Adenine nucleotide translocation in yeast mitochondria. Effect of inhibitors of mitochondrial biogenesis on the ADP translocase

1. Optimal test conditions for adenine nucleotide translocation in Candida utilis mitochondria are a standard medium, consisting of 0.63 M mannitol, 2 mM EDTA (or ethylene glycol tetraacetic acid, EGTA), 10 mM morpholinopropane sulfonic acid (pH 6.8), and a temperature of 0 °C.

2. Adenine nucleotide translocation in C. utilis mitochondria is an exchange-diffusion process. The whole pool of internal adenine nucleotides is exchangeable, ADP being the most readily exchangeable nucleotide. The rate of mitochondrial ADP exchange, but not the K_m value, depends on growth conditions. At 0 °C, the rate is about 3 to 4 nmoles ADP/min per mg protein for mitochondria obtained from yeast grown in the presence of 1.5% glucose; it rises to 11.5 nmoles when glucose is replaced by 3% ethanol in the growth medium. The K_m value for ADP is 2 μM. The Q₁₀ is about 2 between 0 and 20 °C. Among other exchangeable adenine nucleotides are ATP, dADP and the methylene and the hypophosphate analogues of ADP. Unlike mammalian mitochondria, C. utilis mitochondria are able to transport external UDP by a carboxyatractyloside-sensitive process.

3. Under conditions of oxidative phosphorylation (phosphate and substrate present in an aerated medium), added ADP is exchanged with internal ATP. A higher ATP/ADP ratio was found in the extramitochondrial space than in the intramito-chondrial space. The difference between the calculated phosphate potentials in the two spaces was 0.9–1.7 kcal/mole.

4. Atractyloside, carboxyatractyloside, bongkrekic acid and palmityl-CoA inhibit mitochondrial adenine nucleotide translocation in C. utilis as they do in mammalian mitochondria, but 2 to 4 times less efficiently. The inhibition due to atractyloside or palmityl-CoA is competitive with respect to ADP whereas that due to bongkrekic acid and carboxyatractyloside is non-competitive. Carboxyatractyloside and atractyloside inhibitions are additive. The apparent K_d for the binding of [³⁵S]-carboxyatractyloside and [¹⁴C]bongkrekic acid is 10–15 nM and the concentration of sites 0.4–0.6 nmole/mg protein in both cases. [³⁵S]Carboxyatractyloside binding is competitively displaced by atractyloside and vice versa.

5. Binding of [¹⁴C]ADP has been carried out with mitochondria depleted of their endogenous adenine nucleotides by incubation with phosphate and Mg²⁺ at 20 °C. The amount of bound [¹⁴C]ADP which is atractyloside removable is 0.08–0.16 nmole/mg protein.

6. The rate of ADP transport is quite different in mitochondria isolated from C. utilis, according to whether it is grown on glucose, or on ethanol or in the presence of chloramphenicol; for instance, it decreases by 10 times when 3% ethanol in the growth medium is replaced by 10% glucose and by 5 times when chloramphenicol is added to the medium. These variations are accompanied by parallel variations in cytochrome aa₃. The number of atractyloside-sensitive ADP binding sites is not modified by the above conditions of culture, nor the number of [³⁵S]carboxyatractyloside binding sites. The affinity for ADP is apparently not significantly modified, nor the size of the endogenous adenine nucleotide pool. In contrast to glucose repression or chloramphenicol inhibition, semi-anaerobiosis in C. utilis lowers significantly the mitochondrial binding capacity for carboxyatractyloside. Strict anaerobiosis in S. cerevisiae results in a practical loss of the cytochrome oxidase activity, and also of the carboxyatractyloside and ADP binding capacity. Transition from anaerobiosis to aerobiosis restores the cytochrome oxidase activity and the ADP and carboxyatractyloside binding capacities.     

The influence of pH on the inhibition of oxidative phosphorylation and electron transport by triethyltin

The ability of triethyltin to inhibit oxidative phosphorylation and electron transport in tightly coupled rat liver mitochondria is very dependent on the pH and the ionic constitution of the assay medium.

