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Reconstitution of mitochondrial oligomycin and dicyclohexylcarbodiimide-sensitive ATPase 总被引:5,自引:0,他引:5
1. Oligomycin and dicyclohexylcarbodiimide-sensitive ATPase was isolated from beef-heart mitochondria and treated with 3.5 M NaBr in order to remove F1. The residue, called F0, was found to consist of seven components. Five of these are stained by Coomassie blue after dodecylsulfate-polyacrylamide-gel electrophoresis. Two of them correspond to the oligomycin-sensitivity-conferring protein and coupling factor F6, with apparent molecular weights of 21,000 and 9,400, respectively. Three additional polypeptides of molecular weights 23,000, 10,500 and 8,600 were not identified with known proteins. Two components not stained with Coomassie blue were detected by autoradiography of the gels of F0 preincubated with [14C]dicyclohexylcarbodiimide. These two components probably represent monomeric and oligomeric forms of the dicyclohexylcarbodiimide-binding protein. 2. F0 induced an oligomycin and dicyclohexylcarbodiimide-sensitive enhancement of K+ + valinomycin-driven proton translocation across the membrane of artificial phospholipid vesicles. 3. The interaction of F0 with purified, soluble beef heart F1 was investigated. F0 was capable of binding F1 and conferring oligomycin and dicyclohexylcarbodiimide sensitivity and cold stability on its ATPase activity. Furthermore F0 was found to diminish the specific activity of F1-ATPase. A comparison of these effects at varying F0/F1 ratios shows that F0 binds F1 in both an oligomycin-sensitive and an oligomycin-insensitive manner, and that both types of binding involve a conferral of cold stability and a decrease in specific activity. High F0/F1 ratios favoured in oligomycin-sensitive type of binding, indicating that F1 binds preferentially to oligomycin-sensitivity-conferring sites. Treatment of ATPase complex with trypsin resulted in an F0 with a decreased proportion of oligomycin-sensitivity-conferring binding sites and a diminished ability to lower the specific activity an cold lability of F1. 4. Reconstitution of F0 treated with trypsin and F1, oligomycin-sensitivity-conferring protein and F6 showed that at a constant amount of F1 bound, both oligomycin-sensitivity-conferring protein and F6 increased the oligomycin sensitivity of ATPase activity. It was therefore concluded that both of these coupling factors are involved in the conferral of oligomycin sensitivity. 5. The effect of the order of addition of F1, oligomycin-sensitivity-conferring protein and F6 to F0 on the reconstitution of oligomycin-sensitive ATPase activity, and of F1 and oligomycin-sensitivity-conferring protein to submitochondrial particles on the reconstitution of respiratory control, was investigated. The highest values of oligomycin sensitivity and respiratory control were obtained when F1 was added as the first component, indicating that F1 plays a directing role in the organisation of the components. 相似文献
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The characteristics of the oligomycin and DCCD inhibitions of energy linked reactions supported by ATP, indicate that the inhibition is increased by the presence of ATP. It is proposed that such an effect can be attributed to an energy linked change in the structure of some component of the ATPase, facilitating oligomycin interaction with the membrane. 相似文献
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The addition of oligomycin in the presence of Ca2+ increased the ADP pool in mitochondrial suspension. It is suggested that oligomycin inhibition of Ca2+-induced mitochondrial respiratory activation is the function of the increased endogenous ADP pool. Low ADP concentrations (5–20 μM) produce the same inhibitory effect as oligomycin. The increase of ADP levels in the presence of glucose plus hexokinase resulted in the inhibition of Ca2+-induced respiration, while the addition of phosphoenol pyruvate plus pyruvate kinase followed by a reduction in ADP levels, reversed the oligomycin inhibitory effect. One of the essential stages of ADP accumulation in mitochondrial suspensions in the presence of oligomycin and Ca2+ is proposed to be the formation of ADP from AMP and ATP, effected by adenylate kinase. 相似文献
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Topography of oligomycin sensitivity conferring protein in the mitochondrial adenosinetriphosphatase-ATP synthase 总被引:1,自引:0,他引:1
