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1.
The ratio between the amount of oligomycin-sensitivity-conferring protein (OSCP) and the amount of the alpha and beta subunits of F1-ATPase in the mitochondria has been determined by a method combining electrophoresis, electrotransfer and immunotitration with monoclonal antibodies. The peptides separated in SDS-polyacrylamide gel electrophoresis were blotted to nitrocellulose sheets by electrotransfer. The nitrocellulose sheets were incubated with 125I-labelled purified monoclonal antibodies specific to various peptides. The 125I-labelled immune complexes were located by immunodecoration using peroxidase-conjugated second antibodies and the blotted peptides were revealed with H2O2 and alpha-naphthol. The amount of immune complex present on the nitrocellulose was determined by counting the radioactivity present on the spots. The amount of peptide blotted is directly proportional to the amount of protein loaded on the electrophoresis. By comparing standard curves made with the isolated proteins to the values obtained in the presence of various amounts of the membrane-protein complex, one can calculate the content of this peptide in the membrane. It was found that the mitochondrial membrane contains 2 mol of OSCP per mol of F1. 相似文献
2.
We have measured the inhibitory potencies of several local anesthetics (procaine, lidocaine, tetracaine and dibucaine) and related compounds (chlorpromazine, procainamide and propranolol) on the ATPase activities of bovine heart submitochondrial particles and purified F 1 extracted from these particles. All of these agents cause inhibition of ATPase in F 1 as well as in submitochondrial particles. A linear relationship is found between the log of the octanol/water partition coefficients and the log of the concentrations required for 50% inhibition of F 1. Sedimentation velocity ultracentrifugation and polyacrylamide gel electrophoresis showed that 1.0 mM tetracaine caused partial dissociation of the F 1 complex. Complete reversibility of the enzyme inhibitory effects was demonstrated, however. This work shows that local anesthetics can affect protein structure and enzyme activity without the mediation of lipid. 相似文献
3.
The pea cotyledon mitochondrial F 1-ATPase was released from the submitochondrial particles by a washing procedure using 300 mM sucrose /2 mM Tricine (pH 7.4). The enzyme was purified by DEAE-cellulose chromatography and subsequent sucrose density gradient centrifugation. Using polyacrylamide gel electrophoresis under non-denaturing conditions, the purified protein exhibited a single sharp band with slightly lower mobility than the purified pea chloroplast CF 1-ATPase. The molecular weights of pea mitochondrial F 1-ATPase and pea chloroplast CF 1-ATPase were found to be 409 000 and 378 000, respectively. The purified pea mitochondrial F 1-ATPase dissociated into six types of subunits on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Most of these subunits had mobilities different from the subunits of the pea chloroplast CF 1-ATPase. The purified mitochondrial F 1-ATPase exhibited coupling factor activity. In spite of the observed differences between CF 1 and F 1, the mitochondrial enzyme stimulated ATP formation in CF 1-depleted pea chloroplast membranes. Thus, the mitochondrial F 1 was able to substitute functionally for the chloroplast CF 1 in reconstituting photophosphorylation. 相似文献
4.
Site-directed mutagenesis was used to generate three mutations in the uncB gene encoding the a-subunit of the F 0 portion of the F 0F 1-ATPase of Escherichia coli. These mutations directed the substitution of Arg-210 by Gln, or of His-245 by Leu, or of both Lys-167 and Lys-169 by Gln. The mutations were incorporated into plasmids carrying all the structural genes encoding the F 0F 1-ATPase complex and these plasmids were used to transform strain AN727 ( uncB402). Strains carrying either the Arg-210 or His-245 substitutions were unable to grow on succinate as sole carbon source and had uncoupled growth yields. The substitution of Lys-167 and Lys-169 by Gln resulted in a strain with growth characteristics indistinguishable from a normal strain. The properties of the membranes from the Arg-210 or His-245 mutants were essentially identical, both being proton impermeable and both having ATPase activities resistant to the inhibitor DCCD. Furthermore, in both mutants, the F 1-ATPase activities were inhibited by about 50% when bound to the membranes. The membrane activities of the mutant with the double lysine change were the same as for a normal strain. The results are discussed in relation to a previously proposed model for the F 0 (Cox, G.B., Fimmel, A.L., Gibson, F. and Hatch, L. (1986) Biochim. Biophys. Acta 849, 62–69). 相似文献
5.
