The mechanism of inhibition by oligomycin of oxidative phosphorylation in mitochondria

1. The concentration of specific oligomycin-binding sites in rat-liver mitochondria is 0.12 nmole/mg protein, whereas at least 10-times more oligomycin can be bound non-specifically.

2. The activity of oligomycin-inhibited processes in intact mitochondria and submitochondrial particles cannot be restored by treatment with egg lecithin or mitochondrial phospholipids.

3. Analysis of the kinetics of inhibition of State-3 respiration by oligomycin reveals that (i) after a certain lag period the inhibition by oligomycin is pseudo-first order with respect to the respiratory-control ratio, defined as the ratio of the respiratory rate at time t to that of the final inhibited site, (ii) the value of the pseudo-first-order rate constant (k₀) is dependent on the oligomycin: protein ratio, phospholipid: protein ratio, pH and temperature, (iii) the effects of various substrates and inhibitors of electron transfer on the kinetics of oligomycin inhibition can be explained by their effects on respiratory control.

4. A detailed model is proposed for the interaction of oligomycin with mitochondria. It is proposed that two conformations of the oligomycin-sensitive site are present, and that oligomycin specifically binds to the conformation that is involved in the induction of respiratory control.     

Measurement of the rate ATP synthesis in bovine anterior pituitary slices

A method is described which uses firely luciferase for the measurement of the specific activity of [^{3 2}P] orthophosphate in the γ-position in ATP. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) which uncouples respiration and oxidative phosphorylation and at 0.1 μ/ml reduced pituitary ATP concentration by 35%, decreased the rate of incorporation of [^{3 2}P] orthophosphate into ATP by 40%. Ouabain caused a 50% reduction in the specific activity of ATP after 30 min incubation. Four stimuli of growth hormone secretion, p-chloromercuribenzoate, prostaglandin E₂, 3-isobutyl-1-methylxanthine and Ba²⁺ did not alter the rate of incorporation of [^{3 2}P] orthophosphate into ATP; a fith stimulato, high K⁺, decreased the rate of incorporatio. These results suggest that overall. ATP utilisation is not greatly increased under conditions of stimulated secretion.     

New type of delayed light emission from photosynthetic bacteria

A new type of short-wavelength delayed light emission from the photosynthetic bacteria Rhodospirillum rubrum and Rhodopseudomonas spheroides was found. The emission and action spectra suggest that magnesium protoporphyrin IX or a similar compound is the emitter. Anaerobic conditions were necessary to detect this long-lived emission. Some inhibitors of the primary processes of photosynthesis affected the intensity and the decay rate of the delayed light emission.     

Ureidosuccinic acid permeation in Saccharomyces cerevisiae

Some strains of Saccharomyces cerevisiae exhibit a specific transport system for ureidosuccinic acid, which is regulated by nitrogen metabolism. Ureidosuccinic acid uptake occurs with proline but with ammonium sulfate as nitrogen source it is inhibited. The V for transport is 20–25 μmol/ml cell water per min. The apparent K_m is 3 · 10^-5. For the urep1 mutant (ureidosuccinic acid permease less) the internal concentration never exceeds the external one.In the permease plus strain ureidosuccinic acid can be concentrated up to 10 000 fold and the accumulated compound remains unchanged in the cells. Energy poisons such as dinitrophenol, carbonyl cyanide-m-chlorophenyl-drazone (CCCP) or NaN₃ inhibit the uptake. No significant efflux of the accumulated compound occurs even in the presence of these drugs.The specificity of the permease is very strict, only amino acids carrying an α-N-carbamyl group are strongly competitive inhibitors.The high concentration capacity of the cells and the lack of active exit of the accumulated compound support the hypothesis of a carrier mediated active transport system.     

Permeability to cobalt ions and action of colicin K in a mutant that overproduces cardiolipin

A mutant of Escherichia coli that produces excess cardiolipin becomes less capable of transporting Co²⁺. Cardiolipin therefore does not act as an ionophore under these conditions. Colicin K brings about the typical increase in permeability to Co²⁺ in the mutant.     

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.

Mitochondrial oxygen affinity as a function of redox and phosphate potenitals

1. The conditions under which mitochondria might catalyse a net reversal of oxidative phosphorylation are analysed.

2. Rat-liver mitochondria, incubated under such conditions, show a strongly diminished affinity for oxygen.

3. The velocity of respiration under these conditions is a hyperbolic function of the oxygen concentration.

4. The K_m for oxygen is less than 0.1 μM at low phosphate potential, irrespective of substrate, and 1–3 μM under reversal conditions.

