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PubMed2006

Multiple voxel 1H MR spectroscopy of phosphorylase-b kinase deficient patients (GSD IXa) showing an accumulation of fat in the liver that resolves with aging.

BACKGROUND/AIMS: Phosphorylase-b deficient patients suffer from glycogen storage disease (GSD IXa) leading to liver enlargement which usually resolves during puberty and adolescence. This pathology has not yet been documented by (1)H MR spectroscopy (MRS) investigation. METHODS: MRS of eight GSD IXa patients was performed in this study to assess whether or not liver fat content is elevated in GSD IXa and decreases with aging. An improvement in our MRS method compared with previous liver fat MRS studies is that we measured a plane of liver voxels at once rather than a single MRS voxel, yielding a reliable determination of liver fat content. RESULTS: Fat contents of 3.4-10% were observed in young GSD IXa patients, as compared with 0.5-0.9% in controls, these dropped to control levels in patients past age 40 (r = -0.82; P < 0.01). CONCLUSIONS: Liver fat content is increased in glycogen storage disease (GSD IXa) and normalizes with ageing. Assessing liver fat levels in this population is a novel and interesting concept. This could potentially enhance the understanding of liver function in that 20% of the population who has increased liver fat.

Adipose Tissue↗
PubMed1974

Phosphorylation of troponin and the effects of interactions between the components of the complex.

1. The troponin complex from skeletal muscle contains approximately 1 mol of phosphate/80000g of complex, covalently bound to the troponin T component. 2. On prolonged incubation of the troponin complex or troponin T with phosphorylase kinase the phosphate content of troponin T was increased to approx. 3mol/mol. 3. On prolonged incubation of troponin I with phosphorylase kinase up to 1.6mol of phosphate/mol were incorporated. 4. Phosphorylation of troponin I was greatly inhibited by troponin C owing to the strong interaction between these proteins. Thus in the troponin complex troponin T was the main substrate for phosphorylase kinase. The phosphorylation of isolated troponin T was also inhibited by troponin C. 5. Troponin I was phosphorylated when the troponin complex was incubated with a bovine cardiac 3':5'-cyclic AMP-dependent protein kinase. Troponin T either in its isolated form or in the troponin complex was not phosphorylated by bovine protein kinase to any significant extent under the conditions used. 6. If the troponin complex was dephosphorylated to 0.2mol/mol, or phosphorylated up to 2.5mol/mol there was no significant effect on the ability of normal concentrations to confer Ca(2+) sensitivity on the adenosine triphosphatase of densensitized actomyosin.

Actomyosin↗
PubMed1983

The protein phosphatases involved in cellular regulation. 1. Classification and substrate specificities.

The protein phosphatase activities involved in regulating the major pathways of intermediary metabolism can be explained by only four enzymes which can be conveniently divided into two classes, type-1 and type-2. Type-1 protein phosphatases dephosphorylate the beta-subunit of phosphorylase kinase and are potently inhibited by two thermostable proteins termed inhibitor-1 and inhibitor-2, whereas type-2 protein phosphatases preferentially dephosphorylate the alpha-subunit of phosphorylase kinase and are insensitive to inhibitor-1 and inhibitor-2. The substrate specificities of the four enzymes, namely protein phosphatase-1 (type-1) and protein phosphatases 2A, 2B and 2C (type-2) have been investigated. Eight different protein kinases were used to phosphorylate 13 different substrate proteins on a minimum of 20 different serine and threonine residues. These substrates include proteins involved in the regulation of glycogen metabolism, glycolysis, fatty acid synthesis, cholesterol synthesis, protein synthesis and muscle contraction. The studies demonstrate that protein phosphatase-1 and protein phosphatase 2A have very broad substrate specificities. The major differences, apart from the site specificity for phosphorylase kinase, are the much higher myosin light chain phosphatase and ATP-citrate lyase phosphatase activities of protein phosphatase-2A. Protein phosphatase-2C (an Mg2+-dependent enzyme) also has a broad specificity, but can be distinguished from protein phosphatase-2A by its extremely low phosphorylase phosphatase and histone H1 phosphatase activities, and its slow dephosphorylation of sites (3a + 3b + 3c) on glycogen synthase relative to site-2 of glycogen synthase. It has extremely high hydroxymethylglutaryl-CoA (HMG-CoA) reductase phosphatase and HMG-CoA reductase kinase phosphatase activity. Protein phosphatase-2B (a Ca2+-calmodulin-dependent enzyme) is the most specific phosphatase and only dephosphorylated three of the substrates (the alpha-subunit of phosphorylase kinase, inhibitor-1 and myosin light chains) at a significant rate. It is specifically inhibited by the phenathiazine drug, trifluoperazine. Examination of the amino acid sequences around each phosphorylation site does not support the idea that protein phosphatase specificity is determined by the primary structure in the immediate vicinity of the phosphorylation site.

