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PubMed1984

Comparison of the phosphorylation of rabbit skeletal muscle phosphorylase kinase by cAMP-dependent protein kinase and cAMP-independent glycogen synthase (casein) kinase-1.

We have previously reported that rabbit skeletal muscle phosphorylase kinase is phosphorylated by glycogen synthase (casein) kinase-1 (CK-1) primarily on the beta subunit (beta = 1 mol of PO4; alpha = 0.2 mol of PO4) when the reaction was carried out in beta-glycerophosphate. The resultant enzyme activation was 16-fold (Singh, T. J., Akatsuka, A., and Huang, K.-P. (1982) J. Biol. Chem. 257, 13379-13384). In the present study we found that in Tris-Cl buffer CK-1 catalyzes the incorporation of greater than 2 mol of PO4/monomer into each of the alpha and beta subunits. Phosphorylase kinase activation resulting from the higher level of phosphorylation remained 16-fold. 32P-Labeled tryptic peptides from the alpha and beta subunits were analyzed by isoelectric focusing. Cyclic AMP-dependent protein kinase (A-kinase) phosphorylates a single major site in each of the alpha and beta subunits at 1.5 mM Mg2+. In addition to these two sites, A-kinase phosphorylates at least three other sites in the alpha subunit at 10 mM Mg2+. CK-1 also catalyzes the phosphorylation of multiple sites in both the alpha and beta subunits. Of the two major sites phosphorylated by CK-1 in the beta subunit, one of these sites is also recognized by A-kinase. At least three sites are phosphorylated by CK-1 in the alpha subunit. One of these sites is recognized by CK-1 only after a prior phosphorylation of phosphorylase kinase by A-kinase at a single site in each of the alpha and beta subunits at 1.5 mM Mg2+. The roles of the different phosphorylation sites in phosphorylase kinase activation are discussed.

Animals↗
PubMed1980

Regulation of liver phosphorylase phosphatase by glutathione disulfide.

The mechanism of inactivation of rabbit liver phosphorylase phosphatase by glutathione disulfide (GSSG) was investigated. The catalytic subunit of phosphorylase phosphatase was inactivated by GSSG and other disulfides. Inactivation by GSSG was a concentration-dependent process and resulted in the formation of an inactive, stable enzyme species. The inactivated enzyme could be reactivated by addition of various sulfhydryl compounds, including glutathione (GSH). Homogeneous phosphorylase phosphatase contains, per mol of catalytic subunit (Mr = 33,000), two sulfhydryl groups, one of which reacted with GSSG to form inactive enzyme. Binding studies with [glycine-2-3H]GSSG revealed simultaneous incorporation of 3H radioactivity into the catalytic subunit and stoichiometric loss of catalytic activity. Treatment of the 3H-labeled enzyme with GSH was accompanied by release of 3H radioactivity from the enzyme and restoration of enzyme activity. The results suggest that inactivation of phosphorylase phosphatase by GSSG results from the formation of a mixed disulfide between GSSG and one of the two sulfhydryl groups in the catalytic subunit.

Animals↗
PubMed1982

Rabbit kidney cortex phosphorylase phosphatases: evidence for complexes between high molecular weight forms and heat-stable inhibitor proteins.

Two forms of high molecular weight phosphorylase phosphatase have been partially resolved by gel filtration chromatography of rabbit kidney cortex extracts. Two heat-stable inhibitor proteins co-eluted with the phosphatase peaks. Phosphorylase phosphatase and heat-stable inhibitor activity also co-migrated on gel electrophoresis of cortex extracts. When extracts were heated to 95 degrees for 5 minutes prior to gel filtration or electrophoresis, phosphorylase phosphatase inhibitor activity eluted at a lower molecular weight and a higher mobility, respectively. Storing cortex extracts at -20 degrees for 6 months resulted in partial conversion of both phosphatase and inhibitor activities to lower molecular weight forms which co-eluted on gel filtration. The two inhibitor peaks from gel filtration chromatography were heat-treated and characterized. Both inhibitor peaks had molecular weight of 25,000 to 35,000. The inhibitory activity of one of the peaks was increased about 3.5-fold by incubation with cyclic AMP-dependent protein kinase and ATP, and required higher concentrations of TCA to be precipitated. Hence, one of the inhibitor peaks resembled rabbit muscle inhibitor -1, while the other peak may represent an inhibitor similar to rabbit muscle inhibitor -2. These results represent the first indication that low molecular weight heat-stable inhibitor proteins may be bound to high molecular weight phosphorylase phosphatases in the cell.