The enzyme F 1-ATPase is a rotary nanomotor in which the central γ subunit rotates inside the cavity made of α 3β 3 subunits. The experiments showed that the rotation proceeds in steps of 120° and each 120° step consists of 80° and 40° substeps. Here the Author proposes a stochastic wave mechanics of the F 1-ATPase motor and combines it with the structure-based kinetics of the F 1-ATPase to form a chemomechanic coupled model. The model can reproduce quantitatively and explain the experimental observations about the F 1 motor. Using the model, several rate-limited situations about γ subunit rotation are proposed, the effects of the friction and the load on the substeps are investigated and the chemomechanic coupled time during ATP hydrolysis cycle is determined. 相似文献
6.
It has been claimed (Andreu, J.M., Warth, R. and Muñoz, E. (1978) FEBS Lett. 86, 1–5) that the F 1-ATPase of Micrococcus lysodeikticus is a glycoprotein containing mannose and glucose as the principal sugars. Even after extensive purification of M. lysodeikticus F 1-ATPase by DEAE-Sephadex A25 chromatography, carbohydrate contents varying from 2.7 to 10.8% have been found. Concanavalin A-reactive components corresponding to the succinylated lipomannan have been detected and separated from the ATPase in purified F 1 preparations by immunoelectrophoresis (rocket and two-dimensional) through agarose gels containing concanavalin A. Passage of the purified F 1-ATPase through concanavalin A-Sepharose 4B columns removed the carbohydrate component(s) without loss of the specific activity of the ATPase. Mannose was the only sugar detectable by gas-liquid chromatography of the F 1-ATPase before Con A-Sepharose 4B chromatography and it was completely eliminated after chromatography. No qualitative or quantitative changes in the subunit (, β, γ, δ and ε) profiles were detectable when the sodium dodecyl sulfate polyacrylamide gels were scanned by densitometry of F 1-ATPase before and after Con A-Sepharose 4B chromatography. We conclude that there is no evidence of carbohydrate covalently linked to this F 1-ATPase and that this membrane protein is not a glycoprotein. The presence of carbohydrate is attributable to contamination with lipomannan. 相似文献
7.
Specific mgi mutations in the α, β or γ subunits of the mitochondrial F 1-ATPase have previously been found to suppress ρ 0 lethality in the petite-negative yeast Kluyveromyces lactis. To determine whether the suppressive activity of the altered F 1 is dependent on the F 0 sector of ATP synthase, we isolated and disrupted the genes KlATP4, 5 and 7, the three nuclear genes encoding subunits b, OSCP and d. Strains disrupted for any one, or all three of these genes are
respiration deficient and have reduced viability. However a strain devoid of the three nuclear genes is still unable to lose
mitochondrial DNA, whereas a mgi mutant with the three genes inactivated remains petite-positive. In the latter case, ρ 0 mutants can be isolated, upon treatment with ethidium bromide, that lack six major F 0 subunits, namely the nucleus-encoded subunits b, OSCP and d, and the mitochondrially encoded Atp6, 8 and 9p. Production of
ρ 0 mutants indicates that an F 1-complex carrying a mgi mutation can assemble in the absence of F 0 subunits and that suppression of ρ 0 lethality is an intrinsic property of the altered F 1 particle.
Received: 7 April 1998 / Accepted: 10 June 1998 相似文献
8.
