Biomedical subjects
Publications and source records attributed to N Sone.
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H+-Translocating ATPase, which catalyzes ATP synthesis in biomembranes, is composed of a head piece (F1) and a membrane moiety (F0). Using highly-purified F0 from a thermophilic bacterium PS3 (TF0), the following results were obtained. 1. Inhibition by N,N'-dicyclohexylcarbodiimide (DCCD) of H+ conduction through TF0 followed pseudo-first-order kinetics. The second-order rate constant for inhibitor-enzyme interaction was 5 times 10(3) M(-1)-min(-1). 2. H+ conductivity blocked by DCCD was proportional to the amount of DCCD incorporated in the band 8 protein of TF0. When only one-third of the band 8 protein was labeled with DCCD, TF0 hardly transported any H+. 3. By extracting TF0 with chloroform-methanol, the band 8 protein was obtained as a proteolipid. Polyacrylamide gel electrophoresis with dodecyl sulfate and urea showed that the molecular weight was about 6,000. 4. The amino acid composition of band 8 protein indicated that this protein contained an extremely high percentage of hydrophobic amino acids (0.29 in polarity) and was devoid of histidine, tryptophan, cysteine, and lysine. Its minimum molecular weight was 6,500. 5. The role of band 8 protein (DCCD-binding protein) in H+ conduction through TF0 is discussed on the basis of these results.
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The H+-translocating ATPase complex from the thermophilic bacterium PS3 (TF0-F1) is composed of a water-soluble part with ATP-hydrolyzing activity (TF1) and a membrane moiety with H+-conducting activity (TF0). TF0 was obtained by treating TF0-F1 with urea and removing contaminations on a carboxymethyl-cellulose column. This TF0 contained only two kinds of subunits, band 6 protein (13,500 daltons) and band 8 protein (5400 daltons), and it was active in H+ conduction and TF1 binding when reconstituted into proteoliposomes (TF0 vesicles). The binding of TF1 to TF0 present in vesicles restored energy-transducing activities, such as ATP-32Pi exchange, dicyclohexylcarbodiimide-sensitive ATPase, and ATP-dependent enhancement of 8-anilinonaphthalene-1-sulfonate fluorescence. Treatments such as protease digestion and chemical modification with acetic anhydride, succinic anhydride, or diazobenzenesulfonic acid destroyed the TF1-binding activity, which was caused by band 6 protein. Band 8 protein was a proteolipid that reacted specifically with dicylcohexyl-carbodiimide and seemed to play a central role in H+ conduction through the membrane.
1. The membrane-integrated portion (TF0) of the proton translocating ATPase complex (TF0-F1) of the thermophilic bacterium PS3 was highly purified. Its proton-conducting activity was investigated in vesicles reconstituted from TF0 and phospholipids (TF0 vesicles). 2. The rate of proton conduction through TF0 was proportional to the membrane potential imposed (6H+ uptake/s/TF0 molecule with 103 mV at pH 8.0). The pH profile of the rate revealed that a proton, not a hydroxy ion, was the true substrate conducted and that there was a monoprotic proton binding site in TF0 (pKa = 6.8). The temperature coefficient of proton conductance of TF0 showed a considerable variation depending on the phospholipids of the vesicles with respective transition temperatures. 3. Passive proton conduction through TF0 was inhibited stoichiometrically by addition of either the soluble ATPase portion (TF1) of TF0-F1, or an energy transfer inhibitor dicyclohexylcarbodiimide or an antibody against TF0. 4. The proton conductance of TF0 was concluded to represent its intrinsic activity in the original TF0-F1 complex.
1. Five subunits (alpha, beta, gamma, delta, and epsilon) of an ATPase from a thermophilic bacterium PS3 were purified in the presence of 8 M urea by ion exchange chromatography. Then the ATPase activity was reconstituted by mixing the subunit solutions and incubating them at 20-45 degrees, at pH 6.3 to 7.0. 2. Mixtures containing beta + gamma or alpha + beta + delta regained ATP-hydrolyzing activity, but mixtures of alpha + beta and beta + delta did not. Combinations not including beta were all inactive. 3. The ATPase activity reconstituted from alpha + beta + delta was thermolabile and insensitive to NaN3, whereas the activities obtained from mixtures containing beta and gamma were thermostable and sensitive to NaN3, like the native ATPase. 4. The assemblies containing both beta and gamma subunits had the same mobility as the native ATPase molecule on gel electrophoresis, those without the gamma subunit moved more rapidly toward the anode. 5. Subunits epsilon and delta did not inhibit the ATPase activity of either the assembly (alpha + beta + gamma) or the native ATPase.