Animals↗
PubMed1982

[cAMP-dependence of phosphorylation of the phosphorylase b kinase of the skeletal muscles of rats during physical exercise and training].

During relative rest of trained rats the phosphorylase b kinase activity is increased by 24% (pH 6.8). Physical load causes an increase of the phosphorylase b kinase activity of untrained and trained rats by 44 and 33%, respectively. The degree of phosphorylase b kinase phosphorylation by a homologous soluble cAMP-dependent protein kinase from the muscles of trained rats at rest is 1.9 times that of the control group. The cAMP-dependent phosphorylation of phosphorylase b kinase of untrained and trained rats under physical load is increased 2.5-fold. The data obtained are indicative of the regulatory role of cAMP-dependent phosphorylation in biochemical adaptation of skeletal muscles when their function is increased.

Animals↗
PubMed1983

Time course of alpha1-adrenergic and vasopressin actions on phosphorylase activation, calcium efflux, pyridine nucleotide reduction, and respiration in hepatocytes.

Calcium efflux from rat liver perfused with nonrecirculating medium was observed at 1.4 s following 10(-6) M (-)epinephrine infusion, when the perfusate Ca2+ was 60 microM. Net calcium efflux was also seen in livers perfused with 1.3 microM Ca2+ at approximately 8 s. In isolated rat hepatocytes, phosphorylase, a cytosolic enzyme, was activated significantly at 3 s and maximally at approximately 15 s by phenylephrine (10(-5) M), epinephrine (10(-6) M), and vasopressin (10(-8) M). Hexose phosphates were elevated at between 3 and 6 s with vasopressin. Phenylephrine and vasopressin stimulated hepatocyte respiration relatively slowly. The effects took 10 s to become evident, were dependent on the presence of Ca2+, and were probably the result of increased total cellular reduced pyridine nucleotide observed at 5 s. The slowness of the increase in respiration indicates that it cannot be the cause of the Ca2+ mobilization, but is more likely to be a consequence of it. From these studies, it is proposed that, following binding of catecholamines to alpha 1-adrenergic receptors, Ca2+ is first mobilized from the plasma membrane resulting in an elevation of the free Ca2+ ion concentration in the cytosol (Charest, R., Blackmore, P. F., Berthon, B., and Exton, J. H. (1983) J. Biol. Chem. 258, 8769-8773) which stimulates phosphorylase kinase and, hence, phosphorylase. These events begin to occur within the first 2 to 3 s. Following this, the concentration of reduced pyridine nucleotide(s) increases at 5 s resulting in the stimulation of respiration seen at 10 s. These events occur more slowly than the mobilization of cell Ca2+ and activation of phosphorylase, and may be secondary to the rise in cytosolic Ca2+. The time at which mitochondrial Ca2+ decreases is not known, but it accounts for most of the Ca2+ mobilized.

Animals↗
PubMed1984

A probable mechanism for tolbutamide mediated activation of glycogen phosphorylase in the isolated rat heart.

Previous studies have shown that 0.6 mM tolbutamide stimulates the rate of glycogenolysis and transiently increases % phosphorylase a activity in the isolated rat heart. Since tolbutamide has been reported to activate adenylate cyclase, one possible mechanism for the conversion of phosphorylase b to the a form is through a cAMP mediated process. However, we failed to detect any drug-induced changes in tissue cAMP content of rat and rabbit hearts or in basal, Gpp(NH)p stimulated and isoproterenol-stimulated adenylate cyclase activity of sarcolemma prepared from rat, rabbit or dog ventricles. Since tolbutamide can alter calcium transport across cell membranes, we investigated the possibility that the drug's effects on phosphorylase were linked to an elevation in calcium concentration. It was found that tolbutamide was not able to activate phosphorylase when the calcium channel blocker verapamil was present in the perfusate. In addition, the sulfonylurea increased binding of [3H]-verapamil to isolated sarcolemma suggesting that tolbutamide is able to unmask previously inactive calcium channels and thereby stimulate calcium movement into the cell.

