The pathway of inorganic-phosphate efflux from isolated liver mitochondria during adenosine triphosphate hydrolysis

1. The distribution of P_i between mitochondria and suspending medium during uncoupler-stimulated hydrolysis of ATP by rat liver mitochondria [Tyler (1969) Biochem. J. 111, 665–678] has been reinvestigated, by using either mersalyl or N-ethylmaleimide as inhibitors of P_i transport and either buffered sucrose/EDTA or LiCl/EGTA solutions as suspending medium. More than 75% of the total P_i liberated was retained in mitochondria treated with either inhibitor at all ATP concentrations tested (0.2–2.5mm). With low ATP concentrations and mersalyl-treated mitochondria incubated in sucrose/EDTA, virtually all the P_i liberated was retained in the mitochondria. 2. Larger amounts of P_i appeared in the suspending medium during ATPase activity, despite the presence of N-ethylmaleimide, when LiCl/EGTA was used as suspending medium compared with sucrose/EDTA. Two sources of this P_i were identified: (a) a slow efflux of P_i from mitochondria to suspending medium despite the presence of N-ethylmaleimide; (b) a slow ATPase activity insensitive to carboxyatractyloside, which was stimulated by added Mg²⁺, partially inhibited by oligomycin or efrapeptin and strongly inhibited by EDTA. 3. It is concluded that liver mitochondria preparations contain two distinct forms of ATPase activity. The major activity is associated with coupled mitochondria of controlled permeability to adenine nucleotides and P_i and is stimulated strongly by uncoupling agents. The minor activity is associated with mitochondria freely permeable to adenine nucleotides and P_i, is unaffected by uncoupling agents and is activated by endogenous or added Mg²⁺. 4. When mitochondria treated with mersalyl were incubated in buffered sucrose solution, almost all the P_i liberated was recovered in the suspending medium, unless inhibitors of P_i-induced large-amplitude swelling such as EDTA, EGTA, antimycin, rotenone, nupercaine or Mg²⁺ were added. Thus the loss of the specific permeability properties of the mitochondrial inner membrane associated with large-amplitude swelling also influences the extent of P_i retention during ATPase activity. 5. The results confirm the previous conclusion (Tyler, 1969) that the P_i transporter provides the sole pathway for P_i efflux during uncoupler-stimulated ATP hydrolysis by mitochondria. It is concluded that more recent hypotheses concerning the influence of Mg²⁺ on mersalyl inhibition of the P_i transporter [Siliprandi, Toninello, Zoccaroto & Bindoli (1975) FEBS Lett. 51, 15–17] and a postulated role of the adenine nucleotide exchange carrier in P_i efflux [Reynafarje & Lehninger (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4788–4792] are erroneous and should be discarded.     

The Stoichiometry between CO(2) and H Fluxes Involved in the Transport of Inorganic Carbon in Cyanobacteria

The pH of the medium during CO₂ uptake into the intracellular inorganic carbon (C_i) pool of a high CO₂-requiring mutant (E₁) and wild type of Anacystis nidulans R2 was measured. Experiments were performed under conditions where photosynthetic CO₂ fixation is inhibited. There was an acidification of the medium during CO₂ uptake in the light and an alkalization during CO₂ efflux after darkening. A one to one stoichiometry existed between the amounts of H⁺ appearing in the medium and CO₂ taken up into the intracellular C_i pool, regardless of the carbon species transported. The results indicate that (a) CO₂ is taken up simultaneously with an efflux of equimolar H⁺, probably produced as a result of CO₂ hydration during transport and (b) HCO₃⁻ produced by hydration of CO₂ in the medium was transported into the cells without accompanying net flux of H⁺ or OH⁻. The influx and efflux of C_i during C_i transport produced nonequilibrium between CO₂ and HCO₃⁻ in the medium, with the concentration of HCO₃⁻ being higher than that expected under equilibrium conditions. The nonequilibrium was present even under the conditions where the influx of C_i is compensated by its efflux. The direction of this nonequilibrium suggested that efflux of HCO₃⁻ occurs during uptake of C_i.     

