ADP-ATP Exchange Research Articles

Oxidative phosphorylation as a function of temperature

1. The activation energy of succinate oxidation by rat-liver mitochondria changes at a temperature of about 17° in State 3 as well as in the uncoupled state. 2. Over the whole temperature range investigated (0–23°) the rate of phosphorylation of intramitochondrial ADP during succinate oxidation exceeds that of added ADP. 3. The activation energy of the ADP-ATP and P i-ATP exchange reactions and of the 2,4-dinitrophenol-induced ATPase also changes at about 17°. 4. The temperature coefficients of the State-3 oxidation and of the P i-ATP and ADP-ATP exchange reactions are similar and, at temperatures below 17°, are high in comparison with that of the phosphorylation of intramitochondrial ADP. 5. The translocation of ADP and ATP through the inner membrane is rate-limiting for the process of oxidative phosphorylation in rat-liver mitochondria.

Fractionation and comparative studies of enzymes in aqueous extracts of spinach chloroplasts

Water and dilute salt solutions were employed gor the hypotonic rupture and repeated extraction of proteins from spinach chloroplasts. The extracts were partially fractionated with ammonium sulfate, and the fractions so obtained were resolved by sucrose-gradient centrifugation and by electrophoresis on acrylamide gels. These techniques physically separated several enzymes of phosphate metabolism which are interdependent upon common substrates, co-factors, and reaction products, and which may function critically in photophosphorylation or its reversal. These enzymes are a Ca ++-dependent ATPase ( S 20 w 12.7; DTT stimulated; active as coupling factor in photophosphorylation; morphologically identical to quantasomes), a discretely different Mg ++-dependent ATPase ( S 20 w 6.0; entirely different from mitochondrial ATPases; does not require activation but is stabilized by reductants), and ATP-ADP exchange enzyme ( S 20 w 7.0), adenylate kinase ( S 20 w 4.2), and an alkaline inorganic pyrophosphatase (associated with fructose diphosphatase activity). In addition, a similar interacting system of CO 2-fixing enzymes, comprised of ribulose diphosphate carboxylase, ribose phosphate isomerase, and phosphoribulokinase was separated. The fractionation and isolation conditions described were chosen to preserve all of these activities, and to provide an environment suited to a uniform comparison of their physical and enzymatic properties. These properties are compared with those of enzymes having similar functions, purified from chloroplasts by other means.

An improved procedure for preparation of inner membrane vesicles from rat liver mitochondria by treatment with digitonin

A method for the preparation of inner membrane vesicles from rat liver mitochondria that represents a substantial improvement over one previously described (Hoppel, C. and Cooper, C., Biochem. J. 107, 367 (1968)) is presented. The inner membrane preparation is further fractionated by differential centrifugation and although all of the fractions are qualitatively similar with regard to enzyme content only the fraction sedimenting between 50–100,000 g shows considerable structural uniformity. Electron micrographs obtained by both sectioning and negative staining are presented and the former show that the bounding membrane is trilaminar and that material appears to be present within the vesicles. Certain inner membrane enzymes are recovered in high yields from mitochondria with a large increase in specific activity. The specific activity of succinic dehydrogenase is increased 7.4-fold and the recovery from the mitochondria is 61% while rotenone-sensitive NADH-cytochrome c reductase showed a 12.2-fold increase in specific activity and the recovery was 98%. The specific activity for phosphate uptake with 3-hydroxybutyrate as substrate is approximately 80% of that of the intact mitochondria. The preparation is virtually free of adenylate kinase and nucleoside diphosphokinase and the P i-ATP and ADP-ATP exchange reactions have the same rates.

Mathematical Model of an Enzymatic Reaction Application to the Study of the ADP-ATP Exchange Reaction

This paper present the construction of the mathematical model of enzymatic reactions, and the simulation on digital computer of a enzymatic “ping-pong” reaction. The results of these simulations are applied to the identification of a complex enzymatic “ping-pong” reaction = the ADP-ATP exchange reaction eatalysed by a nucleoside diphosphate kinase enzyme.

