Cross-reconstitution of isolated F1-ATPase from potato tuber mitochondria with F1-depleted beef heart and yeast submitochondrial particles

Cross-reconstitution of isolated potato mitochondrial F1-ATPase with F1-depleted beef heart and yeast submitochondrial particles is reported. Potato F1 binds to the heterologous membrane and confers oligomycin sensitivity on the ATPase activity of the reconstituted system. Binding of F1 is promoted by the presence of Mg2+ with the maximal stimulatory effect at 20 mM. Mg2+ increase the sensitivity to oligomycin of the reconstituted system consisting of potato F1 and yeast membranes, however, they do not influence oligomycin sensitivity of potato F1 and beef heart membranes.     

Modulators of transmembrane calcium exchange in myometrium mitochondria change their hydrodynamic diameter

The influence of modulators of calcium exchange in mitochondria--oligomycin, Mg2+ and ruthenium red (RuR)--on the myometrium mitochondria size and granularity was studied in the work. The study of the mitochondria size was carried out using the photon correlation spectroscopy. It was shown that the average hydrodynamic diameter was 655 +/- 14 nm (n = 5; control). The addition of oligomycin (1 microg/ml)--the inhibitor of ATP-synthase F0-component, increases the mitochondria average hydrodynamic diameter to 913 +/- 75 nm (n = 5), that is by 39% more than the control. In the presence of RuR (10 microM) (Ca2(+)-uniporter inhibitor) and Mg2+ (7 mM) the mitochondria average hydrodynamic diameter increases to 788 +/- 28 and 788 +/- 38 nm (n = 5) respectively, that is by 17% more than the control. Using flow cytometry it was shown, that oligomycin (1 microg/ml) causes the increase of side scattering of the mitochondria. Addition of RuR (10 microM) and Mg2+ (7 mM) does not lead to significant changes in side scattering of the mitochondria. So it was shown that oligomycin significantly increases mitochondria granularity, but Mg2+ and RuR have no influence on this parameter     

Role of Mg2+ in lipid-protein interaction in reconstituted procine heart mitochondrial H+-ATPase

During reconstitution of pig heart mitochondrial H+-ATPase in soybean phospholipid liposomes by the cholate dialysis method, Mg2+ greatly enhances 32Pi-ATP exchange activity, ATPase activity and the sensitivity to oligomycin of the reconstituted enzyme complex. The effect of Mg2+ on the fluidity of the reconstituted proteoliposomes was measured by means of a fluoursecent probe. 1-anilinonaphthalene ?e-8-sulfonate, and spin-label probes, 5-nitroxide stearate, 12-nitroxide stearate and 16-nitroxide stearate. A difference in fluidity seems to be localized near the polar faces of the lipid bilayers of the reconstituted proteolipsomes. Fluidity was less in the presence of Mg2+ than it is absence. The conformations of the Mg2+-containing proteoliposomes was higher. We postulate that Mg2+ may play a role in altering the fluidity of the proteoliposomes, which would favor the formation of a conformation of the reconstituted H+-ATPase with higher activity.     

Energy-dependent accumulation of Ca2+ by human embryonic lung fibroblasts.

Human embryonic fibroblasts accumulate Ca2+ in the presence of extracellular ATP and Mg2+, the uptake being maximal at 3 mM ATP. Iodoacetic acid, oligomycin and temperatures of 2 degrees C all inhibit the ATP-potentiated uptake suggesting that an active process may be involved in the transport of Ca2+ into these cells under certain conditions.     

Kinetic mechanism of ATP synthesis catalyzed by mitochondrial Fo x F1-ATPase

Initial rates of succinate-dependent ATP synthesis catalyzed by submitochondrial particles from bovine heart substoichiometrically coupled with oligomycin were found to have hyperbolic dependencies on contents of Mg x ADP, free Mg2+, and phosphate. The results suggest that Mg x ADP complex and free phosphate are true substrates of the enzyme; and an unordered ternary complex of Fo x F1-ATPase, Mg x ADP, and phosphate is generated during the catalysis. The presence of free Mg2+ is required for the reaction. Mg2+ was a noncompetitive activator of ATP synthesis relative to Mg x ADP and a competitive activator relative to phosphate. The decrease in steady-state values of Deltamu(H)+ (by the inhibition of succinate oxidase with malonate) results in the decreased value of Vmax and in a slight decrease in Km for the substrates and Mg2+ without changes in affinity for the substrates. Based on these results, a kinetic scheme of ATP synthesis is proposed.     