Animals↗
PubMed1987

Predicted calmodulin-binding sequence in the gamma subunit of phosphorylase b kinase.

A basic, amphiphilic alpha helix is a structural feature common to a variety of inhibitors of calmodulin and to the calmodulin-binding domains of myosin light chain kinases. To aid in recognizing this structural feature in sequences of peptides and proteins we have developed a computer algorithm which searches for sequences of appropriate length, hydrophobicity, helical hydrophobic moment, and charge to be considered as potential calmodulin-binding sequences. Such sequences occurred infrequently in proteins of known crystal structure. This algorithm was used to find the most likely site in the catalytic (gamma) subunit of phosphorylase b kinase for interaction with calmodulin (the delta subunit). A peptide corresponding to this site (residues 341-361 of the gamma subunit) was synthesized and found to bind calmodulin with approximately an 11 nM dissociation constant. A variant of this peptide in which an aspartic acid at position 7 in its sequence (347 of the gamma subunit) was replaced with an asparagine was found to bind calmodulin with approximately a 3 nM dissociation constant.

Algorithms↗
PubMed1980

Interaction between alpha and beta adrenergic receptors and cholinergic receptors in isolated perfused rat heart: effects on cAMP-protein kinase and phosphorylase.

The ability of acetylcholine to antagonize catecholamine-induced activation of myocardial cyclic AMP dependent protein kinase and glycogen phosphorylase activity was assessed using isolated perfused rat hearts. Perfused hearts were treated with either saline, epinephrine, epinephrine plus phentolamine or isoproterenol. After 1 minute of infusion of the indicated drug a second infusion containing acetylcholine was started. After an additional minute hearts were frozen and analyzed for cyclic nucleotide content and enzyme activity. In the presence of the alpha receptor blocking agent, phentolamine, epinephrine is a more effective activator of protein kinase than in its absence. Under these conditions the antagonistic action of acetylcholine on protein kinase activation is more pronounced. In the presence of epinephrine plus phentolamine or in the presence of isoproterenol the antagonistic action of acetylcholine on phosphorylase activity can be accounted for by a reduction in cyclic AMP-protein kinase. This same action of acetylcholine on epinephrine-stimulated phosphorylase in the aabsence of phentolamine, however, cannot be totally accounted for by a reduction in cyclic AMP content or in protein kinase activity.

Acetylcholine↗
PubMed1983

The calmodulin-dependent glycogen synthase kinase from rabbit skeletal muscle. Purification, subunit structure and substrate specificity.

A calmodulin-dependent glycogen synthase kinase distinct from phosphorylase kinase has been purified approximately equal to 5000-fold from rabbit skeletal muscle by a procedure involving fractionation with ammonium sulphate (0-33%), and chromatographies on phosphocellulose, calmodulin-Sepharose and DEAE-Sepharose. 0.75 mg of protein was obtained from 5000 g of muscle within 4 days, corresponding to a yield of approximately equal to 3%. The Km for glycogen synthase was 3.0 microM and the V 1.6-2.0 mumol min-1 mg-1. The purified enzyme showed a major protein staining band (Mr 58 000) and a minor component (Mr 54 000) when examined by dodecyl sulphate polyacrylamide gel electrophoresis. The molecular weight of the native enzyme was determined to be 696 000 by sedimentation equilibrium centrifugation, indicating a dodecameric structure. Electron microscopy suggested that the 12 subunits were arranged as two hexameric rings stacked one upon the other. Following incubation with Mg-ATP and Ca2+-calmodulin, the purified protein kinase underwent an 'autophosphorylation reaction'. The reaction reached a plateau when approximately equal to 5 mol of phosphate had been incorporated per 58 000-Mr subunit. Both the 58 000-Mr and 54 000-Mr species were phosphorylated to a similar extent. Autophosphorylation did not affect the catalytic activity. The calmodulin-dependent protein kinase initially phosphorylated glycogen synthase at site-2, followed by a slower phosphorylation of site-1 b. The protein kinase also phosphorylated smooth muscle myosin light chains, histone H1, acetyl-CoA carboxylase and ATP-citrate lyase. These findings suggest that the calmodulin-dependent glycogen synthase kinase may be a enzyme of broad specificity in vivo. Glycogen synthase kinase-4 is an enzyme that resembles the calmodulin-dependent glycogen synthase kinase in phosphorylating glycogen synthase (at site-2), but not glycogen phosphorylase. Glycogen synthase kinase-4 was unable to phosphorylate any of the other proteins phosphorylated by the calmodulin-dependent glycogen synthase kinase, nor could it phosphorylate site 1 b of glycogen synthase. The results demonstrate that glycogen synthase kinase-4 is not a proteolytic fragment of the calmodulin-dependent glycogen synthase kinase, that has lost its ability to be regulated by Ca2+-calmodulin.