Biosynthesis of the Purines: XXXI. BINDING OF FORMYLGLYCINAMIDE RIBONUCLEOTIDE AND ADENOSINE TRIPHOSPHATE TO FORMYLGLYCINAMIDE RIBONUCLEOTIDE AMIDOTRANSFERASE

Abstract 14C-Formylglycinamide ribonucleotide (FGAR) and γ-32P-ATP (or 14C-ATP) bind to FGAR amidotransferase to form a complex, which may be isolated by Sephadex gel filtration and characterized. The formation of this complex is dependent on the presence of three components, FGAR, ATP, and magnesium ions, but does not require glutamine. The binding sites for FGAR and ATP on the enzyme molecule may be distinguished from that of glutamine. The binding ratio of either FGAR or ATP to the enzyme expressed in molar quantity was approximately 0.7 under the conditions used at pH 6.5. The ratio of FGAR to ATP bound to the enzyme was unity. The analysis of the radioactive materials obtained from the complex of FGAR and γ-32P-ATP with the enzyme indicated that the terminal phosphoanhydride bond of ATP was cleaved to ADP and a phosphate moiety, which remained bound to the enzyme presumably in ester linkage. Furthermore, when the enzyme was incubated with 14C-ATP in the presence of MgCl2, an amount of 14C-ADP nearly equivalent to the moles of enzyme present was obtained. FGAR did not affect this reaction. These results and others including observations on an ATP-ADP exchange reaction indicate strongly that the enzyme itself is phosphorylated in a step of the over-all reaction catalyzed by FGAR amidotransferase. Azaserine and iodoacetate, both of which reacted at the glutamine-binding site, reduced and enhanced, respectively, the stability of the FGAR-ATP-enzyme complex. However, these compounds did not affect the true binding ratios of the substrates. Although both compounds inhibited the reaction in which glutamine was the nitrogen donor, azaserine stimulated and iodoacetate partially inhibited the reaction with ammonium chloride as the nitrogen donor. A mechanism of the reaction catalyzed by FGAR amidotransferase has been discussed on the basis of the above information. The over-all reaction has been broken down into partial reactions, which, however, operate in a highly coordinated manner with one another.

Trypsin digestion of fragmented sarcoplasmic reticulum

Short incubation of fragmented sarcoplasmic reticulum with trypsin produces inhibition of ATP dependent calcium accumulation, while ATPase is activated. The addition of trypsin to loaded vesicles causes a rapid calcium leak, suggesting that certain easily digestible peptidic bonds may contribute to the maintenance of semipermeability in sarcoplasmic membranes. A small amount of calcium is still taken up by partially digested membranes, independently of the presence of ATP. This uptake is due to binding to the membrane itself and is competitively inhibited by other cations. ATPase is inhibited by longer digestions, while obvious structural changes appear throughout the thickness of the membrane. This indicates that either ATPase is extended into the deeper layer of the membrane, or it is very resistant to trypsin attack. A parallel pattern of inhibition is produced by trypsin on ATPase and ADP-ATP exchange, indirectly suggesting interdependence of these two activities. Depending on the time of digestion, more or less marked alterations of the membrane structure can be observed on electronmicroscopy (negative staining with phosphotungstic acid): disappearance of an outer granular layer, irregular thickness of the membrane, opening of the vesicles and aggregation. Suspensions of fragmented sarcoplasmic reticulum digested with trypsin, undergo a rapid decrease in turbidity, likely due to a change in the state of aggregation of the vesicles.

Effect of ouabain on ion transport mechanisms in the isolated turtle bladder.

Effect of ouabain on ion transport mechanisms in the isolated turtle bladder.

The Atractyloside-sensitive Nucleotide Binding Site in a Membrane Preparation from Rat Liver Mitochondria

