FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2057988567> ?p ?o ?g. }

• W2057988567 endingPage "22840" @default.
• W2057988567 startingPage "22833" @default.
• W2057988567 abstract "Oxygen free radicals may act as second messengers in signal transduction pathways and contribute to inflammatory diseases. We studied the action in vitro of radiolytically generated hydroxyl radicals (⋅OH) and superoxide radicals (O⨪2) on the cAMP-dependent protein kinases, I and II (PKAI and -II, respectively). The effects of the gasses O2 and N2O used to produce O⨪2 or⋅OH radicals by γ-radiolysis of the water were also studied. PKAI is more sensitive than PKAII to oxygen gas (10 mmsodium formate) and to hydroxyl and superoxide radicals. Hydroxyl radicals decreased the kinase phosphotransferase activities stimulated either by cAMP or its site-specific analogs for both PKAI and PKAII; however, PKAI was more affected. The binding of [3H]cAMP and of 8-N3-[32P]cAMP to RI regulatory subunits was decreased. ⋅OH caused a loss of tryptophan 260 fluorescence at site A of PKAI and of bityrosine production. Superoxide radicals affected only PKAI. O⨪2 modified both cAMP-binding sites A and B of the regulatory subunit but had a smaller effect on the catalytic subunit. The catalytic subunit was more sensitive to radicals when free than when part of the holoenzymes during exposure to the oxygen free radicals. These results suggest that oxygen free radicals alter the structure of PKA enzymes. Thus, oxidative modifications may alter key enzymes, including cAMP-dependent protein kinases, in certain pathological states. Oxygen free radicals may act as second messengers in signal transduction pathways and contribute to inflammatory diseases. We studied the action in vitro of radiolytically generated hydroxyl radicals (⋅OH) and superoxide radicals (O⨪2) on the cAMP-dependent protein kinases, I and II (PKAI and -II, respectively). The effects of the gasses O2 and N2O used to produce O⨪2 or⋅OH radicals by γ-radiolysis of the water were also studied. PKAI is more sensitive than PKAII to oxygen gas (10 mmsodium formate) and to hydroxyl and superoxide radicals. Hydroxyl radicals decreased the kinase phosphotransferase activities stimulated either by cAMP or its site-specific analogs for both PKAI and PKAII; however, PKAI was more affected. The binding of [3H]cAMP and of 8-N3-[32P]cAMP to RI regulatory subunits was decreased. ⋅OH caused a loss of tryptophan 260 fluorescence at site A of PKAI and of bityrosine production. Superoxide radicals affected only PKAI. O⨪2 modified both cAMP-binding sites A and B of the regulatory subunit but had a smaller effect on the catalytic subunit. The catalytic subunit was more sensitive to radicals when free than when part of the holoenzymes during exposure to the oxygen free radicals. These results suggest that oxygen free radicals alter the structure of PKA enzymes. Thus, oxidative modifications may alter key enzymes, including cAMP-dependent protein kinases, in certain pathological states. Reactive oxygen species (ROS) 1The abbreviations used are: ROSreactive oxygen speciesMOPS3-(N-morpholino)propanesulfonic acidMES4-morpholineethanesulfonic acid8-N3-[32P]cAMP8-azidoadenosine 3′:5′-mono[32P]phosphate8-AHA-cAMP8-aminohexylamino-cAMPPKAcAMP-dependent protein kinasePKAI and PKAIIcAMP-dependent protein kinase I and II, respectivelyN 6-Bnz-cAMPN 6-benzoyl-cAMPPAGEpolyacrylamide gel electrophoresis. including the superoxide radical (O⨪2), hydrogen peroxide (H2O2), and the hydroxyl radical (⋅OH) are generated in several cell types in response to stimulation by various hormones and cytokines (1Matsubara T. Ziff M. Basic Life Sci. 1986; 137: 3295-3298Google Scholar, 2Meier B.H.H. Radeke S. Selle M. Young H. Sies K. Resh H. Habermehl G.G. Biochem. J. 1989; 263: 539-545Crossref PubMed Scopus (574) Google Scholar, 3Zoccarato F. Deana R. Cavallini L. Alexandre A. Eur. J. Biochem. 1989; 180: 473-478Crossref PubMed Scopus (19) Google Scholar, 4Garett I.R. Boyce B.F. Oreffo R.O.C. Bonewald L. Poser J. Mundy G.R. J. Clin. Invest. 