Photocleavage of muscle glycogen phosphorylase by vanadate.

Glycogen phosphorylase is progressively degraded during irradiation with near UV light in the presence of vanadate. The pattern of protein cleavage by monovanadate is characterised by fewer peptides than that by decavanadate, which leads to fragmentation in a ligand dependent way. In both instances, the initial cleavage releases a peptide of 82,000 daltons which accounts for the N-terminal portion of the subunit, including the regulatory phosphorylation site.     

Insulin regulation of hepatic glycogen synthase and phosphorylase.

The relative roles of insulin and glucose in the regulation of hepatic glycogen synthase and phosphorylase were studied in hepatocytes from fed rats. Elevation of extra-cellular glucose led to a rapid decrease in phosphorylase a activity followed by a slower increase in glycogen synthase I activity. A reciprocal and coordinate relationship between phosphorylase inactivation and synthase activation in response to glucose was observed; following initial glucose-induced inactivation of phosphorylase, there was a highly significant linear inverse relationship between residual phosphorylase activity and glycogen synthase activation. Insulin led to a further decrease in phosphorylase activity and a 30-50% additional increase in glycogen synthase activity over that caused by glucose. The effects of insulin required the presence of glucose and served to augment acute glucose stimulation of glycogen synthase and inhibition of phosphorylase. Insulin did not perturb the reciprocal and coordinate relationship between phosphorylase inactivation and synthase activation in response to glucose. The results suggest that the ability of insulin to activate hepatic glycogen synthase can be entirely accounted for by its ability to inactivate phosphorylase.     

The influence of vasopressin and related peptides on glycogen phosphorylase activity and phosphatidylinositol metabolism in hepatocytes.

The relative abilities of seven vasopressin-like peptides to activate hepatic glycogen phosphorylase and stimulate phosphate incorporation into phosphatidylinositol were compared. Although the individual peptides differed in their potencies, the concentrations required to stimulate phosphatidylinositol metabolism were always greater (about 10 times) than those needed to activate phosphorylase. The molecular specificity of the hepatic vasopressin receptor and the role of vasopressin-stimulated phosphatidylinositol turnover are discussed.     

The interaction of muscle glycogen phosphorylase b with glycogen

Interaction of muscle glycogen phosphorylase b (EC 2.4.1.1) with glycogen was studied by sedimentation, stopped-flow and temperature-jump methods. The equilibrium enzyme concentration was determined by sedimentation in an analytical ultracentrifuge equipped with absorption optics and a photoelectric scanning system. The maximum adsorption capacity of pig liver glycogen is 3.64 mumol dimeric glycogen phosphorylase b per g glycogen, which corresponds to 20 dimeric enzyme molecules per average glycogen molecule of Mr 5.5 X 10(6). Microscopic dissociation constants were determined for the enzyme-glycogen complex within the temperature range from 12.7 to 30.0 degrees C. Enzyme-glycogen complexing is accompanied by increasing light scattering and its increment depends linearly on the concentration of the binding sites on a glycogen particle that are occupied by the enzyme. Complex formation and relaxation kinetics are in accordance with the proposed bimolecular reaction scheme. The monomolecular dissociation rate constant of the complex increases as the temperature increases from 12.7 to 30.0 degrees C, whereas the bimolecular rate constant changes slightly and is about 10(8) M-1 X S-1. These data point to the possibility of diffusional control of the complex formation.     

Kinetics of the reaction between muscle glycogen phosphorylase B and glycogen

Kinetics of glycogen binding by glycogen phosphorylase b has been studied by stopped flow and temperature jump methods. This reaction is followed by increase in light scattering whose amplitude depends upon the enzyme binding sites concentration of glycogen particles occupied by the enzyme. It has been shown that the complex formation has the first order with respect to enzyme and glycogen concentrations. Relaxation kinetics is compatible with proposed bimolecular reaction scheme. Microscopic rate constants of the forward and reverse reactions of glycogen binding by glycogen phosphorylase b are determined in temperature range from 12,7 to 30 degrees C. The possibility of diffusional control of the binding rate is discussed.     

Adenovirus-mediated gene transfer of interferon alpha inhibits hepatitis C virus replication in hepatocytes

Recently we reported that on-site interferon (IFN)-alpha production in the liver using an adenovirus vector can achieve a substantial confinement of IFN-alpha in the target organ and can improve liver fibrosis in a rat liver cirrhosis model. However, the major therapeutic effect of IFN for hepatitis C virus (HCV)-associated liver diseases is its antiviral effect on HCV. As a prelude to the in vivo HCV infection experiment using a primate animal model, here we examined the antiviral effect of IFN-alpha gene transfer into HCV-positive hepatocytes in vitro. The non-neoplastic human hepatocyte cell line PH5CH8 was inoculated with HCV-positive serum. Successful in vitro HCV replication and thus the validity of this model was confirmed by a strong selection for HCV variants determined by sequence analysis of the hypervariable region and an increase of HCV RNA estimated by real time TaqMan RT-PCR. One day after the inoculation of HCV, PH5CH8 cells were infected with adenoviral vectors encoding human IFN-alpha cDNA. HCV completely disappeared 9 days after the adenoviral infection, which is linked to the increase of 2('),5(')-oligoadenylate synthetase activity, suggesting that IFN-alpha produced by gene transfer effectively inhibits HCV replication in hepatocytes. This study supports the development of IFN-alpha gene therapy for HCV-associated liver diseases.     

