Adrenergic regulation of pyruvate kinase and gluconeogenesis in hepatocytes from phosphorylase kinase-deficient (gsd/gsd) rats.
Explore the source record for details and available documents.
Animals↗
SEARCH · PubMed Health
Results for “PHOSPHORYLASE KINASE”
Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Purification of several proteins involved in glycogen metabolism.
Several well-established procedures for the isolation of enzymes involved in glycogen metabolism have been modified such that all the enzymes can now be isolated from the same muscle preparation. The purified proteins are the catalytic subunit of cyclic AMP-dependent protein kinase, its thermostable inhibitor, glycogen phosphorylases a and b, and phosphorylase kinase. Phosphorylase kinase is separated by acid precipitation of the muscle extract. The other proteins are purified from the acid supernatant by chromatography on DEAE-cellulose. Further purification of each protein to homogeneity is then achieved using previously described methods. The proposed protocol saves sample tissue, and considerably reduces the work involved in obtaining muscle samples.
Acetates↗
Heparin, a powerful inhibition of type II casein kinases, stimulates the phosphorylation of some protein substrates by the catalytic subunit of cAMP-dependent protein kinase.
Heparin, which has been shown to behave as a very effective and specific inhibitor of type II casein kinases, exhibits a stimulatory effect on the phosphorylation rate of pyruvate kinase and phosphorylase kinase, but not of histones, by the catalytic subunit of cAMP-dependent protein kinase. When pyruvate kinase is the substrate the phosphorylation rate is approximately doubled by heparin concentrations around 100 micrograms/ml, but just 2 to 4 micrograms heparin per ml are sufficient to induce a half maximal effect. No stimulation by heparin can be observed replacing the protein substrates with two synthetic peptides reproducing the phosphorylatable sites of pyruvate kinase and of the gamma subunit of phosphorylase kinase. These data support the hypothesis that heparin accelerates phosphorylation by rendering the phosphorylatable sites more readily accessible to the protein kinase.
Animals↗
Adult-onset exercise intolerance due to phosphorylase b kinase deficiency.
Muscle-specific phosphorylase b kinase deficiency is an unusual form of glycogen storage disorder. The majority of patients are male with an age at diagnosis between 15 to 36 years. Clinical features include exercise intolerance, myalgia and muscle weakness. A forearm ischaemic exercise test is usually normal and histochemical staining for myophosphorylase positive. The demonstration of reduced muscle phosphorylase b kinase activity by biochemical assay confirms the diagnosis. We report a 36 year old male with phosphorylase b kinase deficiency and symptom onset in adult life.
Adult↗
Mechanism of calmodulin inhibition of cAMP-dependent protein kinase activation of phosphorylation kinase.
The activation of phosphorylase kinase (EC 2.7.1.38; ATP:phosphorylase b phosphotransferase) by the catalytic subunit of cAMP-dependent protein kinase (EC 2.7.1.37; ATP:protein phosphotransferase) is inhibited by calmodulin. The mechanism of that inhibition has been studied by kinetic measurements of the interactions of the three proteins. The binding constant for calmodulin with phosphorylase kinase was found to be 90 nM when measured by fluorescence polarization spectroscopy. Glycerol gradient centrifugation studies indicated that 1 mol of calmodulin was bound to each phosphorylase kinase. Phosphorylation of the phosphorylase kinase did not reduce the amount of calmodulin bound. Kinetic studies of the activity of the catalytic subunit of cAMP-dependent protein kinase on phosphorylase kinase as a function of phosphorylase kinase and calmodulin concentrations were performed. The results of those studies were compared with mathematical models of four different modes of inhibition: competitive, noncompetitive, substrate depletion, and inhibition by a complex between phosphorylase kinase and calmodulin. The data conform best to the model in which the inhibitory species is a complex of phosphorylase kinase and calmodulin. The complex apparently competes with the substrate, phosphorylase kinase, which does not have exogenous calmodulin bound to it. In contrast, the phosphorylation of the synthetic phosphate acceptor peptide, Kemptide, is not inhibited by calmodulin.
Adenosine Triphosphate↗
Synthase activation is not a prerequisite for glycogen synthesis in the starved liver.