1990; 85: 632-639Crossref PubMed Scopus (683) Google Scholar, 5Finkel T. Curr. Opin. Cell Biol. 1998; 10: 248-253Crossref PubMed Scopus (1014) Google Scholar). The oxygen radicals generated in turn appear to act as second messengers in the activation of the transcription factor NF-κB (5Finkel T. Curr. Opin. Cell Biol. 1998; 10: 248-253Crossref PubMed Scopus (1014) Google Scholar) and in the oxidative modification of several important proteins in various cell types (6Schreck R. Bauerle P.A. Trends Cell Biol. 1991; 1: 39-42Abstract Full Text PDF PubMed Scopus (445) Google Scholar, 7Stadtman E.R. Trends Biol. Sci. 1986; 11: 11-12Abstract Full Text PDF Scopus (247) Google Scholar, 8Abate C. Patel L. Rauscher F.J. Curran T. Science. 1990; 249: 1157-1161Crossref PubMed Scopus (1377) Google Scholar, 9Chevalier M. Lin E.C. Levine R.L. J. Biol. Chem. 1990; 265: 42-46Abstract Full Text PDF PubMed Google Scholar, 10Landgraf W. Regulla S. Meyer H.E. Hofman F. J. Biol. Chem. 1991; 266: 16305-16311Abstract Full Text PDF PubMed Google Scholar). H2O2 influences the activity of a number of enzymes involved in cellular signaling pathways, including the lowkm cAMP phosphodiesterase (11Ueda M. Robinson W. Smith M. Kono T. J. Biol. Chem. 1984; 259: 9520-9525Abstract Full Text PDF PubMed Google Scholar), the soluble guanylate cyclase (12Thomas G. Ramwell P. Biochem. Biophys. Res. Commun. 1986; 139: 102-108Crossref PubMed Scopus (49) Google Scholar), the protein kinase C (13Gopalakrishna R. Anderson W.B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6758-6762Crossref PubMed Scopus (371) Google Scholar, 14Gopalakrishna R. Anderson W.B. Arch. Biochem. Biophys. 1991; 285: 382-387Crossref PubMed Scopus (80) Google Scholar), the MAP kinase pathway (15Stevenson M.A. Pollock S.S. Coleman N. Calderwood S.K. Cancer Res. 1994; 54: 12-15PubMed Google Scholar), growth factor receptor tyrosine kinase, and tyrosine phosphatase (16Rao G.N. Oncogene. 1996; 13: 713-719PubMed Google Scholar). reactive oxygen species 3-(N-morpholino)propanesulfonic acid 4-morpholineethanesulfonic acid 8-azidoadenosine 3′:5′-mono[32P]phosphate 8-aminohexylamino-cAMP cAMP-dependent protein kinase cAMP-dependent protein kinase I and II, respectively N 6-benzoyl-cAMP polyacrylamide gel electrophoresis. The elevation of intracellular levels of cAMP and subsequent activation of cyclic AMP-dependent protein kinases (PKA) are events that contribute to the regulation of a variety of cell functions including gene expression, cellular metabolism, and cell proliferation and differentiation. The inactive PKA holoenzyme is a tetramer consisting of two regulatory (R) subunits and two catalytic (C) subunits. There are two major types of PKA (PKAI and PKAII), which display different biochemical properties due to differences in their R subunits (RI and RII). RI and RII differ in molecular weight, antigenicity, amino acid sequence of the N-terminal domain, ability to be autophosphorylated, and affinity for cAMP analogs. Genetic studies have revealed numerous types of PKA subunit (RIα, RIβ, RIIα, RIIβ, Cα, Cβ, Cγ). PKA is activated by the binding of four cAMP molecules. They bind to two asymmetric sites (designated A and B), on each monomer in a positive cooperative fashion. This results in the dissociation of the holoenzyme to release active C subunit and dimers of the R subunits (for a review see Refs. 17Taylor S.S. Buechler J.A. Yonemoto W. Annu. Rev. Biochem. 1990; 59: 971-1005Crossref PubMed Scopus (959) Google Scholar and 18Tasken K. Solberg R. Bente Foss K. Skälhegg B.S. Hansson V. Janhsen T. Hardie G. Hanks S. Protein-Serine Kinases. Academic Press, London1995: 58-63Google Scholar). The cAMP-dependent protein kinases in psoriatic cells are abnormal (19Evain-Brion D. Raynaud F. Plet A. Laurent P. Leduc B. Anderson W.B. Proc. Natl. Acad. Sci. 1986; 83: 5272-5276Crossref PubMed Scopus (44) Google Scholar). Both 8-N3-[32P]cAMP binding to the regulatory subunits RI and RII and the cAMP-dependent protein kinase activities are low (20Raynaud F. Gerbaud P. Enjolras O. Gorin I. Donnadieu M. Anderson W.B. Evain-Brion D. Lancet. 1989; : 1153-1156Abstract PubMed Scopus (19) Google Scholar). Indeed, the loss of cAMP binding activity correlates with the severity of the disease (20Raynaud F. Gerbaud P. Enjolras O. Gorin I. Donnadieu M. Anderson W.B. Evain-Brion D. Lancet. 