Vesicles were reconstituted from a purified dicyclohexyl-carbodiimide-sensitive ATPase complex (TF0-F1) and phospholipids of a thermophilic bacterium PS3. These vesicles synthesized ATP from ADP and Pi with energy from an electrochemical proton gradient (delta-micronH+) formed by a pH gradient and an electrical potential across their membranes. Maximal ATP synthesis was achieved by incubating the vesicles in malonate at pH 5.5 with valinomycin, and then rapidly transferring them to a solution of pH 8.4 and 150 mM K+. Under these conditons ATP synthesis continued at a decreasing rate for 30 s at 40 degrees. Appreciable formation of ATP (40 to 150 nmol/mg of TF0-F1) occurred at an initial delta-micronH+ above 205 mV and moderate formation at an initial value above 180 mV. ATP hydrolysis by the vesicles produced a delta-micronH+, and the additions of 32Pi and hexokinase to them resulted in 32Pi esterification. Analysis of the time courses of 32Pi esterification and decays of the pH difference and membrane potential, followed using 9-aminoacridine and 8-anilinonaphthalene-1-sulfonate, respectively, as probes, showed a relationship between delta-micronH+ and the rate of ATP synthesis. These results demonstrate that purified TF0-F1 is itself a reversible H+-translocating ATPase of oxidative phosphorylation.
A carrier protein mediating alanine transport was purified from the membranes of the thermophilic bacterium PS3, by ion exchange chromatography in the presence of both Triton X-100 and urea. The alanine carrier was recovered in the nonadsorbed fraction from either DEAE- or CM-cellulose columns, suggesting that its isoelectric point was in the neutral pH region. The final preparation contained virtually no electron transfer components, ATPase, or NADH dehydrogenase. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the final preparation consisted of two major protein components with molecular weights of 36,000 and 9,400. Active transport of alanine after incorporation of the alanine carrier into reconstituted proteoliposomes was driven not only by an artificial membrane potential generated by potassium ion diffusion via valinomycin but also by mitochondrial cytochrome oxidase incorporated into the same liposomes and supplemented with both cytochrome c and ascorbic acid. The membrane-integrated portion (TFo) of the ATPase complex uncoupled alanine transport by conducting protons across the membrane.
Purified dicyclohexylcarbodiimide-sensitive ATPase (TF0-F1) from thermophilic bacterium PS3 is composed of a water soluble part with ATP hydrolytic activity (TF1) and a water insoluble moiety (TF0). All of the five subunits (alpha, beta, gamma, delta, and epsilon) of TF1 were isolated. TF1 was reconstituted from the five subunits, which catalyzed an ATP-32Pi exchange and an ATP-driven enhancement of fluorescence of 1-anilinonaphthalene-8-sulfonate, when adsorbed on proteoliposome inlaid with TF0 (TF3-vesicles). Subunit epsilon and/or delta became firmly bound to TF0-vesicles and there was no preferential sequence in the binding. Both subunits were required for binding of the remaining subunits of TF1 to TF0-vesicles, but they did not modify the high H+ -permeability of TF0-vesicles. The addition of gamma but they did not modify the high H+-permeability of TFO-vesicles. The addition of gamma subunit together with epsilon and delta subunits caused a marked decrease of H+ -permeability of TF0-vesicles, similar to that induced by TF1. We conclude tentatively that the epsilon and delta subunits connect TF0 and the other subunits forming a part of a proton pathway, gamma is a gate of proton flow coupled to ATP hydrolysis (or synthesis), and alpha and beta subunits contain the active site for energy transformation. A possible model of subunit structure of TF1 is proposed.