1. 1. In an assay medium containing Cl⁻ at an alkaline pH, above 7.1, triethyltin inhibited both the ADP stimulated rate of oxygen uptake and the dinitrophenol-induced ATPase (EC 3.6.1.3) but had no effect on the dinitrophenol-stimulated rate of oxygen uptake. If the pH was reduced to below 6.9 the pattern of inhibition changed and both the ADP and dinitrophenol-stimulated rates of oxygen uptake were inhibited by triethyltin.

2. 2. In the absence of Cl⁻ in the medium triethyltin inhibited both the ADP-stimulated rate of oxygen uptake and dinitrophenol-induced ATPase and had no effect on the dinitrophenol-stimulated rate of oxygen uptake at either pH 7.4 or 6.6.

3. 3. In either the presence or absence of Cl⁻ the ability of triethyltin to inhibit ATP synthesis appears to markedly decrease as the pH is lowered from 7.4 to 6.6.

4. 4. The significance of these observations is discussed in relation to the operation of a Cl⁻/OH⁻ antiport in the coupling membrane.

Mechanism of respiration-driven proton translocation in the inner mitochondrial membrane. Analysis of proton translocation associated with oxidation of endogenous ubiquinol

A study is presented of the kinetics and stoichiometry of fast proton translocation associated to aerobic oxidation of components of the mitochondrial respiratory chain.

1. 1. Aerobic oxidation of ubiquinol and b cytochromes is accompanied in EDTA particles, obtained by sonication of beef-heart mitochondria, by synchronous proton uptake.

2. 2. The rapid proton uptake associated to oxidation of ubiquinol and b cytochromes is greatly stimulated by valinomycin plus K⁺, but is unaffected by carbonyl cyanide p-trifluoromethoxyphenylhydrazone.

3. 3. 4 gion H⁺ are taken up per mol ubiquinol oxidized by oxygen. This H⁺/2e− ratio, measured in the rapid anaerobic-aerobic transition of the particles is unaffected by carbonyl cyanide p-trifluoromethoxyphenylhydrazone.

4. 4. In intact mitochondria aerobic oxidation of oxygen-terminal electron carriers is accompanied by antimycin-insensitive synchronous proton release, oxidation of ubiquinol and reduction of b cytochromes. The amount of protons released is in excess with respect to the amount of ubiquinol oxidized.

5. 5. It is concluded that electron flow along complex III, from ubiquinol to cytochrome c, is directly coupled to vectorial proton translocation. The present data suggest that there exist(s) between ubiquinol and cytochrome c one (or two) respiratory carrier(s), whose oxido-reduction is directly linked to effective transmembrane proton translocation.

Transport of glutamate in rat-liver mitochondria

The transport of glutamate across the membrane of rat-liver mitochondria has been studied.

1. 1. Glutamate can be transported across the mitochondrial membrane in exchange for OH⁻ (or together with H⁺).

2. 2. Intramitochondrial glutamate is not extruded from the mitochondria by addition of aspartate when the mitochondria are preloaded with glutamate.

3. 3. N-Ethylmaleimide is a specific inhibitor of the movement of glutamate across the mitochondrial membrane.

Fuscin, an inhibitor of mitochondrial SH-dependent transport-linked functions

1. 1. Fuscin, a mould metabolite, is a colored quinonoid compound which reacts readily with −SH groups to give colorless addition derivatives.

2. 2. Binding of fuscin to mitochondria has been monitored spectrophotometrically. Fuscin binding is prevented by −SH reagents such as N-ehylmaleimide, N-Methylmaleimide, mersalyl or p-chloromercuribenzoate. Conversely, fuscin prevents the binding of −SH reagents as shown with N-[¹⁴C]ethylmaleimide. Once bound to mitochondria, fuscin is not removable by washing of mitochondria.