Abstract Treatment of rat liver mitochondria with the nonionic detergent Lubrol-WX results in loss of 70% of the protein in soluble form. The vesicular membrane preparation recovered after this treatment is freely penetrated by sucrose and no longer can retain a pool of internal adenine nucleotides; however, it retains the capacity to bind adenine nucleotides in an atractyloside-sensitive reaction. The atractyloside-sensitive site was found to be specific for ADP and ATP; AMP is not bound. The affinity of the preparation for ADP and ATP at the atractyloside-sensitive sites is very high (Kd about 1 µm). The maximum amount of nucleotide bound to these sites has been estimated as about 0.2 nmole per mg of total mitochondrial protein. The affinity for atractyloside is also extremely high; Ki is less than 1 µm. ADP or ATP bound to the atractyloside-sensitive sites is readily exchangeable with free nucleotides in the medium. Atractyloside cannot displace nucleotides from their binding sites. Furthermore, since no competition between nucleotide and atractyloside is apparent, atractyloside and ADP are probably bound at different specific sites on the molecule. When atractyloside is bound first, neither ATP nor ADP can be bound at the nucleotide site. On the other hand, when the nucleotide site is occupied first, subsequent binding of atractyloside at its site prevents the loss or exchange of the bound ADP (or ATP). These properties suggest that the specific ADP-ATP exchange diffusion carrier in the membrane may undergo conformational changes on binding atractyloside so that nucleotides cannot be bound or, if they have already been bound, they cannot be lost.

Isolation and reactions of a phosphorylated form of phosphoryl transferase from beef heart mitochondria

Phosphoryl transferase, a mitochondrial protein which increases the phosphorylative capacity of poorly phosphorylating submitochondrial particles and catalyzes an ATP-ADP exchange reaction, is phosphorylated during oxidation either of succinate or pyruvate-malate. Inhibitors of oxidative phosphorylation and electron transfer, as well as uncouplers of oxidative phosphorylation, inhibit the phosphorylation of the transferase when phosphorylation is mediated by electron transfer. The protein is also phosphorylated by ATP, the donor group being specifically the terminal phosphate of ATP. The transphosphorylation reaction is not inhibited by inhibitors of electron transfer and coupled phosphorylation, nor by uncouplers of oxidative phosphorylation. The phosphoryl form of the transferase can phosphorylate ADP in the presence of hexokinase, glucose, and magnesium ion, but the transfer is only 50% complete. During this transfer reaction a portion of the protein-bound phosphate becomes transformed to an acid-stable form. Phosphorus is released from phosphoryl transferase as inorganic orthophosphate at pH 4 and 10 and by heat, but is relatively stable at pH 7.5 at 0 °. Hydroxylamine also induces release of protein-bound phosphorus as inorganic phosphate. The possible role of the phosphoryl group of the transferase in oxidative phosphorylation is discussed.

Light-triggered, thiol-dependent ADP-ATP exchange activity in spinach chloroplasts

Light-triggered, thiol-dependent ADP-ATP exchange activity in spinach chloroplasts

Relationships between Erythrocyte Membrane Phosphorylation and Adenosine Triphosphate Hydrolysis

Abstract Human erythrocyte membranes were incubated with a low concentration of terminally labeled ATP with the aim of detecting a phosphorylated intermediate of adenosine triphosphatase activity. A large portion of material labeled briefly at 37° turned over and had properties consistent with participation in sodium ion-stimulated ATPase activity, including the following: (a) magnesium ions present, addition of sodium ions stimulated an increase in both 32P bound to trichloracetic acid-precipitated membrane material and the rate of ATP hydrolysis. (b) The Na+-activated bound 32P turned over and correlated quantitatively with the amount of bound 32P sensitive to hydroxylamine, in agreement with properties of phosphorylated intermediate of ATPase of other preparations. (c) Addition of potassium ions stimulated breakdown of the 32P bound in the presence of sodium ions. At 0°, a large portion of material labeled briefly was sensitive to hydroxylamine. Its turnover was apparent upon addition of excess ATP or ADP, but not guanosine triphosphate, deoxyguanosine diphosphate, AMP, or 3-phosphoglycerate. The addition of ethylenediamine tetraacetate allowed dephosphorylation of the bound 32P to proceed, and it was shown that the approximate rate of turnover was similar to the rate of ATP hydrolysis. Although the addition of sodium ions stimulated an increase in bound 32P and in ATPase activity at 2 µm ATP concentration, manifestations of altered sensitivity to sodium and potassium ions at 0° have been attributed to temperature-induced changes in conformation of catalytic sites. The relationship of membrane (14C)ADP-ATP exchange activity to ATPase activity was investigated. It was found that at least 30% of the exchange activity was associated with material which could be separated from ATP hydrolytic activity and which was not labeled with 32P after incubation with terminally labeled ATP. This activity did not appear to be related to other membrane activities tested, including nucleoside diphosphokinase, adenylate kinase, and phosphoglycerate kinase.