1. A stable ATPase [EC 3.6.1.3] complex (TF0-F1) from the thermophilic bacterium PS3 was reconstituted into vesicles capable of energy transformation,measured as ATP-dependent enhancement of fluorescence of 8-anilinonoaphthalene-1-sulfonate. 2. The factors necessary for obtaining highly active vesicles were investigated. Cholate and deoxycholate were both required for solubilization of TF0-F1 and P-lipids, and removal of the detergents by dialysis resulted in vesicle formation. Medium of around pH 8 and low ionic strength containing 2.5 mM MgSO4 was found suitable for dialysis. The optimal temperature for reconstitution was 30 degrees with soybean P-lipids and 45 degree with PS3 P-lipids. The optimal ratio of protein to lipid was about 1/50. 3. The vesicles obtained under these conditions were mainly 100-200 nm in diameter, covered with 9.5 nm spheres, and had a bouyant density of 1.06 in sucrose andan internal volume of about 0.5 mul per mg of P-lipids.
the toxic effects of CH3HgCL on mitochondria of mammalian organs including human and rat liver were examined. [203Hg]CH3HGCl was bound mainly to mitochondrial proteins. The binding was not effected by the energy state of mitochondria. The state 3 respiration, oxidative phosphorylation and 32Pi-ATP exchange reaction were inhibited by 10 to 50 nmol of CH3HgCl per mg of mitochondrial protein, while NADH-and succinate-dehydrogenase and ATPase were more resistant to it The difference spectrum of the treated mitochondria indicated that the point of inhibition was located after flavin and before cytochrome b. Mitochondrial swelling was induced by CH3HgCl, in accordance with previous morphological observations in vivo. The swelling, stimulation of ATPase and energy-dependent H+ extrusion cauded by CH3HgCl were equally dependent on K+. Under these conditions, uptake of K+ by mitochondria was increased and the membrane potential was dissipated. Unlike the case with other organomercuric compounds, transport of phosphate was not inhibited by CH3HgCl. When tested on liposomes, CH3HgCl itself was not lipid-soluble, as some organomercuric compounds are, and was not an uncoupler or a K+-carrier. It was concluded that protein bound CH3HgS-induced K+ uptake into mitochondria and the resulting loss of membrane potential was the major cause of uncoupling, though at higher concentrations, the electron transport system was also inhibited.
Proteoliposome vesicles containing both bacteriorhodopsin of Halobacterium halobium and H+-translocating ATPase [EC 3.6,1.3] of a thermophilic bacterium, PS3, (TF0-F1) were reconstituted by either the dialysis method or the sonication method. Generation of the electrochemical proton gradient (deltamuH+) in these vesicles was measured using 9-aminoacridine for estimation of the chemical (deltapH) component and 8-anilinonaphthalene sulfonate for the electrical (deltaphi) component). In illuminated bacteriorhodopsin-vesicles the deltamuH+ reached 180-190 mV when reconstituted by the dialysis method and 210-220 mV when reconstituted by the sonication method. Vesicles reconstituted from both TF0-F1 and bacteriorhodopsin by the dialysis method generated a deltapH+ of about 200 mV on addition of ATP, while vesicles prepared by the sonication method generated very little deltamuH+, if any. These vesicles generated similar deltamuH+ on illumination to that found in bacteriorhodopsin-vesicles. Using vesicles reconstituted from both TF0-F1 and bacteriorhodopsin by the dialysis method, light dependent ATP synthesis was measured in relation to deltamuH+ formation. It was necessary to generate a deltamuH+ of above 170 mV for demonstration of appreciable formation of ATP and the greater the deltamuH+, the faster the rate of ATP synthesis.
Stable membrane proteins and lipids are convenient to study biomembranes. Two stable proton translocating proteins were purified and reconstituted into vesicles capable of proton translocation. One was a thermostable ATPase (TF0-F1) of thermophilic bacterium PS3 and the other was rhodopsin of Halobacterium halobium. TF0-F1 was composed of a proton pump moiety (TF1) and a proton channel moiety (TF0). TF1 was the first membrane ATPase which was crystallized and reconstituted from its five polypeptides. Like TF0 and TF1, the rhodopsin in purple membrane was highly stable against dissociating agents, acids and alkali. Phospholipids of these biomembranes were also stable and contained no unsaturated fatty acyl groups. The molecular species of the phospholipids of PS3 were determined by mass chromatography. Measurements were made of the difference in electrochemical potential of protons (deltamicronH+) across the membrane of the reconstituted vesicles. The deltamicronH+ attained was 312 mV in TF0-F1 vesciles and was 230 mV in the rhodopsin vesicles. To conclude that electron transport components are not necessary for ATP synthesis in energy yielding biomembranes, two experiments were performed: The ATP synthesis was observed i) on acid-base treatment of TF0-F1 vesicles, and ii) on illumination of the rhodopsin-TF0-F1 vesicles.
