Studies of mitochondrial calcium movements using chlorotetracycline

1. The association of calcium with isolated rat liver mitochondrial membranes under various metabolic conditions was monitored using the fluorescent chelate probe, chlorotetracycline. Chlorotetracycline fluorescence increased markedly during energized calcium uptake in the absence of a permeant anion. Uncoupler and a respiratory chain inhibitor caused a rapid decrease in chlorotetracycline fluorescence when added either before or after calcium. During calcium uptake experiments concentrations of calcium exceeding 100 μM caused a transient fluorescence increase followed by an extensive decrease in fluorescence.

2. Changes in the chlorotetracycline-associated fluorescence of the mitochondrial suspensions were correlated with the uptake of exogenous ⁴⁵Ca. A positive correlation was observed between fluorescence and energized ⁴⁵Ca uptake in the absence of permeant anions. Addition of the permeant anion, phosphate, caused an extensive decrease in chlorotetracycline fluorescence but an enhanced uptake of exogenous ⁴⁵Ca.

3. The interaction of endogenous mitochondrial calcium with the fluorescent chelate probe was studied under a number of experimental conditions using mitochondria labeled during preparation with ⁴⁵Ca. Endogenous ⁴⁵Ca was lost rapidly from the mitochondria upon treatment with uncoupler, antimycin A, and A23187. Potassium phosphate and EGTA had no effect on the endogenous calcium as measured by either the ⁴⁵Ca content of the mitochondria or the fluorescence of the probe.

4. Mitochondria treated with antimycin A lost most of their endogenous ⁴⁵Ca within 3 min; subsequent energization of the mitochondria resulted in a partial uptake of the released ⁴⁵Ca but caused nearly a complete return of the chlorotetracycline fluorescence to the original level. Addition of phosphate did not change the fluorescence level but resulted in an almost complete accumulation of the ⁴⁵Ca previously released.

5. Following this energized uptake of ⁴⁵Ca, EGTA, p-trifluoromethoxyphenyl hydrazone of carbonyl cyanide, A23187 and calcium chloride all caused a nearly complete loss of the ⁴⁵Ca from the mitochondria and, with the exception of calcium chloride, caused an extensive decrease in the fluorescence level. Hence, the apparent location and/or properties of the endogenous calcium in this rat liver mitochondrial system were altered significantly by manipulation of the energetic state of the mitochondrial membrane.     

Absorption changes of carotenoids and bacteriochlorophyll in energized chromatophores of Rhodospirillum rubrum

The energization of Rhodospirillum rubrum chromatophores by the light, ATP, PP_i, by dark electron transfer via energy-coupling sites of the redox chain, by the combination of KC1 and valinomycin causes absorption changes of carotenoids and bacteriochlorophyll. These changes due to the absorption-band shifts of the pigments are sensitive to the uncoupler p-trifluoromethoxycarbonyl cyanide phenylhydrazone (FCCP) but not to the combination of KC1 and nigericin, which abolishes fluorescence changes of atebrin. Dithionite and ferricyanide depress the light-induced absorption changes of bacteriochlorophyll but have no inhibitory effect on the PP_i-induced changes. Analysis of bacteriochlorophyll absorption changes in the infra-red region shows that the photooxidation of bacteriochlorophyll reaction centers with the negative peak in the region of 890 nm is accompanied by red and blue shifts of bacteriochlorophyll absorption bands. These shifts are due to a transmembrane electrochemical gradient of H⁺ and a local electric field arising as a result of oxidation of the reaction centers. It appears that the superposition of the (1) red shift which is characterized by negative and positive peaks at 865 and 895 nm, respectively, and (2) photobleaching of bacteriochlorophyll reaction centers in the region of 890 nm cause overall absorption changes with the negative peak at 865 nm.     

Kinetics of cytochrome c oxidase from yeast. Membrane-facilitated electrostatic binding of cytochrome c showing a specific interaction with cytochrome c oxidase and inhibition by ATP (With an appendix on the occurrence of two-dimensional diffusion of cytochrome c on the membrane surface)