Kinetics of ATP-dependent Mg2+ flux in mitochondria.

ATP-dependent Mg2+ accumulation in isolated mitochondria occurs predominantly in the matrix and inner membrane compartments. In mitochondria contaminated with lysosomes, the time course and magnitude of ATP-dependent Mg2+ accumulation are influenced by various cytoplasmic substances, besides substrates of the citric acid cycle. Removal of lysosomes by treatment of the mitochondrial preparation with low concentrations of digitonin, which does not damage the mitoplast, eliminates the modifying influence of cytoplasmic components on Mg2+ flux. In lysosome-free mitochondria, the kinetics of Mg2+ flux is dependent only on the concentration of ATP, of Mg2+, and on the availability of site specific reducing substrates of the electron transport system. Oligomycin at concentrations sufficient to inhibit phosphorylation coupled electron transport and ATP synthesis does not modify Mg2+ flux, which is dependent on added ATP. Site specific inhibitors of the electron transport system inhibit the augmenting effect of oxidizable substrates on Mg2+ uptake, even when electron transfer is inhibited by oligomycin. Atractyloside, by inhibiting the action of externally added ATP, diminishes Mg2+ flux. Ruthenium red is a powerful inhibitor of ATP dependent Mg2+ flux. Uncouplers not only inhibit Mg2+ uptake, but induce Mg2+ efflux. From the time course of Mg2+ flux, a first-order rate constant of egress of Mg2+ and other kinetic constants were calculated and a kinetic model was derived which describes the bi-directional movement of Mg 2+ in mitoplasts.     

Is there a plasma membrane-located anion-sensitive ATPase? IV. Distribution of the enzyme in rat pancreas.

The intracellular localization of anion-sensitive Mg2+-ATPase in rat pancreas was studied by differential centrifugation, density gradient centrifugation and by the use of inhibitors of mitochondrial Mg2+-ATPase. The anion-sensitive MG2+-ATPase appears to be localized almost exclusively in a mitochondrial (15 min, 15 000 times g) fraction which shows two peaks after density gradient centrifugation. Both peaks coincide with the highest levels of cytochrome c oxidase activity, but not with alkaline phosphatase, (Na+ plus K+)-ATPase and leucine aminopeptidase activities or RNA. They appear to be equal sensitive to inhibition by oligomycin, aurovertin D and the rat liver mitochondrial inhibitor protein, at least when 1 mM EDTA is present in the isolation media. We conclude that no significant plasma membrane-located anion-sensitive Mg2+-ATPase activity is present in rat pancreas.     

Matrix free Mg2+ changes with metabolic state in isolated heart mitochondria

The concentration of free Mg2+ in the matrix of isolated heart mitochondria has been monitored by using the fluorescent probe furaptra (mag-fura-2). Beef heart mitochondria respiring in a KCl medium in the absence of external Mg2+ maintain free matrix Mg2+ near 0.50 mM. Addition of Pi under these conditions decreases free Mg2+ by 0.12-0.17 mM depending on the substrate. This decrease in free Mg2+ appears to reflect changing ligand availability in the matrix. The decrease is prevented when the Pi transporter is blocked by mersalyl. Addition of ADP to initiate state 3 respiration causes a marked increase in free matrix Mg2+ (0.1-0.2 mM) that persists as long as ATP formation is taking place; free Mg2+ then returns to the base level. This cyclic change is blocked by oligomycin and carboxyatractyloside and appears to reflect to a large extent the decrease in matrix Pi that accompanies oxidative phosphorylation. Exchange of external ADP for matrix ATP may also contribute to the increase in free matrix Mg2+. Addition of an uncoupler promotes anion efflux and increases free matrix Mg2+. Similar changes in free Mg2+ on addition of Pi, ADP, or uncoupler are seen when extramitochondrial Mg2+ is buffered from 0.5 to 2 mM, but the basal free matrix Mg2+ increases as external Mg2+ concentration increases in this range. Free matrix Mg2+ also increases when total mitochondrial Mg2+ is increased by respiration-dependent uptake in the presence of Pi. It is concluded that matrix free Mg2+ changes significantly with changing ligand availability and that such changes may contribute to the regulation of Mg2(+)-sensitive matrix enzymes and membrane transporters.     