Adenylyl Cyclases↗
PubMed1980

Role of uridine phosphorylase for antitumor activity of 5'-deoxy-5-fluorouridine.

5'-Deoxy-5-fluorouridine (5'-DFUR) was parenterally and orally effective on various transplantable tumors and its activity was better than that of other fluorinated pyrimidines. However, like 5-fluorouracil and 2'-deoxy-5-fluorouridine (FUdR), 5'-DFUR was ineffective on L1210 leukemia resistant to 5-fluorouracil, suggesting that it would exert its antitumor activity through converted 5-fluorouracil. In tissue culture, 5'-DFUR inhibited the growth of various tumor cells similarly to ther fluorinated pyrimidines. However, 5'-DFUR was unique in that uridine completely reversed its inhibitory effect. Enzymological study clarified that uridine inhibited the conversion of 5'-DFUR to 5-fluorouracil by a uridine phosphorylase, in parallel to its reverse effect on cell growth inhibition by 5'-DFUR. Furthermore, a subline of L1210 leukemia resistant to 5'-DFUR but not to 5-fluorouracil was found to lack the uridine phosphorylase. These results indicate that 5'-DFUR is a depot form of 5-fluorouracil which can be promptly activated by uridine phosphorylase. In addition, the uridine phosphorylase was found to be abundant in sarcoma-180 solid tumor, leading to a significantly higher concentration of converted 5-fluorouracil in this tumor than in other normal tissues. This provides a good explanation for the high chemotherapeutic index of 5'-DFUR against this tumor, which may be applicable also for other tumors.

Animals↗
PubMed1984

Activated states of phosphorylase kinase as detected by the chemical cross-linker 1,5-difluoro-2,4-dinitrobenzene.

When phosphorylase kinase from rabbit skeletal muscle was activated by phosphorylation and then cross-linked with 1,5-difluoro-2,4-dinitrobenzene at pH 6.8, dimers of beta subunits were formed that were not observed during cross-linking of nonphosphorylated enzyme under the same conditions. The ability to form these dimers was due to phosphorylation of the beta subunit because when enzyme phosphorylated in the alpha and beta subunits was incubated with a protein phosphatase relatively specific for the beta subunit (Ganapathi, M.K., Silberman, S.R., Paris, H., and Lee, E.Y.C. (1981) J. Biol. Chem. 256, 3213-3217), the ability to form the cross-linked beta dimers was lost. Significant amounts of two complexes also judged to be dimers of beta subunits were observed when nonphosphorylated phosphorylase kinase was cross-linked after preincubation with Ca2+ plus Mg2+ ions, after proteolysis by chymotrypsin, or when it was cross-linked at pH 8.2, three conditions known to stimulate the activity of the nonphosphorylated enzyme. From these results, we conclude that 1,5-difluoro-2,4-dinitrobenzene can serve as a structural probe for activated states of phosphorylase kinase. The activation is associated with a conformational change in which two beta subunits either move closer together or have a reactive group on one, or both, of them unmasked. Our results suggest that the diverse mechanisms listed above for stimulating phosphorylase kinase activity cause a common conformational change to occur.

Animals↗
PubMed1982

Effects of alloxan diabetes on the turnover of rat liver glycogen synthase. Comparison with liver phosphorylase.