To evaluate the contribution of phosphorylase and synthase interconversion as well as the availability of substrates to the onset of liver glycogen synthesis, this process was studied in rats starved overnight and refed for 4 h. On feeding, phosphorylase kinase and phosphorylase were inactivated in a cAMP-independent way, but the proportion of synthase a was unchanged and associated with increased hexoses 6-phosphate (glucose and fructose 6-phosphate), uridine diphosphoglucose (UDPG), and fructose 2,6-bisphosphate concentrations. These findings serve to support a "push" mechanism whereby substrate availability for synthase a concerted with phosphorylase inactivation provokes glycogen deposition. Anesthesia was compulsory for liver sampling and analysis. If such experiments were carried out in conscious rats killed by decapitation, artefactual cAMP-dependent phosphorylase activation and synthase inactivation were observed in starved animals. The phosphorylase activation persisted in refed animals but by a cAMP-independent mechanism.
Animals↗
Complex formation between phosphorylase b and phosphorylase b kinase. A new evidence for protein interactions in the phosphorylase system.
Explore the source record for details and available documents.
Animals↗
Cyclic nucleotides and changes in protein kinase activity ratio in the ischemic and nonischemic myocardium.
Following coronary artery ligation (CAL), levels of cAMP and the activity ratio of cAMP-dependent protein kinase, of phosphorylase kinase, and of phosphorylase are significantly elevated in both ischemic and nonischemic areas of the canine left ventricle. The aerobic level of cAMP was found to be 0.4 to 0.6 pmol/mg myocardium only after a precooled clamp or a cryobiopsy device was employed to guarantee tissue freezing in situ. Maximal changes in response to ischemia are observed within 2 min in both parts of the heart. Twenty minutes after the onset of ischemia, different responses have been found in the nonischemic and ischemic tissue. Whereas the levels of cAMP and the activity ratio of protein kinase, of phosphorylase kinase, and of phosphorylase returned to aerobic values in the nonischemic area, these parameters remained elevated in the ischemic area. The changes in the levels of myocardial cAMP and in the cAMP-dependent protein kinase activity ratio following CAL could be prevented by propranolol.
Animals↗
Influence of fructose on the glycogen synthase and phosphorylase systems in rat liver.
Fructose and glucose, when administered as a single, large intravenous dose (500 mg/kg) produced opposite effects on key regulatory enzymes of glycogen metabolism in intact normal fed animals. Glucose rapidly stimulated glycogen synthase phosphatase activity and increased the proportion of glycogen synthase in the active (I) form as expected; fructose reduced synthase phosphatase activity and the proportion of synthase in the I form. Glucose also stimulated a reduction in the proportion of phosphorylase in the active (a) form, whereas fructose stimulated an increase in the proportion of phosphorylase in thea form. The effect of fructose was not mediated by an increase in cyclic adenylate (cAMP) concentration nor by a conversion of phosphorylase kinase b to phosphorylase kinase a. As expected, the concentration of ATP decreased significantly. The increase in proportion of phosphorylase in the a form may be due to stimulation of phosphorylase kinase b activity by a decrease in the intracellular ATP:Mg++ ratio or by increase in intracellular Ca++ concentration. The mechanism of the fructose-induced change in synthase phosphatase activity and in synthase I activity is unknown.
Animals↗
Ganglioside-modulated protein phosphorylation in muscle. Activation of phosphorylase b kinase by gangliosides.
Gangliosides have profound effects on protein phosphorylation in skeletal muscle. Addition of GT1b to guinea pig muscle extract stimulated the phosphorylation of a 98-kDa protein 4-8-fold. In contrast, Ca2+ stimulated the phosphorylation of this protein and two other proteins with apparent Mr of 107,000 and 145,000, respectively. Addition of GT1b in the presence of Ca2+ further enhanced the phosphorylation of the 98-kDa protein but completely inhibited the phosphorylation of both the 107- and the 145-kDa proteins. The nature of the ganglioside-modulated 98-kDa protein has been characterized. Results on the pH activity profiles and the requirements of Ca2+ for phosphorylation suggest that this phosphoprotein may correspond to glycogen phosphorylase. Phosphorylation of purified rabbit muscle phosphorylase b by nonactivated phosphorylase kinase was stimulated by GT1b. This stimulation was in part due to an activation of the kinase activity. Autophosphorylation of highly purified phosphorylase kinase was increased 4-10-fold in the presence of GT1b. Polysialogangliosides were more potent than monosialogangliosides in stimulating the autocatalytic activity, whereas asialo-GM1, colominic acid, N-acetylneuraminic acid, and phosphatidylserine were ineffective. The effects of gangliosides were dose-dependent. At physiological pH, the concentrations of GT1b required for half-maximal stimulation of the autophosphorylation of phosphorylase kinase were 6.4 microM in the absence of Ca2+ and 1.3 microM when the divalent cation was present. These findings suggest that gangliosides may play a role as biomodulators in the regulation of glycogenolysis in muscle.