The steady-state kinetics of purified yeast cytochrome c oxidase were investigated at low ionic strength where the electrostatic interaction with cytochrome c is maximized. In 10 mM cacodylate/Tris (pH 6.5) the oxidation kinetics of yeast iso-1-cytochrome c were sigmoidal with a Hill coefficient of 2.35, suggesting cooperative binding. The half-saturation point was 1.14 μM. Horse cytochrome c exhibited Michaelis-Menten kinetics with a higher affinity (K_m = 0.35 μM) and a 100% higher maximal velocity.In 67 mM phosphate the Hill coefficient for yeast cytochrome c decreased to 1.42, and the species differences in Hill coefficients were lessened. Under the latter conditions, a yeast enzyme preparation partially depleted of phospholipids was activated on addition of diphosphatidylglycerol liposomes. When the enzyme was incorporated into sonicated yeast promitochondrial particles the apparent K_m for horse cytochrome c was considerably lower than the value for the isolated enzyme.ATP was found to inhibit both the isolated oxidase and the membrane-bound enzyme. With the isolated enzyme in 10 mM cacodylate/Tris, 3 mM ATP increased the half-saturation point with yeast cytochrome c 3-fold, without altering the maximal velocity or the Hill coefficient. 67 mM phosphate abolished the inhibition of the isolated oxidase by ATP.The increase in affinity for cytochrome c produced by binding the oxidase to the membrane was not observed in the presence of 3 mM ATP, with the result that the membrane-bound enzyme was more sensitive to inhibition by ATP. ADP was a less effective inhibitor than ATP, and did not prevent the inhibition by ATP.It is proposed that non-specific electrostatic binding of cytochrome c to phospholipid membranes, followed by rapid lateral diffusion, is responsible for the dependence of the affinity on the amount and nature of the phospholipids and on the ionic strength.ATP may interfere with the membrane-facilitated binding of cytochrome c by a specific electrostatic interaction with the membrane or by binding to cytochrome c.     

On the, stoichiometry between uncouplers of oxidative phosphorylation and respiratory chains. The catalytic action of SF 6847 (3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile)

Titration of State 4 rat-liver mitochondria at pH 7.2 with the uncoupler 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF 6847) at various concentrations of mitochondria and using various substrates indicates that under optimal conditions less than 0.2 molecule of 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile per respiratory chain is sufficient to induce complete uncoupling. This result suggests that there is not a stoichiometric relationship between uncoupler molecules and cytochrome c oxidase, involved in oxidative phosphorylation, or between the former and phosphorylation assemblies.

Experiments on the release by 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile of azide-inhibited respiration of State 3 mitochondria and titrations with 5-chloro-3-tert-butyl-2′-chloro-4′-nitrosalicylanilide (S₁₃) of State 4 mitochondria at various mitochondrial concentrations confirm this conclusion.     

Characterization of a mutant of Schizosaccharomyces pombe lacking cytochrome b-566

1. A chromosomal respiration-deficient mutant of the petite-negative yeast Schizosaccharomyces pombe was isolated. Its mitochondria show respiration rates of about 7% of the wild-type respiration with NADH and succinate as substrate, and 45% with ascorbate in the presence of tetramethyl-p-phenylenediamine. Oxidation of NADH and succinate is insensitive to antimycin and cyanide and that of ascorbate is much less sensitive to cyanide than the wild type.

2. The amounts of cytochromes c₁ and aa₃ are similar in the mutant and wild type. Cytochrome b-566 could not be detected in low-temperature spectra after reduction with various substrates or dithionite. A b-558 is, however, present.

3. The b-cytochromes in the mutant are not reduced by NADH or succinate during the steady state even after addition of ubiquinone-1. QH₂-3: cytochrome c reductase activity is very low and succinate oxidation is highly stimulated by phenazine methosulphate.

4. Antimycin does not bind to either oxidized or reduced mitochondrial particles of the mutant.

5. In contrast to the b-cytochromes of the wild type, b-558 in the mutant reacts with CO.

6. Cytochromes aa₃, c and c₁ are partly reduced in aerated submitochondrial particles isolated from the mutant and the EPR signal of Cu (II), measured at 35°K, is detectable only after the addition of ferricyanide. In the mutant, a signal with a trough at g = 2.01 is found, in addition to the signal at g = 1.98 found in the wild type.

7. The ATPase activity of particles isolated from the mutant is much lower than in the wild type but is still inhibited by oligomycin.     

An ATPase from dog gastric mucosa: changes of outer pH in suspensions of membrane vesicles accompanying ATP hydrolysis

A microsomal Mg-ATPase from the gastric mucosa of dog, cat and frog has a K_m for ATP in the region 20–25 μM and by the value of this coefficient can be differentiated from the mitochondrial Mg-ATPase. The microsomal Mg-ATPase from dog gastric mucosa can be stimulated by gramicidin, nigericin and valinomycin in a KCl medium. This Mg-ATPase seems to be located in the ion impermeable membrane of microsomal vesicles and ATP hydrolysis driven changes of the outer pH can be observed. The data are consistent with the ATP hydrolysis driven entry of H⁺ ions across the vesicle membrane.     