Rat liver glycogen synthase shows almost a 2-fold increase in activity 8 days after onset of alloxan diabetes. Immunological and catalytic criteria indicate that the change in activity is associated with an increase in the amount of enzyme in the diabetic. Apparent rates of degradation were determined for isolated glycogen synthase and phosphorylase from the livers of 2-, 5-, and 8-day diabetic, insulin-treated diabetic and normal rats using the double isotope ([3H]leucine and [14C]leucine) labeling method (Arias, I. M., Doyle, D., and Schimke, R. T. (1969) J. Biol. Chem. 244, 3303-3315). Relative rates of enzyme synthesis and degradation were determined by comparing the 3H incorporation and 3H/14C ratios of the isolated enzymes to the isotope labeling of a liver fraction representing the average of liver proteins. Glycogen synthase showed a gradual increase in the rate of degradation through the course of diabetes with an average relative rate of degradation in the 8-day diabetic 1.8 times greater than the normal. The relative rate of synthesis for glycogen synthase in the diabetic was 2.2- to 2.5-fold greater than the normal. Phosphorylase from 5- and 8-day diabetic rats had relative rates of degradation 4.0-5.3 times greater than enzyme from the normal. In the diabetic, the rate of degradation of phosphorylase was greater than for synthase while the opposite was observed in the normal rat. The relative rate of synthesis for phosphorylase from diabetic rats was approximately 4.5-fold greater than normal. The increased concentration of glycogen synthase in the diabetic liver is because of an increased rate of synthesis and not a decreased rate of enzyme degradation.

Animals↗
PubMed1982

Deficiency of methylthioadenosine phosphorylase in human leukemic cells in vivo.

Cells from 20 patients with leukemia and 9 with solid tumors were assayed for the enzyme methylthioadenosine phosphorylase, which function in both purine and polyamine metabolism in rapidly dividing cells. As determined by autoradiography of viable cells, and by direct enzymatic analysis, samples from one individual with pre-T-cell acute lymphoblastic leukemia and one with common acute lymphoblastic leukemia were methylthioadenosine phosphorylase deficient. In contrast, other leukemias of similar antigenic phenotype, as well as normal peripheral blood lymphocytes, thymic lymphocytes, and normal bone marrow cells, had substantial methylthioadenosine phosphorylase activity. This evidence suggests that the complete absence of methylthioadenosine phosphorylase distinguishes some leukemic cells in vivo from their nonmalignant counterparts.

Adolescent↗
PubMed1982

Rabbit skeletal muscle phosphorylase kinase. Interactions between subunits and influence of calmodulin on different complexes.

Aspects of the molecular interaction and subunit structure of rabbit skeletal muscle phosphorylase kinase, (alpha beta gamma delta)4, were investigated. Exogenous addition of the delta subunit (calmodulin) stimulated the activities of nonactivated phosphorylase kinase and the alpha gamma delta complex, but not the gamma delta complex. This stimulatory effect does not seem to affect the activity-pH profile of the native kinase and is expressed at least partly through an interaction of the calmodulin with the alpha subunit, resulting in an increase in the apparent Vm parameters. Low concentrations of trifluoperazine had only slight effect on the activities of the three forms of kinase, whereas high concentrations caused nonspecific inactivation. The effects of trifluoperazine and EGTA on the activities of the alpha gamma delta and gamma delta complexes were additive; together, they inactivated the two complexes to about 10% of their original activities. The delta subunit remained tightly bound to phosphorylase kinase and the alpha gamma delta complex, even in the presence of 8 M urea, but less so with the gamma delta complex. The nonactivated kinase is more stable towards heat inactivation at 37 degrees C than the alpha gamma delta complex, whereas the gamma delta complex is least stable. Similar to the holoenzyme, limited trypsin digestion activated the alpha gamma delta complex. The pH 6.8/8.2 activity ratio of this complex increased from 0.5 to 0.9, with concomitant degradation of the alpha subunit. In contrast, the gamma delta complex is fairly inert to limited proteolysis. By using sucrose density gradient ultracentrifugation and pretreatment of the gamma delta complex with EGTA, results indicated that the gamma subunit may be a Ca2+-independent kinase, which has about 30% of the original phosphorylase kinase activity.

Animals↗
PubMed1981

Rat liver phosphorylase kinase. Stimulation by heparin.