Animals↗
Severe phenotype of phosphorylase kinase-deficient liver glycogenosis with mutations in the PHKG2 gene.
Phosphorylase kinase-deficient liver glycogenosis manifests in infancy with hepatomegaly, growth retardation, and elevated plasma aminotransferases and lipids. It can be caused by mutations in three different genes of phosphorylase kinase subunits: PHKA2, PHKB, and PHKG2. It is usually a benign condition, often with complete resolution of symptoms during puberty. A minority of patients displays a more severe phenotype with symptomatic fasting hypoglycemia and abnormal liver histology that may progress to cirrhosis. Three patients with liver cirrhosis in childhood analyzed previously all had PHKG2 mutations. This suggested that this genotype may generally cause a more severe clinical manifestation, but to date PHKG2 mutations have been identified in only seven patients. Here, we report mutation analysis in three new patients with liver phosphorylase kinase deficiency and recurrent hypoglycemia, liver fibrosis, and lack of glucagon response but no overt cirrhosis. In all three patients, PHKG2 mutations were found (H89fs[insC], E157K, D215N, W300X). Three of these mutations are novel, bringing the total number of distinct human PHKG2 mutations to 11, found in 10 patients. We conclude that liver phosphorylase kinase deficiency with a severe phenotype, with or without cirrhosis, is indeed often caused by PHKG2 mutations. These patients require active measures to maintain normoglycemia (raw cornstarch, nocturnal tube feeding), which may also alleviate growth retardation and the development of abnormal liver histology.
Female↗
Phosphorylase b kinase deficiency in man: a review.
Phosphorylase b kinase is involved in the activation of glycogen phosphorylase and is thus involved in the breakdown of glycogen. The enzyme exists as several tissue specific isoenzymes of which the muscle enzyme (rabbit) has been most characterized. It is a multimeric protein composed of four subunits, alpha, beta, gamma and delta. The four subunits are coded on different chromosomes, the alpha, beta and gamma subunit genes being on the X, 16 and 17 chromosomes respectively. The delta subunit is a calmodulin and confers calcium sensitivity on phosphorylase b kinase. Tissue specificity of the enzyme is conferred, at least in some cases, by variation in the gamma subunit. Seven different clinical types of phosphorylase b kinase deficiency have been described. The most common type is X-linked and affects the liver only; other types affect liver, muscle and liver, muscle or heart and have an autosomal recessive mode of inheritance, while in some types the mode of inheritance is not clear. Diagnosis based on the study of erythrocytes or leukocytes can be misleading due to the tissue specific nature of the enzyme, and liver or muscle biopsies may be required.
Diagnosis↗
Characteristics of the dephosphorylated form of phosphorylase purified from rat liver and measurement of its activity in crude liver preparations.
The phosphorylated form of liver glycogen phosphorylase (alpha-1,4-glucan : orthophosphate alpha-glucosyl-transferase, EC 2.4.1.1) (phosphorylase a) is active and easily measured while the dephosphorylated form (phosphorylase b), in contrast to the muscle enzyme, has been reported to be essentially inactive even in the presence of AMP. We have purified both forms of phosphorylase from rat liver and studied the characteristics of each. Phosphorylase b activity can be measured with our assay conditions. The phosphorylase b we obtained was stimulated by high concentrations of sulfate, and was a substrate for muscle phosphorylase kinase whereas phosphorylase a was inhibited by sulfate, and was a substrate for liver phosphorylase phosphatase. Substrate binding to phosphorylase b was poor (KM glycogen = 2.5 mM, glucose-1-P = 250 mM) compared to phosphorylase a (KM glycogen = 1.8 mM, KM glucose-1-P = 0.7 mM). Liver phosphorylase b was active in the absence of AMP. However, AMP lowered the KM for glucose-1-P to 80 mM for purified phosphorylase b and to 60 mM for the enzyme in crude extract (Ka = 0.5 mM). Using appropriate substrate, buffer and AMP concentrations, assay conditions have been developed which allow determination of phosphorylase a and 90% of the phosphorylase b activity in liver extracts. Interconversion of the two forms can be demonstrated in vivo (under acute stimulation) and in vitro with little change in total activity. A decrease in total phosphorylase activity has been observed after prolonged starvation and in diabetes.