Animals↗
PubMed2000

Identification of binding sites on protein targeting to glycogen for enzymes of glycogen metabolism.

The activation of protein phosphastase-1 (PP1) by insulin plays a critical role in the regulation of glycogen metabolism. PTG is a PP1 glycogen-targeting protein, which also binds the PP1 substrates glycogen synthase, glycogen phosphorylase, and phosphorylase kinase (Printen, J. A., Brady, M. J., and Saltiel, A. R. (1997) Science 275, 1475-1478). Through a combination of deletion analysis and site-directed mutagenesis, the regions on PTG responsible for binding PP1 and its substrates have been delineated. Mutagenesis of Val-62 and Phe-64 in the highly conserved (K/R)VXF PP1-binding motif to alanine was sufficient to ablate PP1 binding to PTG. Phosphorylase kinase, glycogen synthase, and phosphorylase binding all mapped to the same C-terminal region of PTG. Mutagenesis of Asp-225 and Glu-228 to alanine completely blocked the interaction between PTG and these three enzymes, without affecting PP1 binding. Disruption of either PP1 or substrate binding to PTG blocked the stimulation of PP1 activity in vitro against phosphorylase, indicating that both binding sites may be important in PTG action. Transient overexpression of wild-type PTG in Chinese hamster ovary cells overexpressing the insulin receptor caused a 50-fold increase in glycogen levels. Expression of PTG mutants that do not bind PP1 had no effect on glycogen accumulation, indicating that PP1 targeting is essential for PTG function. Likewise, expression of the PTG mutants that do not bind PP1 substrates did not increase glycogen levels, indicating that PP1 targeting glycogen is not sufficient for the metabolic effects of PTG. These results cumulatively demonstrate that PTG serves as a molecular scaffold, allowing PP1 to recognize its substrates at the glycogen particle.

Alanine↗
PubMed1981

Rate dependence of isoproterenol-stimulated phosphorylase a formation in mouse heart.

In mouse right ventricular strips, field-stimulated to contract isometrically in an oxygenated bicarbonate-buffered physiological salt solution at 22--24 degrees C, isoproterenol (3 microM) stimulation of phosphorylase a accumulation was approximately a linear function of the rate of contraction from 0.2 to 5 Hz. In muscles incubated in the absence of isoproterenol, the phosphorylase a activity did not increase when the contraction frequency was varied over this range. At a low frequency of stimulation (0.2 Hz), phosphorylase a activity was not increased after a 5-min exposure to 3 microM isoproterenol, as compared to a 4-fold increase in phosphorylase a activity at a high frequency (3.-3 Hz). Isoproterenol (3 microM) increased tissue cyclic AMP content and the activated form of phosphorylase kinase activity to similar extents at both frequencies. N6,O2'-dibutyryl cyclic AMP increased the phosphorylase a activity at both frequencies but the increase at 3.3 Hz was approximately 3-fold greater than at 0.2 Hz. Verapamil did not block isoproterenol-stimulated phosphorylase a activity at 3.3 Hz at a concentration (0.6 microM) that inhibited the increased sensitivity to the inotropic action of isoproterenol seen at high frequencies of contraction. Isoproterenol stimulation of phosphorylase a accumulation did not correlate with developed tension. It is proposed that the difference in isoproterenol stimulation of phosphorylation b to a conversion at 0.2 and 3.3 Hz primarily results from a difference in Ca++ control of the activity of the activated form of phosphorylase kinase.

Animals↗
PubMed1982

In vitro inhibition of protein catabolism by alpha 2-macroglobulin in plasma from a patient with posttraumatic acute renal failure.