Sodium-Potassium-activated Adenosine Triphosphatase of Electrophorus Electric Organ: V. PHOSPHORYLATION BY ADENOSINE TRIPHOSPHATE-32P

Abstract Electric organ microsomes from Electrophorus electricus can be phosphorylated by ATP. The properties of this reaction have been studied and compared with the properties of the sodium-potassium-activated adenosine triphosphatase and the Na+-dependent ATP-ADP exchange reaction which occur in the same microsomal preparation. Only sodium ion, among the monovalent cations tested, will increase the extent of microsomal phosphorylation. The phosphorylation also appears to be specific for ATP. The Na+-dependent, ATP-specific phosphorylation is not prevented by treatment with N-ethylmaleimide or oligomycin. Under some conditions, ouabain inhibits the phosphorylation. Following phosphorylation in the presence of sodium and ATP, potassium and most monovalent cations decrease the extent of phosphorylation. This reduction is prevented by N-ethylmaleimide or oligomycin, which, in an earlier report, were shown to inhibit Na+-K+-ATPase and to activate a Na+-dependent ATP-ADP exchange. Other workers had reported that hydroxylamine dephosphorylates brain microsomes without inhibiting Na+-K+-ATPase, thus questioning the relevance of the Na+-dependent phosphorylation reaction. With electric organ microsomes, hydroxylamine will replace K+ to some extent in the activation of ATP hydrolysis. Hydroxylamine will also reduce the extent of phosphorylation of native microsomes. However, under comparable conditions, hydroxylamine does not dephosphorylate acid-denatured microsomes. Thus any hydroxylaminolysis of the microsomal phosphate which may occur is slow relative to the turnover rate of the intact enzyme. These observations are consistent with the hypothesis that the Na+-dependent phosphorylation and exchange reactions are manifestations of the Na+-K+-ATPase and represent the initial step in a sequence of reactions to achieve hydrolysis of ATP. This sequence of reactions is probably related to the mechanism of active cation transport across cell membranes.

Localization of oligomycin-sensitive ADP-ATP exchange activity in rat liver mitochondria

Intact rat liver mitochondria catalyze an ADP-ATP exchange which is partially inhibited by oligomycin and dinitrophenol ( Wadkins and Lehninger, 1963; Guillory and Slater, 1965). It has been postulated that the dinitrophenol- and oligomycin-sensitive ADP-ATP exchange reaction is catalyzed by the enzyme(s) participating in ATP formation during oxidative phosphorylation ( Wadkins and Lehninger, 1958; 1963). However, the presence of large amounts of adenylate kinase and nucleoside diphosphokinase, which catalyze oligomycin-insensitive ADP-ATP exchanges, has rendered further investigation of the oligomycin-sensitive exchange difficult. In this communication it is shown that when rat liver mitochondria are fractionated with digitonin the oligomycin-sensitive ADP-ATP exchange activity is recovered with the inner membrane-matrix fraction, while all of the nucleoside diphosphokinase and adenylate kinase activity is released with the outer membrane. The exchange activity found in the inner membrane-matrix fraction is localized in the inner membrane and is completely inhibited by oligomycin, dinitrophenol, and atractyloside when assayed in the absence of added Mg ++. Thus, the oligomycin-sensitive ADP-ATP exchange activity observed in intact mitochondria does not appear to be catalyzed by nucleoside diphosphokinase or adenylate kinase.