Site-directed mutants of glycogen phosphorylase are altered in their interaction with phosphorylase kinase

Glycogen phosphorylase is found in resting muscle as phosphorylase b, which is inactive without AMP. Phosphorylation by phosphorylase kinase (PhK) produces phosphorylase a, which is active in the absence of AMP. PhK is the only kinase that can phosphorylate phosphorylase b, which in turn is the only physiological substrate for PhK. We have explored the reasons for this specificity and how these two enzymes recognize each other by studying site-directed mutants of glycogen phosphorylase. All mutants were assayed for changes in their interaction with a truncated form of the catalytic subunit of phosphorylase kinase, gamma(1-300). Five mutations (R69K, R69E, R43E, R43E/R69E, and E501A), made at sites that interact with the amino terminus in either phosphorylase b or a, showed little difference in phosphorylation by gamma(1-300) compared to wild-type phosphorylase b. Five mutations, made at three sites in the amino-terminal tail of phosphorylase (K11A, K11E, I13G, R16A, and R16E), however, produced decreases in catalytic efficiency for gamma(1-300), compared to that for phosphorylase b. R16E was the poorest substrate for gamma(1-300), giving a 47-fold decrease in catalytic efficiency. The amino terminus, and especially Arg 16, are very important factors for recognition of phosphorylase by gamma(1-300). A specific interaction between Lys 11 of phosphorylase and Glu 110 of gamma(1-300) was also confirmed. In addition, I13G and R16A were able to be phosphorylated by protein kinase A, which does not recognize native phosphorylase.     

Enzyme kinetics of muscle glycogen phosphorylase b

Interest in the kinetics of glycogen phosphorylase has recently been renewed by the hypothesis of a glycogen shunt and by the potential of altering phosphorylase to treat type II diabetes. The wealth of data from studies of this enzyme in vitro and the need for a mathematical representation for use in the study of metabolic control systems make this enzyme an ideal subject for a mathematical model. We applied a two-part approach to the analysis of the kinetics of glycogen phosphorylase b (GPb). First, a continuous state model of enzyme-ligand interactions supported the view that two phosphates and four ATP or AMP molecules can bind to the enzyme, a result that agrees with spectroscopic and crystallographic studies. Second, using minimum error estimates from continuous state model fits to published data (that agreed well with reported error), we used a discrete state model of internal molecular events to show that GPb exists in three discrete states (two of which are inactive) and that state transitions are concerted. The results also show that under certain concentrations of substrate and effector, ATP can activate the enzyme, while under other conditions, it can competetively inhibit or noncompetitively inhibit the enzyme. This result is unexpected but is consistent with spectroscopic, crystallographic, and kinetic experiments and can explain several previously unexplained phenomena regarding GPb activity in vivo and in vitro.     

Intron/exon structure of the human gene for the muscle isozyme of glycogen phosphorylase

The intron/exon organization of the human gene for glycogen phosphorylase has been determined. The segments of the polypeptide chain that corresponds to the 19 exons of the gene are examined for relationships between the three-dimensional structure to the protein and gene structure. Only weak correlations are observed between domains of phosphorylase and exons. The nucleotide binding domains that are found in phosphorylase and other glycolytic enzymes are examined for relationships between exons of the genes and structures of the domains. When mapped to the three-dimensional structures, the intron/exon boundaries are shown to be widely distributed in this family of protein domains.     

The effect of solvents on nucleotide regulation of glycogen phosphorylase.

The activity of glycogen phosphorylase is controlled by two nucleotide sites. We have found that organic solvents affect the regulatory properties of phosphorylase by altering the binding at these two sites. At the activator site, the Ka for AMP is lowered 10-fold in the presence of 10% 1,2-dimethoxyethane while, at the inhibitor site, the Ki for caffeine is increased 6-fold. The stimulation of activity by organic solvents is highly dependent on the enzyme's activity state. Phosphorylase b, which has a requirement for a nucleotide activator, loses this requirement in the presence of organic solvents, while the active form of the enzyme, phosphorylase a, is only slightly stimulated by organic solvents. The activation profile obtained with rabbit liver phosphorylase suggests that differences in the properties of this enzyme from rabbit muscle phosphorylase might be explained by a change in the relative affinity for AMP at the two nucleotide sites. The results also suggest that 1,2-dimethoxyethane may be useful to determine accurately the activities of different forms of liver phosphorylase.     