Transport of NAD in Percoll-Purified Potato Tuber Mitochondria: Inhibition of NAD Influx and Efflux by N-4-Azido-2-nitrophenyl-4-aminobutyryl-3'-NAD

A mechanism by which intact potato (Solanum tuberosum) mitochondria may regulate the matrix NAD content was studied in vitro. If mitochondria were incubated with NAD⁺ at 25°C in 0.3 molar mannitol, 10 millimolar phosphate buffer (pH 7.4), 5 millimolar MgCl₂, and 5 millimolar α-ketoglutarate, the NAD pool size increased with time. In the presence of uncouplers, net uptake was not only inhibited, but NAD⁺ efflux was observed instead. Furthermore, the rate of NAD⁺ accumulation in the matrix space was strongly inhibited by the analog N-4-azido-2-nitrophenyl-4-aminobutyryl-3′-NAD⁺. When suspended in a medium that avoided rupture of the outer membrane, intact purified mitochondria progressively lost their NAD⁺ content. This led to a slow decrease of NAD⁺-linked substrates oxidation by isolated mitochondria The rate of NAD⁺ efflux from the matrix space was strongly temperature dependent and was inhibited by the analog inhibitor of NAD⁺ transport indicating that a carrier was required for net flux in either direction. It is proposed that uptake and efflux operate to regulate the total matrix NAD pool size.     

Nature of the light-induced h efflux and na uptake in cyanobacteria

We investigated the nature of the light-induced, sodium-dependent acidification of the medium and the uptake of sodium by Synechococcus. The rate of acidification (net H⁺ efflux) was strongly and specifically stimulated by sodium. The rates of acidification and sodium uptake were strongly affected by the pH of the medium; the optimal pH for both processes being in the alkaline pH range. Net proton efflux was severely inhibited by inhibitors of adenosine triphosphatase activity, energy transfer, and photosynthetic electron transport, but was not affected by the presence of inorganic carbon (C_i). Light and C_i stimulated the uptake of sodium, but the stimulation by C_i was observed only when C_i was present at the time sodium was provided. Amiloride, a potent inhibitor of Na⁺/H⁺ antiport and Na⁺ channels, stimulated the rate of acidification but inhibited the rate of sodium uptake. It is suggested that acidification might stem from the activity of a light dependent proton excreting adenosine triphosphatase, while sodium transport seems to be mediated by both Na⁺/H⁺ antiport and Na⁺ uniport.     

Energy-linked activities in reconstituted yeast adenosine triphosphatase proteoliposome. Adenosine triphosphate formation coupled with electron flow between ascorbate and ferricyanide.

(1) Conditions are described wherein the yeast oligomycin-sensitive adenosine triphosphatase (ATPase) complex can be reconstituted together with phospholipids to yield extremely high rates of ATP-³²P_j exchange. The vesicles so formed exhibit proton uptake upon addition of Mg²⁺-ATP and a relatively slow decay of the proton gradient. (2) The stimulation of ATP-³²P_i exchange by valinomycin + K⁺ reported previously (Ryrie, I. J. (1975) Arch. Biochem. Biophys. 168, 704–711) is apparently not simply due to a diffusion potential. The findings suggest that an electroimpelled, valinomycin-dependent migration of K⁺ may occur together with the electrogenic movements of protons during ATP hydrolysis and synthesis to establish optimal energized conditions for ATP-³²P_i exchange. (3) An artificial oxidative phosphorylation system in the reconstituted vesicles is described: [³²P]ATP formation from ADP and ³²P_i is shown to be linked with electron flow between external ascorbate and internal ferricyanide where a permeable proton carrier, such as phenazine methosulfate, is used to establish a proton gradient. That the yeast ATPase is capable of net ATP synthesis has also been demonstrated in a light-dependent reaction using ATPase proteoliposomes reconstituted together with bacteriorhodopsin.     

Phosphate transport and its relationship to cation movements in Ehrlich Lettré ascites tumor cells.

