Hydrolase Regulates NAD+ Metabolites and Modulates Cellular Redox
2009; Elsevier BV; Volume: 284; Issue: 17 Linguagem: Inglês
10.1074/jbc.m809790200
ISSN1083-351X
AutoresLei Tong, Susan Lee, John M. Denu,
Tópico(s)Sirtuins and Resveratrol in Medicine
ResumoAlthough the classical redox functions of co-enzyme NAD+ are firmly established in metabolism, there are numerous enzymes that catalyze cleavage of NAD+ to yield free ADP-ribose (ADPr) or related metabolites, whose functions remain largely unknown. Here we show that the Nudix (nucleoside diphosphate linked to another moiety X) hydrolase Ysa1 from Saccharomyces cerevisiae is a major regulator of cellular ADPr and O-acetyl-ADP-ribose (OAADPr). OAADPr is the direct product of NAD+-dependent protein deacetylases (sirtuins) and is readily converted to ADPr. Ysa1 cleaves ADPr/OAADPr into ribose phosphate/acetyl-ribose phosphate and AMP. In cells lacking Ysa1 (Δysa1), ADPr and OAADPr levels increased ∼50%, with a corresponding decrease in AMP. Strikingly, Δysa1 cells display higher resistance to exogenous reactive oxygen species (ROS) and 40% lower basal levels of endogenous ROS, compared with wild type. The biochemical basis for these differences in ROS-related phenotypes was investigated, and the results provide evidence that increased ADPr/OAADPr levels protect cells via the following two pathways: (i) lower ROS production through inhibition of complex I of the mitochondrial electron transport chain, and (ii) generation of higher levels of NADPH to suppress ROS damage. The latter occurs through diverting glucose into the pentose phosphate pathway by ADPr inhibition of glyceraldehyde-3-phosphate dehydrogenase, a central enzyme of glycolysis. Although the classical redox functions of co-enzyme NAD+ are firmly established in metabolism, there are numerous enzymes that catalyze cleavage of NAD+ to yield free ADP-ribose (ADPr) or related metabolites, whose functions remain largely unknown. Here we show that the Nudix (nucleoside diphosphate linked to another moiety X) hydrolase Ysa1 from Saccharomyces cerevisiae is a major regulator of cellular ADPr and O-acetyl-ADP-ribose (OAADPr). OAADPr is the direct product of NAD+-dependent protein deacetylases (sirtuins) and is readily converted to ADPr. Ysa1 cleaves ADPr/OAADPr into ribose phosphate/acetyl-ribose phosphate and AMP. In cells lacking Ysa1 (Δysa1), ADPr and OAADPr levels increased ∼50%, with a corresponding decrease in AMP. Strikingly, Δysa1 cells display higher resistance to exogenous reactive oxygen species (ROS) and 40% lower basal levels of endogenous ROS, compared with wild type. The biochemical basis for these differences in ROS-related phenotypes was investigated, and the results provide evidence that increased ADPr/OAADPr levels protect cells via the following two pathways: (i) lower ROS production through inhibition of complex I of the mitochondrial electron transport chain, and (ii) generation of higher levels of NADPH to suppress ROS damage. The latter occurs through diverting glucose into the pentose phosphate pathway by ADPr inhibition of glyceraldehyde-3-phosphate dehydrogenase, a central enzyme of glycolysis. NAD+ is well known for its role as a hydride-transferring co-enzyme in many oxidation-reduction reactions of metabolism. However, NAD+ is also a substrate for NAD+ glycohydrolases, ADP-ribose transferases, poly(ADP-ribose) polymerases (PARPs), 2The abbreviations used are: PARP, poly(ADP-ribose) polymerase; ADPr, ADP-ribose; OAADPr, O-acetyl-ADP-ribose; Nudix, nucleoside diphosphate linked to another moiety X; ROS, reactive oxygen species; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TRPM2, transient receptor melastin-related ion channel 2; PARG, poly(ADP-ribose) glycohydrolase; ADH, alcohol dehydrogenase; PGK, phosphoglycerate kinase; PPP, pentose phosphate pathway; HPLC, high pressure liquid chromatography; ddH2O, double distilled H2O; LC-MS/MS, liquid chromatography-tandem mass spectrometry; DHE, dihydroethidium. cyclic ADP-ribose synthases (1.Belenky P. Bogan K.L. Brenner C. Trends Biochem. Sci. 2007; 32: 12-19Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar, 2.Kim H. Jacobson E.L. Jacobson M.K. Science. 