The phosphorylation of intramitochondrial AMP: A suggestion for the compartmentation of endogenous Pi pool

1. The mitochondrial level of AMP gradually diminishes during incubation of mitochondria with glutamate but does not with succinate. This decline of AMP, associated with stoichiometric increase in ADP and/or ATP, is accelerated by the addition of electron acceptors or 2,4-dinitrophenol, while arsenite, arsenate and rotenone are inhibitory. These results are in agreement with the view that AMP is phosphorylated to ADP in the inner space of rat liver mitochondria via succinyl-CoA synthetase (succinate: CoA ligase (GDP), EC 6.2.1.4) and GTP:AMP phosphotransferase dependent on the oxidation of 2-oxoglutarate, which is promoted by the transfer of electron from NADH to the respiratory chain.2. Studies of the periodical changes of chemical quantities of adenine nucleotides as well as of their labelling with ³²P_i reveals the following characteristics concerning mitochondrial phosphorylation. (i) In contrast to the mass action ratio of ATP to ADP, the ratio of ADP to AMP is not affected by the intramitochondrial concentration of P_i. (ii) ³²P_i, externally added, is incorporated into ADP much more slowly than into γ-phosphate of ATP. (iii) Conversely, ATP loses its radioactivity from γ-phosphate position more rapidly than [³²P]ADP when ³²P-labelled mitochondria are incubated with non-radioactive P_i.3. In order to elucidate the above characteristic properties of phosphorylation, a hypothetical scheme is proposed which postulates the two separate compartments in the intramitochondrial pool of P_i; one readily communicates with external P_i and is utilized for the phosphorylation of ADP in oxidative phosphorylation, while the other less readily communicates with external P_i and serves as the precursor of ADP via succinyl-CoA synthetase and GTP:AMP phosphotransferase.     

The characterization of myosin–product complexes and of product-release steps during the magnesium ion-dependent adenosine triphosphatase reaction

Evidence is presented that the myosin subfragment-1–ADP complex, generated by the addition of Mg²⁺ and ADP to subfragment 1, is an intermediate within the myosin Mg²⁺-dependent adenosine triphosphatase (ATPase) turnover cycle. The existence of this species as a steady-state intermediate at pH8 and 5°C is demonstrated by fluorescence measurements, but its concentration becomes too low to measure at 21°C. This arises because there is a marked temperature-dependence on the rate of the process controlling ADP dissociation from subfragment 1 (rate=1.4s⁻¹ at 21°C, 0.07s⁻¹ at 5°C). In the ATPase pathway this reaction is in series with a relatively temperature-insensitive process, namely an isomerization of the subfragment-1–product complex (rate=0.055s⁻¹ at 21°C, 0.036s⁻¹ at 5°C). By means of studies on the P_i inhibition of nucleotide-association rates, a myosin subfragment-1–P_i complex was characterized with a dissociation equilibrium constant of 1.5mm. P_i appears to bind more weakly to the myosin subfragment-1–ADP complex. The studies indicate that P_i dissociates from subfragment 1 at a rate greater than 40s⁻¹, and substantiates the existence of a myosin-product isomerization before product release in the elementary processes of the Mg²⁺-dependent ATPase. In this ATPase mechanism Mg²⁺ associates as a complex with ATP and is released as a complex with ADP. In 0.1m-KCl at pH8 1.0mol of H⁺ is released/mol of subfragment 1 concomitant with the myosin-product isomerization or P_i dissociation, and 0.23 mol of H⁺ is released/mol of subfragment when ATP binds to the protein, but 0.23 mol of H⁺ is taken up again from the medium when ADP dissociates. Within experimental sensitivity no H⁺ is released into the medium in the step involving ATP cleavage.     

Does AMP participate in photosynthetic phosphorylation?

Osmotically disrupted chloroplasts catalyze a rapid, light and AMP and ATP dependent ³²P_i incorporation into ATP. Light does not stimulate [¹⁴C] AMP incorporation into ATP in this system. AMP in the presence of P_i inhibits electron flow in a manner analogous to ADP inhibition in the absence of P_i. The inhibition of AMP + P_i is reversed on addition of ADP.     