1989; : 1153-1156Abstract PubMed Scopus (19) Google Scholar). This alteration of PKA in psoriatic fibroblasts may be due to oxidative modification (21Raynaud F. Evain-Brion D. Gerbaud P. Marciano D. Gorin I. Liapi C. Anderson W.B. Free Radical Biol. Med. 1997; 22: 623-632Crossref PubMed Scopus (34) Google Scholar), and the oxidative state of psoriatic cells might be modified (22Grossman R.M. Krueger J. Yourish D. Granelli-Piperno A. Murphy D. May L. Kupper T.S. Sehgal P.B. Gottlieb A.B. Proc. Natl. Acad. Sci. 1989; 86: 6367-6373Crossref PubMed Scopus (751) Google Scholar, 23Ohta Y. Katayama I. Funato T. Yokozeki H. Nishiyama S. Hirano T. Kishimoto T. Nishioka K. Arch. Dermatol. Res. 1991; 283: 351-356Crossref PubMed Scopus (62) Google Scholar, 24Löntz W. Sirsjö A. Liu W. Lindberg M. Rollman O. Törma H. Free Radical Biol. Med. 1995; 18: 349-355Crossref PubMed Scopus (63) Google Scholar). Thus, oxidative modifications may modulate PKA activity and be the basis of alteration in key enzyme activities, including PKA, in certain pathological states such as psoriasis. We present an in vitro analysis of the ability of particular oxygen free radicals generated by γ-radiolysis of water to alter the enzymatic properties of PKAI and PKAII. We have also studied the effects of the gasses N2O and O2 (in the presence of 10 mm sodium formate) in aqueous solutions that generate 90% hydroxyl radicals (⋅OH) or 100% anion superoxide radicals (O⨪2), respectively. We report specific alterations in cAMP binding to the regulatory subunits and specific changes in cAMP-dependent protein kinase activities in response to the oxygen free radicals studied. PKA type I was extracted from rabbit skeletal muscle, which contains mostly the RIα isoform (25Pariset C. Feinberg J. Dacheux J.-L. Oyen O. Janhsen T. Weinman S. J. Cell Biol. 1989; 109: 1195-1205Crossref PubMed Scopus (38) Google Scholar). PKA type II and C subunit were extracted from bovine heart muscle. PKA type II contains essentially the RIIα isoform (26Löffner F. Lohmann S.M. Walckhoff B. Walter U. Hamprecht B. Brain Res. 1986; 363: 205-221Crossref PubMed Scopus (50) Google Scholar). All of these products were obtained from Sigma-Aldrich (Saint Quentin Fallavier, France). Oxygen radical species were homogeneously generated in aqueous protein solutions by γ-radiolysis with a 60Co source (performed at the Laboratoire de Chimie Physique CNRS, URA 400, Université René Descartes, Paris, France). A 1-ml solution of 10 mm-sodium phosphate buffer, pH 7.4, containing 5 μg of the indicated protein was kept cold on ice, saturated for 30 min with the appropriate gas, and then irradiated in closed vessels. The saturation of the solution with N2O or O2 gas in the absence or in the presence of 10 mm sodium formate allowed the generation of 90% hydroxyl radicals (⋅OH), or 100% anion superoxide radicals (O⨪2), respectively. The doses were delivered at a rate of 2.5 ± 0.1 grays/min, as measured by Fricke's method (27Fricke H. Hart F.J. Attix F.H. Roesh W. Radiation Dosimetry. Academic Press, Inc., New York1966: 167-239Google Scholar). A single dose of 900 grays was delivered to each protein solution, such that the ratio of oxygen radical to protein (nmol of radicals/nmol of protein) was between 2500 (⋅OH) and 5040 (O⨪2), according to the nature of the free radical (in steady-state kinetic conditions). Cyclic AMP-dependent protein kinase activities were measured using kemptide as the phosphate acceptor (a specific substrate for cAMP-dependent protein kinases) (28Roskoski R. Methods Enzymol. 1983; 99: 3-6Crossref PubMed Scopus (691) Google Scholar). One μg of PKAI or PKAII was used to catalyze the transfer of 32P from ATP (5000 pmol; 5 × 105 cpm) to 100 μm of kemptide in the presence of 50 mm MOPS, pH 7, 250 μg/ml bovine serum albumin, 10 mm MgCl2, 100 μm ATP, and the appropriate concentration of cAMP or its analogs (10−9 to 10−3m) in a total volume of 50 μl. C subunit activity was determined in the same reaction mixture in the absence of cAMP and its analogs. The phosphorylation reaction was allowed to proceed at 37 °C for 10 min with continuous agitation. The reaction then was terminated by spotting 25 μl of the reaction mixture onto phosphocellulose P-81 strips (Whatman), which were immediately dropped into ice-cold 0.5% phosphoric acid (10 ml/paper strip). The strips were washed three times in 0.5% phosphoric acid with swirling. Radioactivity retained on the P-81 papers was counted in Ready Safe scintillation solvent (Beckman, Fullerton, CA). The background count that was obtained in the absence of enzyme was subtracted from all experimental values. PKA activity was calculated by subtracting the activity measured in the presence of cAMP or its analog from the activity measured in the absence of cAMP. The polyclonal antibodies against bovine skeletal muscle RIα (25Pariset C. Feinberg J. Dacheux J.