The topographical organization of oligomycin sensitivity conferring protein (OSCP) in the mitochondrial adenosinetriphosphatase (ATPase)-ATP synthase complex has been studied. The accessibility of OSCP to monoclonal antibodies has been qualitatively visualized by using the protein A-gold electron microscopy immunocytochemistry or quantitatively estimated by immunotitration of OSCP in depolymerized or intact membranes. Besides, OSCP cannot be labeled by 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID) which selectively labels the hydrophobic core of membrane proteins. These observations demonstrate an external location of OSCP on the inner face of the inner mitochondrial membrane. The position of OSCP relative to other peptides of the complex has been analyzed by cross-linking experiments using either zero length N-(ethoxycarbonyl)-2-ethoxydihydroquinoline or 11-A span dimethyl suberimidate cross-linkers in the ATPase-ATP synthase complex. The OSCP cross-linked products were identified either by immunocharacterization with anti-alpha, anti-beta, or anti-OSCP monoclonal antibodies or by their molecular weight. OSCP was cross-linked with either the alpha- or beta-subunits of F1 or to a subunit of Mr 24 000. Other types of cross-linking were obtained by the labeling of OSCP with [cysteamine-35S]-N-succinimidyl 3-[[2-((2-nitro-4-azidophenyl)amino)ethyl]dithio]propionate ([35S]SNAP) and reconstitution of SNAP-OSCP with F1 in urea-treated submitochondrial particles. Under these conditions, OSCP is found to be adjacent to two other peptides of molecular weight close to 30 000. A comparison is made between the topology and the organization of the b-subunit of Escherichia coli and OSCP, suggesting an analogy between OSCP and the hydrophilic part of the b-subunit. 相似文献
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The mitochondrial F1F0 complex is highly sensitive to macrolide antibiotics and especially targeted by oligomycins. These compounds bind to the membrane-embedded sector F0 and block proton conductance through the inner membrane, thus inhibiting both ATP synthesis and hydrolysis. Oligomycin sensitivity is universally recognized as a clue of the functional integrity and matching between F0 and F1. Since oligomycin binding implies multiple interactions with amino acid residues of F0, amino acid substitutions often affect the inhibition efficiency. Moreover, variegated factors spanning from membrane properties to xenobiotic incorporation and detachment of the oligomycin-insensitive F1 sector can alter the oligomycin sensitivity of the enzyme complex. The overview on the multiple factors involved strengthens the link between altered oligomycin sensitivity and physiopathological conditions associated with defective ATPases. An improved understanding of the mechanisms involved may also favor drug design to counteract oxidative damage, which stems from most mitochondrial dysfunctions. 相似文献
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On the identification of the uncoupler binding protein of bovine mitochondrial oligomycin sensitive ATPase 总被引:1,自引:0,他引:1
G A Blondin 《Biochemical and biophysical research communications》1980,96(2):587-594
The 55,000 dalton polypeptide component of the membrane sector of the mitochondrial oligomycin sensitive ATPase has been purified by recycling chromatography on BioGel P-100. The amino acid composition of the purified polypeptide differs significantly from that of the α-subunit of F1 with which it shares a similar apparent molecular weight. However, the amino acid composition of the former is identical to that of the Factor B polypeptide, which is known to occur in oligomeric forms. Evidence is presented which suggests that the mitochondrial uncoupler binding proteins and the various oligomeric forms of the Factor B polypeptide, including the 55,000 dalton species described in the present report, are identical. 相似文献
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Beef heart mitchondrial oligomycin sensitivity conferring protein (OSCP) labeled with [14C]-N-ethylmaleimide ([14C]OSCP) at the only cysteine residue, Cys-118, present in the sequence [Ovchinnikov, Y. A., Modyanov, N. N., Grinkevich, V. A., Aldanova, N. A., Trubetskaya, O. E., Nazimov, I.V., Hundal, T., & Ernster, L. (1984) FEBS Lett. 166, 19-22] exhibits full biological activity in a reconstituted F0-F1 system [Dupuis, A., Issartel, J. P., Lunardi, J., Satre, M., & Vignais, P. V. (1985) Biochemistry 24, 728-733]. The binding parameters of [14C]OSCP with respect to the F0 sector of submitochondrial particles largely depleted of F1 and OSCP (AUA particles) have been explored. In the absence of added F1, a limited number of high-affinity OSCP binding sites were detected in the AUA particles (20-40 pmol/mg of particles); under these conditions, the low-affinity binding sites for OSCP were essentially not saturable. Addition of F1 to the particles promoted high-affinity binding for OSCP, with an apparent Kd of 5 nM, a value 16 times lower than the Kd relative to the binding of OSCP to F1 in the absence of particles. Saturation of the F1 and OSCP binding sites of AUA particles was attained with about 200 pmol of both F1 and OSCP added per milligram of particles. The oligomycin-dependent inhibition of F1-ATPase bound to AUA particles was assayed as a function of bound OSCP. At subsaturating concentrations of F1, the dose-effect curves were rectilinear until inhibition of ATPase activity by oligomycin was virtually complete, and maximal inhibition was obtained for an OSCP to F1 ratio of 1 (mol/mol).