Measurements were made of the difference in the electrochemical potential of protons (delta-mu H+) across the membrane of vesicles restituted from the ATPase complex (TF0.F1) purified from a thermophilic bacterium and P-lipids. Two fluorescent dyes, anilinonaphthalene sulfonate (ANS) and 9-aminoacridine (9AA) were used as probes for measuring the membrane potential (delta psi) and pH difference across the membrane (delta pH), respectively. In the presence of Tris buffer the maximal delta psi ans no delta pH were produced, while in the presence of the permeant anion NO-3 the maximal delta pH and a low delta psi were produced by the addition of ATP. When thATP concentration was 0.24 mm, the delta psi was 140-150 mV (positive inside) in Tris buffer, and the delta pH was 2.9-3.5 units (acidic inside) in the presence of NO-3. Addition of a saturating amount of ATP produced somewhat larger delta psi and delta pH values, and the delta -muH+attained was about 310mV. By trapping pH indicators in the vesicles during their reconstitution it was found that the pH inside the vesicles was pH 4-5 during ATP hydrolysis. The effects of energy transfer inhibitors, uncouplers, ionophores, and permeant anions on these vesicles were studied.
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1. Thermostable membrane vesicles which were capable of active transport of alanine dependent on either respiration or an artificial membrane potential were isolated from the thermophilic aerobic bacterium PS3. 2. Uptake of alanine was dependent on the oxidation of ascorbate-phenazine methosulfate or on generated or exogenous NADH, but succinate and malate failed to drive the uptake. The optimum temperature for respiration-driven uptake of alanine was 45 to 60 degrees. 3. Potassium ion-loaded vesicles were prepared by incubating vesicles at 55 degrees in 0.5 M potassium phosphate. The addition of valinomycin elicited rapid and transient uptake of alanine under the test conditions. Uptake of alanine in response to valinomycin was progressively enhanced by the addition of dicylohexylcarbodiimide, but was completely abolished in the presence of a proton conductor or synthetic permeable cation. The effect of dicyclohexylcarbodiimide was dependent on its concentration and was maximal at a concentration of 0.4 mM. 4. The proton permeability of membrane vesicles was reduced by the addition of dicyclohexylcarbodiimide. A small but significant difference was found in the initial rates of proton uptake in the presence of dicyclohexylcarbodiimide with and without alanine. The results suggest that protons alanine are transported simultaneously in a stoichiometric ratio of 1 : 1. 5. The uptake of alanine was also driven by a pH gradient induced by an instantaneous pH drop in a suspension of alkali-loaded vesicles. Thus, alanine accumulation was driven not only by an electrical potential but also by a pH gradient. 6. Addition of ATP resulted in the inhibition of alanine uptake dependent on artificial membrane potential. ATP hydrolysis by membrane ATPase created a membrane potential which was inside-positive, and this might decrease the effective membrane potential (generated by K+ efflux mediated by valinomycin) available to drive alanine uptake.
1. A stable membrane-bound ATPase [EC 3.6.1.3] (TF0-F1) capable of proton translocation in reconstituted vesicles was purified from the thermophilic bacterium PS3 cultured in medium containing L-[U-14C]amino acids. 2. TF0-F1 was composed of a catalytic moiety (TF1) and a hydrophobic moiety (TF0). TF1 contained 3 polypeptide chains with molecular weights of 56,000, 3 of 53,000, 1 of 32,000, 1 of 15,500, and 1 of 11,000. TF0 contained 1 chain of 19,000, 2 of 13,500, and 5 of 5,400 daltons. TF1 was dissociated into subunits much less readily than F1. 3. TF1 consisted of 95A particles arrayed in hexagonal microcrystals. TF0-F1 consisted of a sphere (TF1) and a stalk plus base (TF0) which was buried in the membrane of the proton translocating vesicles. 4. Vesicles capable of energy transformation were formed when TF1 came in contact with the surface of liposomes containing TF0. On addition of phospholipids, the helix content of TF0 increased 3-fold. The role of F0 in forming channels for protons is discussed. 5. The amino acid compositions of TF0, TF1, and TF0-F1 were compared. TF0 was not hydrophobic, despite its interaction with phospholipids. The phospholipid composition and other properties of the proton translocating vesicles were examined. Vesicles reconstituted from a mixture of phosphatidylethanolamine, phosphatidylgly-cerol, and cardiolipin in the same ratio as in the membranes had the highest activity.
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