-L. Oyen O. Janhsen T. Weinman S. J. Cell Biol. 1989; 109: 1195-1205Crossref PubMed Scopus (38) Google Scholar) and against rat heart RIIα (26Löffner F. Lohmann S.M. Walckhoff B. Walter U. Hamprecht B. Brain Res. 1986; 363: 205-221Crossref PubMed Scopus (50) Google Scholar) were prepared as reported previously. The polyclonal antibody against bovine heart catalytic subunit C was a generous gift from Dr. S. Lohmann (Labor für Klinische Biochemie, Medizinische Universitätsklinik, Würzburg, Germany). Protein kinase preparations (5 μg of PKAI or PKAII or 0.25 μg of catalytic subunit solution) were heated at 100 °C for 5 min in electrophoresis sample buffer and subjected to SDS-PAGE on a 10% gel (minigel). Proteins were transferred electrophoretically to a nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany) using a semidry blotting apparatus (Bio-Rad). The membranes were soaked for 1 h in phosphate-buffered saline containing 0.1% Tween 20 and 5% skim milk. The blots were probed with the specified antibody (anti-RIα (diluted 1:100), anti-RIIα (1:1000), or anti-C (1:500)) by incubation in phosphate-buffered saline containing 0.1% Tween 20 and 5% skim milk overnight at 4 °C and then washed with phosphate-buffered saline containing 0.1% Tween 20 for 30 min. Bound anti-RI antibody was detected by chemiluminescence (ECL, Amersham Pharmacia Biotech, Les Ulis, France) after incubation with the anti-rabbit peroxidase-coupled secondary antibody. Bound anti-RIIα and anti-C antibodies were revealed by a goat anti-rabbit IgG conjugated to alkaline phosphatase (1:7500, Promega, Lyon, France) and NBT and BCIP as substrates. RI and RII regulatory subunits were photoaffinity-labeled as described previously (29Raynaud F. Leduc B. Anderson W.B. Evain-Brion D. J. Invest. Dermatol. 1987; 89: 105-110Abstract Full Text PDF PubMed Google Scholar) in a reaction mixture (80 μl) containing 10 mm MES, pH 6.2, 10 mm MgCl2, 1.0 μm8-N3-[32P]cAMP, and 10 μg of PKA. Where indicated, 100 μm cAMP was included to block 8-N3-[32P]cAMP binding to determine nonspecific labeling. Mixtures were incubated for 60 min in the dark at 4 °C and then irradiated for 10 min with a UV lamp to allow irreversible photoaffinity binding of 8-N3-[32P]cAMP to the RI and RII subunits. The irradiated samples were pipetted into 20 μl of stop solution (9% SDS, 15% (v/v) glycerol, 6 mm EDTA, 250 mmTris-HCl, pH 8) and heated at 100 °C for 2 min. Then, 2 μl of 2-mercaptoethanol and 5 μl of 0.1% bromphenol blue in 50% (v/v) glycerol was added, and the samples were electrophoresed in 8.75% polyacrylamide slab gels containing SDS. The gels were dried and autoradiographed at −80 °C using Cronex 4 DuPont medical x-ray film. Band intensities on autoradiograms were quantitated by scanning with a microdensitometer. Ten μg of protein labeled with 8-N3-[32P]cAMP was applied to each gel lane to allow comparison between the different lanes and autoradiograms. Care was taken not to overexpose the x-ray films. Under these conditions, peak heights obtained by scanning were proportional to the total radioactivity of the corresponding bands as estimated by scintillation counting as described by Walter (30Walter U. Uno I. Liu A.Y.C. Greengard P. J. Biol. Chem. 1977; 252: 6494-6500Abstract Full Text PDF PubMed Google Scholar). Labeling of RI and RII regulatory subunits was calculated by integrating the areas under the curves and subtracting nonspecific labeling (in the presence of 0.1 mm cAMP). Cyclic AMP binding activity was determined by the Millipore filtration method (Ref. 31Gilman A.G. Proc. Natl. Acad. Sci. U. S. A. 1970; 67: 305-312Crossref PubMed Scopus (3358) Google Scholar, as modified in Ref. 32Gill G.N. Garren L.D. Biochem. Biophys. Res. Commun. 