Changes in membrane potential during calcium ion influx and efflux across the mitochondrial membrane

1. A depolarisation of the membrane of rat liver mitochondria, as measured with the safranine method, is seen during Ca²⁺ uptake. The depolarisation is followed by a slow repolarisation, the rate of which can be increased by the addition of EGTA or phosphate.2. Plots relating the initial rate of calcium ion (Ca²⁺) uptake and the decrease in membrane potential (Δψ) to the Ca²⁺ concentration show a half-maximal change at less than 10 μM Ca²⁺ and a saturation above 20 μM Ca²⁺.3. Plots relating the initial rate of Ca²⁺ uptake to Δψ are linear.4. Addition of Ca²⁺ chelators, nitriloacetate or EGTA, to deenergized mitochondria equilibrated with Ca²⁺ causes a polarisation of the mitochondrial membrane due to a diffusion potential created by electrogenic Ca²⁺ efflux.5. If the extent of the response induced by different nitriloacetate concentrations is plotted against the expected membrane potential a linear plot is obtained up to 70 mV with a slope corresponding to two-times the extent of the response induced by valinomycin in the presence of different potassium ion gradients. This suggests that the Ca²⁺ ion is transferred across the membrane with one net positive charge in present conditions.     

Interaction of a synthetic polyanion with rat liver mitochondria

The effect of a polyanion (a copolymer of methacrylate, malaete and styrene in a 1:2:3 proportion with an average molecular weight of 10 000) on respiration, ATPase activity and ADP/ATP exchange activity of rat liver mitochondria and submitochondrial particles has been studied.The polyanion (at 17–150 μg/ml concentration, 100 μg polyanion corresponding to 0.83 μequiv. of carboxylic groups) inhibits the oxidation of succinate and NAD-linked substrates in state 3 in a concentration-dependent manner. The extent of this inhibition can be decreased by elevating the concentration of ADP. State 4 respiration is not affected by the polyanion. It has also a slight inhibitory effect on the oxidation of the above mentioned substrates in the uncoupled state (a maximum inhibition of 37% at 166 μg/ml polyanion concentration), which is unaffected by ADP. The strong inhibition of state 3 respiration can be relieved by 2,4-dinitrophenol to the low level observed in the uncoupled state. Ascorbate+TMPD oxidation is slightly inhibited in state 3, while it is not inhibited at all in the uncoupled state.The polyanion, depending on its concentration, strongly inhibits also the DNP-activated ATPase activity of mitochondria (50% inhibition at 40 μg/ml polyanion concentration).The ATPase activity of sonic submitochondrial particles is also inhibited. However, this inhibition is incomplete (reaching a maximum of 65%) and higher concentrations of the polyanion are required than to inhibit the ATPase activity of intact mitochondria.The polyanion inhibits the ADP/ATP translocator activity of mitochondria, measured by the “back exchange” of [2-³H]ADP. After a short preincubation of the mitochondria with the polyanion, the concentration dependence of the inhibition by the polyanion corresponds to that of the DNP-activated ATPase activity of intact mitochondria.It is concluded that, in intact mitochondria, the polyanion has at least a dual effect, i.e. it partially inhibits the respiratory chain between cytochrome b and cytochrome c, and strongly oxidative phosphorylation by blocking the ADP/ATP translocator.     

Influence of vanadate on calcium fluxes and net movement of calcium in intact squid axons

Intracellular vanadate at a concentration of 100 μM inhibits the uncoupled efflux of Ca²⁺ from intact axons but has little effect on the exchange fluxes and on the Ca²⁺-dependent Na⁺ efflux. External vanadate has no effect on the Ca²⁺ efflux. In addition and most importantly intracellular vanadate inhibits the Ca²⁺ efflux in the presence of external Na⁺ and Ca²⁺ suggesting that the uncoupled efflux is operative under physiological conditions. Measurements of the net movements of Ca²⁺ under near physiological conditions have confirmed this conclusion.     

Effect of calmodulin antagonists on Ca2+ uptake by boar spermatozoa

Calcium uptake by washed boar sperm suspensions is markedly stimulated by the calmodulin antagonists trifluoperazine and calmidazolium. Both ⁴⁵Ca²⁺ uptake and net Ca²⁺ uptake are increased by these drugs. Drug stimulated Ca²⁺ uptake is blocked by verapamil (1 mM), by ruthenium red (25 μM) and by carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. Calmodulin antagonists do not slow ATP-dependent Ca²⁺ extrusion from plasma membrane vesicles, and they do not inhibit plasma membrane Ca²⁺-ATPase. It is proposed that calmodulin is involved in the control of Ca²⁺ entry in boar spermatozoa. Most entering Ca²⁺ in uncapacitated spermatozoa is sequestered by mitochondria or rapidly extruded by plasma membrane pumps. In contrast to the uptake mechanism, ATP-dependent Ca²⁺ extrusion does not appear to be regulated by calmodulin.