Rapid proteolytic digestion of three subunits of phosphorylase kinase was shown in plasma ultrafiltrates from a patient who was admitted with multiple traumatic injuries, respiratory insufficiency, hemorrhagic shock and acute renal failure. The observed cleavage of phosphorylase kinase may be a consequence of protease-antiprotease imbalance. After an initial determination of 80 mg/dl the alpha 2-macroglobulin values were too low to be detected. Addition of purified alpha 2-macroglobulin to the ultrafiltrates resulted in complete inhibition of phosphorylase kinase digestion in vitro. Aprotinin did not inhibit proteolytic digestion. Daily dialysis did not ameliorate the observed protein catabolism of this patient. These findings may have clinical application in hypercatabolic states, if alpha 2-macroglobulin becomes available in a form suitable for human use.

Acute Kidney Injury↗
PubMed1983

Mechanisms of enhanced phosphorylase activation in the hyperthyroid rat heart.

Enhanced phosphorylase activation in hearts from hyperthyroid animals has been well documented. To elucidate the mechanisms responsible for the enhanced phosphorylase a formation, hearts from euthyroid and hyperthyroid rats were perfused by the Langendorff method with calcium (3.75 mM), isoproterenol, dibutryl cAMP and trifluoperazine, an inhibitor of calcium-calmodulin dependent enzymes. Comparative biochemical analyses revealed increased phosphorylase a formation in hearts from both euthyroid and hyperthyroid animals following exposure to calcium, dibutryl cAMP and isoproterenol. Hearts from hyperthyroid rats had an increased sensitivity to threshold concentrations of isoproterenol for both cAMP formation and phosphorylase b to a conversion. At higher concentrations of isoproterenol (10(-8) M and 3 x 10(-8) M), no significant differences in cAMP formation were noted between euthyroid and hyperthyroid animals in spite of persistently increased phosphorylase a levels in the hyperthyroid state. Trifluroperazine had no effect on basal phosphorylase a levels but significantly inhibited phosphorylase a formation in both groups following calcium or isoproterenol stimulation. However, enhanced phosphorylase a formation was still present in the hearts from hyperthyroid rats following trifluoperazine preperfusion. Determinations of phosphorylase kinase activity revealed a specific activity in the hyperthyroid animals twice that of the euthyroid controls. At least two mechanisms, an increased sensitivity to beta-adrenergic agents and increased cardiac phosphorylase kinase activity, may mediate the enhanced phosphorylase a formation found in hearts from hyperthyroid rats.

Animals↗
PubMed1977

Role of 3':5'-cyclic-AMP-dependent protein kinase in regulation of protein synthesis in reticulocyte lysates.

The initiation inhibitor of reticulocyte lysates has been shown by others to be associated with a 3':5'-cyclic-AMP-independent protein kinase that catalyzes the phosphorylation of the small (38,000 daltons) subunit of the polypeptide chain initiation factor eIF-2. This factor forms a ternary complex with Met-tRNAi and GTP which, on interaction with a 40S ribosome, gives rise to a 40S complex. Ternary complex formation is inhibited by prior incubation of partially purified eIF-2 with reticulocyte inhibitor and ATP. The relation between phosphorylation and inactivation of eIF-2 is indicated by the lack of inhibition when ATP is omitted. Translation in hemin-containing reticulocyte lysates is also inhibited by cyclic-AMP-dependent protein kinases or their catalytic subunits. They act by converting proinhibitor (inactive eIF-2 kinase) present in lysates to inhibitor (active eIF-2 kinase). This reaction is analogous to the conversion of inactive phosphorylase kinase to active phosphorylase kinase.

Animals↗
PubMed1978

The modulator-dependent protein kinase. A multifunctional protein kinase activatable by the Ca2+-dependent modulator protein of the cyclic nucleotide system.

A protein kinase which depends on the simultaneous presence of Ca2+ and the modulator protein for its histone phosphorylation activity has been demonstrated in rabbit skeletal muscle and partially purified. The purified enzyme was not activated by cAMP, cGMP, or incubation with trypsin. Nor was the enzyme inhibited by the protein inhibitor of cAMP-dependent protein kinase. In addition to histone, myosin light chains and phosphorylase kinase served as substrates for the protein kinase, and their phosphorylation also depended on the presence of Ca2+ and the modulator protein. The phosphorylation of phosphorylase kinase was accompanied with a marked activation of the enzyme. The results suggest that the protein kinase has multiple functions and may be involved in the mediation of Ca2+ effects in many biological processes. It is proposed that this enzyme be designated as the modulator-dependent protein kinase. The modulator-dependent protein kinase may be identical to the myosin light chain kinase; chicken gizzard light chain kinase has been shown activatable by the modulator protein (Dabrowska, R., Sherry, J. M. F., Aramatorio, D. K., and Hartshorne, D. J. (1978) Biochemistry 17, 253-258).