1970; 39: 335-343Crossref PubMed Scopus (263) Google Scholar). The incubation mixtures (225 μl) containing buffer A (50 mm Tris-HCl buffer, pH 7.4, 10 mmMgCl2, 6 mm theophylline, 4 mg/ml bovine serum albumin), a standard series of concentrations of [3H]cAMP (Centre d'Energie Atomique, Saclay, France, 30 Ci/mmol; 0.2–7 pmol) and 10 μg of the different types of PKA were incubated for 2 h at 4 °C. The reaction was terminated by adding 4 ml of buffer A without bovine serum albumin. The samples were immediately loaded onto Millipore filters (0.45 μm, Millipore Corp., HAWPO 2500). The filters were washed twice with 4 ml of buffer A without bovine serum albumin. Radioactivity retained on the filters was determined by counting in Ready Safe scintillation solvent (Beckman, Fullerton, CA). The blank value (nonspecific binding in presence of 0.1 mm cAMP) was subtracted from the total binding value. RII was phosphorylated by incubation of 2 μg of PKAII holoenzyme in the presence of 5 nm of the exogenous catalytic subunit of PKA in 20 mm MOPS, 20 mm MgCl2, 84 mm Tris-HCl (pH 7.4), 100 μm ATP, and 0.5 μCi of [γ-32P]ATP in a final volume of 15 μl. RII was also phosphorylated by the endogenous catalytic subunit of PKAII in the presence of 0.1 mm cAMP to dissociate the holoenzyme. For RII phosphorylation by casein kinase II, 15 nm of casein kinase II (a generous gift from Claude Cochet, INSERM U.244, Grenoble, France) was added to the incubation reaction, and 50 μm H-89 (Seikagaku Corp., Coger, Paris, France) was also added to inhibit the phosphorylation of RII by the endogenous catalytic subunit. After 10 min at 37 °C, the reaction was terminated by the addition of 5 μl of stop solution (200 mm Tris-HCl, pH 6.8, 8% SDS, 20% glycerol, 5% 2-mercaptoethanol, and 0.01% bromphenol blue) and heating at 100 °C for 5 min. Samples were then processed for SDS-polyacrylamide gel electrophoresis and autoradiography. Fluorescence was measured at 23 °C using 100 μl of 0.1 μm PKAI and PKAII in a quartz cuvette in a Perkin-Elmer spectrofluorimeter. For tryptophan fluorescence, the excitation wavelengh was 290 nm with a bandpass of 10 nm. The excited samples were scanned from 300 to 450 nm using an emission bandpass of 2.5 nm. For tyrosine fluorescence, the excitation wavelengh was 280 nm with a bandpass of 2.5 nm, and the excited samples were scanned from 300 to 400 nm using an emission band of 2.5 nm. For bityrosine fluorescence, the excitation wavelength was 325 nm with a bandpass of 2.5 nm, and the excited samples were scanned from 360 to 470 nm using an emission bandpass of 4 nm. Fluorescence was titrated three times independently. For cAMP titrations, PKAI was incubated with each of a series of concentrations of cAMP (0–100 μm) (33Gibson R.M. Ji-Buechler Y. Taylor S. J. Biol. Chem. 1997; 272: 16343-16350Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The excitation and emission wavelengths were 295 and 341 nm, respectively. Bands on autoradiograms from nonsaturated autoradiography of Western blots were scanned densitometrically. The results are expressed as relative optical density units. The peak heights obtained by scanning were proportional to the amount of protein loaded onto the gel, and values were corrected according to the variations in the controls. To produce superoxide or hydroxyl radicals selectively during H2O radiolysis with γ-irradiation, aqueous solutions of PKA were saturated either with O2 (in the presence of sodium formate) or N2O alone. The phosphotransferase activities of rabbit skeletal muscle PKAI and of bovine heart muscle PKAII holoenzymes exposed to these oxygen free radical-generating systems were measured as the phosphorylation of a specific substrate in the presence and absence of cAMP. The various conditions of free radical generation did not alter the basal transferase activities of PKA in the absence of cAMP. However, the basal phosphotransferase activity of PKAI saturated with O2(in the presence of sodium formate) was higher than that in controls. Incubation of PKAI in water saturated with O2 (in the presence of sodium formate) caused a 50% decrease in theVmax of the cAMP-dependent phosphotransferase activity without modification of theKact (i.e. the concentration of cAMP required for half-maximal kinase activation; control/O2 = 3 × 10−7m) (Fig. 1 A). In contrast, the cAMP-dependent phosphotransferase activity of PKAII was increased by the same conditions (Fig. 1 B). A decrease in cAMP-dependent phosphotransferase activity of both type I and