Adenosine Monophosphate↗
Diurnal variations in the activities of the glycogen metabolizing enzymes in mouse liver.
The glycogen level in mouse liver was maximal during the night and decreased to the lowest level during the light period. The peak activity of phosphorylase alpha was observed during the light hours and thus paralleled the decline of hepatic glycogen concentrations. The period of rapid glycogen synthesis (1800-2200 hr) was immediately preceded by maximum glycogen synthase alpha activity. Significant diurnal rhythms for phosphorylase kinase and phosphorylase phosphatase activities were also observed and appear to play a role in regulating the diurnal rhythm of phosphorylase alpha activity.
Animals↗
Purification and characterization of a novel protein phosphatase highly specific for ribosomal protein S6.
Ribosomal protein S6 is the principal phosphoprotein of the eucaryotic ribosome that becomes multiply phosphorylated on serine residues in response to a wide variety of mitogenic stimuli. In this paper the principal protein phosphatases able to dephosphorylate S6 were characterized in Xenopus laevis ovary and eggs. Two enzymes termed peak I and peak II were found to account for most S6 phosphatase activity in both oocytes and eggs. The peak I enzyme had an apparent Mr of 200,000 on gel filtration, dephosphorylated the beta subunit of phosphorylase kinase and phosphorylase a, and was inhibited by inhibitor 1 and inhibitor 2, suggesting it was similar to protein phosphatase 1. The peak II enzyme was purified over 12,000-fold and had an apparent Mr = 55,000 on glycerol gradient centrifugation. This phosphatase could dephosphorylate all sites in S6 but was unable to dephosphorylate phosphorylase a or phosphorylase kinase. However, it was inhibited by nanomolar concentrations of inhibitor 1 and inhibitor 2. These results indicate the peak II enzyme represents a new class of highly specific protein phosphatase and suggest that inhibition of dephosphorylation in cellular extracts by inhibitor 1 and inhibitor 2 is not a sufficient criterion for implicating protein phosphatase 1 in a cellular process.
Animals↗
Regulation of glycogen metabolism in liver by the autonomic nervous system. VI. Possible mechanism of phosphorylase activation by the splanchnic nerve.
The effects of autonomic-nerve stimulation on the activities of phosphorylase (EC 2.4.1.1), dephospho-phosphorylase kinase (EC 2.7.1.38) and phosphorylase phosphatase (EC 3.1.3.17), and on the concentration of adenosine 3', 5'-monophosphate in rabbit liver were investiaged. Results were compared with the effects of epinephrine and glucagon on these enzymes. 1. The acitivity of liver phosphorylase increased rapidly and markedly on electrical stimulation of the splanchnic nerve, or after intraportal administration of epinephrine or glucagon. The activity was not affected by vagal stimulation. 2. The activity of dephospho-phosphorylase kinase increased about 2--3-fold 1 min after injections of epinephrine and glucagon, glucagon causing more activation than epinephrine. The enzyme activity was not altered by splanchnic-nerve, or vagal stimulation. 3. Injections of epinephrine and glucagon caused marked elevation of liver adenosine 3', 5'-monophosphate within a few minutes. With epinephrine, the nucleotide concentration rose to a maximum after 1 min and amounted to about 3-fold increase, while with glucagon the maximum increase of approximately 8-fold increase was observed after 2 min. Stimulation of the splanchnic nerve for 10 min did not affect the adenosine 3', 5'-monophosphate level in the liver. Vagal stimulation also had no effect on the level. 4. The activity of phosphorylase phosphatase decreased promptly (within 30 s) and markedly on splanchnic-nerve stimulation, but did not change significantly on administration of epinephrine of glucagon. A small but insignificant increase in phosphatase activity wasobserved upon vagal stimulation. 5. The effect of Ca-2+ on purified dephospho-phosphorylase kinase was studied. The activity was found to depend partially on free Ca-2+ at low Ca-2+ concentrations (1-10-minus 7--1-10-minus 5 M). 6. These results suggest that the rise in hepatic phosphorylase content upon splanchnic-nerve stimulation, unlike that induced by epinephrine and glucagon, is not mediated by adenosine 3', 5'-monophosphate and subsequent activation of dephospho-phosphorylase kinase, but rather by inactivation of phosphorylase phosphatase. The possible existence of a new factor in this mechanism is discussed.