1993; 261: 1330-1333Crossref PubMed Scopus (235) Google Scholar), and sirtuins (3.Imai S. Armstrong C.M. Kaeberlein M. Guarente L. Nature. 2000; 403: 795-800Crossref PubMed Scopus (2817) Google Scholar, 4.Yang T. Sauve A.A. AAPS J. 2006; 8: E632-E643Crossref PubMed Scopus (143) Google Scholar), all of which cleave the glycosidic bond of NAD+ to produce nicotinamide and an ADP-ribosyl product. Notably, sirtuins catalyze NAD+-dependent lysine deacetylation to generate nicotinamide, deacetylated lysine, and OAADPr (5.Jackson M.D. Denu J.M. J. Biol. Chem. 2002; 277: 18535-18544Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 6.Tanner K.G. Landry J. Sternglanz R. Denu J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14178-14182Crossref PubMed Scopus (500) Google Scholar). OAADPr has been proposed to act as a second messenger, signaling to other processes that NAD+-dependent protein deacetylation has occurred (7.Hoff K.G. Wolberger C. Nat. Struct. Mol. Biol. 2005; 12: 560-561Crossref PubMed Scopus (18) Google Scholar, 8.Liou G.G. Tanny J.C. Kruger R.G. Walz T. Moazed D. Cell. 2005; 121: 515-527Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 9.Kustatscher G. Hothorn M. Pugieux C. Scheffzek K. Ladurner A.G. Nat. Struct. Mol. Biol. 2005; 12: 624-625Crossref PubMed Scopus (245) Google Scholar). The biological functions and in vivo metabolism of OAADPr and free ADPr are largely unknown. Through a quantitative microinjection assay of starfish oocytes, both ADPr and OAADPr caused a delay/block in oocyte maturation, suggesting ADPr/OAADPr may have specific biological activity (10.Borra M.T. O'Neill F.J. Jackson M.D. Marshall B. Verdin E. Foltz K.R. Denu J.M. J. Biol. Chem. 2002; 277: 12632-12641Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). In mammalian cells, intracellular ADPr/OAADPr can activate the TRPM2 (transient receptor melastatin-related ion channel 2) nonselective cationic channel (11.Kuhn F.J. Luckhoff A. J. Biol. Chem. 2004; 279: 46431-46437Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 12.Grubisha O. Rafty L.A. Takanishi C.L. Xu X. Tong L. Perraud A.L. Scharenberg A.M. Denu J.M. J. Biol. Chem. 2006; 281: 14057-14065Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 13.Perraud A.L. Takanishi C.L. Shen B. Kang S. Smith M.K. Schmitz C. Knowles H.M. Ferraris D. Li W. Zhang J. Stoddard B.L. Scharenberg A.M. J. Biol. Chem. 2005; 280: 6138-6148Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). TRPM2 contains a conserved intracellular Nudix hydrolase domain (referred to as NudT9H) that directly binds ADPr/OAADPr, but it is incapable of cleaving the ligand because a major catalytic residue is missing (11.Kuhn F.J. Luckhoff A. J. Biol. Chem. 2004; 279: 46431-46437Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 14.Dunn C.A. O'Handley S.F. Frick D.N. Bessman M.J. J. Biol. Chem. 1999; 274: 32318-32324Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Although still disputed, ADPr binding to NudT9H appears to be required for the well known oxidative stress activation of the channel (13.Perraud A.L. Takanishi C.L. Shen B. Kang S. Smith M.K. Schmitz C. Knowles H.M. Ferraris D. Li W. Zhang J. Stoddard B.L. Scharenberg A.M. J. Biol. Chem. 2005; 280: 6138-6148Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 15.Perraud A.L. Schmitz C. Scharenberg A.M. Cell Calcium. 