type II PKA was observed after incubation in the presence of N2O (Fig. 1, C and D), and the Kact of cAMP for PKAII but not that for PKAI was modified (for PKAII, Kact(C) = 3 × 10−8m/ Kact(N2O)= 3 × 10−7m). We determined the effects of a series of doses of radiation on the cAMP-dependent activities of PKAII. The effect of radiation was dose-dependent. The maximal effect on the cAMP-dependent phosphotransferase activity of PKAII was observed with 900 grays in the presence of 50% of superoxide and 50% of hydroxyl anions (21Raynaud F. Evain-Brion D. Gerbaud P. Marciano D. Gorin I. Liapi C. Anderson W.B. Free Radical Biol. Med. 1997; 22: 623-632Crossref PubMed Scopus (34) Google Scholar). This dose was used for all of the following studies. Under these experimental conditions, incubation of the holoenzymes in aqueous solutions saturated with O2 (in the presence of sodium formate) or N2O and irradiated to produce the ROS (O⨪2 or ⋅OH), respectively, the protein patterns of the holoenzymes were not noticeably modified as ascertained by Coomassie Blue staining after SDS-PAGE (Fig.2). The carboxyl termini of the RI and RII regulatory subunits of PKAI and PKAII contain two tandem homologous cAMP binding domains, called sites A and B. These two cAMP binding sites have different specificities for cAMP analogs. Site A has a faster off-rate and a preference forN 6-substituted analogs, whereas site B has a slower off-rate and exhibits a preference for C-2- and C-8-substituted cAMP analogs. We investigated whether these two cAMP binding sites are differentially altered by ROS. The phosphotransferase activities of PKAI and PKAII both before and after irradiation were measured in the presence of each of a series of concentrations of cAMP or its analogs, the N-6-substitutedN 6-Bnz-cAMP and the C-8-substituted 8-AHA-cAMP (see “Materials and Methods”; Figs.3 and 4). The maximum activity of cAMP-dependent kinase obtained in the presence of gas but without irradiation was taken as the 100% value.Figure 4In vitro effects of hydroxyl radicals generated by γ-radiation on purified rabbit PKAI and bovine PKAII. Preparations of PKA were saturated with N2O in closed vessels without irradiation (hollow squares, A–C for PKAI; D–F for PKAII) or with γ-radiation to generate ⋅OH (solid diamonds; A and D). See the legend to Fig. 2 for further details. The maximal value of cAMP-dependent kinase activity obtained in the presence of N2O without irradiation was used as the 100% value. Results are the mean of two experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Superoxide radical treatment of PKAI caused a 55% decrease in stimulation of PKAI phosphotransferase activity by both cAMP and 8-AHA-cAMP (Fig. 3, A and C). The apparent activation constant Kact was increased (from Kact(O2) = 3 × 10−7m/ Kact(O2-irr)= 10−6/3 × 10−5m). Interestingly, the ability of the A site-selective analogN 6-Bnz-cAMP to activate PKAI was completely abolished by exposure to superoxide radicals (Fig.3 B). In contrast, exposure of PKAII to superoxide radicals only slightly reduced stimulation of phosphotransterase activity by cAMP and the cAMP analogs (Fig. 3, D–F). Exposure of PKAI to hydroxyl radicals also resulted in a significant increase in phosphotransferase activity in the presence of cAMP (Fig. 4 A), N 6-Bnz-cAMP (Fig.4 B), and 8-AHA-cAMP (Fig. 4 C). Exposure of PKAII to hydroxyl radicals had similar effects to those on PKAI: a significant decrease in phosphotransferase activities (Fig. 4,D–F). The Kact values were also increased under these conditions, most substantially for PKAI activation by N 6-Bnz-cAMP ( Kact(O2) = 10−8m/ Kact(O2-irr)= 3 × 10−7m). The effects of ROS on cAMP binding to the RI and RII regulatory subunits of PKAI and PKAII were determined by Millipore filtration assays with [3H]cAMP or affinity labeling with 8-N3-[32P]cAMP. The first of these techniques only allows determination of affinity and binding capacity of site B. 8-N3-[32P]cAMP binds covalently to both sites A and B on RI but only to site B on RII. We also tried to estimate protein-bound [3H]cAMP by ammonium sulfate precipitation according to the method of Ogreid and Doskeland (34Ogreid D. Doskeland S.O. FEBS Lett. 1980; 121: 340-344Crossref PubMed Scopus (26) Google Scholar), but we were unable to discriminate between sites