3. 3. High affinity-fuscin binding sites (K_d = 1 μM, N = 4–8 nmoles/mg protein) are present in whole mitochondria obtained from rat heart, rat liver, pigeon heart or yeast (Candida utilis). They are lost upon sonication but are still present in digitonin inner membrane + matrix vesicles. On the other hand, lysis of mitochondria by Triton X-100 does not increase the number of high affinity binding sites indicating that all these sites are accessible to fuscin in whole mitochondria. The number of fuscin high affinity sites appears to correlate with the glutathione content of mitochondrial preparations.

4. 4. Fuscin as well as N-ethylmaleimide and avenaciolide are penetrant SH-reagents;

5. 5. Fuscin interferes with the ADP-stimulated respiration of mitochondria on NAD-linked substrates, several functions of the mitochondrial respiratory apparatus being inhibited by fuscin in a non-competitive manner, but to various extents: (a) The electron transfer chain (K_i in the range of 0.1 mM); (b) the lipoamide dehydrogenase system (K_i = 5–10 μM); (c) the transport systems of phosphate (K_i ≈ 20 μM) and of glutamate (K_i = 3–5 μM); (d) the ADP transport, indirectly (K_i ≈ 10 μM).

6. 6. Like N-ethylmaleimide, fuscin inhibits the glutamate-OH⁻ carrier, the inhibition of that carrier bringing about an apparent increase of aspartate entry in glutamate-loaded mitochondria by the glutamate-aspartate carrier.

7. 7. The inhibition of phosphate transport by fuscin probably accounts for the inhibition of the reduction of endogenous NAD by succinate in intact pigeon heart mitochondria.

8. 8. By binding the −SH groups of mitochondrial membrane specifically unmasked by addition of micromolar amounts of ADP, fuscin, like N-ethylmaleimide, prevents the functioning of ADP translocation.

9. 9. Because of their specific and analogous effects on some well defined mitochondrial functions such as glutamate transport and ADP transport, fuscin and N-ethylmaleimide can be distinguished from other −SH reagents. The lipophilic nature of fuscin and N-ethylmaleimide which accounts for the accessbility of these compounds to hydrophobic sites in the mitochondrial membrane or on the matrix side of this membrane may be partly responsible for their characteristic inhibitory effects on mitochondrial functions.

Short-term effects of 3,4-methylen-dioxy-metamphetamine (MDMA) on 5-HT(1A) receptors in the rat hippocampus

The first effects of 3,4-methylen-dioxy-metamphetamine (MDMA, “ecstasy”), on serotonin 1A (5-HT_1A) receptors in rat hippocampus were determined by means of [³H]-8-hydroxy-dipropylamino-tetralin ([³H]-8-OH-DPAT) and 5′guanosine-(γ-[³⁵S]-thio)triphosphate ([³⁵S]-GTPγS) binding as well as inhibition of forskolin (FK)-stimulated adenylyl cyclase (AC) activity. The study was completed by [³⁵S]-GTPγS functional autoradiography experiments carried out in frontal sections of rat brain, including the hippocampal region. Results showed that MDMA was either able to displace [³H]-8-OH-DPAT binding (K_i  500 nM) or to reduce the number of specific sites (B_max) without affecting K_d. The drug also failed to change the [³⁵S]-GTPγS binding or to inhibit AC velocity, underlying its behavior as a non-competitive 5-HT_1A receptor antagonist. Further, MDMA (1 or 100 μM), partially antagonized either [³⁵S]-GTPγS binding stimulation of the agonists 5CT and 8-OH-DPAT or the AC inhibition induced by 5CT and DP-5CT. However, in contrast to binding studies, in AC assays the amphetamine displayed an effect also on EC₅₀, always being less potent than the reference antagonist WAY100,635. In functional autoradiography, MDMA behaved either as a partial 5-HT_1A antagonist in limbic areas or, added alone, as an agonist, increasing the coupling signal presumably through 5-HT release from synapses. Interestingly, the selective 5-HT re-uptake inhibitor (SSRI) fluoxetine had no effect on MDMA [³⁵S]-GTPγS binding activation. This latter finding indicates that the amphetamine can release 5-HT via alternative mechanisms to 5-HT transporter binding, probably via membrane synaptic receptors or vesicular transporters. The release of other transmitters is not excluded. Therefore, our results encourage at extending the study of MDMA biochemical profiles, in the attempt to elucidate those amphetamine-induced pathways with a potential for neurotoxicity or psycho-stimulant activity.     

