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At least 19 recordsLinked to original sources
PubMed1979

The interplay between covalent and non-covalent regulation of glycogen phosphorylase. The role of different effectors of phosphorylase b on the phosphorylase b to a conversion rate.

Glycogen phosphorylase b is converted to glycogen phosphorylase a, the covalently activated form of the enzyme, by phosphorylase kinase. Glc-6-P, which is an allosteric inhibitor of phosphorylase b, and glycogen, which is a substrate of this enzyme, are already known to have respectively an inhibiting and activating effect upon the rate of conversion from phosphorylase b to phosphorylase a by phosphorylase kinase. In the former case, this effect is due to the binding of glucose-6-phosphate to glycogen phosphorylase b. In order to investigate whether or not the rate of conversion of glycogen phosphorylase b to phosphorylase a depends on the conformational state of the b substrate, we have tested the action of the most specific effectors of glycogen phosphorylase b activity upon the rate of conversion from phosphorylase b to phosphorylase a at 0 degrees C and 22 degrees C : AMP and other strong activators, IMP and weak activators, Glc-6-P, glycogen. Glc-1-P and phosphate. AMP and strong activators have a very important inhibitory effect at low temperature, but not at room temperature, whereas the weak activators have always a very weak, if even existing, inhibitory effect at both temperatures. We confirmed the very strong inhibiting effect of Glc-6-P at both temperatures, and the strong activating effect of glycogen. We have shown that phosphate has a very strong inhibitory effect, whereas Glc-1-P has an activating effect only at room temperature and at non-physiological concentrations. The concomitant effects of substrates and nucleotides have also been studied. The observed effects of all these ligands may be either direct ones on phosphorylase kinase, or indirect ones, the ligand modifying the conformation of phosphorylase b and its interaction with phosphorylase kinase. Since we have no control experiments with a peptidic fragment of phosphorylase b, the interpretation of our results remains putative. However, the differential effects observed with different nucleotides are in agreement with the simple conformational scheme proposed earlier. Therefore, it is suggested that phosphorylase kinase recognizes differently the different conformations of glycogen phosphorylase b. In agreement with such an explanation, it is shown that the inhibiting effect of AMP is mediated by a slow isomerisation which has been previously ascribed to a quaternary conformational change of glycogen phosphorylase b. The results presented here (in particular, the important effect of glycogen and phosphate) are also discussed in correlation with the physiological role of the different ligands as regulatory signals in the in vivo situation where phosphorylase is inserted into the glycogen particle.

Adenosine Monophosphate↗
PubMed1986

The behavior of hepatic phosphorylase b kinase, phosphorylase a and b after administration of glucagon to patients with glycogen storage disease type VIa.

In the patients with glycogen storage disease (GSD) type VIa and different serum glucose response to glucagon, the activities of hepatic phosphorylase b kinase, phosphorylase a and b were estimated before and after the intravenous administration of glucagon. 3 min after the administration of glucagon an increase in the activities of phosphorylase b kinase and phosphorylase a was found in liver tissue of all patients except one. These enzymatic activities, however, did not exceed the values of these enzymes in the control liver biopsies without glucagon loading. After the intravenous administration of glucagon an unsuspected increase of phosphorylase b activity was observed in the control liver tissues and in patients with GSD type VIa, except one. In vitro investigations revealed that an increase of hepatic phosphorylase b activity occurs during its conversion to phosphorylase a. We suppose that this phosphorylase b represents a partially phosphorylated form of this enzyme (an intermediate form) that is due to the action of the active phosphorylase b kinase. The correlations between the activities of phosphorylase b kinase, phosphorylase a and an intermediate form of phosphorylase b and hepatic glycogen degradation after administration of glucagon has been discussed.

Child, Preschool↗
PubMed1976

The interaction of ligands with chemically modified phosphorylase b.