A and B. 3H-Labeled cyclic AMP binding to the regulatory subunits of PKAI was determined in the presence of oxygen (sodium formate) without irradiation (Fig. 5 A,black diamonds), in the presence of N2O alone (Fig. 5 B, black diamonds), and after exposure to superoxide (Fig.4 C, black diamonds) or hydroxyl radicals (Fig. 5 D, black diamonds). The same experiment was performed with PKAII (Fig.6, A–D). The presence of the different gasses did not affect the Kd of PKAII for cAMP binding ( Kd (C) = Kd (O2) = Kd (N2O) = 3.6 × 10−6m; Fig. 6, A andB; Table I). However, incubation of PKAI in the presence of O2 modified cAMP binding to RI ( Kd (C) = 6.2 × 10−7m, Kd (O2) = 1.8 × 10−7m; Fig. 5 A, Table I). Incubation of PKAI in the presence of N2O resulted in decreased cAMP binding to RI ( Kd (N2O) = 9.5 × 10−7m; Fig. 5 B).Figure 6Effects of ROS on the saturation curves of specific [3H]cAMP binding to RII from purified bovine PKAII. The PKAII solutions (A and B, respectively) were saturated with O2 in the presence of 10 mm sodium formate in closed vessels and irradiated (solid diamonds) or not (hollow squares). Solutions of PKAI and PKAII (C andD, respectively) were saturated with N2O in closed vessels and irradiated (solid diamonds) or not (hollow squares). PKAI and PKAII (10 μg of protein) were incubated for 2 h at 4 °C with various amounts of [3H]cAMP in the absence (total binding) or in the presence (nonspecific binding) of 0.1 mm unlabeled cAMP. Specific binding was calculated by subtracting nonspecific from total binding. The Scatchard plots derived from each saturation curve are shown in the insets.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IThe Kd values of [3 H]cAMP binding for PKAI and PKAII and K values of cAMP transferase activity in various conditionsKact of cAMPCO2O2irr.airr., irradiated.N2ON2O irr.mPKAI3 × 10−73 × 10−710−6/3 × 10−53 × 10−63 × 10−6PKAII3 × 10−83 × 10−710−73 × 10−710−6Kd of [3H]cAMPCO2O2 irr.N2ON2O irr.mPKAI6.2 × 10−71.8 × 10−73.6 × 10−79.5 × 10−713.5 × 10−7PKAII3.6 × 10−64 × 10−62.7 × 10−63.3 × 10−62.4 × 10−6a irr., irradiated. Open table in a new tab The binding of [3H]cAMP to PKAI was reduced by treatment with superoxide radicals (Fig. 5 C). There was no change in the binding capacity under these conditions, but a difference in the affinity of RI for cAMP was observed ( Kd (O2-irr) = 3.6 10−7m, Kd (O2) = 1.8 × 10−7m; Fig. 5 C). There was no significant difference between cAMP binding to RII in the presence and absence of superoxide radicals (Fig. 6 C). Hydroxyl radicals modified [3H]cAMP binding to RI only (Fig. 5 D). Exposure of PKAI to hydroxyl radicals only affected affinity for [3H]cAMP ( Kd (N2O-irr)= 13.5 × 10−7m (RI), Kd (N2O) = 9.5 × 10−7m). Treatment of PKAII with hydroxyl radicals resulted in altered binding capacity for [3H]cAMP, although the affinity was not substantially changed ( Kd (N2O-irr)= 2.4 × 10−6m, Kd (N2O) = 3.3 × 10−6m). RII bound 10 times more [3H]cAMP than RI, as has been previously reported. TheKd values of [3H]cAMP binding for PKAI and PKAII and Kact values of cAMP transferase activity are shown in Table I. Photoaffinity labeling with 8-N3-[32P]cAMP gave different results with RI and RII. Two amino acid residues are affinity-labeled in RI (Trp260 in site A and Tyr371 in site B of RI) (35Su Y. Dostmann W.R.G. Herberg F.W. Durick K. Xuong N. Ten Eyck L. Taylor S.S. Varughese K.I. Science. 1995; 269: 807-813Crossref PubMed Scopus (346) Google Scholar). The ligand binds covalently to the RII subunit, which is modified at a single residue (Tyr381 in site B). Thus, the ligand does not bind covalently to the A binding site of RII (35Su Y. Dostmann W.R.G. Herberg F.W. Durick K. Xuong N. Ten Eyck L. Taylor S.S. Varughese K.I." @default.
• W2057988567 created "2016-06-24" @default.
• W2057988567 creator A5013876454 @default.
• W2057988567 creator A5021872359 @default.
• W2057988567 creator A5040998061 @default.
• W2057988567 creator A5041111465 @default.
• W2057988567 creator A5052097146 @default.
• W2057988567 creator A5072361054 @default.
• W2057988567 date "1998-08-01" @default.
• W2057988567 modified "2023-10-18" @default.
• W2057988567 title "In Vitro Effects of Oxygen-derived Free Radicals on Type I and Type II cAMP-Dependent Protein Kinases" @default.
• W2057988567 cites W1512161195 @default.