High-affinity calcium-stimulated, magnesium-dependent adenosine triphosphatase in Trypanosoma cruzi.

1. A high-affinity (Ca2+ + Mg2+)-ATPase and a low-affinity Mg(2+)-ATPase were identified in the 105,000 g fraction from epimastigote forms of Trypanosoma cruzi, the agent of Chagas' disease (Tulahuen strain). 2. Activities were conserved after enzyme solubilization with deoxycholate. 3. The Ca(2+)-stimulated ATPase activity was (a) lower than that of the Mg(2+)-ATPase; (b) inhibited by p-chloromercurobenzoate and orthovanadate and (c) insensitive to oligomycin. 4. Optimal stimulation by Ca2+ was observed at pH 6.5-6.8 in the presence of 1 mM MgCl2 and 0.1 M KCl. 5. The Mg(2+)-ATPase was insensitive to p-chloromercurobenzoate and orthovanadate and did not require KCl for activity. 6. Kinetic analysis of the (Ca2+ + Mg2+)-ATPase yielded a half-maximal stimulating concentration of 1.1 microM for Ca2+ and a Km of 66 microM for ATP. 7. The (Ca2+ + Mg2+)-ATPase clearly differed from the Ca(2+)- or Mg(2+)-ATPases previously characterized in the same strain of T. cruzi (Frasch et al., 1978; Comp. Biochem. Physiol. 60B, 271-275).     

Three aspecific ATPases in Trichomonas vaginalis.

1. Three aspecific ATPases were found in the sedimentible fractions of Trichomonas vaginalis. 2. One, with a pH optimum of 5.5, was equally activated by Ca2+ or Mg2+, moderately stable, preferred nucleotide diphosphates as substrates, and was inhibited by vanadate, oligomycin, nitrate and Na+. 3. A second, with a pH optimum of 7.5, was activated by Mg2+, preferred guanosine diphosphate as substrate, and was the least stable and most subject to inhibitors (vanadate, oligomycin, NEM, NBD-Cl, azide and Cl-). 4. The third, pH optimum 8.0, was activated by Ca2+, was latent and the most stable, reacted equally well with nucleotide tri- or diphosphates, and was the least susceptible to inhibitors (vanadate and NEM). 5. All exhibited proton-translocating ability.     

Rat liver plasma membrane Ca2+- or Mg2+-activated ATPase. Evidence for proton movement in reconstituted vesicles

The Ca2+- or Mg2+-activated ATPase from rat liver plasma membrane was partly purified by treatments with sodium cholate and lysophosphatidylcholine, and by isopycnic centrifugation on sucrose gradients. The ATPase activity had high sensitivity to detergents, poor nucleotide specificity and broad tolerance for divalent cations. It was insensitive to mitochondrial ATPase inhibitors such as oligomycin and to transport ATPase inhibitors such as vanadate and ouabain. Using the cholate dialysis procedure, the partly purified enzyme was incorporated into asolectin vesicles. Upon addition of Mg2+-ATP, fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine (ACMA) was observed. The quenching was abolished by a protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Asolectin vesicles or purified ATPase alone failed to promote quenching. These data suggest that the Ca2+- or Mg2+-activated ATPase from rat liver plasma membrane is able of H+-translocation coupled to ATP hydrolysis.     