Rat liver phosphorylase kinase activity was increased up to 7-fold by low concentrations of heparin, but other glycosaminoglycans were relatively ineffective. A half-maximal effect was seen with 0.5 units of heparin/ml, corresponding to 150 nM. The stimulation was rapid, i.e. occurring within 15 s, and was prevented or reversed by addition of the heparin-binding protein antithrombin III. The action of heparin was also sharply reduced by prior activation of the enzyme by MgATP. In the absence of heparin, the time course of the phosphorylase kinase reaction was concave upward, whereas in the presence of heparin, a constant rate was seen. The stimulatory effect of heparin was inversely proportional to the concentration of phosphorylase b. The data suggest that heparin reversibly stimulates a low activity form of liver phosphorylase kinase to a degree similar to that found with activation by phosphorylation.

Animals↗
PubMed1975

[A further study of the nature of phenotypical reversions in thymidine phosphorylase deletion mutants of Escherichia coli K-12].

Thymidine dependent (thy) strains of Escherichia coli carrying deletions for thymidine phosphorylase (tpp gene) formed phenotypical reversions for the ability of growth at the medium containing thymine as a source of thymidilate. The ability of the thy tpp strains to utilyze thymine for growth is due to mutations of the regulatory genes cytR and udpR, which control the uridine phosphorylase activity. This enzyme in constitutive amounts catalyzes conversion of thymine to thymidine in bacterial cells with a block of deoxyribose catabolism (deoxyribomutase-negative strains). The udpR mutant by contrast to cytR mutants reported by Munch-Petersen et al. (1972) are shown to contain low, inducible levels of cytidine deaminase and deo-enzymes. The udpR mutation is recessive with respect to wild type allele. It is supposed that the product of udpR gene is a specific repressor of uridine phosphorylase (udp) gene. The udpR mutation is closely linked (90% contransduction) to uridine phosphorylase (udp) gene, and is located approximately at 75 min, on the E. coli chromosome.

Chromosome Mapping↗
PubMed1994

[Retarded conformational transitions in muscle glycogen phosphorylase b induced by specific ligands].

The effect of specific ligands on the initial rate of muscle glycogen phosphorylase b digestion by trypsin has been studied. The kinetics of tryptic proteolysis were followed by measuring the decrease in phosphorylase b fluorescence intensity at 335 nm (excitation at 290 nm). The kinetic curves were linear at least in the region 0-400 s (0.02 M HEPES, pH 6.8; 37 degrees C). An allosteric activator (AMP) and allosteric inhibitors (flavins) protected the enzyme from tryptic digestion when trypsin was added to the enzyme preincubated with the ligand. Differences were found between the kinetic curves of trypsinolysis initiated by addition of the trypsin-ligand mixture to phosphorylase b an by addition of trypsin to the enzyme preincubated with the ligand for 10 min. It is concluded that the specific ligands under study (AMP, flavins, and the substrate--glucose 1-phosphate) induce relatively slow conformational changes in the phosphorylase b molecule with the half-conversion time of several minutes.

Adenosine Monophosphate↗
PubMed1993

Isolation of an autoinhibitory region from the regulatory beta-subunit of phosphorylase kinase.

An equimolar mixture of the regulatory alpha- and beta-subunits of phosphorylase kinase has been shown to inhibit its catalytic gamma-subunit (Paudel, H.K., and Carlson, G.M. (1987) J. Biol. Chem. 262, 11912-11915). The possible presence of an autoinhibitory sequence within those regulatory subunits has been evaluated by peptide isolation and characterization following chemical and proteolytic cleavage of an isolated equimolar mixture of those subunits; the peptides generated were tested for their ability to inhibit the activity of a complex of the gamma-subunit and calmodulin. An isolated inhibitory fragment, hereafter referred to as I-peptide, was sequenced and found to correspond to residues 420-436 of the beta-subunit (KRNPGSQKRFPSNCGRD). This sequence showed homology with the kinase's natural substrate, phosphorylase b. A synthetic peptide based on this sequence was constructed and used to study the mechanism of inhibition. Kinetic analysis of the inhibition of the gamma-subunit-calmodulin complex by the I-peptide revealed a competitive pattern versus the homologous substrate phosphorylase b and an uncompetitive pattern versus MgATP, suggesting an ordered binding of substrates, with the nucleotide binding first. In addition to its ability to inhibit, the I-peptide was also a substrate for the gamma-subunit-calmodulin complex, with a relatively good Km but poor Vmax. The parallel inhibition of free gamma-subunit and the gamma-subunit-calmodulin complex by progressively increasing concentrations of I-peptide provided further evidence that the peptide inhibits by interacting directly with the catalytic subunit and not with the stimulatory calmodulin molecule. The results of this study are consistent with previous findings from this laboratory showing that the conformation of the beta-subunit changes following activation of phosphorylase kinase through a variety of mechanisms.