The role of the ADP-ATP exchange enzyme in oxidative phosphorylation

1. A nucleoside diphosphate kinase (EC 2.7.4.6) has been isolated from liver mitochondria that is rather specific for ATP as phosphate donor. It catalyses an ADP-ATP exchange reaction. 2. The isolated enzyme is inhibited by ADP in concentrations higher than 1 mM. The reaction is stimulated by both ferri- and ferrocytochrome c, but the inhibition by high ADP concentrations is not relieved. 3. The isolated enzyme is insensitive to 2,4-dinitrophenol and oligomycin. It appears to become sensitive to 2,4-dinitrophenol but not to oligomycin on addition of freshly prepared digitonin particles. The inhibition is due to the formation of ADP, catalysed by the adenosine triphosphatase in the digitonin particles. 4. The oligomycin-sensitive ADP-ATP exchange activity in the mitochondria is not affected by extraction of the nucleoside diphosphate kinase. 5. The isolated enzyme has no effect either on the P/O ratio or on the ATP-driven nicotinamide nucleotide transhydrogenase of submitochondrial particles. 6. A role of this enzyme in oxidative phosphorylation seems improbable.

The ATP-ADP exchange catalyzed by phosphoryl transferase

Purified phosphoryl transferase catalyzes an ATP-ADP exchange reaction. Evidence is presented which excludes the possibility that the exchange activity of the preparation is due to contaminating adenylate kinase. The rate of the exchange reaction is affected by the molar ratio of ATP to ADP. The reaction is not highly specific either for the divalent metal ion or for the nucleoside triphosphate which serves as phosphoryl donor. Neither inhibitors of oxidative phosphorylation and electron transfer nor uncouplers of oxidative phosphorylation affect the rate of the exchange reaction significantly. Hydroxylamine in high concentration inhibits the exchange reaction. Phosphoryl transferase does not catalyze several other types of reactions which might be expected to involve protein-bound phosphate as intermediates, such as the reactions catalyzed by pyrophosphatase, alkaline phosphatase and succinylthiokinase, respectively. A previously described protein fraction from mitochondria which inhibited the increase in P:O ratio induced by the phosphoryl transferase also inhibits the ATP-ADP exchange reaction catalyzed by the transferase. The possible role of phosphoryl transferase in the transfer of a phosphoryl group from ATP, formed during oxidative phosphorylation, to exogenous ADP is discussed.

Studies on ATP citrate lyase of rat liver. 3. The reaction mechanism.

The mode of action of ATP citrate lyase [EC 4.1.3.8] was investigated using a single homogeneous preparation from rat liver. First, the enzyme was incubated with labelled citrate and after incubation the protein portion was isolated through a column of Sephadex G-50. It was found that an enzyme-citrate complex is formed in the presence of both ATP and Mg++. When this enzyme-citrate complex was isolated and then incubated with CoA, the formation of acetyl-CoA was demonstrated in the absence of added ATP. When 8-14C-ATP and γ-32P-ATP were separately incubated with the enzyme, radioactivity associated with protein was obtained only with γ-32P-ATP, but not with 8-14C-ATP. In accord with this, a rapid ATP-ADP exchange, but not an ATP-P1 exchange, was observed. These two reactions were solely dependent on the presence of Mg++. Therefore, the reaction involves the formation of an enzyme-phosphate complex.

A light-triggered adenosine triphosphate-phosphate exchange reaction in chloroplasts.

Chloroplasts were triggered by light in the presence of dithiol reagents to catalyze an ATP-Pi exchange reaction in a following dark period. This light-triggered ATP-Pi exchange was related to photophosphorylation, and to light-triggered ATPase by the similarity in requirements for magnesium ions, phenazine methosulfate and flavin mononucleotide, and the inhibition by uncouplers and electron transport inhibitors. The ATP-Pi exchange reaction showed substrate specificity for ATP and was competitively inhibited by ADP. The light-triggered condition decayed in the dark with a half-life of several minutes. The rate of the decay was not affected by uncouplers or energy transfer inhibitors. The light-triggering phase required the presence of a dithiol-reagent, and was stimulated by magnesium and electron transfer catalysts. The dark phase did not require the presence of dithiol reagents. The energy transfer inhibitors DIO-9 and phloridzin as well as the uncoupler atebrin and the inhibitor n-butyl-3,5-diiodo-4-hydroxybenzoate inhibited only the dark phase. The uncoupler, NH4CI, and the inhibitor 4,5,6,7-tetrabromo-2-trifluoromethylbenzinamidazol inhibited both the light-triggering phase and the dark phase. The overall pH optimum for ATP-Pi exchange was about 8. However, the pH optimum of the light stage alone was about 9 and that of the dark stage — about 7.5. Chloroplast fragments which retain their capacity for photophosphorylation possessed light-triggered ATP-Pi exchange activity but had no light-triggered ADP-ATP exchange.