(1) a photoactivable derivative of 5-adenylyl imidodiphosphate (Ado PP[NH] P), was synthesized. (2) Binding of 3H]NAP 4-Ado PP[NH] P to soluble ATPase from beef heart mitochrondria (F 1) was studied in the absence of photoirradiation, and compared to that of . The photoactivable derivative of Ado PP[NH] P was found to bind to F 1 with high affinity, like Ado PP[NH] P. Once had bound to F 1 in the dark, it could be released by Ado PP[NH] P, ADP and ATP, but not at all by NAP 4 or AMP. Furthermore, preincubation of F 1 with unlabeled Ado PP[NH] P, ADP, or ATP prevented the covalent labeling of the enzyme by upon photoirradiation. (3) Photoirradiation of F 1 by resulted in covalent photolabeling and concomitant inactivation of the enzyme. Full inactivation corresponded to the binding of about 2 mol . Photolabeling by NAP 4-Ado PP[NH] P was much more efficient in the presence than in the absence of MgCl 2. (4) Bound was localized on the α- and β-subunits of F 1. At low concentrations (less than 10 μM), bound was predominantly localized on the α-subunit; at concentrations equal to, or greater than 75 μM, both α- and β-subunits were equally labeled. (5) The extent of inactivation was independent of the nature of the photolabeled subunit (α or β), suggesting that each of the two subunits, α and β, is required for the activity of F 1. (6) The covalently photolabeled F 1 was able to form a complex with aurovertin, as does native F 1. The ADP-induced fluorescence enhancement was more severely inhibited than the fluorescence quenching caused by ATP. The percentage of inactivation of F 1 was virtually the same as the percentage of inhibition of the ATP-induced fluorescence quenching, suggesting that fluorescence quenching is related to the binding of ATP to the catalytic site of F 1. 相似文献
9.
Interaction of mitochondrial F 1-ATPase with the isolated natural inhibitor protein resulting in the inhibition of multi-site ATP hydrolysis is accompanied by the loss of activity at low ATP concentrations when single-site hydrolysis should occur. Catalytic site occupancy by [ 14C]nucleotides in F 1-ATPase during steady-state [ 14C]ATP hydrolysis, which is saturated in parallel with single-site catalysis, is prevented after blocking the enzyme with the inhibitor protein. 相似文献
12.
F 1-ATPase catalyzes ATP hydrolysis to drive the central γ-shaft rotating inside a hexameric cylinder composed of alternating α and β subunits. Experiments showed that the rotation of γ-shaft proceeds in steps of 120° and each 120°-rotation is composed of an 80° substep and a 40° substep. Here, based on the previously proposed models, an improved physical model for chemomechanical coupling of F 1-ATPase is presented, with which the two-substep rotation is well explained. One substep is driven by the power stroke upon ATP binding, while the other one resulted from the passage of γ-shaft from previous to next adjacent β subunits via free diffusion. Using the model, the dynamics and kinetics of F 1-ATPase, such as the rotating time of each substep, the dwell time at each pause and the rotation rate, are analytically studied. The theoretical results obtained with only three adjustable parameters reproduce the available experimental data well. 相似文献
13.
F 1-ATPase (F 1) is a reversible ATP-driven rotary motor protein. When its rotary shaft is reversely rotated, F 1 produces ATP against the chemical potential of ATP hydrolysis, suggesting that F 1 modulates the rate constants and equilibriums of catalytic reaction steps depending on the rotary angle of the shaft. Although the chemomechanical coupling scheme of F 1 has been determined, it is unclear how individual catalytic reaction steps depend on its rotary angle. Here, we report direct evidence that the ATP-binding rate of F 1 increases upon the forward rotation of the rotor, and its binding affinity to ATP is enhanced by rotation. 相似文献
14.