2003; 33: 519-531Crossref PubMed Scopus (140) Google Scholar). Cell stress via puromycin treatment led to TRPM2-mediated cell death that was dependent on sirtuin deacetylases, presumably from the production of OAADPr (12.Grubisha O. Rafty L.A. Takanishi C.L. Xu X. Tong L. Perraud A.L. Scharenberg A.M. Denu J.M. J. Biol. Chem. 2006; 281: 14057-14065Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Increasing evidence suggests that free ADPr may function as a cellular signal. ADPr can be produced from the coordinate actions of PARPs and poly(ADP-ribose) glycohydrolase (PARG), which cleave ADPr polymers to free ADPr (16.Miwa M. Sugimura T. J. Biol. Chem. 1971; 246: 6362-6364Abstract Full Text PDF PubMed Google Scholar, 17.Bonicalzi M.E. Haince J.F. Droit A. Poirier G.G. Cell. Mol. Life Sci. 2005; 62: 739-750Crossref PubMed Scopus (110) Google Scholar). Under massive genotoxic stress, hyper-stimulation of the NAD+-dependent PARPs depletes cellular NAD+, which is linked to catastrophic ATP loss and cell death (18.Berger N. Radiat. Res. 1985; 101: 4-15Crossref PubMed Scopus (681) Google Scholar, 19.Schreiber V. Dantzer F. Ame J.C. de Murcia G. Nat. Rev. Mol. Cell Biol. 2006; 7: 517-528Crossref PubMed Scopus (1589) Google Scholar). The mechanism by which PARP1 hyperactivity in the nucleus impairs ATP production in mitochondria is unclear. The fact that PARP1 and poly(ADP-ribose) are localized in the nucleus adds a perplexing aspect. However, recent data suggest that PARP1-induced loss of ATP requires PARG (20.Gao H. Coyle D.L. Meyer-Ficca M.L. Meyer R.G. Jacobson E.L. Wang Z.Q. Jacobson M.K. Exp. Cell Res. 2007; 313: 984-996Crossref PubMed Scopus (48) Google Scholar). Under conditions of PARP1 hyperactivation, it has been suggested that the PARG-dependent production of ADPr can exit the nucleus and interfere with ATP production in mitochondria (21.Cipriani G. Rapizzi E. Vannacci A. Rizzuto R. Moroni F. Chiarugi A. J. Biol. Chem. 2005; 280: 17227-17234Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 22.Dumitriu I.E. Voll R.E. Kolowos W. Gaipl U.S. Heyder P. Kalden J.R. Herrmann M. Cell Death Differ. 2004; 11: 314-320Crossref PubMed Scopus (27) Google Scholar). Thus ADPr could be the molecular signal released from the nucleus of cells undergoing massive poly(ADP-ribosyl)ation and rapidly triggers mitochondrial dysfunction. In support for ADPr/OAADPr as potential signaling molecules, the existence of enzymes capable of metabolizing these compounds suggests that their cellular concentrations may be subject to tight regulation (23.Ono T. Kasamatsu A. Oka S. Moss J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 16687-16691Crossref PubMed Scopus (93) Google Scholar, 24.Rafty L.A. Schmidt M.T. Perraud A.L. Scharenberg A.M. Denu J.M. J. Biol. Chem. 2002; 277: 47114-47122Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). To understand the biological roles played by ADPr/OAADPr, it is essential to elucidate the degradation pathways that can modulate their levels. Previously we described the ability of several conserved members of the Nudix hydrolase family to hydrolyze in vitro the diphosphate linkage in ADPr/OAADPr, generating ribose phosphate or acetyl-ribose phosphate and AMP (10.Borra M.T. O'Neill F.J. Jackson M.D. Marshall B. Verdin E. Foltz K.R. Denu J.M. J. Biol. Chem. 2002; 277: 12632-12641Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 24.Rafty L.A. Schmidt M.T. Perraud A.L. Scharenberg A.M. Denu J.M. J. Biol. Chem. 