In view of the importance of P_i in the control of cell metabolism, it was of interest to study the mechanism and regulation of P_i uptake by ascites tumor cells. For this purpose, the incorporation of ³²P_i into Ehrlich Lettré cells was compared when competitive anions and inhibitors which alter cation movements were present. Anions such as sulfanilate (35 mm) and succinate (30 mm) decrease ³²P_i uptake by ca. 35%, suggesting that transport is mediated by a protein similar to the 100,000 M_r anion carrier isolated from erythrocyte membranes. Furosemide, a diuretic which bears a structural analogy to sulfanilate inhibitors of anion transport, also decreases ³²P_i incorporation at concentrations as low as 2 × 10^?5m. This inhibitor blocks cation exchange in ascites tumor cells, and from the present data, it is suggested that a possible function of the furosemidesensitive cation exchange protein is to facilitate anion transport. Ouabain, known to inhibit (Na⁺ + K⁺)-ATPase and its dephosphorylation, stimulates the rate of incorporation of ³²P_i into cells and also raises the net inorganic phosphate level. The stimulation of ³²P_i incorporation is decreased by sulfanilate or succinate. In contrast to the effects of ouabain, addition of 10 mm K⁺, which is known to stimulate (Na⁺ + K⁺)-ATPase and its dephosphorylation, decreases ³²P_i incorporation. These observations suggest that anion transport and energy-dependent Na⁺ and K⁺ movements may be closely coupled to the intact cell.     

PHOSPHORUS UPTAKE KINETICS OF SPIROGYRA FLUVIATILIS (CHAROPHYCEAE) IN FLOWING WATER1,2

The inorganic phosphorus (P_i) uptake kinetics of Spirogyra fluviatilis Hilse were examined as a function of phosphorus cell quota (Q^P) and flow velocity in a laboratory stream apparatus. Short-term uptake and the acclimation of the uptake mechanism to flow were measured by the disappearance of P_i pulses in a recirculating flow cell. Short-term P_i uptake was biphasic. When the alga was P-deficient, Phase 1 and 2 half-saturation constants and maximum uptake rates were 11.0 and 47.2 μg P·L^?1 and 473 and 803 μg P·g dry wt^?1 h^?1, respectively. Flowing water altered short-term uptake when the alga was P-deficient, but not when it was P-replete. When Q^P was less than 0.21%, increases in flow velocity from 3 to 15 cm·s^?1 enhanced uptake with maximum uptake for any P_i pulse at 12 and 15 cm·s^?1. At 22 and 30 cm·s^?1, uptake was reduced by 12% or more relative to the maxima. If, however, the alga was cultivated at 22 and 30 cm·s^?1 and short-term P_i uptake was measured at 12 cm·s^?1, uptake was on average 33% greater than when the alga was cultivated at the latter velocity. Apparently, the alga could adjust short-term uptake to compensate for the suboptimal conditions of the faster velocities. Long-term P_i uptake and net phosphorus efflux were estimated by a non-steady state application of the Droop equation. Long-term uptake of very low P_i concentrations was not reduced by fast flowing water. Instead, uptake increased proportionately with flow velocity. Maximum phosphorus efflux from S. fluviatilis was 3% of cellular P per hour and occurred when Q^P was greater than 0.2%. At lower Q^P, the hourly efflux rate was typically less than 1%. Flowing water did not greatly enhance efflux, although when P_i was undetectable, efflux did tend to increase slightly with velocity. The data show that the effects of flowing water on P_i uptake were varied and not always beneficial. If the effects of flowing water on nutrient acquisition by other lotic algae are similarly varied and complex, flow may be an important determinant of nutrient partitioning among benthic algae in streams.     

Orthophosphate flux across the membrane of Tetrahymena pyriformis W

Tetrahymena pyriformis W suspended in a buffered glucose solution accumulated orthophosphate [³²P] from the external solution at a measurable rate. The uptake of orthophosphate by the organisms was linear with respect to time when corrections were made to account for a constant efflux observed during the one hour time course of the experiments. Such corrections were based on the measured lowering of the relative specific activity of the suspension medium and led to the derivation of the expressions for the influx and efflux coefficients. The derived expressions for the coefficients are based solely upon the isotopic measurements and by means of these equations it is possible to describe the observed net inward flux of orthophosphate in quantitative terms. The dependence of the uptake of orthophosphate on the external concentration of orthophosphate followed Michaelis-Menten kinetics, and the temperature coefficient (18–28°) of 1.7 for the process fell into the range normally associated with a chemical reaction. The kinetic pattern, per se, does not distinguish a membrane transport mechanism from metabolic incorporation of P_i. Deviations from the expected pattern of uptake were observed at low temperatures.     