Phosphorylase b which had been inactivated with 5-diazo1H-tetrazole was specifically labelled with 4-iodoacetamidosalicylic acid (a fluorescent probe) or with N-(1-oxyl-2,2,6,6,-tetramethyl-4-piperidinyl)iodoacetamide (a spin label probe) so that the binding of ligands and accompanying conformational changes could be determined by fluorescence or electron spin resonance changes, respectively. The allosteric effector, AMP, causes conformational changes similar to those caused in the native enzyme. The affinity of binding of phosphate or AMP to the inhibited protein is the same as for the unmodified protein. The heterotropic interactions between glucose-1-phosphate or glycogen and AMP are much less in the inactivated enzyme than in unmodified phosphorylase. Using a light scattering assay, it is shown that the modified enzyme binds to glycogen less strongly than the native protein. Phosphorylase b which had been inactivated by carbodimide in the presence of glycine ethyl ester, resulting in the modification of one or more carboxyl groups, was labelled with the spin label probe described above. The modified enzyme has an affinity for AMP similar to that of the native enzyme. AMP binding to the modified enzyme is tightened by glycogen, weakened by glucose-6-phosphate and is unaffected by glucose-1-phosphate. The actions of 5-diazo-1H-tetrazole and carbodimide on phosphorylase are discussed in the light of the above observation.

Adenosine Monophosphate↗
PubMed2002

Three-dimensional structure of phosphorylase kinase at 22 A resolution and its complex with glycogen phosphorylase b.

Phosphorylase kinase (PhK) integrates hormonal and neuronal signals and is a key enzyme in the control of glycogen metabolism. PhK is one of the largest of the protein kinases and is composed of four types of subunit, with stoichiometry (alphabetagammadelta)(4) and a total MW of 1.3 x 10(6). PhK catalyzes the phosphorylation of inactive glycogen phosphorylase b (GPb), resulting in the formation of active glycogen phosphorylase a (GPa) and the stimulation of glycogenolysis. We have determined the three-dimensional structure of PhK at 22 A resolution by electron microscopy with the random conical tilt method. We have also determined the structure of PhK decorated with GPb at 28 A resolution. GPb is bound toward the ends of each of the lobes with an apparent stoichiometry of four GPb dimers per (alphabetagammadelta)(4) PhK. The PhK/GPb model provides an explanation for the formation of hybrid GPab intermediates in the PhK-catalyzed phosphorylation of GPb.

Animals↗
PubMed1982

Multiple activities on phosphorylase kinase. 2. Different specificities toward the protein substrates phosphorylase b, troponin, and phosphorylase kinase.

Phosphorylase kinase exhibits three kinds of enzymatic activities. A partial activity, A0, catalyzes the phosphorylation of phosphorylase b, troponin I, and phosphorylase kinase itself (autophosphorylation); A1 can utilize only phosphorylase b and phosphorylase kinase as the substrate, whereas A2 can utilize only phosphorylase b and troponin T. Stimulation of A1 by Ca2+ coincides with an increase in the number of sites that can undergo self-phosphorylation ranging from ca. 35 to ca. 70 mol of phosphate incorporated/1.28 X 10(6) g of proteins. Inhibition of A0 and A1 by millimolar Ca2+ is accompanied by a decrease in substrate availability during self-phosphorylation. NH4Cl (150 mM) strongly inhibits the availability of troponin as a substrate. In the course of self-phosphorylation, the activities A0 and A1 are both stimulated moderately by an increase in pH; however, only A1 shows some inhibition by 150 mM NH4Cl. Millimolar Ca2+ inhibits A1 and A2 as measured by self-phosphorylation or troponin phosphorylation, as observed with the phosphorylation of phosphorylase b [Kilimann, M. W., & Heilmeyer, L. M. G., Jr. (1982) Biochemistry (preceding paper in this issue)]. The rate of self-phosphorylation varies as a function of substrate concentration (Km = 68 nM at 10 mM Mg2+ and 184 microM Ca2+, pH 9.0). The data indicate that both Ca2+ activation and inhibition seem to be mediated by phosphorylase kinase itself rather than by the substrates.