2002; 277: 47114-47122Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Here we examine the biochemical and cellular functions of the Nudix hydrolase Ysa1 (14.Dunn C.A. O'Handley S.F. Frick D.N. Bessman M.J. J. Biol. Chem. 1999; 274: 32318-32324Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) from Saccharomyces cerevisiae. We determined that Ysa1 is the major ADPr Nudix hydrolase and an important regulator of cellular ADPr/OAADPr levels. A Δysa1 strain displays increased resistance to both exogenously and endogenously generated ROS. Basal level of ROS decreased by 40% in the Ysa1 deletion strain. We provide biochemical evidence that increased ADPr/OAADPr levels protect cells via the following two pathways: (i) lower ROS production through the inhibition of complex I of the electron transport chain, and (ii) generation of higher NADPH levels to suppress ROS damage. The latter occurs by diverting glucose into the pentose phosphate pathway by ADPr inhibition of glycolysis. Yeast Strains—Parental strains BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0), BY4742 (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0), Δysa1 (Δysa1::Kanr), with BY4741 and BY4742 as parental strains, and TAP-Ysa1 strain with BY4742 as parental strain were purchased from Open Biosystems (Huntsville, AL). The TAP tag was inserted at the C-terminal end of the coding region of YSA1 gene, consisting of a calmodulin-binding peptide, a tobacco etch virus protease cleavage site, and two IgG binding domains of Staphylococcus aureus protein A (25.Ghaemmaghami S. Huh W.K. Bower K. Howson R.W. Belle A. Dephoure N. O'Shea E.K. Weissman J.S. Nature. 2003; 425: 737-741Crossref PubMed Scopus (3008) Google Scholar). Cultivation and Harvest Conditions—For the copper resistance spot test experiment, cells were spotted on copper containing YMD (2% (w/v) glucose, 0.67% (w/v) yeast nitrogen base without amino acids, supplemented with histidine (20 mg/liter), leucine (40 mg/liter), lysine (40 mg/liter), and uracil (20 mg/liter)), 2% (w/v) agar plates. For all other experiments, yeast strains were cultivated on 2% (w/v) agar plates or in liquid culture with rich YPD medium (1% (w/v) yeast extract, 2% (w/v) tryptone, 2% (w/v) glucose) at 30 °C. Liquid cultures were harvested by centrifugation at 1,600 × g for 20 min at 4 °C and rinsed once with ddH2O, and the pellet was stored at -20 °C. Cell Lysis—Packed yeast cell volume was estimated based on cell concentration of 3 × 107 cell/ml with A600 of 1 (26.Ausubel F.M. Short Protocols in Molecular Biology. 5th Ed. 2. John Wiley, Inc., New York2002: 9-13Google Scholar), average cell size of 66 μm3 (27.Roskams J. Rogers L. Lab. Ref.: A Handbook of Recipes, Reagents, and Other Reference Tools for Use at the Bench (Section 7A). Cold Spring Harbor, New York2002: 196Google Scholar), and the harvested liquid culture size. The pellet was resuspended in 1–3 volumes of lysis buffer (either 20 mm Tris-HCl, pH 7.9, 10 mm MgCl2, 1 mm EDTA, 5% (v/v) glycerol, 1 mm dithiothreitol, 0.3 m ammonium sulfate, 1 mm phenylmethylsulfonyl fluoride for protein extraction; 10% (v/v) trifluoroacetic acid in water for LC-MS/MS analysis of ADPr/OAADPr; or as indicated below for determination of NAD+, NADH, AMP, ATP, and NADPH), resulting in a 2–4-fold dilution of the final cellular metabolite concentrations. This cell paste was mixed with an equal volume of glass beads and vortexed at maximum speed for 10 min at 4 °C. The resulting cell extract was spun at 15,800 × g for 30 min at 4 °C. The clear supernatant was collected and stored at -20 °C. ADPr/OAADPr/NAADPr Metabolizing Assays and HPLC Analysis—To determine the degradation of