The effect of univalent cations of activities catalyzed bovine-liver propionyl-CoA carboxylase

The propionyl-CoA carboxylase (propionyl-CoA:Co 2 ligase (ADP), EC 6.4.1.3) activity of a highly purified preparation (1400 units/mg of protein at pH 8.5 and 37°) was activated by K +, NH 4 +, Rb +, or Cs +; Na +; Li +, or tetramethyl ammonium ion showed little or no activation. The maximal velocity of carboxylation was enhanced 7- to 9-fold in the presence of K +; however, the values of K m for propionyl CoA, bicarbonate, or Mg 2+; were the same in the absence or presence of K +. The P i-ATP and ADP-ATP exchange reactions and transcarboxylase activities catalyzed by the enzyme were also stimulated by K +. The propionyl-CoA carboxylase activity was activated maximally by Mg 2+ or Mn 2+ and was optimal at pH 8 to ph 8.5. However, the P i-ATP and ADP-ATP exchange activities of the enzyme were activated by Mn 2+; Mg 2+ was much less effective than Mn 2+, and the optimal rates of the exchange reactions occurred between pH 6.8 and pH 7.0. These properties of the exchange activities of the enzyme and certain other features reported here suggest that the ‘exchange enzyme’ of Chiga and Plaut 2 is identical with propionyl-CoA carboxylase. A method of separation of propionyl-CoA and methylmalonyl-CoA by thin-layer chromatography has been described.

Properties of a Nucleoside Diphosphokinase from Liver Mitochondria and Its Relationship to the Adenosine Triphosphate-Adenosine Diphosphate Exchange Reaction

A nucleoside diphosphokinase which also catalyzes an adenosine triphosphate-adenosine diphosphate exchange reaction has been extracted from bovine liver mitochondria and purified by conventional methods of protein fractionation. A comparison of the catalytic properties of the two activities indicates that both are catalyzed by the same enzyme, which is different in several respects from the nucleoside diphosphokinase previously isolated by others from rat liver and from erythrocytes. Evidence is presented that the enzyme herein described also catalyzes the dinitrophenol-sensitive ATP-ADP exchange reaction when bound to intact mitochondria and to phosphorylating submitochondrial particles.

Properties of an Oligomycin-sensitive Adenosine Diphosphate-Adenosine Triphosphate Exchange Reaction in Intact Beef Heart Mitochondria

Abstract Intact beef heart mitochondria having high acceptor control ratios catalyze an oligomycin-sensitive adenosine diphosphate-ATP exchange reaction in the presence of 6.0 mm Mg++ at a rate of about 400 to 600 mµmoles per min per mg of mitochondrial protein, or somewhat higher than the rate of oxidative phosphorylation in beef heart mitochondria. The oligomycin-sensitive component of the total ADP-ATP exchange activity is saturated with Mg++ at much lower concentrations (∼1.0 mm) than the oligomycin-insensitive component (∼8.0 mm). Gramicidin, valinomycin, aurovertin, azide, arsenate, and atractyloside inhibit the ADP-ATP exchange, to an extent not exceeding the inhibition by oligomycin, whereas the adenylate kinase activity is insensitive to these agents. Beef heart mitochondria also catalyze uridine diphosphate-UTP, cytidine diphosphate-CTP, guanosine diphosphate-GTP, and ADP-dCTP exchanges, but these are insensitive to oligomycin and occur at less than 10% of the rate of the ADP-ATP exchange. Freezing and thawing of beef heart mitochondria, which has been reported not to cause loss of oxidative phosphorylation as measured by the P:O ratio, caused loss of the oligomycin-sensitive ADP-ATP exchange, loss of acceptor control of respiration, and gain of ATPase activity. It is suggested that the loss of oligomycin-sensitive ADP-ATP exchange activity during preparation of phosphorylating submitochondrial particles is caused by a molecular transformation of the ATP-synthesizing system so that the affinity for ADP is decreased, with a resulting gain of ATPase activity.