Ammonium Chloride↗
PubMed1991

Phosphorylase b kinase and phosphorylase a phosphatase activities in contracting vascular smooth muscle: stimulation by fatty acid.

The activities of phosphorylase b kinase and phosphorylase a phosphatase were determined during the phases of KCl-induced contraction in porcine carotid artery. Phosphorylase b kinase exhibited a biphasic pattern with activity increasing 70% above basal levels during the early phase of active force generation (45 s into contraction) followed by a decline in activity during the phase of steady-state tension maintenance. Phosphorylase a phosphatase was stimulated simultaneously with phosphorylase b kinase, with activity increasing 100% over basal levels at 45 s into contraction, but remaining elevated at 30 min. Incubation of arteries in 0.5 mM palmitate resulted in a 30% increase in basal activity of phosphorylase b kinase and 117% augmentation of basal phosphatase activity, with no further increase in activity of either enzyme with contraction. The results indicate that both the kinase and phosphatase are subject to regulation during contractile activation of the muscle, possibly by similar but not identical mechanisms.

Animals↗
PubMed1985

Comparative study of the interactions AMP-phosphorylase b and AMP analogues-phosphorylase b.

The different effects induced by AMP and its analogues on the tertiary structure and the coenzyme environment of phosphorylase b were studied by titration of the slowly reacting thiol groups and by quenching of the coenzyme fluorescence, respectively, to determine the possible differences that activate phosphorylase b. The following results were obtained: The coenzyme environment depends upon the nucleotide studied. AMP, when bound to its first site, opens the coenzyme pocket. The slow cysteines were shielded by the nucleotides against their DTNB titration depending on the nucleotide studied. The enzyme difference spectra in presence of the nucleotide showed that the negative band of 260 nm is similar for all nucleotides possessing the same base, but the positive band obtained in the presence of AMP was diminished when other nucleotides were present.

Adenosine Monophosphate↗
PubMed1970

Conversion of glycogen phosphorylase b to a by non-activated phosphorylase b kinase: an in vitro model of the mechanism of increase in phosphorylase a activity with muscle contraction.

Phosphorylase b kinase activity, as present in resting muscle in the non-activated form, appears to be ample to account for the fast appearance of phosphorylase a observed with muscle contraction. The kinase activity is repressed by free ATP and stimulated by free Mg(2+). Phosphorylase b kinase activity increases greatly when the Mg(2+):ATP ration exceeds 1. It is proposed that the breakdown of ATP that occurs during muscle contraction may represent the triggering factor for the observed in vivo conversion of phosphorylase b into a.

Adenosine Triphosphate↗
PubMed1986

Activation of hepatic glycogen phosphorylase b in vivo by sodium sulphate in normal (Wistar) and phosphorylase b kinase-deficient (gsd/gsd) rats.

Sulphate ions have been known for some years to enhance the activity of hepatic glycogen phosphorylase b in vitro. Here we report that intravenous injections of 4.92 mmol of Na2SO4/kg body wt. to rats induced marked hepatic glycogenolysis in vivo, accompanied by polyuria, glycosuria and a mild hyperglycaemia. These effects were observed both in normal (Wistar) rats and in gsd/gsd rats that lacked hepatic phosphorylase kinase. In both rat strains the activity of glycogen phosphorylase in liver extracts was enhanced by pretreatment of the animals with Na2SO4, but in phosphorylase kinase-deficient livers the enhancement was solely in phosphorylase b activity, whereas both the a and b forms of the enzyme were activated in normal livers. Hepatic glycogenolysis was also induced by perfusing rat livers, both normal and gsd/gsd, with 25 mM-Na2SO4. Under these conditions both the rat strains showed only enhanced activities of glycogen phosphorylase b. This suggested that the increased activity of phosphorylase a in the extracts of normal livers after Na2SO4 administration in vivo was due to a hormonally mediated conversion of the b form into the a form. The activation of glycogen phosphorylase b was stable to dilution and appeared to be due to a long-lasting structural change in the enzyme or very tight binding of an activator.

Animals↗