Two Ca2+ transport systems are distinguished on the basis of their Mg2+ dependency in a post-nuclear particulate fraction of bovine adrenal cortex

Inositol 1,4,5-trisphosphate (InsP3) is a second messenger responsible for Ca2+ release from an internal store whose nature and location remains undefined. To get more information on this intracellular Ca2+ store, a post-nuclear particulate fraction was prepared from bovine adrenal cortex and its Ca2+ uptake and release activities were monitored with the fluorescent indicator Fura-2. In the presence of Mg2+ (2 mM), the particulate preparation showed high ATP-dependent Ca2+ sequestering activity and decreased the ambient Ca2+ concentration to about 150 nM. In the absence of Mg2+, Ca2+ was still sequestered but less efficiently, reaching a level around 170 nM. In the presence of Mg2+, the Ca2+ released by a maximal dose of InsP3 (2 microM) was completely resequestered whereas in the absence of Mg2+, no resequestration occurred even after complete degradation of InsP3. The use of selective agents such as oligomycin, saponin, ionomycin and biliary salts indicated that Ca2+ was stored in three different pools which are distinct from the mitochondria and from inside-out membrane vesicles. Our data also indicate that InsP3 releases Ca2+ from a pool which is filled up by a Mg2(+) -dependent Ca2+ ATPase.     

Properties of the N,N'-dicyclohexylcarbodiimide resistant ATPase of Streptococcus cremoris

1. The specific activity of the membrane-bound ATPase of Streptococcus cremoris HA was 1.30 mumol Pi/mg protein/min. 2. Km for ATP as substrate was 0.8 mM. 3. The pH optimum was 8.0 at +37 degrees C. 4. The ATPase was maximally activated with Mg2+/ATP molar ratio of 1:2. 5. Cations activated the enzyme in order: Mg2+ greater than Co2+ greater than Mn2+ greater than Zn2+ greater than Ca2+ greater than K+ greater than Na+. 6. The enzyme was inhibited by oligomycin (27-77%), sodium azide (13-33%) and ouabain (15-22%). N,N'-dicyclohexylcarbodiimide had no effect on the enzyme activity.     

Is there a plasma membrane-located anion-sensitive ATPase? III. Identity of the erythrocyte enzyme with (Ca2+ + Mg2+)-ATPase.

The characteristics of the anion-sensitive Mg2+-ATPase activity of the rabbit erythrocyte have been studied in a lyophilized ghost preparation. The enzyme appears to be different from the anion-sensitive Mg2+-ATPase activity of other tissues in many parameters, such as optimal pH, effects of various anions, oligomycin sensitivity and effects of Triton X-100. The enzyme is insensitive towards inhibition by irreversibly bound 4,4'-diisothiocyano-dihydrostilbene-2,2'-disulfonic acid (H2DIDS). This excludes a relationship between the enzyme and the "band 3" protein, which is thought to be involved in the anion exchange over the erythrocyte membrane. From the effects of ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA), CaCl2, chlorpromazine and ruthenium red it is concluded that the enzyme activity does not represent a separate entity but is part of the (Ca2+ + Mg2+)-ATPase system of the erythrocyte membrane. A reported stimulatory effect of carbonic anhydrase is attributed to a contamination of the carbonic anhydrase preparation by calcium and/or (Ca2+ + Mg2+)-ATPase activator protein.     

Permeability of yeast mitochondrial internal membrane: structure-activity relationship]