Animals↗
PubMed1983

Separation and identification of type 1 and type 2 protein phosphatases from rabbit reticulocyte lysates.

Protein phosphatase type 1 and type 2 activities (designated PP-1 and PP-2, respectively) from rabbit reticulocyte lysates have been identified and characterized based on criteria previously established for similar activities in rabbit skeletal muscle and rabbit liver. These include (a) chromatographic separation on DEAE-cellulose, (b) substrate specificity toward glycogen phosphorylase a and the alpha- and beta-subunits of phosphorylase kinase, (c) differential sensitivity to the heat-stable protein phosphatase inhibitors-1 and -2, and (d) sensitivity to MgATP. When total lysate phosphatases are assayed in the presence of 1 mM MnCl2, protein phosphatase type 2 represents 84% of lysate phosphorylase phosphatase activity. However, when phosphatase assays are carried out with MgATP concentrations similar to those in the lysate, type 2 activity is diminished, and the levels of type 1 (41%) and type 2 (59%) phosphatase activities are comparable. A small proportion (6%) of total lysate phosphatase is tightly bound to the ribosomes, where type 1 phosphatase predominates. At least five species of protein phosphatases can be identified in lysates. These constitute two forms of protein phosphatase type 1, one of which (designated FC) is dependent on MgATP and a lysate activator protein FA; both FC and FA have been identified previously in skeletal muscle. Three species of protein phosphatase type 2 have been identified and designated PP-2B, PP-2A1, and PP-2A2 based on criteria recently established for rabbit skeletal muscle and rabbit liver phosphatases, which display similar phosphatase profiles. Lysate protein phosphatases types 1, FC, 2A1, and 2A2 can all act on phosphorylase a and the alpha- (type 2) or beta-(type 1) subunit of phosphorylase kinase. PP-2B, a Ca2+/calmodulin-dependent phosphatase, specifically dephosphorylates the alpha-subunit of phosphorylase kinase, but does not act on phosphorylase alpha. The heat-stable protein phosphatase inhibitor-2 from skeletal muscle completely blocks the activity of the two type 1 phosphatases (PP-1, FC), but has no effect on the three species of type 2 protein phosphatase. A preliminary assay of the two heat-stable phosphatase inhibitors in lysates indicates significant levels of inhibitor-2, but little or no detectable inhibitor-1.

Animals↗
PubMed1976

Glycogen storage diseases.

Each of 12 types of glycogen storage disease (GSD O-XI) is delineated by clinical, biochemical and histologic features that allow its identification in future patients. GSD II occurs in 2 forms that are not both encountered in the same family. GSD IIa is the infantile fatal form with cardiomegaly, increased cardiac glycogen concentration and cardiac failure; GSD IIb is the adult form with clinically normal heart and normal cardiac glycogen concentration. Nonetheless, the heart muscle of both forms is equally deficient in acid alpha-glucosidase activity, and this raises questions as to the latter's role in the pathophysiology of GSD II. The appearance of hepatocytes in GSD IIa becomes normal after the administration of alpha-glucosidase. Using electron microscopy of uncultured amniotic fluid cells, the prenatal diagnosis of GSD IIa is feasible within one day after the amniocentesis. GSD VI and IX are instances of benign hepatomegaly except when GSD IX and III occur in the same child; one such patient died suddenly at home. There are 2 modes of inheritance in GSD IX: one (GSD IXa) is autosomal recessive, the other one (GSD IXb) is X-linked recessive. In either form the Km of the remaining liver phosphorylase kinase is normal. Both forms of GSD IX have the normal blood sugar response to glucagon, whereas GSD VI does not. Equally, the glucagon tolerance curve is flat in GSD XI although in vitro activity of glycolytic enzymes is normal. The in vivo administration of glucagon in GSD XI is followed by the normal increase of both urinary 3'5'-AMP and hepatic phosphorylase activity. GSD V may have increased activity of muscle phosphorylase kinase. Deficiencies of debrancher, liver phosphorylase and liver phosphorylase kinase can occur singly or in combination. Before any novel treatment of GSD is initiated, one should obtain tissue for the biochemical determination of the exact type of GSD. This is so because the clinical signs may not indicate the type with the necessary precision, and because some types are compatible with normal life and thus may not require therapy, especially if the latter is unproved and potentially dangerous.

Bone and Bones↗