Inhibition of photophosphorylation by ATP and the role of magnesium in photophosphorylation

ATP and pyrophosphate at high concentration (> 1 mM) inhibited photophosphorylation of isolated spinach chloroplasts in the normal salt medium and did not cause stimulation of electron transport. The inhibition of photophosphorylation by ATP or pyrophosphate was shown to be abolished by the addition of excess MgCl₂, ADP and phosphate. It has been demonstrated that the rates of photophosphorylation in the absence and presence of ATP or pyrophosphate are determined similarly by the concentrations of magnesium-ADP (Mg · ADP^?) and magnesium-phosphate (Mg · P_i) complexes.It is highly probable that Mg · ADP^? and Mg · P_i, but not free ADP and free phosphate, are the active form of the substrates of photophosphorylation. This is in support of the view that ATP inhibits photophosphorylation by decreasing the concentration of Mg²⁺ which is available for the formation of the complex with ADP and phosphate.     

Kinetics of glycogen phosphorylase a from the muscle of the blue crab, Callinectes danae

The kinetics of purified glycogen phosphorylase a from the muscle of the blue crab (Callinectes danae) were studied in the direction of glycogen synthesis, and in the direction of glycogen degradation with P_i or arsenate as substrates. The effects of AMP, UDPG, G-6-P, glucose, and arsenate on the appropriate systems were studied. AMP is an activator of the enzyme. Inhibition by UDPG with respect to P_i changes from noncompetitive to competitive when AMP is added; it changes from noncompetitive to mixed with respect to glycogen when AMP is added. G-6-P is a competitive inhibitor of G-1-P and arsenate. Inhibition by glucose with respect to glycogen changes from noncompetitive to competitive when AMP is added in the direction of glycogen breakdown; it is noncompetitive with respect to P_i. Arsenate is a competitive inhibitor with respect to P_i. The K_m for AMP increases in the presence of UDPG, and decreases with increasing concentrations of P_i or glycogen. We propose a model in which the enzyme bears three interacting sites: an active site, an activator (AMP) site, and an inhibitor (glucose) site. The active site has three subsites: one for P_i, one for glycogen, and one for a glucose moiety which may be part of the substrates or inhibitors.     

Affinity labelling of rat liver acetyl-CoA car☐ylase by a 2′,3′-dialdehyde derivative of ATP

The interaction of rat liver acetyl-CoA car☐ylase with a 2′,3′-dialdehyde derivative of ATP (oATP) has been studied. The degree of the enzyme inactivation has been found to depend on the oATP concentration and the incubation time. ATP was proved to be the only substrate which protected the inactivation. Acetyl-CoA did not effect inactivation, while HCO₃⁻ accelerated the process. K_i values for oATP in the absence and presence of HCO₃⁻ were 0.35 ± 0.04 and 0.5 ± 0.06 mM , and those of the modification constant (k_mod) were 0.11 and 0.26 min⁻¹ respectively. oATP completely inhibited the [¹⁴C]ADP ⇌ ATP exchange and did not effect the [¹⁴C]acetyl-CoA ⇌ malonyl-CoA exchange. Incorporation of ∼1 equivalent of [³H]oATP per acetyl-CoA car☐ylase subunit has been shown. No recovery of the modified enzyme activity has been observed in Tris or β-mercaptoethanol containing buffers, and treatment with NaB³H₄ has not led to³H incorporation. The modification elimination of the ATP triphosphate chain. The results indicated the affinity modification of acetyl-CoA car☐ylase by oATP. It was shown that the reagent apparently interacted selectively with the ɛ-amino group of lysine in the ATP-binding site to form a morpholine-like structure.     