ADPr/OAADPr/NAADPr within cell extracts, 1 mm of each small molecule was incubated with yeast cell extracts at 37 °C for 30 min in 50 mm Tris, pH 7.5, 10 mm MgCl2. The reactions were quenched with 1% (v/v) trifluoroacetic acid and loaded onto Shimadzu HPLC (LC 2010) and separated by C18 analytical reverse phase column (Grace Vydac, 201SP104) at a flow rate of 0.5 ml/min. The column was equilibrated with buffer A (0.05% (v/v) trifluoroacetic acid in water) for 5 min, and thereafter the reaction mixture was eluted in a 20-min gradient to 8% buffer B (0.02% (v/v) trifluoroacetic acid in acetonitrile), followed by a 20-min gradient to 40% B. The column was then rinsed with 100% buffer B for 10 min and re-equilibrated with buffer A for 10 min before the next injection. ADPr/OAADPr/NAADPr and their respective degradation products were monitored at 260 nm. Halo Assay—The halo assay was performed following the protocol described in Guisbert et al. (28.Guisbert K. Duncan K. Li H. Guthrie C. RNA (N. Y.). 2005; 11: 383-393Crossref PubMed Scopus (81) Google Scholar). Briefly, yeast cells were grown to saturation, and then 50 μl of the culture was rapidly mixed with 4 ml of 0.5% (w/v) agar at 50 °C and poured on top of standard YPD plates. Next, three dilutions of hydrogen peroxide (1.5, 2.9, and 4.4 m) were applied onto the plates as 5-μl drops. The plates were incubated at 30 °C for 1 day before being photographed. H2O2 Survival Test—Yeast cells were grown to A600 of 0.6 before hydrogen peroxide was added to a final concentration of 3 mm. At each time point, the treatment was quenched by pelleting the cells and rinsing the cell pellets with ddH2O. Next, equal numbers of cells were plated onto standard YPD plates for recovery, and the plates were incubated at 30 °C for 2 days before the number of colonies were counted, using Epi Chem II Darkroom Imager (UVP). Copper Treatment—For the spot test, yeast cells were grown overnight at 30 °C with constant shaking at 200 rpm. After rinsing with ddH2O, the cultures were serially diluted from A600 = 1–10-5. Each dilution (5 μl) was spotted on YMD plates, with the indicated concentrations of CuSO4, and incubated at 30 °C for 2 days. For the survival test, cells were grown to A600 of 0.6, and CuSO4 was added to the liquid culture to a final concentration of 9 mm. Control cultures without CuSO4 were also grown in parallel. After incubation at 30 °C for 24 h, cells were pelleted, rinsed with ddH2O, and normalized before being plated onto YPD agar plates for recovery. The plates were incubated at 30 °C for 2 days before being photographed. Assessment of Cellular ROS and Mitochondrial Membrane Potential (ΔΨm)—Cellular ROS and mitochondrial membrane potential were measured using staining with dihydroethidium (DHE) or rhodamine 123 (Rho123), respectively. For ROS staining, cells were incubated in the presence of 5 μg/ml DHE for 15 min, and ROS oxidized DHE into ethidium. For mitochondrial membrane potential, cells were incubated with 2.5 μg/ml Rho123 for 35 min. In each experiment, 10,000 cells were analyzed per cell culture by FACSCalibur flow cytometer (BD Biosciences) equipped with multicolor analysis (λex 488 nm/λem 605 nm for ethidium; λex 507 nm/λem 529 nm for Rho123). Unstained cells served as controls. Ysa1 Cellular Localization—Nuclei and mitochondria were isolated as described previously (29.Amati B.B. Gasser S.M. Cell. 1988; 54: 967-978Abstract Full Text PDF PubMed Scopus (187) Google Scholar, 30.Andrew A.J. Song J.Y. Schilke B. Craig E.A. Mol. Biol. Cell. 2008; 19: 5259-5266Crossref PubMed Scopus (23) Google Scholar). Anti-TAP antibody was purchased from Open Biosystems (Huntsville, AL). Anti-Isu1 