Synthesis of enzyme-bound ATP was demonstrated with purified TF 1 (F 1-ATPase from thermophilic bacterium PS3) from medium inorganic phosphate (P i) and enzyme-bound ADP in the presence of organic solvents such as dioxane, ethanol, dimethylformamide, methanol, acetone, acetonitrile or ethyleneglycol. The optimal concentrations of dimethylformamide, ethanol or methanol were 50%, 30% and 40% and the half-maximal concentrations of P i were 13 mM, 20 mM and 18 mM, respectively. Thus it is evident that the effect of dimethylsulfoxide on TF 1 to form enzyme-bound ATP [8] is not due to a specific interaction between dimethylsulfoxide and the enzyme, but to a decrease in polarity of the medium. In the presence of methanol, the dependence of ATP synthesis on various divalent metal ions was compared to that for the ATP-hydrolyzing activity and the ATP-driven proton-translocating activity of TF 1. While Mn 2+, Co 2+, Zn 2+ and Cd 2+ are as effective as Mg 2+ for the ATP-hydrolyzing activity of TF 1, Zn 2+ and Cd 2+ are either less or not effective for proton translocation and for ATP synthesis. This result appears to be consistent with the idea that the TF 1-ATP complex formed in organic solvents represents one of the intermediates in the reaction sequences of ATP synthesis by H +-ATPase using the proton gradient. 相似文献
15.
The expression, localization and activity of the serum- and glucocorticoid-induced protein kinase, Sgk-1, are regulated by multiple hormonal and environmental cues including cellular stress. Biochemical fractionation and indirect immunofluorescence demonstrated that sorbitol induced hyperosmotic stress stimulated expression and triggered the localization of endogenous Sgk-1 into the mitochondria of NMuMG mammary epithelial cells. The immunofluorescence pattern of endogenous Sgk-1 was similar to that of a green fluorescent linked fusion protein linked to the N-terminal Sgk-1 fragment that encodes the mitochondrial targeting signal. In the presence or absence of cellular stress, exogenously expressed wild type Sgk-1 efficiently compartmentalized into the mitochondria demonstrating the mitochondrial import machinery per se is not stressed regulated. Co-immunoprecipitation and GST-pull down assays identified the IF-1 mitochondrial matrix inhibitor of the F 1F 0-ATPase as a new Sgk-1 binding partner, which represents the first observed mitochondrial target of Sgk-1. The Sgk-1/IF-1 interaction requires the 122-176 amino acid region within the catalytic domain of Sgk-1 and is pH dependent, occurring at neutral pH but not at slightly acidic pH, which suggests that this interaction is dependent on mitochondrial integrity. An in vitro protein kinase assay showed that the F 1F 0-ATPase can be directly phosphorylated by Sgk-1. Taken together, our results suggest that stress-induced Sgk-1 localizes to the mitochondria, which permits access to physiologically appropriate mitochondrial interacting proteins and substrates, such as IF-1 and the F 1F 0-ATPase, as part of the cellular stressed induced program. 相似文献
16.
Sepsis and ensuing multiple organ failure continue to be the most leading cause of death in critically ill patients. Despite hepatocyte-related dysfunctions such as necrosis, apoptosis as well as mitochondrial damage are observed in the process of sepsis, the molecular mechanism of pathogenesis remains uncertain. We recently identified one of the differentially expressed genes, mitochondrial ATPase inhibitor protein (IF1) which is down-regulated in late septic liver. Hence, we further hypothesized that the variation of IF1 protein may be one of the causal events of the hepatic dysfunction during late sepsis. The results showed that the elevated mitochondrial F 0F 1-ATPase activity is concomitant with the decline of intramitochondrial ATP concentration in late septic liver. In addition, the key finding of this study showed that the mRNA and the mitochondrial content of IF1 were decreased in late sepsis while no detectable IF1 was found in cytoplasm. When analyzed by immunoprecipitation, it seems reasonable to imply that the association capability of IF1 with F 1-ATPase β-subunit is not affected. These results confirm the first evidence showing that the suppression of IF1 expression and subsequent elevated mitochondrial F 0F 1-ATPase activity might contribute to the bioenergetic failure in the liver during late sepsis. 相似文献
17.