Isotopic probes of catalytic steps of myosin adenosine triphosphatase

A new approach to the direct estimation of the value of the off constant for dissociation of ATP from myosin subfragment 1 (S1) has been developed. From measurements of the extremely slow rate of release of [³²P]-ATP formed from ³²P_i by S1 catalysis and the amount of rapidly formed [³²P]-ATP tightly bound to S1, the value of the off constant is approximately 2.8 × 10⁻⁴ sec⁻¹ at pH 7.4. The concentration dependencies for P_i ⇌ H¹⁸ OH exchange and for ³²P_i incorporation into myosin-bound ATP give direct measurements of the dissociation constant of P_i from S1. Both approaches show that the enzyme has a very low affinity for P_i, with an apparent K_d of > 400 mM. Measurement of the average number of water oxygens incorporated into P_i released from ATP by S1-catalyzed hydrolysis in the presence of Mg²⁺ suggests that the hydrolytic step reverses an average of at least 5.5 times for each ATP cleaved. With the Ca²⁺-activated hydrolysis, less than one oxygen from water appears in each P_i released. This finding is indicative of a possible isotope effect in the attack of water on the terminal phosphoryl group of ATP.     

Requirement of medium ADP for the steady-state hydrolysis of ATP by the proton-translocating Paracoccus denitrificans Fo·F1-ATP synthase

F_o·F₁-ATP synthase in inside-out coupled vesicles derived from Paracoccus denitrificans catalyzes P_i-dependent proton-translocating ATPase reaction if exposed to prior energization that relieves ADP·Mg²⁺-induced inhibition (Zharova, T.V. and Vinogradov, A.D. (2004) J. Biol. Chem.,279, 12319–12324). Here we present evidence that the presence of medium ADP is required for the steady-state energetically self-sustained coupled ATP hydrolysis. The initial rapid ATPase activity is declined to a certain level if the reaction proceeds in the presence of the ADP-consuming, ATP-regenerating system (pyruvate kinase/phosphoenol pyruvate). The rate and extent of the enzyme de-activation are inversely proportional to the steady-state ADP concentration, which is altered by various amounts of pyruvate kinase at constant ATPase level. The half-maximal rate of stationary ATP hydrolysis is reached at an ADP concentration of 8 × 10⁻⁶ M. The kinetic scheme is proposed explaining the requirement of the reaction products (ADP and P_i), the substrates of ATP synthesis, in the medium for proton-translocating ATP hydrolysis by P. denitrificans F_o·F₁-ATP synthase.     

Relationships of inosine triphosphate and bicarbonate effects on F1 ATPase to the binding change mechanism

Two interesting previously reported properties of mitochondrial F₁ ATPase have been confirmed and have been examined by¹⁸O exchange measurements to assess if they are consistent with sequential participation of catalytic sites during ATP hydrolysis. These are the ability of HCO ₃ ^– to increase reaction rate with apparent loss of cooperative interaction between subunits and the ability of ITP to accelerate the hydrolysis of a low concentration of ATP. The effect of HCO ₃ ^– was tested at concentrations of ATP lower than previous measurements. The activation disappeared when ATP was reduced to 0.1 µM. The HCO ₃ ^– activation at higher ATP concentrations did not change the extent of reversal of the cleavage of tightly bound ATP at the catalytic site, as measured by the average number of water oxygens incorporated with each P_i formed when 5 or 10 µM ATP is hydrolyzed. The data are consistent with sequential site participation with HCO ₃ ^– acceleration of ADP departure after a binding change that stops¹⁸O exchange and loosens ADP binding.When ITP concentration was lowered during net ITP hydrolysis by F₁ ATPase an increase in water oxygen incorporation into P_i formed is observed, as noted previously for ATP hydrolysis. The acceleration of the cleavage of a constant low concentration of [-¹⁸O]ATP by concomitant hydrolysis of increasing concentrations of ITP was accompanied by a decrease in water oxygen incorporation with each P_i formed from the ATP. These results add to evidence for the binding change mechanism for F₁ ATPase with sequential participation of catalytic sites.     