(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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Enzymes in the respiratory chain are increasingly seen as potential targets against multi-drug resistance of human pathogens and cancerous cells. However, a detailed understanding of the mechanism and specificity determinants of known inhibitors is still lacking. Oligomycin, for example, has been known to be an inhibitor of the membrane motor of the mitochondrial ATP synthase for over five decades, and yet little is known about its mode of action at the molecular level. In a recent breakthrough, a crystal structure of the S. cerevisiae c-subunit ring with bound oligomycin revealed the inhibitor docked on the outer face of the proton-binding sites, deep into the transmembrane region. However, the structure of the complex was obtained in an organic solvent rather than detergent or a lipid bilayer, and therefore it has been unclear whether this mode of recognition is physiologically relevant. Here, we use molecular dynamics simulations to address this question and gain insights into the mechanism of oligomycin inhibition. Our findings lead us to propose that oligomycin naturally partitions into the lipid/water interface, and that in this environment the inhibitor can indeed bind to any of the c-ring proton-carrying sites that are exposed to the membrane, thereby becoming an integral component of the proton-coordinating network. As the c-ring rotates within the membrane, driven either by downhill proton permeation or ATP hydrolysis, one of the protonated, oligomycin-bound sites eventually reaches the subunit-a interface and halts the rotary mechanism of the enzyme. 相似文献
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C Montecucco R Bisson F Dabbeni-Sala A Pitotti H Gutweniger 《The Journal of biological chemistry》1980,255(21):10040-10041
The interaction of bovine heart mitochondrial oligomycin-sensitive ATPase (Serrano, R., Kranner, B. L., and Racker, E. (1976) J. Biol. Chem. 251, 2453-2461) with phospholipids has been examined by labeling the subunits exposed to lipids with photoreactive radioactive phospholipids. A subunit of Mr = 29,000 and some polypeptides in the range of 6,000 to 13,000 daltons were labeled. F1-ATPase subunits did not interact with the photoactive probes. This result is compared with the different pattern of labeling obtained with another mitochondrial ATPase preparation (Galante, Y.M., Wong, S. Y., and Hatefi, Y. (1979) J. Biol. Chem. 254, 12372-12378), which is devoid of the 29,000 component. 相似文献
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V Petronilli B Persson M Zoratti J Rydstr?m G F Azzone 《Biochimica et biophysica acta》1991,1058(2):297-303
The effect of inhibitors of proton pumps, of uncouplers and of permeant ions on the relationship between input force, delta mu H+, and output flows of the ATPase, redox and transhydrogenase H(+)-pumps in submitochondrial particles was investigated. It is concluded that: (1) The decrease of output flow of the transhydrogenase proton pump, defined as the rate of reduction of NADP+ by NADH, is linearily correlated with the decrease of input force, delta mu H+, in an extended range of delta mu H+, independently of whether the H(+)-generating pump is the ATPase or a redox pump, or whether delta mu H+ is depressed by inhibitors of the H(+)-generating pump such as oligomycin or malonate, or by uncouplers. (2) The output flows of the ATPase and of the site I redox H(+)-pumps exhibit a steep dependence on delta mu H+. The flow-force relationships differ depending on whether the depression of delta mu H+ is induced by inhibitors of the H(+)-generating pump, by uncouplers or by lipophilic anions. (3) With the ATPase as H(+)-consuming pump, at equivalent delta mu H+ values, the output flow is more markedly inhibited by malonate than by uncouplers; the latter, however, are more inhibitory than lipophilic anions such as ClO4-. With redox site I as proton-consuming pump, at equivalent delta mu H+ values, the output flow is more markedly inhibited by oligomycin than by uncouplers; again, uncouplers are more inhibitory than ClO4-. (4) The results provide further support for a delocalized interaction of transhydrogenase with other H(+)-pumps. 相似文献
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Cytoplasmic transfer of oligomycin resistance during protoplast fusion of Saccharomycopsis lipolytica 总被引:1,自引:1,他引:0 下载免费PDF全文
Mitotic segregation of oligomycin resistance and oligomycin sensitivity was observed among the prototrophic progeny of protoplast fusion between drug-resistant and drug-sensitive complementary auxotrophs of Saccharomycopsis lipolytica. The transfer of oligomycin resistance by protoplast fusion without karyogamy suggests a cytoplasmic inheritance of this drug resistance determinant. 相似文献
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Sergey A. Novgorodov Tatjana I. Gudz Yulia E. Kushnareva Dmitry B. Zorov Yury B. Kudrjashov 《FEBS letters》1990,270(1-2):108-110
The effect of oligomycin and cyclosporine A on the induction of non-specific permeability of the inner mitochondrial membrane by Ca2+ was under study. Both oligomycin and cyclosporine A were able to prevent the activation of non-specific permeability, but cyclosporine A was the only agent which could restore initial permeability of the inner mitochondrial membrane. The effect of cyclosporine A was shown not to be mediated through redistribution of Ca2+ between different mitochondrial subpopulations 相似文献