A method has been developed to allow the level of F 0F 1ATP synthase capacity and the quantity of IF 1 bound to this enzyme be measured in single biopsy samples of goat heart. ATP synthase capacity was determined from the maximal mitochondrial ATP hydrolysis rate and IF 1 content was determined by detergent extraction followed by blue native gel electrophoresis, two-dimensional SDS-PAGE and immunoblotting with anti-IF 1 antibodies.Anaesthetized open-chest goats were subjected to ischemic preconditioning and/or sudden increases of coronary blood flow (CBF) (reactive hyperemia). When hyperemia was induced before ischemic preconditioning, a steep increase in synthase capacity, followed by a deep decrease, was observed. In contrast, hyperemia did not affect synthase capacity when applied after ischemic preconditioning. Similar effects could be produced in vitro by treatment of heart biopsy samples with anoxia (down-regulation of the ATP synthase) or high-salt or high-pH buffers (up-regulation). We show that both in vitro and in vivo the same close inverse correlation exists between enzyme activity and IF 1 content, demonstrating that under all conditions tested the only significant modulator of the enzyme activity was IF 1. In addition, both in vivo and in vitro, 1.3-1.4 mol of IF 1 was predicted to fully inactivate 1 mol of synthase, thus excluding the existence of significant numbers of non-inhibitory binding sites for IF 1 in the F 0 sector. 相似文献
19.
(1) Incubation of the beef heart mitochondrial ATPase, F 1 with Mg-ATP was required for the binding of the natural inhibitor, IF 1, to F 1 to form the inactive F 1-IF 1 complex. When F 1 was incubated in the presence of [ 14C]ATP and MgCl 2, about 2 mol 14C-labeled adenine nucleotides were found to bind per mol of F 1; the bound 14C-labeled nucleotides consisted of [ 14C]ADP arising from [ 14C]ATP hydrolysis and [ 14C]ATP. The 14C-labeled nucleotide binding was not prevented by IF 1. These data are in agreement with the idea that the formation of the F 1-IF 1 complex requires an appropriate conformation of F 1. (2) The 14C-labeled adenine nucleotides bound to F 1 following preincubation of F 1 with Mg-[ 14C]ATP could be exchanged with added [ 3H]ADP or [ 3H]ATP. No exchange occurred between added [ 3H]ADP or [ 3H]ATP and the 14C-labeled adenine nucleotides bound to the F 1-IF 1 complex. These data suggest that the conformation of F 1 in the isolated F 1-IF 1 complex is further modified in such a way that the bound 14C-labeled nucleotides are no longer available for exchange. (3) 32P i was able to bind to isolated F 1 with a stoichiometry of about 1 mol of P i per mol of F 1 (Penefsky, H.S. (1977) J. Biol. Chem. 252, 2891–2899). There was no binding of 32P i to the F 1-IF 1 complex. Thus, not only the nucleotides sites, but also the P i site, are masked from interaction with external ligands in the isolated F 1-IF 1 complex. 相似文献
20.
1. When irradiated 8-azido-ATP becomes covalently bound (as the nitreno compound) to beef-heart mitochondrial ATPase (F 1) as the triphosphate, either in the absence or presence of Mg 2+, label covalently bound is not hydrolysed. 2. In the presence of Mg2+ the nitreno-ATP is bound to both the and β subunits, mainly (63%) to the subunits. 3. After successive photolabelling of F1 with 8-azido-ATP (no Mg2+) and 8-azido-ADP (with Mg2+) 4 mol label is bound to F1, 2 mol to the and 2 mol to the β subunits. 4. When the order of photolabelling is reversed, much less 8-nitreno-ATP is bound to F1 previously labelled with 8-nitreno-ADP. It is concluded that binding to the -subunits hinders binding to the β subunits. 5. F1 that has been photolabelled with up to 4 mol label still contains 2 mol firmly bound adenine nucleotides per mol F1. 6. It is concluded that at least 6 sites for adenine nucleotides are present in isolated F1. 相似文献
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