Effect of ATP/ADP/phosphate potential on the maximal steady-state uptake of Ca2+ by skeletal sarcoplasmic reticulum

The ability of the Ca²⁺-Mg²⁺ ATPase pump of skeletal SR to produce and maintain a Ca²⁺ gradient was studied as a function of the ATP/ADP/P_i ratio. The internal free Ca²⁺ concentration [Ca²⁺]_i was monitored by changes in fluorescence of CTC. Increasing ADP concentrations in the medium reduce the maximal [Ca²⁺]_i concentration achieved. The inclusion or the omission of 4×10^–4 M P_i or doubling the absolute ATP and ADP concentrations at a constant ATP/ADP ratio does not affect the level obtained. The level depends primarily on the ATP/ADP ratio. The [Ca²⁺] concentration shows a 1.5 power dependence on the ATP/ADP ratio. Further, [Ca²⁺]_i achieved at steady state does not depend on whether the pump had been working in the forward or the reverse direction prior to testing. Analysis shows that the levels of Ca²⁺ achieved are much lower than the levels predicted thermodynamically under the assumption of ideal coupling between Ca²⁺ transport and ATP hydrolysis with a stoichiometry of 2:1. Under this condition the osmotic energy of the [Ca²⁺]_i/[Ca²⁺]_o ratio was shown to be 48% as large as the free energy of hydrolysis of ATP, giving an overall thermodynamic efficiency of 48%. Analysis shows that maximal steady-state uptake is determined by the balance between the rates of uptake by the pump and rates of leak processes (intrinsic or extrinsic to the pump). Comparison with other studies shows that the [Ca²⁺]_i achieved results in trans-inhibition of the pump by tying up the Ca²⁺ translocator in the inwardly oriented phosphorylated form. The absence of an effect of P_i can be taken as evidence that the dissociation of Ca²⁺ from the inwardly oriented translocator on the phosphoylated enzyme must precede the dephosphorylation of the enzyme.     

ATP synthesis catalyzed by a V-ATPase: an alternative pathway for energy conservation operating in plant vacuoles?

The electrochemical H⁺ gradient generated in tonoplast vesicles isolated from maize seeds was found to be able to drive the reversal of the catalytic cycle of both vacuolar H⁺-pumps (Façanha and de Meis, 1998). Here we describe the reversibility of the vacuolar V-type H⁺-ATPase (V-ATPase) even in the absence of the H⁺ gradient in a water-Me₂SO co-solvent mixture, resulting in net synthesis of [γ-³²P]ATP from [³²P]P_i and ADP. The water-Me₂SO (5 to 20 %) media promoted inhibition of both PP_i hydrolysis and synthesis reactions whereas it slightly affected the ATP hydrolysis and clearly stimulated the ATP synthesis, which was unaffected by uncoupling agents (FCCP, Triton X-100 or NH₄⁺). This effect of Me₂SO on the ATP⇔³²P exchange reaction seems to be related to a decrease of the apparent K_m of the V-ATPase for P_i. The results are in accordance to the concept that the energetics of ATP synthesis catalysis depends on the solvation energies interacting in the enzyme microenvironment. A possible physiological significance of this phenomenon for the metabolism of desiccation-tolerant plant cells is discussed.Key words: bind energy, proton pumps, proton gradient, DMSO, corn seeds, V₁V₀-ATPase, membrane bound H⁺-pyrophosphatase     

Synthesis and possible character of a high-energy intermediate in bacterial photophosphorylation

1. In photophosphorylation with chromatophores from Rhodospirillum rubrum, evidence is presented for the synthesis of activated precursors of ATP in the energy-conversion system coupled to photosynthetic electron transport. 2. A significant amount of ATP is synthesized when a reaction mixture containing chromatophores and ADP is illuminated and then incubated with P_i in the dark. ATP is not synthesized to an appreciable extent, either when a reaction mixture containing chromatophores and P_i is illuminated and then incubated with ADP in the dark, or when one containing chromatophores alone is illuminated and then incubated with ADP and P_i in the dark. The amount of ATP thus synthesized is influenced markedly by concentrations of ADP. 3. The chromatophores illuminated with ADP, if allowed to stand in the dark at 30°, gradually lose the ability to form ATP with P_i in the dark. No loss of the ability occurs when the chromatophores illuminated with ADP are allowed to stand in the dark at 13° or in a frozen state. 4. Mg²⁺ is absolutely required for chromatophores to form ATP in the dark after illumination in the presence of ADP, and for the chromatophores to achieve ATP formation with P_i in the dark. 5. Antimycin A, 2-heptyl-4-hydroxyquinoline N-oxide and o-phenanthroline strongly inhibit the light-dependent acquisition of the ability to form ATP with P_i in the dark, but not the consequent ATP formation with P_i in the dark. Arsenate, 2,4-dinitrophenol, quinacrine hydrochloride, quinine hydrochloride and pyrophosphate inhibit the former or the latter, or both. Oligomycin inhibits the former somewhat more than the latter. 6. From these findings it is suggested that a high-energy intermediate is formed in photosynthetic ATP formation, and that its formation is dependent on ADP but not P_i.     