Thermal sensitivity of chinese hamster V79 cells related to time after plating and cell-cycle redistribution

1. 1.|Cultured Chinese hamster V79 cells were trypsinized plated and grown attached or inoculated into spinner flasks and grown in suspension from exponnential growth to plateau-phase growth and their thermal sensitivity was measured after various growth times.

2. 2.|For attached cells, cultures were trypsinized and replated either 2 h before or after heating and the results were qualitatively the same: the thermal sensitivity remained approximately the same for the first 20 h and then increased and reached a maximum at 40–70 h. During this time, cells were in an exponential growth phase, and little or no change was observed in the cell-cycle age distribution as measured by flow cytometry (FMF) or [³H]thymidine pulse ([³H]Tdr) labelling.

3. 3.|At longer times after plating, cells grew into plateau phase, and thermal sensitivity decreased and became less than for the cells in exponential growth phase at the beginning of the experiment.

4. 4.|FMF and [³H]Tdr labelling showed that cells were accumulating in G₁ phase as the population density increased and that this accumulation was maximum at about 120–140 h as cells grew into plateau phase. This would account for the decrease in heat sensitivity and the increase in radiosensivity observed in plateau-phase cells.

5. 5.|For cells cultured in suspension there was no change in thermal sensitivity while cells were in exponential growth phase, As cell entered plateau phase, thermal resistance increased and most of the cell population had accumulated in G₁ as measured by FMF.

Specificity of nucleotide binding and coupled reactions utilising the mitochondrial ATPase

1. 1. Tightly bound ATP and ADP, found on the isolated mitochondrial ATPase, exchange only slowly at pH 8, but the exchange is increased as the pH is reduced. At pH 5.5, more than 60% of the bound nucleotide exchanges within 2.5 min.

2. 2. Preincubation of the isolated ATPase with ADP leads to about 50% inhibition of ATP hydrolysis when the enzyme is subsequently assayed in the absence of free ADP. This effect, which is reversed by preincubation with ATP, is absent on the membrane-bound ATPase. This inhibition seems to involve the replacement of tightly bound ATP by ADP.

3. 3. Using these two findings, the binding specificity of the tight nucleotide binding sites was determined. iso-Guanosine, 2′-deoxyadenosine and formycin nucleotides displaced ATP from the tight binding sites, while all other nucleotides tested did not. The specificities of the tight sites of the isolated and membrane-bound ATPase were similar, and higher than that of the hydrolytic site.

4. 4. The nucleotide specificities of ‘coupled processes’ nucleoside triphosphate-driven reversal of electron transfer, nucleoside triphosphate-³²P_i exchange and phosphorylation were higher than that of the hydrolytic site of the ATPase and similar to that of the tight nucleotide binding sites.

5. 5. The different nucleotide specificities of uncoupled ATP hydrolysis and coupled processes can be explained even if both processes involve a single common site on the ATPase molecule. This model requires that energy can be ‘coupled’ only when it is released/utilised in the nucleotide binding steps of the mechanism.

6. 6. Adenosine β,γ-imidotriphosphate (AMP-PNP) is not a simple reversible inhibitor of the ATPase, since incubation requires preincubation and is not reversed when the compound is diluted out, or by addition of ATP. This compound inhibits the isolated and membrane-bound ATPase equally well. Its guanosine analogue does not act in this way.

7. 7. In submitochondrial particles, ADP inhibited uncoupled hydrolysis of ATP much more effectively than coupled hydrolysis, the latter being measured both directly (from ATP hydrolysis in the absence of uncoupler) or indirectly, by monitoring ATP-driven reduction of NAD⁺ by succinate.

8. 8. The effects of ADP and AMP-PNP were interpreted as providing evidence for two of the intermediates in the proposed scheme for coupled triphosphate hydrolysis.

Interaction of (3H) bongkrekic acid with the mitochondrial adenine nucleotide translocator.