In order to investigate the possible relations between the anionic permeability and the functions (or the structure ) of the inner mitochondrial membrane, three types of organelles isolated from S. cerevisiae were tested: mitochondria (aerobic culture), promitochondria (anaerobic culture) and CAP-mitochondria (aerobic culture with chloramphenicol added). By using the technique of swelling in isoosmotic potassium salts, after a derermination of the isotonic conditions, it was possible to discriminate between an electrogenic (valinomycin induced) or an electroneutral (both valinomycin and uncoupler induced) translocation. 1) Mitochondria: The permeability properties of mitochondria are energy dependent: a) Respiring mitochondria are permeable to Cl-; Mg2+, however, inhibits this translocation. Phosphate transport seems to be exclusively electrogenic and mersalyl sensitive, but swelling inhibition by that thiol reagent is restored by Mg2+. b) Non respiring mitochondria are impermeable to Cl-, but ATP addition restores the permeability. Thiocyanate permeates as the anionic form and acetate as the undissociated form. The phosphate transport, sensitive to mersalyl, seems to be partially electrogenic. 2) Promitochondria: Deficient of respiratory enzymes but containing an oligomycin sensitive ATPase, they are impermeable to Cl- only when Mg2+ is added. In these conditions, an electrogenic phosphate transport, sensitive to mersalyl, is observed. 3) CAP-mitochondria: Although CAP-mitochondria are cytochrome deficient and contain an oligomycin insensitive ATPase, they are also impermeable to Cl- in presence of Mg2+. As in fully differenciated mitochondria, an electroneutral phosphate entry is observed; Mg2+ is required for mersalyl sensitivity.     

Characterization of a Mg2+-stabilized state of the (Na+ and K+)--stimulated adenosine triphosphatase using a fluorescent reporter group

A Mg2+-induced change of the (Na+ and K+)-stimulated adenosine triphosphatase (Na+,K+)-ATPase) from Electrophorus electricus was investigated by kinetics and fluorescence techniques. Binding of Mg2+ to a low affinity site(s) caused inhibition of (Na+,K+)-ATPase activity, an effect which was antagonized by both Na+ and ATP. Mg2+ also caused inhibition of K+-dependent dephosphorylation of the enzyme without inhibiting either (Na+)-ATPase activity or Na+-dependent phosphorylation. Mg2+ also induced a 5 to 6% enhancement in the fluorescence intensity of enzyme labeled with the fluorescent sulfhydryl reagent, 2-(4-maleimidylanilino)naphthalene-6-sulfonate. As in the case of Mg2+ inhibition of activity, the affinity for Mg2+ as an inducing agent for this effect was significantly reduced by both Na+ and ATP, suggesting that the same change was being monitored in both cases. The Mg2+ effect was reduced by both Na+ and ATP, suggesting that the same change was being monitored in both cases. The Mg2+ effect was reduced in magnitude by ouabain and prevented by oligomycin, specific inhibitors of the enzyme. In addition, K+ (and cations that substitute for K+ in supporting activity) induced a 3 to 4% enhancement in fluorescence intensity in the presence of Na+, Mg2+, and ATP, although the K+ and Mg2+ effects appeared to be different on the basis of their excitation spectra. The K+ effect was inhibited by ouabain and occurred with a rate greater than the rate of turnover of the enzyme, permitting its involvement in the catalytic cycle.     

Isolation and identification of the cytoplasmic membrane from Saccharomyces carlsbergensis by radioactive labeling.

The cytoplasmic membrane of Saccharomyces carlsbergensis was isolated by enzymatic digestion of the yeast cell wall, followed by lysis of the protoplasts and fractionation by ultracentrifugation in a discontinuous sucrose density gradient. Location of the cytoplasmic membrane fraction on the sucrose gradient was made by labeling intact protoplasts with [G-3H]dansyl chloride, and was settled at the 50% (wt/vol) sucrose gradient (d = 1.186 g/cm3). Approximately 80% of the radioactivity was found in the membrane fraction prepared in the presence of Mg2+ ions. However, when protease inhibitors were used in the preparation step, the membrane fraction contained over 90% of the total radioactivity. The presence of Mg2+ ions during membrane isolation and purification enhanced the aggregation of membrane components but, at higher concentrations, as well as in the prolonged presence of Mg2+ ions in the membrane suspension, it caused the breakdown of membrane components. The membrane preparation contained Mg2+-adenosine triphosphatase, which was insensitive to oligomycin and ouabain. The distribution of Mg2+-adenosine triphosphatase in different fractions during sucrose gradient is reported.     

Isolation and identification of the cytoplasmic membrane from Saccharomyces carlsbergensis by radioactive labeling.

