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Phosphotransferase Activity of Liver Mitochondria (Oxidative Phosphorylation/Adenine Nucleotide Ni-Oxides/Substrate Specificity/In Vivo Phosphorylation) G

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Phosphotransferase Activity of Liver Mitochondria (Oxidative Phosphorylation/Adenine Nucleotide Ni-Oxides/Substrate Specificity/In Vivo Phosphorylation) G Proc. Nat. Acad. Sci. USA Vol. 71, No. 11, pp. 4630-4634, November 1974 Participation of N1-Oxide Derivatives of Adenine Nucleotides in the Phosphotransferase Activity of Liver Mitochondria (oxidative phosphorylation/adenine nucleotide Ni-oxides/substrate specificity/in vivo phosphorylation) G. JEBELEANU, N. G. TY, H. H. MANTSCH*, 0. BARZU, G. NIACt, AND I. ABRUDAN Department of Biochemistry, Medical and Pharmaceutical Institute, Cluj, * Institute of Chemistry, University of Cluj, Cluj, and t Department of Physical Chemistry, University of Craiova, Craiova, Romania Communicated by Henry Lardy, September 3, 1974 ABSTRACT The modified adenine nucleotides ATP- rivatives of adenine nucleotides once produced become "ac- NO, ADP-NO, and AMP-NO were tested as potential tive" components of the mitochondrial or cellular adenylate substrates and/or inhibitors of mitochondrial phospho- transferases. ADP-NO is not recognized by the translocase pool. membrane; system located in the inner mitochondrial AND METHODS however, it is rapidly phosphorylated to ATP-NO in the MATERIALS outer compartment of mitochondria, by way of the The following commercially available chemicals were used: nucleosidediplhosphate kinase (EC 2.7.4.6) reaction, pro-' de- vitded there is sufficient ATP in the mitochondria. AMP- crystalline bovine serum albumin, glucose-6-phosphate NO is not phosphorylated by liver mitochondria to the hydrogenase (Glc-6-P dehydrogenase; EC 1.1.1.49; BDH corresponding nucleoside diphosphate; it cannot serve as Chemicals, Ltd.), yeast hexokinase (EC 2.7.1.1) (Nutritional substrate for adenylate kinase (EC 2.7.4.3). ATP-NO and Biochemicals, Cleveland), (log muscle lactate dehydrogenase ADP-NO, however, are substrates of this enzyme. The (EC 1.1.1.28), pyruvate kinase (EC 2.7.1.40), adenylate apparent equilibrium constant for the reaction, ADP-NO + ADP ATP-NO + AMP, of 0.908 at pH 7.4 and 5 mM kinase, NAD, NADH, and NADP (Boehrinoer, M\annheim). M2+2 is significantly higher than that of the reaction with Nueleotides were obtained from Sigma Chemical Co., [14C]- natural nucleotides. ADP from Schwarz BioResearch. The nucleotide analogs Although ade iosine Ni-oxide is easily phosphorylated ATP-NO, ADP-NO, and AMP-NO were obtained by gentle to AMP-NO by adenosine kinase [Schnebli et al. (1967) with acid in J. Biol. Chem. 242, 1997-2004], the formation of corre- oxidation of the natural nueleotides permaleic sponding nucleoside triphosplhate in vivo seems also to be aqueous solutions at neutral )1H. The ratio of the UV absorp- limited by adenylate kinase; adenosine Ni-oxide cannot tion A233/A260 was 4.8 for the Ni-oxides, whereas the same replace adenosine in restoring the normal ATP level in ratio was 0.2 for the natural compounds. ethionine-treated rats. Rat and mouse liver mitochondria were isolated as de- of Lowry In an earlier paper we showed that the modified adenine scribed (4). Proteins were measured by the method adenosine N1-oxide (ADP-NO), is et al. (5). The mitochondrial respiration was monitored with a nucleotide, diphosphate with unable to act as phosphate acceptor in the reactions of oxi- Clark type electrode. Mlitochondria loaded [i4C]ADP dative phosphorylation when different substrates are oxiilized were l)repare(l as described by Duee and Vignais (6); radio- activity was measured by liquid scintillation counting. Chro- by intact rat or human liver mitochondria (1). Since the nucleoside oxidative phosphorylation is specific for ADP as phosphate matographic separation of rat liver mitochondrial with intact mitochoildria but not with sonic parti- diphosphate kinase and adenylate kinase was performed essen- acceptor Rat liver mitochondrial (2, 3), our results were explained by the fact that ADP-NO tiallv as described bv Pedersen (7). cles in 2 ml of 10 mM Tris * HCl (pH is not translocated across the inner mitochondrial membrane. protein (50 mg) suspended that the outer compartment 7.4) wvas allowed to stand 1 hr at 00. After centrifugation at Taking into account the fact about 7 of mitochondria contains the nucleosidediphosphate kinase 15,000 X g for 10 min, the supernatant, containing mg of protein, was placed on a Sephadex G-100 column (42 X (EC 2.7.4.6; ATP: nucleosidediphosphate phosphotransferase) M and adenylate kinase (EC 2.7.4.3; ATP :AMP phospho- 1.5 cm) that had been equilibrated with 0.1 phosphate buffer (pH 7.5) and chromatographed with the same buffer. transferase), which are involved in a rather nonspecific kinase of nucleoside mono-, di-, and tri-phosphates, The peak fractions