Chemical labeling by 3H and biosynthetic labeling by 14C of bongkrekic acid (BA) are described. In the rat liver cell, mitochondria are the only subcellular particles to bind [3H]BA with high affinity. The high affinity sites for BA in mitochondria are located in the inner membrane. High affinity binding sites for BA are only displayed at pH below 7; they amount to 0.15-0.20 nmol/mg of protein in rat liver mitochondria and to 1.1-1.3 nmol/mg of protein in rat heart mitochondria. These values are similar to those found for the high affinity atractyloside binding sites and for the carboxyatractyloside binding sites. The kinetic parameters for BA binding to rat heart mitochondria at 20 degrees C are Kd = 10-40 X 10(-9) M, k+1 = 0.7 X 10(5) M-1 s-1, k-1 = 1.4 X 10(-3) M s-1. Binding assays carried out with rat heart mitochondria, under equilibrium conditions, showed that the amount of BA bound to high affinity sites increases with temperature and reaches the maximum value of 1.1-1.3 nmol/mg of protein at 32-35 degrees C. At lower temperatures, and under equilibrium conditions, a significant fraction of high affinity sites remains masked and is not titrated by BA; these masked BA sites are revealed by addition of micromolar concentrations of ADP or by energization of the mitochondria. Carboxyatractyloside added to rat heart mitochondria preloaded with [3H]BA is able to displace part of the bound [3H]BA. Displacement of the bound BA is enhanced by simultaneous additions of carboxyatractyloside plus ADP, or by energization of the mitochondria. The synergistic effect of carboxyatractyloside and ADP on displacement of bound [3H]BA is also observed in isolated inner membrane vesicles from rat liver mitochondria. When BA is preincubated with rat heart mitochondria before addition of [14C]ADP for assay of ADP transport, the inhibition of ADP transport is a mixed-type inhibition. When BA is preincubated with the mitochondria together with a very small concentration of ADP (less than 0.5 muM), the inhibition of [14C]ADP transport is markedly increased (up to ten times) and it becomes typically uncompetitive, which suggests the formation of a ternary complex, carrier-ADP-BA. The transition from a mixed-type inhibition, with high Ki value, to an uncompetitive type of inhibition, with low Ki value, upon addition of ADP, is explained by an ADP-induced conformational change of the ADP translocator.     

The phosphorylation potential generated by respiring mitochondria

1. 1. The phosphorylation potential, ΔG_P = ΔG₀′ + 1.36 log ([ATP]/[ADP][P_i]), where ΔG_O′ is the standard free energy of hydrolysis of ATP at a given pH, and [ATP], [ADP] and [P_i] refer to concentrations in the suspending medium, has been determined in rat-liver mitochondria under various conditions.

2. 2. The ATP/ADP ratio is relatively constant, over a 10-fold range of phosphate concentration. Thus, the phosphate potential is higher at low phosphate concentration. State-4 rat-liver mitochondria in the presence of succinate, oxygen and low concentrations of phosphate in State 4 maintain a phosphorylation potential of 16.1 kcal (67.3 kJ) per mole ATP.

3. 3. High concentrations of ATP inhibit ADP uptake, and it is suggested that this is the reason for the independence of the ATP/ADP ratio on the phosphate concentration. A steady-state ratio is set up dependent upon two processes that are relatively slow compared with State-3 respiration, namely ADP transport and ATP hydrolysis.

4. 4. The phosphorylation potential calculated from the concentrations of total ADP, ATP and P_i within State-4 mitochondria is 4.5 kcal/mole less than that in the suspending medium.

5. 5. It was shown experimentally that the phosphorylation potential cannot be calculated from the ΔG of the redox couple, the respiratory-control ratio and the P:O ratio, as has been suggested in the literature.

6. 6. The measured phosphorylation potential is 83% of that calculated from the span succinate to oxygen, assuming thermodynamic equilibrium, and 95% of that calculated from the span NADH to oxygen.