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 transport of oxaloacetate in isolated mitochondria

The mechanism of mitochondrial oxaloacetate transport has been investigated by measuring the rate and the extent of exchange reactions between intramitochondrial anions and added oxaloacetate. The exchange between oxaloacetate and intramitochondrial oxoglutarate is insensitive to mersalyl at a concentration which completely inhibits the dicarboxylate carrier. Oxaloacetate causes efflux of intramitochondrial P_i, malonate, and malate. Mersalyl inhibits completely the oxaloacetate/P_i exchange, but only partially the oxaloacetate/malonate and the oxaloacetate/malate exchanges. The inhibition of the last two reactions decreases on increasing the time of incubation. Butylmalonate inhibits more than phenylsuccinate the exchange oxaloacetate_out/³²P_iin, whereas phenylsuccinate is a more effective inhibitor than butylmalonate of the oxaloacetate_out/[¹⁴C]oxoglutarate_in exchange. The apparent K_m values ranged from 0.6 to 1.2 mm for the oxaloacetate/oxoglutarate exchange and from 6.5 to 10 mm for the oxaloacetate/P_i exchange. The inhibition of oxoglutarate uptake by oxaloacetate is competitive. Oxaloacetate inhibits the malonate/P_i exchange competitively and it is a noncompetitive inhibitor of the $\text{P}{}_{\mathrm{i}}\text{P}{}_{\mathrm{i}}$ exchange. It is concluded that oxaloacetate may be transported across the mitochondrial membrane by the oxoglutarate carrier and, much less effectively, by the dicarboxylate carrier. The implications of these findings are discussed.     

An Analysis of the Leakages of Sodium Ions into and Potassium Ions out of Striated Muscle Cells

Net sodium influx under K-free conditions was independent of the intracellular sodium ion concentration, [Na]_i, and was increased by ouabain. Unidirectional sodium influx was the sum of a component independent of [Na]_i and a component that increased linearly with increasing [Na]_i. Net influx of sodium ions in K-free solutions varied with the external sodium ion concentration, [Na]_o, and a steady-state balance of the sodium ion fluxes occurred at [Na]_o = 40 mM. When solutions were K-free and contained 10^-4 M ouabain, net sodium influx varied linearly with [Na]_o and a steady state for the intracellular sodium was observed at [Na]_o = 13 mM. The steady state observed in the presence of ouabain was the result of a pump-leak balance as the external sodium ion concentration with which the muscle sodium would be in equilibrium, under these conditions, was 0.11 mM. The rate constant for total potassium loss to K-free Ringer solution was independent of [Na]_i but dependent on [Na]_o. Replacing external NaCl with MgCl₂ brought about reductions in net potassium efflux. Ouabain was without effect on net potassium efflux in K-free Ringer solution with [Na]_o = 120 mM, but increased potassium efflux in a medium with NaCl replaced by MgCl₂. When muscles were enriched with sodium ions, potassium efflux into K-free, Mg⁺⁺-substituted Ringer solution fell to around 0.1 pmol/cm²·s and was increased 14-fold by addition of ouabain.     

Phosphate transport system inParacoccus denitrificans

P_i uptake in cells or spheroplasts ofParacoccus denitrificans is biphasic; only the first rapid phase represents net P_i transport. The second phase is limited by the rate of P_i utilization inside the cell, i.e., mainly by its esterification, and as such it was inhibited by DCCD. The P_i/dicarboxylate antiporter does not seem to be operative, and its inhibitorn-butylmalonate did not exert specific inhibition. P_i transport is inhibited by SH reagents; the most potent inhibitor is PCMB, and mersalyl is much less effective. However, neither inhibitor affects efflux of accumulated P_i. The gradient of potassium ions may be involved in the P_i uptake, which is lowered in the presence of valinomycin. FCCP alone does not release accumulated P_i from spheroplasts unless they are preincubated with SCN^?. The results indicate that P_i enters the cell by symport with protons.     

