Developmental changes in cyclic AMP, protein kinase, phosphorylase kinase, and phosphorylase in liver, heart, and skeletal muscle of the rat.
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Adenosine Triphosphate↗
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Hormonal regulation of adipocyte enzymes. The effects of epinephrine and insulin on the control of lipase, phosphorylase kinase, phosphorylase, and glycogen synthase.
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Adipose Tissue↗
[Kinetics of kinase phosphorylase action in a cascade enzymatic system. I. Theoretical basis of a method for determining phosphorylase kinase activity].
Based on a theoretical analysis of functioning of a monocascade enzymatic system, a method for continuous monitoring of the phosphorylase kinase-catalyzed enzymatic reaction has been developed. The method is based on the ability of the kinase reaction product--the phosphorylated form of glycogen phosphorylase (form a)-to catalyze glycogen phosphorolysis (with inorganic phosphate as the low molecular weight substrate) or synthesis (with glucose 1-phosphate) in the absence of AMP. A turbidimetric method may be used for the monitoring of the reaction of glycogen degradation (or synthesis) by phosphorylase a formed in the course of the kinase reaction. A method to calculate the initial rate of the kinase reaction from an absorbance versus (time)2 plot has been theoretically substantiated.
Animals↗
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+].
Animals↗
Molecular heterogeneity of rabbit heart phosphorylase kinase.
Phosphorylase kinase (ATP: phosphorylase-b phosphotransferase, EC 2.7.1.38) from rabbit heart, when submitted to electrophoresis on Pevikon, separates into two discrete peaks A and B. The two peaks have been analyzed using reelectrophoresis, chromatography on DEAE-cellulose, thermal stability, inactivation by EGTA (ethyleneglycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid) and reaction with an anti-muscle phosphorylase kinase antiserum. It can be concluded that rabbit heart extracts contain two isozymes of phosphorylase kinase. The more negatively charged isozyme seems to be identical with the muscle enzyme. The other isozyme resembles the liver enzyme but differs from the major fraction of the latter by its charge. It is likely that there exist at least three molecular types of phosphorylase kinase.
Animals↗
Calcium-dependent phosphorylation of bovine cardiac C-protein by phosphorylase kinase.
Phosphorylase kinase catalyzed the calcium-dependent phosphorylation of bovine cardiac C-protein. Phosphorylation of C-protein by phosphorylase kinase reached nearly 2 mol [32P]/mol C-protein. Tryptic phosphopeptide mapping and phosphoamino acid analysis indicated that phosphorylase kinase maybe phosphorylating some of the same seryl residues that undergo phosphorylation by cAMP-dependent protein kinase and that C-protein from bovine and chicken heart are structurally different. Bovine cardiac C-protein was not a substrate for a number of calcium and cyclic nucleotide-independent protein kinases, suggesting that phosphorylation of cardiac C-protein is restricted to protein kinases which are modulated by calcium and cAMP.
Animals↗
Two-dimensional electron microscopic analysis of the chalice form of phosphorylase kinase.
Phosphorylase kinase (Mr 1.3 X 10(6], a Ca2+-calmodulin-dependent protein kinase, plays a key role in the initiation of glycogenolysis. After purification on hydroxylapatite, the negatively stained enzyme was used for electron microscopy. In electron micrographs, phosphorylase kinase shows two major molecular forms: a butterfly form (approx. 60%) and a chalice form (approx. 40%). Images of the chalice form of the enzyme were computer-averaged by the method of single particle averaging. The following apparent molecular dimensions were obtained from the averages: total height, 20 nm; maximal width, 18 nm. The chalice form of phosphorylase kinase consists of a major structure termed the cup (11 nm X 18 nm), containing a large accessible cleft, and a minor structure termed the stem (8 nm X 9 nm). A closer examination of the images by averaging of molecular parts revealed two subpopulations of the cup part: a flexed (closed) type and an extended (open) type. The orifice, which can be closed partly by two protrusions (I, I'), is about 6 nm wide when the protrusions are flexed and 9 nm wide when they are extended. It is suggested that the substrates, e.g. phosphorylase b, may be accommodated in the large cleft of the enzyme. While the orientation of the protrusions (I, I') is the most obvious difference between the two types, more structural differences can be detected, suggesting a concerted movement of the protein domains against each other.