7. 7. Based on the measurements of the phosphorylation potential and of the redox potentials and redox states of redox components in mitochondria, ubiquinone and cytochrome b are found at their expected position at the junction of the phosphorylations at Sites 1 and 2. The iron-sulphur centres 2 and 5 and the iron-sulphur centre of succinate dehydrogenase also probably lie at this junction. Cytochrome a₃ lies at its expected junction between phosphorylation Sites 2 and 3. A number of electron carriers (cytochromes c, c₁, and a, the iron-sulphur centre of Complex III and the EPR-detectable copper), however, lie in the ‘no-man's land’ within Site 2.

8. 8. A phosphorylation potential of 16.1 kcal/mole corresponds to a membrane potential of 350 mV in State 4, on the basis of the chemiosmotic hypothesis.

Adenine nucleotide transport in sonic submitochondrial particles. Kinetic properties and binding of specific inhibitors.

1. A procedure for preparation of sonic submitochondrial particles competent for adenine nucleotide transport is described. ADP or ATP transport was assayed, in the presence of oligomycin, in a saline medium made of 0.125 M KCl, 1 mM EDTA, 10 mM 4-morpholinopropane sulfonic acid buffer, pH 6.5. 2. Sonic particles transport ADP and ATP by an exchange diffusion process. Externally added ADP (or ATP) is exchanged with internal ADP and ATP with a stoichiometry of one to one. The V value for ADP transport 5 degrees C was between 2 and 3 nmol/min per mg protein. 3. The transport system in sonic particles is specific for ADP and ATP. It is strongly dependent on temperature. The activation energy between 0 and 9 degrees C is approx. 35 kcal/mol. The optimum pH is 6.5, 4, Like in intact mitochondria, externally added ADP is transported into sonic particles faster at a given concentration than externally added ATP. The V value for ADP transport is 1.5-2 times higher than the V value for ATP transport. 5. The transition from the energized to the deenergized state in sonic particles results in a decrease of the pH gradient across the membrane (internal pH less than external pH) and in a 2-4 fold increase in the Km value for ATP. This latter effect is opposite that found for transport of added ATP in intact mitochondria (Souverijn, J.H.M., Huisman, L.A., Rosing J. and Kemp, Jr., A. (1973) Biochim. Biophys. Acta 305, 185-198). Energization has no effect on the V value of ATP transport in sonic particles. 6. In contrast to intact mitochondria, inhibition of ADP transport in sonic particles by bongkrekic acid does not have any lag-time and does not depend on pH. The inhibition caused by bongkrekic acid is a mixed type inhibition with a Ki value of 1.2 micronM. Atractyloside and carboxyatractyloside do not inhibit ADP transport in sonic particles, unless the particles have been preloaded with these inhibitors during the sonication. 7. Palmityl-CoA added to sonic particles inhibits efficiently ADP transport. The mixed type inhibition found with palmityl-CoA has a Ki value of 1.6 micronM. 8. [3H]Bongkrekic acid binds to sonic particles readily and with high affinity. Bongkrekic acic binding to sonic particles does not depend on pH and it has a saturation plateau, corresponding approximately to 1.3 mol of site per mol of cytochrome a. The number of [3H]atracytloside binding sites is much lower (one-fifth of the bongkrekic acid). External carboxyatractyloside does not compete with [3H]bongkrekic acid for binding to sonic particles. However, when carboxyatractyloside is present inside the particles, it inhibits the binding of [3H]bongkrekic acid.     

Kinetic, binding and ultrastructural properties of the beef heart adenine nucleotide carrier protein after incorporation into phospholipid vesicles