A Biophysical Model of the Mitochondrial ATP-Mg/Pi Carrier

Mitochondrial adenine nucleotide (AdN) content is regulated through the Ca²⁺-activated, electroneutral ATP-Mg/P_i carrier (APC). The APC is a protein in the mitochondrial carrier super family that localizes to the inner mitochondrial membrane (IMM). It is known to modulate a number of processes that depend on mitochondrial AdN content, such as gluconeogenesis, protein synthesis, and citrulline synthesis. Despite this critical role, a kinetic model of the underlying mechanism has not been developed and validated. Here, a biophysical model of the APC is developed that is thermodynamically balanced and accurately reproduces a number of reported data sets from isolated rat liver and rat kidney mitochondria. The model is based on an ordered bi-bi mechanism for heteroexchange of ATP and P_i and includes homoexchanges of ATP and P_i to explain both the initial rate and time course data on ATP and P_i transport via the APC. The model invokes seven kinetic parameters regarding the APC mechanism and three parameters related to matrix pH regulation by external P_i. These parameters are estimated based on 19 independent data curves; the estimated parameters are validated using six additional data curves. The model takes into account the effects of pH, Mg²⁺, and Ca²⁺ on ATP and P_i transport via the APC, and supports the conclusion that the pH gradient across the IMM serves as the primary driving force for AdN uptake or efflux. Moreover, computer simulations demonstrate that extramatrix Ca²⁺ modulates the turnover rate of the APC and not the binding affinity of ATP, as previously suggested.     

Energy-dependent uptake of ochratoxin A by mitochondria.

The interaction of ochratoxin A, a mycotoxin produced by Aspergillus ochraceus, with isolated rat liver mitochondria and plasma membranes has been studied. Cell membranes bind [¹⁴C]ochratoxin A poorly and do not show saturation in the concentration range examined. The uptake of the toxin by mitochondria is saturable, with an apparent K_m at 0 °C of 30 nmol/mg of protein. Sonication or freeze-thawing reduces the extent of incorporation by 88%. Ochratoxin A uptake is energy dependent, resulting in a depletion of intramitochondrial ATP. Uncouplers such as m-chlorocarbonylcyanide phenylhydrazone or the respiratory inhibitors rotenone and antimycin A inhibit uptake 60–85%, while ATP reverses the antimycin and rotenone inhibition. Phosphate transport is sensitive to inhibition by the toxin, as measured by Ca²⁺ plus P_istimulated respiration and [³²P]P_i incorporation. In turn, phosphate inhibits nearly completely [¹⁴C]ochratoxin A uptake at 22 °C and causes a concomitant mitochondrial swelling yet is not incorporated into the matrix space. Thus, the saturable uptake of ochratoxin A is accompanied by a decrease in the energy state and inhibition of P_i transport, which results in deteriorative changes of the mitochondria, as evidenced by large-amplitude swelling.     

Na+-transporting ATPase in the plasma membrane of halotolerant microalga Dunaliella maritima operates as a Na+ uniporter

A fraction of inside-out membrane vesicles enriched in plasma membranes (PM) was isolated from Dunaliella maritima cells. Attempts were made to reveal ATP-driven Na⁺-dependent H⁺ efflux from the PM vesicles to external medium, as detected by alkalization of the vesicle lumen. In parallel experiments, ATP-dependent Na⁺ uptake and electric potential generation in PM vesicles were investigated. The alkalization of the vesicle lumen was monitored with an impermeant pH-sensitive optical probe pyranine (8-hydroxy-1,3,6-pyrenetrisulfonic acid), which was loaded into vesicles during the isolation procedure. Sodium uptake was measured with ²²Na⁺ radioactive label. The generation of electric potential in PM vesicles (positive inside) was recorded with a voltage-sensitive probe oxonol VI. Appreciable Na⁺-and ATP-dependent alkalization of vesicle lumen was only observed in the presence of a protonophore CCCP (carbonyl cyanide-chlorophenylhydrazone). In parallel experiments, CCCP accelerated the ATP-dependent ²²Na⁺ uptake and abolished the electric potential generated by the Na⁺-ATPase at the vesicle membrane. A permeant anion NO^? ₃ accelerated ATP-dependent ²²Na⁺ uptake and promoted dissipation of the electric potential like CCCP did. At the same time, NO^? ₃ inhibited the ATP-and Na⁺-dependent alkalization of the vesicle lumen. The results clearly show that the ATP-and Na⁺-dependent H⁺ efflux from PM vesicles of D. maritima is driven by the electric potential generated at the vesicle membrane by the Na⁺-ATPase. Hence, the Na⁺-transporting ATPase of D. maritima carries only one ion species, i.e., Na⁺. Proton is not involved as a counter-ion in the catalytic cycle of this enzyme.     