Role of CFTR’s intrinsic adenylate kinase activity in gating of the Cl− channel

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl^?channel in the ATP-binding cassette (ABC) transporter protein family. CFTR features the modular design characteristic of ABC transporters, which includes two membrane-spanning domains forming the channel pore, and two ABC nucleotide-binding domains that interact with ATP and contain the enzymatic activity coupled to normal gating. Like other ABC transporters CFTR is an ATPase (ATP + H₂O → ADP + P_i). Recent work has shown that CFTR also possesses intrinsic adenylate kinase activity (ATP + AMP ? ADP + ADP). This finding raises important questions: How does AMP influence CFTR gating? Why does ADP inhibit CFTR current? Which enzymatic activity gates CFTR in vivo? Are there implications for other ABC transporters? This minireview attempts to shed light on these questions by summarizing recent advances in our understanding of the role of the CFTR adenylate kinase activity for channel gating.     

Energy-linked Adenosine Diphosphate Accumulation by Corn Mitochondria: I. General Characteristics and Effect of Inhibitors

Corn mitochondria show respiration-linked net accumulation of [³H]ADP in the presence of phosphate and magnesium, especially if the formation of ATP is blocked with oligomycin. Inhibition of ADP-ATP exchange by carboxyatractyloside also activates ADP accumulation, and addition of carboxyatractyloside or palmitoyl-coenzyme A to oligomycin-blocked mitochondria produces additional ADP uptake. With carboxyatractyloside the accumulated ADP is phosphorylated to ATP. With oligomycin, only a little ATP is formed. Millimolar concentrations of ADP are required for maximum uptake, and the K_m (3.77 millimolar) for ADP translocation is independent of whether oligomycin or carboxyatractyloside is used. This is not true for ADP concentrations in the 0.05 to 0.25 millimolar range. Accumulated [³H]ADP rapidly exchanges with unlabeled AMP, ADP, or ATP, but not with other diphosphate nucleotides or 2 millimolar substrate anions. [³H]AMP is not accumulated, but [³H]ATP is accumulated to about one-half the extent of [³H]ADP. Tricarboxylate substrates inhibit ADP net uptake, and inhibition by citrate is competitive with K_i = 10 millimolar. The evidence suggests the presence of a pathway, carboxyatractyloside-insensitive and different from the translocase, which operates to maintain adenine nucleotides in the matrix.     

Correlation between ATP synthesis and the decay of the carotenoid band shift after single flash activation of chromatophores from Rhodopseudomonas capsulata

ATP synthesis and the acceleration of the decay of the carotenoid absorption band shift after single flash excitation of Rhodopseudomonas capsulata chromatophores were compared. The two processes behave similarly with respect to: (1) ADP and P_i concentration; (2) inhibition by efrapeptin and venturicidin, and (3) inhibition by valinomycin/K⁺ and by ionophores.Taken together with earlier evidence for the electrochromic nature of the carotenoid band shift the data support the contention that positive charge moves outwards across the chromatophore membrane during ATP synthesis and justify the method for determination of the H⁺/ATP ratio (Petty, K.M. and Jackson, J.B. (1979) FEBS Lett. 97, 367–372).The ability of nucleotide diphosphates in the presence of P_i and Mg²⁺ to give rise to the acceleration of the carotenoid shift decay closely correlates with the rate of phosphorylation of the nucleotides in steady-state light. Nucleotide triphosphates enhance the decay in parallel with their rate of hydrolysis.Adenylyl imidodiphosphate is itself without effect on the decay of the carotenoid shift and it does not prevent the ADP-induced acceleration. The analogue does prevent the ATP effect but only after repeated flashes.     