5. The observed kinetics can be accounted for in a simple mechanism for cytochrome oxidase action.     

Anomalies in the kinetics of photosynthetic oxygen emission in sequences of flashes revealed by matrix analysis. Effects of carbonyl cyanide m-chlorophenylhydrazone and variation in time parameters

The model of Kok et al. (Kok, B., Forbush, B. and McGloin, M. (1970) Photochem. Photobiol. 11, 457–475) is considered the best kinetic explanation of the damped oscillations of O₂ evolution induced in higher plants by a sequence of brief saturating flashes. Matrix analysis applied to this model shows that the parameters involved (distribution of S states at zero time, probabilities of transition between states induced by a flash) cannot be completely known from the O₂ yield sequence, Y_n. However, four quantities, with limited content of information, are readily derived from data, without additional assumptions. They are σ₁, σ₂ and σ₃, three quasi-symmetrical functions of the transition coefficients, and $\text{Y}$, a weighed average of four consecutive Y_n values. The extent of misses and double hits and their variations can be qualitatively ascertained by inspection of the relative values of σ₁, σ₂ and σ₃. In a regular sequence (strictly obeying Kok's model), all four quantities should be constant along the time axis.It is shown that actual sequences are seldom regular, in particular in the following conditions: (1) variable flashing frequency, (2) addition of carbonylcyanide m-chlorophenylhydrazone, (3) incomplete deactivation, (4) change of flashing frequency at steady state.In order to account for these anomalies, it is proposed to modify Kok's model by introducing, in parallel to the four state storage entity (S states), a side carrier C, which can reversibly exchange a positive charge with it. In the new model, the transition coefficients are essentially time varying, thus producing a nonregular behaviour of Y_n sequences.     

The effects of phosphate and electron transport on the carbonyl cyanide m-chlorophenylhydrazone-induced ATPase of rat-liver mitochondria

1. The effects of phosphate and electron transport on the ATPase induced in ratliver mitochondria by the uncoupler carbonyl cyanide m-chlorophenylhydrazone have been measured at different uncoupler concentrations and compared with those of ATP, oligomycin and aurovertin.

2. The inhibitory action of respiratory-chain inhibitors on the ATPase activity, which is independent of the actual inhibitor used, is greatly delayed or prevented by the presence of uncoupler, and, in the case of rotenone, can be reversed completely by the subsequent addition of succinate (in the absence of uncoupler). These results can be explained on the basis of the proposal previously made by others that coupled electron transfer causes a structural change in the ATPase complex that results in a decreased affinity of the ATPase inhibitor for the mitochondrial ATPase.

3. Inorganic phosphate specifically stimulates the ATPase activity at high uncoupler concentrations (> 0.2 μM), but has no effect at low concentrations. The stimulation is prevented or abolished by sufficiently high concentrations of aurovertin.

4. Aurovertin prevents the inhibition of the uncoupler-induced ATPase by high uncoupler concentrations.

5. It is proposed that the steady-state concentration of endogenous P_i may be an important regulator of the turnover of the ATPase in intact mitochondria and that the inhibition of ATPase activity by high concentrations of uncoupler is at least partially mediated via changes in the concentration of endogenous P_i.     

Methylammonium uptake by Escherichia coli: evidence for a bacterial NH4+ transport system.

Energy-dependent concentrative uptake of ¹⁴CH₃NH₃⁺ by cells of $\text{Escherichia coli}$ provides preliminary evidence for one or more transport systems for NH₄⁺ uptake. NH₄⁺, but not glutamic acid, inhibited the uptake of ¹⁴CH₃NH₃⁺. Varying the pH for the uptake assays exposed two apparent systems: one maximally functioning at pH 7 that was strongly inhibited by cyanide or by the uncoupler $\text{m}$-chlorophenyl carbonylcyanide hydrazone and another maximally functioning at pH 9 and resistant to cyanide or $\text{m}$-chlorophenyl carbonylcyanide hydrazone. Kinetic analysis showed considerable experimental variability from day to day. Often simple Michaelis-Menten kinetics were not followed, but NH₄⁺ was reproducibly a stronger inhibitor of uptake of ¹⁴CH₃NH₃⁺ than was nonradioactive CH₃NH₃⁺.     

Importance of the flux of phosphate across the inner membrane of kidney mitochondria for the activation of glutaminase and the transport of glutamine

The effect of mersalyl, an inhibitor of phosphate transport across the inner mitochondrial membrane, was investigated on the uncoupled respiration of pig kidney mitochondria in the presence of glutamine as substrate and on the activity of the phosphate-dependent glutaminase in the intact organelles. In addition, the submitochondrial location of the enzyme was reinvestigated.

1. (1) It was found that mersalyl completely inhibits uncoupled respiration of the mitochondria in the presence of glutamine as substrate, whereas respiration with glutamate was not affected. The same amount of mersalyl which inhibits coupled oxidation of glutamine also inhibits coupled oxidation of glutamate and some other substrates.