Effects on Mitochondrial K Flux of pH,K Concentration,and N-Ethyl Maleimide

Based on published evidence that cation transport in mitochondria is not significantly dependent on a membrane potential, it is suggested that the process of mitochondrial cation transport may be nonelectrogenic. These experiments focused on the possibility that K⁺ flux into rat liver mitochondria may be directly coupled, via an energy-linked carrier mechanism, to OH^? influx or H⁺ efflux. The dependence of the unidirectional K⁺ influx on the external K⁺ concentration indicates involvement of a saturable mechanism. Increasing the external pH from 7.0 to 8.0 increases the apparent V_max of the K⁺ influx without significantly altering the apparent K_m for K⁺. The pH dependence is greater in the presence of N-ethyl maleimide, a known inhibitor of the mitochondrial P_i/OH^? exchange mechanism. N-Ethyl maleimide decreases the apparent V_max at pH 7.0 and increases it at pH 8.0. Evidence indicates that both N-ethyl maleimide and a high external P_i concentration may stimulate the K⁺ influx at alkaline external pH (8.0) by preventing net exchanges between endogenous P_i and external OH^?. An apparent first-order dependence of the K⁺ influx on the external OH^? concentration is observed in the presence of N-ethyl maleimide. These results are consistent with a possible role of external OH^? as a cosubstrate of the K⁺ transport mechanism.     

Effects of Intracellular Adenosine-5''-diphosphate and Orthophosphate on the Sensitivity of Sodium Efflux from Squid Axon to External Sodium and Potassium

A study was made of sodium efflux from squid giant axon, and its sensitivity to external K and Na. When sodium efflux from untreated axons was strongly stimulated by K_o, Na_o was inhibitory; when dependence on K_o was low, Na_o had a stimulatory effect. Incipient CN poisoning or apyrase injection, which produces high intracellular levels of ADP¹ and P_i, rendered sodium efflux less dependent on external K and more dependent on external Na. Injection of ADP, AMP, arginine, or creatine + creatine phosphokinase, all of which raise ADP levels without raising P_i levels, had the same effect as incipient CN poisoning. P_i injection had no effect on the K sensitivity of sodium efflux. Axons depleted of arginine and phosphoarginine by injection of arginase still lost their K sensitivity when the ATP:ADP ratio was lowered and regained it partially when the ratio was raised. Rough calculations show that sodium efflux is maximally K_o-dependent when the ATP:ADP ratio is about 10:1, becomes insensitive to K_o when the ratio is about 1:2, and is inhibited by K_o when the ratio is about 1:10. Deoxy-ATP mimicked ADP when injected into intact axons. Excess Mg, as well as P_i, inhibited both strophanthidin-sensitive and strophanthidin-insensitive sodium efflux. An outline is presented for a model which might explain the effects of ADP, P_i and deoxy-ATP.     