Steady-state kinetics of ADP-arsenate and ATP-synthesis in Rhodospirillum rubrum chromatophores

(1) Rates of ATP synthesis and ADP-arsenate synthesis catalyzed by Rhodospirillum rubrum chromatophores were determined with the firefly luciferase method and by a coupled enzyme assay involving hexokinase and glucose-6-phosphate dehydrogenase. (2) V_m for ADP-arsenate synthesis was about 2-times lower than V_m for ATP-synthesis. With saturating [ADP], K(As_i) was about 20% higher than K(P_i). With saturating [anion], K(ADP) was during arsenylation about 20% lower than during phosphorylation. (3) Plots of $\text{1}\text{v}$ vs. $\text{1}\text{[}\text{substrate}\text{]}$ were non-linear at low concentrations of the fixed substrate. The non-linearity was such as to suggest a positive cooperativity between sites binding the variable substrate, resulting in an increased $\text{V}{}_{\mathrm{<mtext>m</mtext>}}\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ ratio. High concentrations of the fixed substrate cause a similar increase in $\text{V}{}_{\mathrm{<mtext>m</mtext>}}\text{K}{}_{\mathrm{<mtext>m</mtext>}}$, but abolish the cooperativity of the sites binding the variable substrate. (4) Low concentrations of inorganic arsenate (As_i) stimulate ATP synthesis supported by low concentrations of P_i and ADP about 2-fold. (5) At high ADP concentrations, the apparent K_i of As_i for inhibition of ATP-synthesis was 2–3-times higher than the apparent K_m of As_i for arsenylation; the apparent K_i of P_i for inhibition of ADP-arsenate synthesis was about 40% lower than the apparent K_m of P_i for ATP synthesis. (6) The results are discussed in terms of a model in which P_i and As_i compete for binding to a catalytic as well as an allosteric site. The interaction between these sites is modulated by the ADP concentration. At high ADP concentrations, interaction between these sites occurs only when they are occupied with different species of anion.     

Exchange between inorganic phosphate and adenosine triphosphate in (Na+,K+)-ATPase

(Na⁺,K⁺)-ATPase is able to catalyze a continuous ATP?P_i exchange in the presence of Na⁺ and in the absence of a transmembrane ionic gradient. At pH 7.6 the Na⁺ concentration required for half-maximal activity is 85 mM and at pH 5.1 it is 340 mM. In the presence of optimal Na⁺ concentration, the rate of exchange is maximal at pH 6.0 and varies with ADP and P_i concentration in the assay medium. ATP?P_i exchange is inhibited by K⁺ and by ouabain.     

Properties of Bound Inorganic Phosphate on Bovine Mitochondrial F1F0-ATP Synthase

Beef-heart mitochondrial F₁F₀-ATP synthase contained six molecules of bound inorganic phosphate (P_i). This phosphate exchanged completely with exogenous ³²P_i when the enzyme was exposed to 30% (v/v) dimethyl sulfoxide (DMSO) and then returned to a DMSO-free buffer (Beharry and Bragg 2001). Only two molecules were replaced by ³²P_i when the enzyme was not pretreated with DMSO. These two molecules of ³²P_i were not displaced from the enzyme by the treatment with 1 mM ATP. Similarly, two molecules of bound ³²P_i remained on the DMSO-pretreated enzyme following addition of ATP, that is, four molecules of ³²P_i were displaced by ATP. The ATP-resistant ³²P_i was removed from the enzyme by pyrophosphate. It is proposed that these molecules of ³²P_i are bound at an unfilled adenine nucleotide-binding noncatalytic site on the enzyme. Brief exposure of the enzyme loaded with two molecules of ³²P_i to DMSO, followed by removal of the DMSO, resulted in the loss of the bound ³²P_i and in the formation of two molecules of bound ATP from exogenous ADP. A third catalytic site on the enzyme was occupied by ATP, which could undergo a P_i ATP exchange reaction with bound P_i The presence of two catalytic sites containing bound P_i is consistent with the X-ray crystallographic structure of F₁ (Bianchet, et al., 1998). Thus, five of the six molecules of bound P_i were accounted for. Three molecules of bound P_i were at catalytic sites and participated in ATP synthesis or P_i ATP exchange. Two other molecules of bound P_i were present at a noncatalytic adenine nucleotide-binding site. The location and role of the remaining molecule of bound P_i remains to be established. We were unable to demonstrate, using chemical modification of sulfhydryl groups by iodoacetic acid, any gross difference in the conformation of F₁F₀ in DMSO-containing compared with DMSO-free buffers.     

