Mitochondrial ATP-Pi exchange complex

An enzyme complex with high ATP-Pi exchange activity has been purified from beef heart mitochondria, using the general procedure which also yields electron transfer complexes I, II, III and IV from the same batch of mitochondria. The ATP-Pi exchange activity of the preparation, designated complex V, is inhibited by various uncouplers, rutamycin, venturicidin, dicyclohexylcarbodiimide, arsenate, azide, adenylyl imidodiphosphate, and valinomycin plus potassium. The ATP-Pi exchange activity of complex V is specific with respect to ATP; ITP, GTP and UTP are essentially ineffective. Complex V is deficient in cytochromes, but 2–3 times enriched as compared to mitochondria with respect to binding sites for the uncoupler 2-azido-4-nitrophenol. As in mitochondria, this binding is competitively inhibited by other uncouplers. Complexes I, III and IV, which in mitochondria contain the three energy coupling sites, do not bind the above uncoupler.     

ATP synthetase: a mixture of factor A and avidin sensitive ATP-Pi exchange activity

The ATP-P_i exchange activity of highly purified preparations of ‘ATP synthetase’ was inhibited by F₁-antiserum, Pullman inhibitor, azide and also by avidin (See You and Hatefi, 1973). The inhibition produced by the first three was relieved in the presence of ADP, and the avidin sensitivity was lost on pretreatment on the avidin with biotin. It is concluded that the ATP-P_i exchange resulted from the combined action of Factor A and a contaminating avidin-sensitive enzyme. The ADP necessary for the exchange reaction catalyzed by the latter was generated by the ATPase activity of Factor A.     

A time lag in the onset of ATP-Pi exchange catalyzed by purified ATP-synthase (CF0-CF1) proteoliposomes and by chloroplasts

The time course of ATP-Pi exchange which is catalyzed by the isolated chloroplast ATP synthase in phospholipid vesicles was studied. The following observations were made. (i) The onset of 32Pi incorporation into ATP lags behind ATP hydrolysis. The lag lasts for about 2 min at 37 degrees C and is followed by a steady-state rate which is constant for more than 30 min. Under the same experimental conditions, ATP hydrolysis shows an initial burst followed by a constant, slower rate. (ii) The initial lag is independent of Mg-ATP concentration in the range 0.2-5 mM and of the presence of ADP. In contrast, the steady-state rate of ATP-Pi exchange has an apparent Km of 0.3 mM for Mg-ATP and is stimulated by ADP. (iii) Increasing the temperature from 30 to 45 degrees C decreases the lag from 6 min to zero. The steady-state rate of ATP-Pi exchange is affected to a much smaller extent by the temperature in this range. (iv) The lag is insensitive to valinomycin or tetraphenylboron, while the steady-state rate is partially inhibited. Nigericin and protonophores affect both the lag and steady-state rate. (v) ATP-induced membrane potential formation, as followed by oxonol VI, does not correlate with the lag in its kinetics and temperature dependence. ATP-induced pH gradient formation could not be detected in the proteoliposome system. (vi) Light-triggered ATP-Pi exchange in chloroplasts shows essentially the same time course as the proteoliposome system, but the lag lasts for only about 20 s at room temperature and is unaffected by a preexisting proton gradient. These results suggest that the initial lag in ATP-Pi exchange does not reflect the time required for the buildup of a protomotive force (delta - mu H+) nor the time required to produce ADP. It is suggested, therefore, that the lag reflects an internal autocatalytic conformational change in the ATP-synthase complex which is initiated by ATP hydrolysis and which converts the enzyme from an "exclusive ATPase state" to a "reversible ATP-synthase state". This slow transition is not directly coupled to a trans-membrane pH or potential gradient.     

Saturation-transfer studies of ATP-Pi exchange in isolated perfused rat liver

The rate of exchange between inorganic phosphate and ATP was measured in isolated perfused rat livers in the direction of ATP synthesis using ³¹P NMR spectroscopy and the saturation-transfer technique. Measurement of ATP hydrolysis was not observable, even after treatment of rats with 100 μg T₃/day per 100 g body wt. When the perfused livers were treated with iodoacetate in order to inhibit glycolysis, NMR measurable exchange between ATP and P_i was eliminated. It is concluded that the inorganic phosphate → ATP conversion detected by saturation transfer is catalyzed by enzymes of the glycolytic pathway and that the mitochondrial ATPase rate is too slow to contribute to the observed effect.     

The energy level associated with the light-triggered Mg-dependent ATPase in spinach chloroplasts

Light-induced Mg²⁺-ATPase activity of chloroplasts and the pH difference (ΔpH) across the thylakoid membrane maintained by this activity are measured simultaneously under varying conditions of preillumination time and dark decay time. It is shown that with increasing ATPase activity, ΔpH reaches a maximal level which is determined by the degree of uncoupling of the thylakoid membrane.     