2. (2) Mersalyl strongly inhibited the activation of glutaminase in intact mitochondria only in the presence of inhibitors of electron transport or of an uncoupler. The addition of a detergent prevented or fully released the inhibition. The effect of mersalyl was observed even when the mitochondria were pre-incubated with phosphate or incubated in the phosphate-free medium. If mersalyl and carbonyl cyanide m-chlorophenylhydrazone (CCCP) were added 3 min after pre-incubation with phosphate the same intramitochondrial concentration of the anion as in control experiments was found, whereas the activity of glutaminase was severely inhibited. These findings suggest that the activation of the enzyme by phosphate in intact nonenergized mitochondria occurs only if the activator moves across the inner mitochondrial membrane.

3. (3) Mersalyl (plus CCCP) markedly decreased [¹⁴C]glutamine- and [³²P]-phosphate-permeable mitochondrial spaces. A close correlation between the decrease of phosphate and glutamine permeable spaces and the inhibition of glutaminase activity was found.

4. (4) If the activation energy of the enzyme was determined with frozen mitochondrial preparations, a discontinuity or break in the Arrhenius plot was observed, whereas the presence of a detergent completely abolished the break. Digitonin or ultrasonic treatment of the mitochondria followed by separation of the membrane and the soluble fraction revealed that glutaminase is a membrane-bound enzyme.

On the basis of these findings it is concluded that there is an association between the transport of phosphate on one side and the transport of glutamine and glutaminase activity on the other. It is possible that the movement of phosphate across the membrane activates the enzyme which facilitates diffusion of glutamine down a concentration gradient. However, the existence of a specific glutamine-phosphate carrier is not ruled out.     

Transient currents carried by the uncoupler,carbonyl cyanide m-chlorophenylhydrazone

The weak acid uncoupler, carbonyl cyanide m-chlorophenylhydrazone, carries protons across lipid membranes. As predicted by the carrier model, at low pH, the current changes immediately following a jump in applied potential and then remains constant. By contrast at high pH, the currents relax from an initial value to a lower value as the carrier anions redistribute in the membrane. These relaxations are slower than those seen with other lipid-soluble anions which presumably explains why they had not been detected previously.     

Energy-linked conformational change of the mitochondrial membrane measured with the NH2-modifying reagent,acrolein

Application of the NH₂-modifying reagent, acrolein (2-propenal, CH₂ = CHCHO), to the mitochondrial membrane gave the information that amino groups in the mitochondrial membrane in the energized state are more accessible to acrolein than those in the non-energized state. This finding was supported by the following experimental results. Addition of acrolein to the respiring mitochondria gives rise to rapid H⁺ production, which is caused by the reaction of the amino groups in the membrane with acrolein, followed by a slow H⁺ consumption, whereas resting mitochondria produce little H⁺. The H⁺ production is stopped by the addition of NaN₃, antimycin A and 2-thenoyltrifluoroacetone but not by oligomycin. During the course of H⁺ production, O₂ consumption and Ca²⁺ uptake remain completely active, indicating that mitochondrial function is unaffected. The subsequent H⁺ consumption may be closely related to the destruction of the transmembrane proton gradient formed by mitochondrial respiration.     

Respiratory components and oxidase activities in Alcaligenes eutrophus

1. Cells of the hydrogen bacterium Alcaligenes eutrophus are broken by gentle lysis using lysozyme treatment in hypertonic sucrose followed by osmotic shock. By this method, 93% of the in vivo activity of the H₂ oxidase is recovered and the ATPase remains particle bound. In contrast, cell disruption in a French pressure cell diminishes the in vivo activity of the H₂ oxidase by 50% and solubilizes the bulk of the ATPase.2. The bacterium contains a periplasmic cytochrome c with bands at 418, 521 and 550 nm (difference spectrum). In addition to cytochrome aa₃, b-560, c-553 and o, low temperature difference spectra of membranes show the presence of two further cytochromes (shoulders at 551 and 553 nm).3. The unsupplemented membrane fraction catalyses the oxidation of hydrogen, NADH, NADPH, succinate, formate and endogenous substrate (NAD linked) at rates 2–3-fold higher than membranes obtained from cells disrupted in a French pressure cell. With the exception of the H₂ oxidase all oxidase activities in lysozyme membranes are sensitive to carbonylcyanide m-chlorophenylhydrazone (20–100% stimulation of oxygen uptake).4. The cytoplasmic fraction contains a B-type cytochrome with absorption maxima at 436 and 560 nm, capable of combining with CO; it contains non-covalently bound protohaem. In alkaline solutions a spectral transition to the haemochrome type with bands at 423, 526 and 556 nm occurs. The addition of NADH to an aerobic suspension of this cytochrome elicits new absorption maxima at 418, 545 and 577 nm (difference spectrum), which are believed to represent an oxygenated form of the reduced cytochrome.