Adenosine Monophosphate↗
Dephosphorylation of distinct sites in myosin light chain by two types of phosphatase in aortic smooth muscle.
Two types of myosin light chain phosphatase from aortic smooth muscle extract were separated by chromatography on heparin-agarose. The phosphatase which appeared in the flow-through fractions had low activity on actomyosin, its apparent molecular mass was 260 kDa and upon ethanol treatment it generated a catalytic subunit with an apparent molecular mass of 36-39 kDa as determined by gel filtration. This phosphatase preferentially dephosphorylated the alpha-subunit of phosphorylase kinase and its phosphorylase phosphatase activity was not inhibited by heparin, inhibitor-1 or inhibitor-2. The phosphatase retained by heparin-agarose had high activity on actomyosin, its apparent molecular mass was 150 kDa and upon ethanol treatment it generated a catalytic subunit with an apparent molecular mass of 39-42 kDa. It preferentially dephosphorylated the beta-subunit of phosphorylase kinase and its phosphorylase phosphatase activity was not inhibited by heparin, inhibitor-1 or inhibitor-2. Myosin light chain was phosphorylated by myosin light chain kinase in peptides AB (Ser-P) and CD (Thr-P), and/or by protein kinase C in peptides E (Ser-P) and F (Thr-P) as determined by one-dimensional phosphopeptide mapping. The catalytic subunit of heparin-agarose flow-through phosphatase preferentially dephosphorylated peptide F over peptides AB, CD and E in both isolated light chain and actomyosin. The catalytic subunit of heparin-agarose bound phosphatase could effectively dephosphorylate all sites in isolated light chain, whereas it was less effective on dephosphorylation of peptide E in actomyosin.
Animals↗
Effect of Mg2+ concentrations on phosphorylation/activation of phosphorylase b kinase by cAMP/Ca(2+)-independent, autophosphorylation-dependent protein kinase.
In a previous report [Yu and Yang, Biochem. Biophys. Res. Commun. 207, 140-147 (1995)], phosphorylase b kinase from rabbit skeletal muscle was found to be phosphorylated and activated by a cyclic nucleotide- and Ca(2+)-independent protein kinase previously identified as an autophosphorylation-dependent multifunctional protein kinase (autokinase) from brain and liver [Yang et al., J. Biol. Chem. 262, 7034-7040, 9421-9427 (1987)]. In this report, the effect of Mg2+ ion concentration on the auto-kinase-catalyzed activation of phosphorylase b kinase is investigated. The levels of phosphorylation and activation of phosphorylase b kinase catalyzed by auto-kinase are found to be dependent on the concentration of Mg2+ ion used. Phosphorylation of phosphorylase b kinase at high Mg2+ ion (> 9 mM) is 2-3 times higher than that observed at low Mg2+ ion (1 mM) and this results in a further 2- to 3-fold activation of the enzyme activity at high Mg2+ ion. Analysis of the phosphorylation stoichiometry of alpha and beta subunits of phosphorylase b kinase at different Mg2+ ion concentrations further reveals that the phosphorylation level of the beta subunit remains almost unchanged, whereas the phosphorylation level of the alpha subunit increases dramatically and correlates with the increased enzyme activity. In similarity with the beta subunit, phosphorylations of myelin basic protein and histone 2A by auto-kinase are also unaffected by Mg2+ ion. Taken together, the results provide initial evidence that Mg2+ ion may specifically render the alpha subunit a better substrate for auto-kinase to cause further phosphorylation/activation of phosphorylase b kinase, representing a new mode of control mechanism for the regulation of auto-kinase involved in the phosphorylation and concurrent activation of phosphorylase b kinase.
Adenosine Triphosphate↗