antibody was a kind gift from the Craig laboratory (University of Wisconsin, Madison). Anti-H3 antibody was a kind gift from Catherine Fox (University of Wisconsin, Madison). Western blots were visualized using Pierce SuperSignal West Pico chemiluminescent substrate kit, according to the manufacturer's instructions. Screening for ADPr/OAADPr Interacting Proteins—Protein (0.5 mg) from whole cell extract was mixed with 100 μl Cibacron blue resin (C1160, Sigma) pre-equilibrated in 20 mm Tris, pH 7, 1 mm 2-mercaptoethanol. The mixture was mixed at 60 rpm at 4 °C for 1 h. Then the resin was washed four times with 1 ml of wash buffer (20 mm Tris, pH 7.5, 1 mm 2-mercaptoethanol, 10 mm NaCl, 10 mm KCl, 1.5 mm MgCl2) and eluted with 200 μl of wash buffer containing the indicated concentrations of ADPr/OAADPr. The wash and elution samples (100 μl) were concentrated, resolved by SDS-PAGE, and stained with SYPRO Ruby (Molecular Probes) protein stain. Anti-Sir2 antibody was a kind gift from Catherine Fox (University of Wisconsin, Madison). Western blot of Sir2 was visualized as described in Ysa1 localization experiment. In-gel Digestion and LC-MS/MS—SDS-PAGE bands were ingel digested with trypsin and analyzed using LC-MS/MS, as described (31.Hallows W.C. Lee S. Denu J.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 10230-10235Crossref PubMed Scopus (659) Google Scholar). Data Base Searching—Data were searched using Mascot and the Swiss Prot S. cerevisiae Database (Feb. 2006). The search parameters included peptide mass tolerance of 1.2 Da, fragment mass tolerance 0.6 Da, trypsin, allowing for five missed cleavages, and carbamidomethyl (C), deamidation (NQ), and oxidation (M) as variable modifications. LC-MS/MS Measurement of ADPr/OAADPr Cellular Concentration—Cell extracts were analyzed by LC-MS/MS as described (32.Lee S. Tong L. Denu J.M. Anal. Biochem. 2008; 383: 174-179Crossref PubMed Scopus (28) Google Scholar). Briefly, cell pellets were spiked with known amounts of isotopic standard 13C-OAADPr before being extracted to correct for sample losses during the processing. Extracts were then diluted with acetonitrile and directly analyzed using HILIC LC-MS/MS. Known concentrations of isotope standard 13C-OAADPr were used to generate a standard curve to quantify concentration of ADPr/OAADPr. Intracellular concentrations of ADPr and OAADPr were calculated from measured concentration, corrected for sample loss, and multiplied by dilution factors in the previously described cell lysis and LC-MS/MS sample preparation step. ADPr Inhibition of GAPDH Activity—The assay mixture (0.3 ml) contained 100 mm potassium phosphate buffer, pH 7.6, 10 mm EDTA, 0.1 mm dithiothreitol, 1 mm glyceraldehyde 3-phosphate, 0.043 to 0.286 mm NAD+, 0.06 to 0.3 mm ADPr, and highly purified GAPDH from S. cerevisiae (Sigma, G5537, 70–140 units/mg, 0.1ADPrcADPr concentrations were determined by subtracting NADH concentrations from the sum of ADPr and NADH concentrations0.105 ± 0.0120.158 ± 0.0110.0088NAD+1.76 ± 0.081.71 ± 0.01>0.1AMP0.749 ± 0.0670.587 ± 0.0260.0431ATP0.904 ± 0.0340.913 ± 0.051>0.1a These data are from Ref. 32.Lee S. Tong L. Denu J.M. Anal. Biochem. 2008; 383: 174-179Crossref PubMed Scopus (28) Google Scholar; 10–3 was used to indicate that OAADPr concentration is in the micromolar rangeb Under our acidic extraction conditions, NADH hydrolyzes to ADPr (33.Bergmeyer H. Methods of Enzymatic Analysis. 3rd Ed. 7. VCH Publishers, Deerfield Beach, FL1985: 253Google Scholar). Thus, this reflects the sum of ADPr and NADH concentrationsc ADPr concentrations were determined by subtracting N
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