Inhibition of anion transport across the mitochondrial membrane by amytal

It has been found that amytal competitively inhibits succinate (+ rotenone) oxidation by intact uncoupled mitochondria. Similar results were obtained in metabolic state 3, the K_i value being 0.45 mM. Amytal did not effect succinate oxidation by broken mitochondria and submitochondrial particles (at a concentration which inhibited succinate oxidation by intact mitochondria). Amytal inhibited the swelling of mitochondria suspended in ammonium succinate or ammonium malate but was without effect on the swelling of mitochondria in ammonium phosphate and potassium phosphate in the presence of valinomycin+carbonylcyanide p-trifluoromethoxyphenylhydrazone.Using [¹⁴C] succinate and [¹⁴C] citrate it has been shown that amytal inhibited the succinate/succinate, succinate/P_i, succinate/malate, and citrate/citrate and citrate/malate exchanges. Amytal inhibited P_i transport across mitochondrial membrane only if preincubated with mitochondria. Other barbiturates: phenobarbital, dial, veronal were found to inhibit [¹⁴C]succinate/anion (P_i, succinate, malonate, malate) exchange reactions in a manner similar to amytal. It is concluded that barbiturates non-specifically inhibit the dicarboxylate carrier system, tricarboxylate carrier and P_i translocator. It is postulated that the inhibition of succinate oxidation by barbiturates is caused mainly by the inhibition of succinate and P_i translocation across the mitochondrial membrane.     

A 31P-NMR study of the cross-membrane pH gradient induced by ATP hydrolysis in mitochondria

³¹P-NMR has been used to study the increase of ΔpH in mitochondria by externally added ATP. Freshly prepared mitochondria was treated with N-ethylmaleimide to inhibit the exchange between internal and external P_i. Upon addition of ATP, phosphocreatine (30 mM) and creatine kinase to a NMR sample of mitochondria suspension (approx. 120 mg protein/ml) at 0°C, an increase of ΔpH by approx. 0.5 pH unit was observed. However the increased ΔpH could not be maintained, but slowly decayed along with the increase of external ADP/ATP ratio. Further addition of valinomycin to the suspension induced a larger ΔpH (approx. 1) which was maintained by the increased rate of internal ATP hydrolysis as seen in the growth of the internal P_i peak intensity in NMR spectra and the concomitant decrease of the external phosphocreatine peak. The external P_i and ATP peaks stayed virtually constant. When carboxyatractyloside was added to inhibit the ATP/ADP translocase, the internal P_i increase was stopped and the ΔpH decayed. These observations in conjunction with those made earlier in respiring mitochondria clearly show the reversible nature of the ATPase function in which the internal ATP hydrolysis is associated with outward pumping of protons.     

The effects of cyclic nucleotides and some related agents on 32Pi labeling of renal polyphosphoinositides in vitro.

When rabbit kidney cortex slices were incubated in the presence of ³²P_i and dibutyrylcyclic AMP (dbcAMP)⁴ a significant decrease in the labeling of phosphatidyl inositol phosphate (DPI) but not phosphatidyl inositol bisphosphate (TPI) was observed. In the presence of 0.3 mm caffeine cyclic AMP (cAMP) produced a similar effect. Caffeine potentiated the inhibitory effect of dbcAMP. At high concentrations (3 mm) caffeine alone decreased the ³²P_i labeling of both DPI and TPI. These decreases in ³²P_i labeling were not mediated by decreases in the labeling of intracellular P_i or ATP as measured by 10-min acid-labile nucleotide phosphate (10′-ALNP). Addition of cyclic GMP (cGMP) to the incubation medium decreased the labeling of DPI and to a lesser extent that of TPI also. Addition of parathyroid hormone (PTH) to the incubation medium (in the absence of exogenous cyclic nucleotides) also decreased the ³²P_i labeling of DPI but not that of TPI. In contrast to the effects of cAMP, dbcAMP, cGMP, PTH, and caffeine, the addition of insulin to the incubation medium resulted in increased ³²P_i labeling of DPI with no effect on TPI labeling. DPI isolated from kidney cortex slices prelabeled with ³²P_i and subsequently incubated with cAMP or dbcAMP contained less label than DPI isolated from slices similarly prelabeled but subsequently incubated in the absence of either cAMP or dbcAMP. These data suggest an increased rate of DPI breakdown in the presence of elevated cAMP or dbcAMP concentrations. This hypothesis was supported by the fact that cAMP stimulated the hydrolysis of DPI but not of TPI by a polyphosphoinositide phosphodiesterase present in the supernatant fraction of rabbit kidney cortex.