Animals↗
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↗
A tentative mechanism of the ternary complex formation between phosphorylase kinase, glycogen phosphorylase b and glycogen.
The kinetics of rabbit skeletal muscle phosphorylase kinase interaction with glycogen has been studied. At pH 6.8 the binding of phosphorylase kinase to glycogen proceeds only in the presence of Mg2+, whereas at pH 8.2 formation of the complex occurs even in the absence of Mg2+. On the other hand, the interaction of phosphorylase kinase with glycogen requires Ca2+ at both pH values. The initial rate of the complex formation is proportional to the enzyme and glycogen concentrations, suggesting the formation of the complex with stoichiometry 1:1 at the initial step of phosphorylase kinase binding by glycogen. According to the kinetic and sedimentation data, the substrate of the phosphorylase kinase reaction, glycogen phosphorylase b, favors the binding of phosphorylase kinase with glycogen. We suggest a model for the ordered binding of phosphorylase b and phosphorylase kinase to the glycogen particle that explains the increase in the tightness of phosphorylase kinase binding with glycogen in the presence of phosphorylase b.
Animals↗
Phosphorylation and inactivation of rabbit skeletal muscle glycogen synthase: distinction between kinase Fa-, phosphorylase kinase-, and glycogen synthase (casein) kinase-1-catalyzed reactions.
Rabbit skeletal muscle glycogen synthase was phosphorylated by kinase Fa, phosphorylase kinase, and cAMP-independent synthase (casein) kinase-1 to determine the differences among these kinase-catalyzed reactions. The stoichiometry of phosphate incorporation, the extent of inactivation, and the sites of phosphorylation were compared. Synthase (casein) kinase-1 catalyzes the highest level of synthase phosphorylation (4 mol/subunit) and inactivation (reduction of the activity ratio to below 0.05). The sites, defined by characteristic tryptic peptides, phosphorylated by synthase (casein) kinase-1 are distinguishable from those by kinase Fa and phosphorylase kinase. In addition, synthase (casein) kinase-1, unlike kinase Fa, does not activate ATP X Mg2+-dependent protein phosphatase. These results demonstrate that synthase (casein) kinase-1 is a distinct glycogen synthase kinase.
Animals↗
Phosphorylase kinase, a metal ion-dependent dual specificity kinase.
Phosphorylase kinase is shown to be a dual specificity kinase. The specificity of phosphorylation is determined by divalent cation. Mg2+ causes seryl phosphorylation of phosphorylase b, but Mn2+ activates tyrosine phosphorylation of angiotensin II. In contrast to seryl phosphorylation, the tyrosine kinase activity of holoenzyme is not regulated by Ca2+. Preincubation of the holoenzyme with Ca2+, Mg2+ and ATP that causes autophosphorylation activates tyrosine kinase activity. The tyrosyl kinase activity is a property of the gamma subunit. Addition of varying amounts of Mn2+ to a truncated form of the gamma subunit of phosphorylase kinase containing MgATP inhibits serine kinase but activates tyrosine kinase activity. This result along with an oxidative reaction caused by Cu2+ and site-directed mutagenesis of the putative catalytic base inhibiting both serine and tyrosine kinase activity suggest that one active site is involved in both activities. Kinetic studies with Mn2+ and ATP show that Km for nucleotide is not changed with a seryl or tyrosyl substrate. The Vm values are different, and the value for tyrosyl phosphorylation is similar to other tyrosyl kinases. We propose two conformations for the active site; one favors seryl phosphorylation, and the second tyrosyl phosphorylation is caused by the binding of divalent cation at a second metal ion binding site.
Adenosine Triphosphate↗
Induction of meiosis by injection of heterologous protein kinase and phosphorylase kinase in Xenopus laevis oocytes.