Amino Acid Sequence↗
PubMed1993

Features of calmodulin that are important in the activation of the catalytic subunit of phosphorylase kinase.

Calmodulin (CaM) is an integral subunit, called delta, of the phosphorylase kinase hexadecamer, and the activity of the isolated catalytic gamma-subunit of the kinase is stimulated by CaM. We report here the first analysis of functionally important features of CaM for activation of the gamma-subunit. A set of genetically engineered CaMs, in which acidic residues in each of the four E-helices of the "EF-hands" were changed to basic lysine residues, was used to probe the relative importance of charge features in each domain of CaM. The maximal activation of the isolated gamma-subunit was diminished by all of the charge reversal mutations. The gamma-subunit was especially sensitive to reversals in the second and third E-helix of CaM (residues 45-47 and 82-84), the latter being present in the central helix. The results suggest the functional importance of electrostatics in the interactions between the delta-subunit (CaM) and the catalytic gamma-subunit of phosphorylase kinase, which is similar to results obtained with CaM-dependent myosin light chain kinase (MLCK) from chicken gizzard and CaM-dependent protein kinase II (CaMPK-II). However, novel features of the interaction between CaM and the gamma-subunit of phosphorylase kinase are the significant contribution of electrostatics throughout the CaM molecule, including residues in both halves and on more than one face of CaM, and the lack of a major effect of the CaM mutations on substrate kinetic parameters, unlike the effects observed with MLCK and CaMPK-II. These results are consistent with a model in which the delta-subunit (CaM) of phosphorylase kinase interacts with an extended region or multiple regions of the gamma-subunit and suggest that the mechanism of CaM activation of the gamma-subunit may have features that are distinct from those of MLCK and CaMPK-II.

Amino Acid Sequence↗
PubMed1977

[Modification of phosphorylase b histidine residues by diethylpyrocarbonate].

Interaction of phosphorylase B with diethylpyrocarbonate was studied. It was found that modification of the histidine residues is accompanied by a change in the adsorption spectrum of phosphorylase B at around 240--245 nm, as well as by inhibition of the enzyme activity. The addition of hydroxylamine to the modified protein results in an elimination of the absorption at round 240 nm and a complete restoration of the enzyme activity. The apparent constants of the phosphorylase B inactivation rates and the rates of the histidine residues modification are shown to be the values of the same order. In the presence of substrate (glucose-1-phosphate) one of the histidine residues (calculated per 1 subunit) does not enter the reaction with diethylpyrocarbonate, a protection of the enzyme against inactivation being observed. In the presence of activator (AMP) four histidine residues are inaccessible for diethylpyrocarbonate; however, the degree of inhibition of the enzyme activity does not change. The phosphorylase B molecule, modified in the presence of AMP, is incapable of dissociating into monomers.

Adenosine Monophosphate↗
PubMed1995

[Association of rabbit skeletal muscle phosphorylase kinase with sarcoplasmic reticulum membranes].

The binding of phosphorylase kinase to sarcoplasmic reticulum has been studied using gel chromatography. The presence of Ca2+, Mg2+ and glycogen was found to be necessary for the maximal binding. The phosphorylase kinase adsorbed on sarcoplasmic reticulum is capable of phosphorylating exogenous phosphorylase b. Phosphorylase kinase was shown to take no part in the phosphorylation of sarcoplasmic reticulum proteins. Exogenous calmodulin initiates the incorporation of [gamma-32P] of ATP into sarcoplasmic reticulum proteins. The data obtained point to a possibility that another Ca(2+)-calmodulin-dependent protein kinase may participate in the phosphorylation of sarcoplasmic proteins.

Animals↗