ATP-dependent spectral response of oxonol VI in an ATP-Pi exchange complex

(1) Energy transduction in an ATPase complex (complex V) has been studied in two reactions catalyzed by this system, i.e., ATP-dependent spectral shift of oxonol VI, and ATP-P_i exchange activity. (2) Aurovertin alone inhibits 50% of the oxonol shift at 2 μM, and no further inhibition occurs at up to 12 μM. In combination with even weakly effective uncouplers, 4 μM aurovertin fully abolishes the oxonol response. No such effects are observed in the presence of oligomycin and uncouplers. (3) No pH gradient is detectable by quenching of 9-amino-6-chloro-2-methoxyacridine; and nigericin is without effect on the oxonol response. Valinomycin is inhibitory even in the absence of added potassium, due to ammonium ions introduced during the purification steps. Thiocyanate inhibits the dye response by only 10–27%, depending on the preparation. The extent of the oxonol response depends on the ATP / ADP ratio rather than the phosphorylation potential. (4) The dye response in the ATPase complex is 4–7-times less sensitive to bile salts than in submitochondrial particles. The inhibition by cardiolipin can be reversed by the addition of phospholipids. (5) The possibility is discussed that the oxonol response in the ATPase complex reflects, at least in part, a more local, ATP-dependent and energy-related process.     

Anion-dependent changes of energy transfer in chloroplasts III. Evidence for inactivation of light-triggered adenosine triphosphatase in chloroplasts by chloride

Washing chloroplasts with a high concentration of Tris-Cl^- buffercaused Cl^- dependent inhibition of photophosphorylation, light-inducedpH rise and light-triggered Mg²⁺-dependent ATPase activity.The inhibition of these activities was largely prevented bythe presence of 10^–4 M ADP or ATP during Tris washing,especially that of Mg²⁺-ATPase activity. The results were interpretedas suggesting that the inactivation of light-triggered ATPaseactivity in chloroplasts by chloride is one of the causes ofthe uncoupling of chloroplasts with Tris washing. (Received April 30, 1974; )     

Uni-site catalysis in thylakoids. The influence of membrane energization on ATP hydrolysis and ATP-Pi exchange

ATP-hydrolysis was measured with thylakoid membranes during continuous illumination. The concentrations of free and enzyme-bound ATP, ADP and Pi were measured using either cold ATP, [gamma-32P]ATP or [14C]ATP. The concentration of free ATP was constant, free ADP and enzyme-bound ATP were below the detection limit. Nevertheless, [gamma-32P]ATP was bound, hydrolyzed and 32Pi was released. The ADP was not released from the enzyme but cold Pi was bound from the medium, cold ATP was resynthesized and released. A quantitative analysis gave the following rate constants: ATP-binding kATP = 2 . 10(5) M-1 s-1, ADP-release: kADP less than 10(-2)s-1, Pi-release: kPi = 0.1 s-1. These rate constants are considerably smaller than under deenergized conditions. The rate constant for the release of ATP can be estimated to be at least 0.2 s-1 under energized conditions. Obviously, energization of the membrane, i.e. protonation of the enzyme leads mainly to a decrease of the rate of ATP-binding, to an increase of the rate of ATP release and to a decrease of the rate of ADP-release.     

A polarographic study of glutamate synthase activity in isolated chloroplasts

Illuminated pea (Pisum sativum) chloroplasts actively catalyzed (glutamine plus alpha-ketoglutarate)-dependent O(2) evolution (average of 12 preparations 10.6 mumole mg chlorophyll per hour). The reaction was specific for glutamine and alpha-ketoglutarate; concentrations of 0.2 mm alpha-ketoglutarate and 0.6 mm glutamine, respectively, effected half-maximum rates of O(2) evolution. The reaction was inhibited by 3-(3,4-dichlorophenyl)-1-1-dimethylurea and did not occur in the dark. After osmotic shock chloroplasts did not catalyze O(2) evolution. The reaction was inhibited by azaserine and glutamate but not by 10 mm ammonia, 2.5 mm methionine sulfoximine, or 5 mm amino-oxyacetate; addition of amino-oxyacetate together with aspartate inhibited O(2) evolution. Arsenate (3 mm) enhanced O(2) evolution. The highest molar ratio for O(2) evolved per mole of alpha-ketoglutarate supplied was 0.40; the corresponding values for glutamine in the absence and presence of 3 mm arsenate were 0.20 and 0.24, respectively. The (glutamine plus alpha-ketoglutarate)-dependent O(2) evolution is attributed to photosynthetically coupled glutamate synthase activity and the activity is sufficient to account for the assimilation of inorganic nitrogen. The low molar ratio for glutamine is discussed.Chloroplasts also catalyzed (aspartate plus alpha-ketoglutarate)-dependent O(2) evolution but this reaction was inhibited by 5 mm amino-oxyacetate and it was insensitive to azaserine and methionine sulfoximine. This reaction was attributed to transaminase and photosynthetically coupled malate dehydrogenase activities.