Meiosis reinitiation has been triggered by injection of beef heart protein kinase or rabbit phosphorylase kinase into Xenopus laevis oocytes. Successful injections are followed by germinal vesicle breakdown, chromosome condensation, formation of a normal meiotic spindle, and appearance of an amplifiable maturation promoting factor. Meiosis reinitiation does not occur when the enzymes are introduced into the oocytes simultaneously with EGTA or after pretreatment with cycloheximide. Antipain, an antiprotease which abolishes the response of oocytes to progesterone, does not suppress the meiosis reinitiation induced by injection of protein kinase or phosphorylase kinase.
Animals↗
Cryoelectron microscopy reveals new features in the three-dimensional structure of phosphorylase kinase.
Phosphorylase kinase (PhK), a regulatory enzyme in the cascade activation of glycogenolysis, is a 1.3-MDa hexadecameric complex, (alphabetagammadelta)(4). PhK comprises two arched octameric (alphabetagammadelta)(2) lobes that are oriented back-to-back with overall D(2) symmetry and connected by small bridges. These interlobal bridges, arguably the most questionable structural component of PhK, are one of several structural features that potentially are artifactually generated or altered by conventional sample preparation techniques for electron microscopy (EM). To minimize such artifacts, we have solved by cryoEM the first three-dimensional (3D) structure of nonactivated PhK from images of frozen hydrated molecules of the kinase. Minimal dose electron micrographs of PhK in vitreous ice revealed particles in a multitude of orientations. A simple model was used to orient the individual images for 3D reconstruction, followed by multiple rounds of refinement. Three-dimensional reconstruction of nonactivated PhK from approximately 5000 particles revealed a bridged, bilobal molecule with a resolution estimated by Fourier shell correlation analysis at 25 A. This new structure suggests that several prominent features observed in the structure of PhK derived from negatively stained particles arise as artifacts of specimen preparation. In comparison to the structure from negative staining, the cryoEM structure shows three important differences: (1) a dihedral angle between the two lobes of approximately 90 degrees instead of 68 degrees, (2) a compact rather than extended structure for the lobes, and (3) the presence of four, rather than two, connecting bridges, which provides the first direct evidence for these components as authentic elements of the kinase solution structure.
Animals↗
[Structure and regulation of the activity of muscle phosphorylase kinase].
Phosphorylase kinase is the key enzyme in the control of glycogen metabolism in skeletal muscles, the heart and the liver. The quaternary structure of the enzyme, the primary structure of the enzyme subunits as well as the kinetic properties and regulation of the skeletal muscle enzyme activity by covalent modification, phosphorylation and some physiological effectors (Ca2+, calmodulin, troponin C) are reviewed.
Animals↗
Glucoamylase-like domains in the alpha- and beta-subunits of phosphorylase kinase.
Phosphorylase kinase is a four-subunit enzyme involved in the regulation of glycogen breakdown. The traditional textbook view is that only the gamma subunit has enzymatic activity, whereas the other three subunits have a regulatory role. Evidence from homology searches and sequence alignments, however, shows that the alpha- and beta-subunits possess amino-terminal glucoamylase-like domains and suggests that they might possess a previously overlooked amylase activity. If true, this would have important implications for the understanding, diagnosis, and management of glycogen storage diseases. There is thus a clear need to test this hypothesis through enzymatic assays and structural studies.
Amino Acid Sequence↗
Direct observation of phosphorylase kinase and phosphorylase b by scanning tunneling microscopy.
The molecular structures of phosphorylase b and phosphorylase kinase have been visualized by scanning tunneling microscopy (STM). STM is a near field technique that can resolve structures at the nanometer level and thus can image individual molecules. Phosphorylase b can be seen in dimeric and tetrameric forms as well as linear and globular aggregates. The linear arrays consist of side by side dimers with the long axis of the dimer perpendicular to the aggregated chain. Individual molecules of phosphorylase kinase appear to be planar, bilobate structures with a 2-fold axis of symmetry and a central depression.
Animals↗
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↗
Differentiation of two catalytic sites on phosphorylase kinase for phosphorylase b and troponin T phosphorylation.
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Animals↗