The cytoplasmic membrane of Saccharomyces carlsbergensis was isolated by enzymatic digestion of the yeast cell wall, followed by lysis of the protoplasts and fractionation by ultracentrifugation in a discontinuous sucrose density gradient. Location of the cytoplasmic membrane fraction on the sucrose gradient was made by labeling intact protoplasts with [G-3H]dansyl chloride, and was settled at the 50% (wt/vol) sucrose gradient (d = 1.186 g/cm3). Approximately 80% of the radioactivity was found in the membrane fraction prepared in the presence of Mg2+ ions. However, when protease inhibitors were used in the preparation step, the membrane fraction contained over 90% of the total radioactivity. The presence of Mg2+ ions during membrane isolation and purification enhanced the aggregation of membrane components but, at higher concentrations, as well as in the prolonged presence of Mg2+ ions in the membrane suspension, it caused the breakdown of membrane components. The membrane preparation contained Mg2+-adenosine triphosphatase, which was insensitive to oligomycin and ouabain. The distribution of Mg2+-adenosine triphosphatase in different fractions during sucrose gradient is reported.     

High affinity Ca2+-stimulated Mg2+-dependent ATPase in rat brain synaptosomes, synaptic membranes, and microsomes

High affinity Ca2+-stimulated Mg2+-dependent ATPase activity of nerve ending particles (synaptosomes) from rat brain tissue appears to be associated primarily with isolated synaptic plasma membranes. The synaptic membrane (Ca2+ + Mg2+)-ATPase activity was found to exhibit strict dependence on Mg2+ for the presence of the activity, a high affinity for Ca2+ (K0.5 = 0.23 microM), and relatively high affinities for both Mg2+ and ATP (K0.5 = 6.0 microM for Mg2+ and KM = 18.9 microM for ATP). These kinetic constants were determined in incubation media that were buffered with the divalent cation chelator trans-cyclohexane-1,2-diamine-N,N,N',N'-tetraacetic acid. The enzyme activity was not inhibited by ouabain or oligomycin but was sensitive to low concentrations of vanadate. The microsomal membrane subfraction was the other brain subcellular fraction with a high affinity (Ca2+ + Mg2+)-ATPase activity which approximated that of the synaptic plasma membranes. The two membrane-related high affinity (Ca2+ + Mg2+)-ATPase activities could be distinguished on the basis of their differential sensitivity to vanadate at concentrations below 10 microM. Only the synaptic plasma membrane (Ca2+ + Mg2+)-ATPase was inhibited by 0.25-10 microM vanadate. The studies described here indicate the possible involvement of both the microsomal and the neuronal plasma membrane (Ca2+ + Mg2+)-ATPase in high affinity Ca2+ transport across membranes of brain neurons. In addition, they suggest a means by which the relative contributions of each transport system might be evaluated based on their differential sensitivity to inhibition by vanadate.     

Further study on the role of Mg2+ in lipid-protein interaction in reconstituted porcine heart mitochondrial H+-ATPase

Porcine heart mitochondrial H+-ATPase was reconstituted by cholate dialysis method in liposomes containing neutral (PC, PE), acidic (PG, PI, PA, PS, DPG) or neutral and acidic phospholipids. The Mg2+ effect on the ATPase activity and its sensitivity to oligomycin, ATP-induced delta psi and delta pH formation was observed for the proteoliposomes containing acidic but not neutral phospholipids. Maleimide spin labels with varying arm lengths or bromoacetamide spin probe were used to monitor the conformational difference of H+-ATPase in the Mg2+-containing and Mg2+-'free' samples. A difference in W/S ratio (weakly immobilized/strongly immobilized component in the ESR spectra) could be detected for the F0.F1-containing and F1-depleted, (F0)-containing proteoliposomes, suggesting conformational difference in the F0-F1 complex and F0 portion induced by the Mg2+ effect. A conformational change of the beta-subunits in the F1 portion was also deduced from the ATP-induced fluorescence quenching of aurovertin-complex for Mg2+-containing samples. The results obtained are in favor of our previous assumption that Mg2+ may play its role by altering the physical state of the lipid bilayer, which would induce a conformational change in F0 (buried in the lipid core), which in turn is transmitted to the catalytic F1, resulting in a higher enzyme activity.