1. ADP/ATP transport has been reconstituted by incorporation of the purified carrier protein in liposomes filled with ATP. The transport was assayed by uptake of [14C]ADP into the liposomes, and by release of ATP as determined by a luminescence technique. [14C]ADP uptake was strictly dependent on internal ATP. 2. The simplest phospholipid system capable of yielding high rates of ADP/ATP transport was a mixture of phosphatidylethanolamine and cariolipin (92: 8, w/w). 3. ADP/ATP transport in the reconstituted system proceeded by exchange-diffusion with a 1/1 stoichiometry. The specificity for aDP and ATP was absolute. The capacity and the rate of exchange depended on the concentration of ATP present in liposomes. The rate of transport at 20 degrees C, at 20 mM internal ATP, routinely ranged between 300 and 1000 nmol of nucleotide exchanged per min/mg of added carrier protein. The apparent Km value for external ADP was around 10 microM. 4. The ADP/ATP exchange in the reconstituted system was rather stable to ageing. It dropped by only 20% after 1 day of ageing at 20 degrees C. Divalent cations (Mg2+, Mn2+, Ca2+) at concentrations higher than 1 to 2 mM had a deleterious effect on ADP/ATP transport, concomitant with the release of internal ATP and accumulation of multilamellar vesicles. 5. Atractyloside behaved as a competitive inhibitor and carboxyatractyloside as a non-competitive inhibitor. Bongkrekic acid required a slightly acidic pH to be inhibitory. The data concerning atractyloside, carboxyatractyloside and bongkrekic acid were similar to those obtained with whole mitochondria, suggesting that the carrier protein in liposomes has the same asymmetrical arrangement as in the mitochondria. 6. The percentage of competent carrier protein in liposomes was calculated from dose-response data concerning the inhibition of ADP/ATP transport by atractyloside or carboxyatractyloside, and from the amount of bound [3H]-atractyloside removable by ADP. By both methods, 3 to 6% of the added carrier protein was found to be competent in ADP/ATP transport, based on the assumption that the binding of one atractyloside or carboxyatractyloside molecule per 30000 molecular weight carrier unit results in complete inhibition of transport. 7. Freeze-fracture electron microscopy showed that the ADP/ATP carrier protein-lipid preparations are formed by small vesicles, most of which give rise to smooth fracture faces (probably pure lipid vesicles). Only a small percentage of the vesicles (2 to 4% depending on the amount of carrier protein added) were clearly particulated. About 90% of the particulated vesicles showed no more than 2 particles per vesicle and only 5% more than 5 particles per vesicle. The distribution of the particles between convex and concave fracture faces was asymmetric; about 2/3 of the protein molecules were anchored at the external surface of the vesicles and only 1/3 at the internal one...     

Affinities of ATP for the dinitrophenol-induced ATPase

1. 1. The effect of Mg²⁺ on ATP-dependent processes catalysed by intact rat-liver mitochondria can be explained quantitatively by the formation of Mg-ATP complexes that cannot act as a substrate for the adenine nucleotide translocator.

2. 2. The dinitrophenol-induced ATPase is characterized by two affinities of ATP: K_m(1) = 6.7 μM and K_m(2) = 63 μM, which contribute to the extent of 70% and 30%, respectively, to the total ATPase activity under the standard conditions employed.

3. 3. K_m(1) of ATP is competitively increased by atractyloside, and is insensitive to changes in cation concentration or to oligomycin or aurovertin.

4. 4. K_m(2) is as sensitive to atractyloside as the K_m(1) and is also insensitive to oligomycin. However, it is increased by decreasing the cation concentration, and disappears in the presence of aurovertin.

5. 5. It is proposed that two conformations of the adenine nucleotide translocator exist, characterized by their different affinities for ATP. The distribution of the enzyme over these two conformations appears to be a function of the energy state of the mitochondria (coupled or uncoupled).

Adenylate Uptake by Proplastids from Cultured Cells of Tobacco {Nicotiana tabacum L. cv. BY2) Indicates That an Adenylate Translocator is Present in All Types of Plastid

The presence of an adenylate translocator in the envelope membranesof proplastids isolated from the cultured cells of tobacco (Nicotianatabacum L. cv. BY2) was examined by means of transport experimentsusing the silicone oil filtering centrifugation technique. Itwas observed that proplastids can import [³H]ATP, [³H]ADP, [³H]AMPand less specifically ADP-[¹⁴C]Glc which can eventually be usedfor starch biosynthesis. The effects of specific inhibitorsof the mitochondrial adenylate translocator, i.e. atractyloside,bongkrekic acid and carboxyatractyloside were tested. Similarto the case of amyloplasts isolated from the cultured cellsof sycamore and chloroplasts isolated from spinach leaves, onlyATP and ADP-Glc uptake were shown to be partially inhibitedby carboxyatractyloside. On the other hand, neither atractylosidenor bongkrekic acid exerted a significant inhibitory effecton adenylate uptake. (Received August 8, 1992; Accepted November 26, 1992)     