containing nucleoside diphosphate interconversion were used for kinetic we now examined the possibility that ATP, which is pro- and adenylate kinase activity experi- kinase was measured duced in the inner mitochondrial compartment, may be ments. Nueleoside diphosphate activity spectrophotometrically at 340 nm with the assay system of coupled to the formation of ATP-NO from ADP-NO or reactions interest in this investigation was stim- Jacobus and Lehninger (8). The forward and reverse from AMP-NO. Our measured ulated by two motives: (a) to explore the role of mito- of adenylate kinase were spectrophotometrically by coupling with the assay system of Adelman et al. (9) and chondria in the phosphorylation of different structural nucleo- nucleotide which properties; Silva Lima and Vignais (10), or by measuring tide analogs, some of possess cytostatic thin-layer chromatography and (b) to determine the degree to which the N1-oxide de- concentration after separation by on DEAE-cellulose (11). The forward reaction of adenylate kinase was considered as 2 ADP A MMP + ATP, while the Abbreviations: ATP-NO, adenosine triphosphate N1-oxide; opposite direction AMP + ATP = 2 ADP was considered adenosine diphosphate Ni-oxide; AMP-NO, adenosine ADP-NO, as the reverse reaction. Since the reaction of ATP-NO with monophosphate N1-oxide; AOPCP, adenosine-5'-methylene- of ADP-NO with Glc-6-P dehydrogenase, glucose-6-phosphate hexokinase as well as the reaction pyruvate diphosphonate; in ex- dehydrogenase. kinase could be rate limiting, we determined separate 4630 Downloaded by guest on September 28, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Ni-Oxides of Adenine Nucleotides 4631 TABLE 1. Km and Vm values of yeast hexokinase and dog muscle pyruvate kinase for A TP, ADP, and their Ni-oxide derivatives Vm Nucle- (,umol/min otide Enzyme Km (mM) per mg) k*/k ATP Hexokinase 0.21 ± 0.03a 143 + 5 1.000 ATP-NO Hexokinase 0.59 ±t 0.08 38 ± 4 0.098 ADP Pyruvate kinase 0.27 i 0.03 189 ± 10 1.000 ADP-NO Pyruvate kinase 1.28 i 0.14 72 4 6 0.078 The reaction medium, in a final volume of 0.7 ml and at 300, contained 50 mM triethanolamine (pH 8.0), 5 mM glucose, 6 mM MgCI2, 0.4 mM NADP, ATP, or ATP-NO, 1.8 units of Glc-6-P dehydrogenase and 0.02 units of hexokinase (hexokinase FIG. 1. Effect of nucleoside mono- and diphosphates on the assay) or 50 mM Tris- HCl (pH 7.4), 80 mM KCl, 8 mM MgC12, respiratory rate of mouse liver mitochondria. The basic respira- 1 mM phosphoenolpyruvate, 0.15 mM NADH, ADP, or ADP- tory medium contained, at 1.5 ml final volume and 23°, 180 mM NO, 3 units of lactate dehydrogenase and 0.04 units of pyruvate sucrose, 50 mM KCl, 25 mM Tris-HCl (pH 7.4), 5 mMi phos- kinase (pyruvate kinase assay). The results represent the mean phate (pH 7.4), 2.5 mM MgCl2, 1 mM EDTA (no EDTA in value of four separate k and k* are the relative experiments; experiments with AMP or AMP-NO), 3 mg of bovine serum Vm/Km ratios for natural nucleotides and their NI-oxide analogs, albumin, and 1.2 mg of mitochondrial protein. At the points respectively. indicated, 5 umol of succinate (S), 0.22 ,umol of ADLP (A), 0.18 a Standard error of the mean. jsmol of ADP-NO (B), 0.12 ,umol of AMP (C), or 0.11 ,umol of AMP-NO (D) were added. The numbers beside the traces indi- periments the Vm and Km of these coupling enzymes, both cate the oxygen consumption expressed as ng-atoms per with ATP or ADP and their Ni-oxide derivatives (Table 1). min/mg of protein. Adenine nucleotides were assayed in the perchloric extracts after neutralization by KOH according to the procedure of if we keep in mind the extremely high affinity of the trans- Adam (12). locase system for ADP and ATP (14, 15). RESULTS The addition of ADP-NO to intact mitochondria has no As shown in Table 2, the exchange between internal [14C]ADP effect on respiration (1). However, addition of ADP-NO to and added ADP-NO is negligible compared to the exchange of respiring mitochondria after the state 3 to state 4 transition ADP or adenosine-5'-methylenediphosl)honate (AOPCP). does result in an immediate stimulation of the rate of respira- Moreover, the competitive action of ADP-NO on the ADP tion (Fig. 1). This enhancement of resliration seems to be exchange is very weak. Both ADP and AOPCP exchanges due to the formation of ATP-NO bv way of nucleosidedi- were atractyloside-sensitive, in agreement with previous phosphate kinase, regenerating ADP that is essential for a data of Duee and Vignais (6). Schlimme and Schafer (13), respiratory stimulation (7). If varying amounts of ADP and using radioactive ATP-NO and ADP-NO obtained by oxi- ADP-NO were added simultaneously to the mitochondrial dation of ['4C]ATP and ['4C]ADP with monoperphtalic acid, suspension with succinate as substrate, the ratio ADP/O described an atractyloside-sensitive translocation of these decreased with increasing concentration of the analog. By nucleotide analogs.
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