Adenovirus-mediated transfer of the SOCS-1 gene to mouse lung confers protection against hyperoxic acute lung injury

Suppressor of cytokine signaling-1 (SOCS-1) is a member of the suppressor of cytokine signaling family of proteins and an inhibitor of interleukin-6 (IL-6) signaling. SOCS-1 has been shown to protect cells from cellular damage and apoptosis induced by tumor necrosis factor (TNF), lipopolysaccharide (LPS), and interferon gamma (IL-γ). However, it is not known whether increased SOCS-1 is protective during pulmonary oxidative stress. Therefore, we hypothesized that increased SOCS-1 in the lungs of mice would be protective in the setting of hyperoxic lung injury. We administered SOCS-1 adenovirus (Ad-SOCS-1) intratracheally into the lungs and exposed the mice to 100% O₂. Mice infected with GFP adenovirus (Ad-GFP) were used as controls. Mice treated with Ad-SOCS-1 had enhanced survival in 100% oxygen compared to Ad-GFP-administered mice. After 3 days of hyperoxia, Ad-GFP mice were ill and tachypnic and died after 4 days. In contrast, all Ad-SOCS-1-treated mice survived for at least 6 days in hyperoxia and 80% survived beyond 7 days. Ad-SOCS-1 transfection protected mouse lungs from injury as indicated by lower lung wet/dry weight, alveolar–capillary protein leakage, reduced infiltration of inflammatory cells, and lower content of thiobarbituric acid-reactive substances in lung homogenate. Our results also indicated that Ad-SOCS-1 significantly inhibits hyperoxia-induced ASK-1 (apoptosis signal-regulating kinase 1) expression. Taken together, these findings show that increased expression of adenovirus-mediated SOCS-1 in the lungs of mice significantly protects against hyperoxic lung injury.     

Epinephrine regulation of skeletal muscle glycogen metabolism. Studies utilizing the perfused rat hindlimb preparation

Studies of rat skeletal glycogen metabolism carried out in a perfused hindlimb system indicated that epinephrine activates phosphorylase via the cascade of phosphorylation reactions classically linked to the beta-adrenergic receptor/adenylate cyclase system. The beta blocker propranolol completely blocked the effects of epinephrine on cAMP, cAMP-dependent protein kinase, phosphorylase, and glucose-6-P, whereas the alpha blocker phentolamine was totally ineffective. Omission of glucose from the perfusion medium did not modify the effects of epinephrine. Glycogen synthase activity in control perfused and nonperfused muscle was largely glucose-6-P-dependent (-glucose-6-P/+glucose-6-P activity ratios of 0.1 and 0.2, respectively). Epinephrine perfusion caused a small decrease in the enzyme's activity ratio (0.1 to 0.05) and a large increase in its Ka for glucose-6-P (0.3 to 1.5 mM). This increase in glucose-6-P dependency correlated in time with protein kinase activation and was totally blocked by propranolol and unaffected by phentolamine. Comparison of the kinetics of glycogen synthase in extracts of control and epinephrine-perfused muscle with the kinetics of purified rat skeletal muscle glycogen synthase a phosphorylated to various degrees by cAMP-dependent protein kinase indicated that the enzyme was already substantially phosphorylated in control muscle and that epinephrine treatment caused further phosphorylation of synthase, presumably via cAMP-dependent protein kinase. These data provide a basis for speculation about in vivo regulation of the enzyme.     

Effects of altered thyroid status on beta-adrenergic actions on skeletal muscle glycogen metabolism

The effects of hypothyroidism on glycogen metabolism in rat skeletal muscle were studied using the perfused rat hindlimb preparation. Three weeks after propylthiouracil treatment, serum thyroxine was undetectable and muscle glycogen and Glc-6-P were decreased. Basal and epinephrine-stimulated phosphorylase a and phosphorylase b kinase activities were also significantly reduced, as were epinephrine-stimulated cAMP accumulation and cAMP-dependent protein kinase activity. Conversely, basal and epinephrine-stimulated glycogen synthase I activities were significantly higher while the Ka of the enzyme for Glc-6-P was lower in hypothyroid animals. Propylthiouracil-treated rats also had increased phosphoprotein phosphatase activities towards phosphorylase and glycogen synthase and decreased activity of phosphatase inhibitor 1. beta-Adrenergic receptor binding and basal and epinephrine-stimulated adenylate cyclase activities were reduced in muscle particulate fractions from hypothyroid rats. Administration of triiodothyronine to rats for 3 days after 3 weeks of propylthiouracil treatment restored the altered metabolic parameters to normal. It is proposed that the decreased beta-adrenergic responsiveness of the enzymes of glycogen metabolism in hypothyroid rat skeletal muscle is due to increased activity of phosphoprotein phosphatases and to reduced beta-adrenergic receptors and adenylate cyclase activity.     

Interaction of muscle glycogen phosphorylase B with F-actin

The binding of rabbit muscle glycogen phosphorylase b to F-actin has been studied by sedimentation in analytical centrifuge in 10 mM Tris-acetate buffer pH 6.8 at 20 degrees C. The adsorption capacity of F-actin is equal to (7.8 +/- 0.9) X 10(-7) mole of glycogen phosphorylase b per 1 g of F-actin; the microscopic dissociation constant for the glycogen phosphorylase-F-actin complex is (5.4 +/- 0.5) X 10(-7) M. It was found that the allosteric activator, AMP, facilitates the adsorption of glycogen phosphorylase b on F-actin, whereas the substrate, Pi, and the inhibitor, ATP, cause an opposite effect.