• W2057988567 cites W1513831681 @default.
• W2057988567 cites W1531328180 @default.
• W2057988567 cites W1563570838 @default.
• W2057988567 cites W1591230411 @default.
• W2057988567 cites W1591268310 @default.
• W2057988567 cites W1600112653 @default.
• W2057988567 cites W1638074023 @default.
• W2057988567 cites W1654460714 @default.
• W2057988567 cites W1759706356 @default.
• W2057988567 cites W1969387049 @default.
• W2057988567 cites W1975772087 @default.
• W2057988567 cites W1979282739 @default.
• W2057988567 cites W1989001888 @default.
• W2057988567 cites W1992200451 @default.
• W2057988567 cites W1993622679 @default.
• W2057988567 cites W1996466550 @default.
• W2057988567 cites W2003073477 @default.
• W2057988567 cites W2020841780 @default.
• W2057988567 cites W2024920268 @default.
• W2057988567 cites W2025332686 @default.
• W2057988567 cites W2029569944 @default.
• W2057988567 cites W2037048256 @default.
• W2057988567 cites W2038873199 @default.
• W2057988567 cites W2040650395 @default.
• W2057988567 cites W2044603696 @default.
• W2057988567 cites W2065365579 @default.
• W2057988567 cites W2070275219 @default.
• W2057988567 cites W2070693959 @default.
• W2057988567 cites W2072569792 @default.
• W2057988567 cites W2076036354 @default.
• W2057988567 cites W2076058833 @default.
• W2057988567 cites W2081748503 @default.
• W2057988567 cites W2088777477 @default.
• W2057988567 cites W2094655396 @default.
• W2057988567 cites W2120331014 @default.
• W2057988567 cites W2132023176 @default.
• W2057988567 cites W2140847067 @default.
• W2057988567 cites W2141524439 @default.
• W2057988567 cites W2177092266 @default.
• W2057988567 cites W2251920431 @default.
• W2057988567 cites W2399866381 @default.
• W2057988567 doi "https://doi.org/10.1074/jbc.273.35.22833" @default.
• W2057988567 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9712918" @default.
• W2057988567 hasPublicationYear "1998" @default.
• W2057988567 type Work @default.
• W2057988567 sameAs 2057988567 @default.
• W2057988567 citedByCount "27" @default.
• W2057988567 countsByYear W20579885672012 @default.
• W2057988567 countsByYear W20579885672013 @default.
• W2057988567 countsByYear W20579885672015 @default.
• W2057988567 crossrefType "journal-article" @default.
• W2057988567 hasAuthorship W2057988567A5013876454 @default.
• W2057988567 hasAuthorship W2057988567A5021872359 @default.
• W2057988567 hasAuthorship W2057988567A5040998061 @default.
• W2057988567 hasAuthorship W2057988567A5041111465 @default.
• W2057988567 hasAuthorship W2057988567A5052097146 @default.
• W2057988567 hasAuthorship W2057988567A5072361054 @default.
• W2057988567 hasBestOaLocation W20579885671 @default.
• W2057988567 hasConcept C139066938 @default.
• W2057988567 hasConcept C178790620 @default.
• W2057988567 hasConcept C184235292 @default.
• W2057988567 hasConcept C185592680 @default.
• W2057988567 hasConcept C18903297 @default.
• W2057988567 hasConcept C202751555 @default.
• W2057988567 hasConcept C2777299769 @default.
• W2057988567 hasConcept C540031477 @default.
• W2057988567 hasConcept C55493867 @default.
• W2057988567 hasConcept C86803240 @default.
• W2057988567 hasConcept C95444343 @default.
• W2057988567 hasConceptScore W2057988567C139066938 @default.
• W2057988567 hasConceptScore W2057988567C178790620 @default.
• W2057988567 hasConceptScore W2057988567C184235292 @default.
• W2057988567 hasConceptScore W2057988567C185592680 @default.
• W2057988567 hasConceptScore W2057988567C18903297 @default.
• W2057988567 hasConceptScore W2057988567C202751555 @default.
• W2057988567 hasConceptScore W2057988567C2777299769 @default.
• W2057988567 hasConceptScore W2057988567C540031477 @default.
• W2057988567 hasConceptScore W2057988567C55493867 @default.
• W2057988567 hasConceptScore W2057988567C86803240 @default.
• W2057988567 hasConceptScore W2057988567C95444343 @default.
• W2057988567 hasIssue "35" @default.
• W2057988567 hasLocation W20579885671 @default.
• W2057988567 hasOpenAccess W2057988567 @default.
• W2057988567 hasPrimaryLocation W20579885671 @default.
• W2057988567 hasRelatedWork W1969404129 @default.
• W2057988567 hasRelatedWork W2061908415 @default.

• next