The extent of the stimulated electrical potential decay under phosphorylating conditions and the H+ATP ratio in Rhodopseudomonas sphaeroides chromatophores following short flash excitation

1. In chromatophores from Rps. sphaeroides, the stimulation by ADP and P_i of the electric potential decay indicated by the carotenoid shift is greater than the stimulation of the decay of the pH change indicated by the colour change of added cresol red under similar conditions. This difference is attributed to H⁺ consumption during the synthesis of ATP. The ratio of H⁺ translocated across the membrane to ATP synthesized was estimated to be approximately 1.7 $\text{H}{}^{\mathrm{+}}\text{ATP}$.2. The stimulation of the electrical potential decay by ADP and P_i was found to be a constant fraction (10%) of the total decay when the flash intensity was varied. No ‘critical’ or ‘threshold’ potential was observed.3. The stimulated electrical potential decay after a second flash, given within a few seconds of the first, was related to the amplitude of the electrical potential produced by the second flash (10%) but neither to the dark time between the flashes, nor to the total extent of the electrical potential above the dark level. These results are consistent with two hypotheses (a) the chromatophores are a mixed population of vesicles, only a small fraction (10%) of which possess an active ATP synthesizing system (b) the activity of the ATP synthesizing system, though driven by a proton motive force, is controlled by electron transport processess. If alternative (a) is correct then the overall single turnover flash yield of 1 ATP per 1470 bacteriochlorophyll measured in (1) would mean that the yield of the active vesicles is approximately 10 ATP per 1470 bacteriochlorophyll or 30 ATP per vesicle.4. The stimulation of the electrical potential decay by ADP and P_i is approximately 40% less in antimycin-treated chromatophores. It is shown that this is probably a consequence of antimycin-inhibited H⁺-release on the inside of the chromatophore vesicles following a flash.     

The thermodynamic efficiency of the Ca2+-Mg2+-ATPase is one hundred percent

The thermodynamic efficiency of the Ca²⁺-Mg²⁺-ATPase of skeletal sarcoplasmic reticulum has been evaluated by comparing the Ca²⁺ gradient established with the ATP/(ADP*P_i) ratio. The evaluation was made at an external Ca²⁺ level (4.7 × 10^–8 M) which is below theK _m value of 7 × 10^–8 M. The Mg-ATP and phosphate concentrations were held constant (0.1 mM) and the ADP concentration was varied. Maximal uptake to an internal free Ca²⁺ concentration of 17 mM was observed at infinite ATP/(ADP*P_i) ratio (absence of ADP). This corresponds to a [Ca²⁺]_i/[Ca²⁺]₀ gradient of 3.6 × 10⁵. A Ca²⁺ gradient one-half as large was observed at an ATP/(ADP*P_i) ratio of 3.5 × 10³ M^–1. The square of the Ca²⁺ gradient is shown to be proportional to the ATP/(ADP*P_i) ratio, for finite values of the latter. The proportionality constant is identical to the equilibrium constant for hydrolysis of ATP (9.02 × 10⁶ M) under these conditions (0.1 mM Mg²⁺, 30°C). The intrinsic thermodynamic efficiency of the pump is shown to be 100%, with a maximal uncertainty of 3%. The efficiency is lower under less optimal conditions, when the pump is inhibited and passive leak processes compete.Dedicated to Prof. Philip George, University of Pennsylvania, whose instruction, research, and example made this contribution possible.