glycogen breakdown, are enhanced by non-saturating concentrations of cosubstrate, without reducing the apparent maximum velocity. First order and hyperbolic kinetics and values of the allosteric constant smaller than 2 were observed. These results suggest a catalytic mechanism different to that shown for mantle Mytilus phosphorylase b.

Adenosine Monophosphate↗