The GABAA receptor complex in the developing chick optic tectum: characterization of benzodiazepine and ionophore-linked convulsant/barbiturate recognition sites

By use of membrane preparations and incubation conditions optimized for each binding site, we have characterized the benzodiazepine and ionophore-linked-convulsant/barbiturate modulatory sites within the chick tectal GABA_A receptor complex. Using [³H]flunitrazepam (FNZ) and [³⁵S]t-butylbicyclophosphorothionate (TBPS), respectively, as specific radioligand probes for the two sites, we have found in each case one single population of high-affinity, saturable, specific binding sites. The apparent dissociation constants (K_d) show no change during tectal development (9 nM for [³H]FNZ, and 25–28 nM for [³⁵S]TBPS) while the respective densities of binding sites at saturation (B_max) experience in both cases a twofold increase between embryonic day 16 and postnatal day 10. Ligand-specific pharmacological profiles and allosteric interactions between the transmitter and modulatory sites appear to be well preserved in the chick tectal membrane preparations employed in this study.     

6'-O-dansyl-gamma-aminobutyryl atractyloside, a fluorescent probe of the ADP/ATP carrier: exploration of conformational changes of the membrane-bound ADP/ATP carrier elicited by substrates and inhibitors

A fluorescent atractyloside analogue, the 6'-O-dansyl-gamma-aminobutyryl atractyloside (DGA), has been used to probe the binding of the inhibitors carboxyatractyloside (CATR) and bongkrekic acid (BA) and nucleotide substrates to the membrane-bound ADP/ATP carrier protein in beef heart mitochondria. Binding and release of DGA were followed by fluorescence responses. Specifically bound DGA was fully released by CATR alone, or by BA in the presence of micromolar amounts of ADP. In the absence of the inhibitors, ADP increased the rate of the specific binding of DGA. The effect of ADP was shared by transportable nucleotides. Non transportable nucleotides were ineffective. These data are consistent with the previously described CATR and BA conformations of the ADP/ATP carrier that are able to bind CATR and BA respectively, the transition between the two conformations being accelerated by micromolar concentrations of transportable nucleotides.     

ATPase complex and oxidative phosphorylation in chloramphenicol-induced megamitochondria from mouse liver

1. 1. Functional properties of the ATPase complex are investigated in megamitochondria isolated from livers of weanling mice fed a diet containing 2% chloramphenicol, as an inhibitor of mitochondrial protein synthesis.

2. 2. Whereas the specific activity of ATPase remains unchanged in chloramphenicol-induced megamitochondria, about 40% of the enzyme activity is resistant to inhibition by oligomycin, triethyltin or venturicidin. It is concluded that the ATPase complex lacks one or more components whose synthesis or accumulation is dependent on mitochondrial translation. The inhibitor-resistant ATPase portion appears tightly bound to the mitochondrial membrane.

3. 3. Respiratory chain phosphorylation is tightly coupled in isolated megamitochondria. ATP synthesis and ATP-P_i exchange are diminished by 40%, as compared to control mitochondria, but both processes are sensitive to oligomycin, triethyltin or venturicidin.

4. 4. The decrease in ATP synthesis and ATP-P_i exchange in megamitochondria correlates quite well with the emergence of inhibitor-resistant ATPase.

5. 5. The following electron transport activities in the megamitochondria are reduced: NADH-cytochrome c reductase, by 60%, cytochrome oxidase, by 80%; the amount of antimycin required to gain complete inhibition of the bc₁-segment is diminished by more than 50%. On the other hand succinate dehydrogenase activity is increased by 50%.

6. 6. Chloramphenicol-induced megamitochondria appear to be a useful system for studying the role of mitochondrial translation in the assembly of mammalian mitochondria.