Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.3.14 (ATP synthase)
7,042 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bovine heart submitochondrial particles depleted of F1 by treatment with urea ("F1-depleted particles') were incubated with soluble F1-ATPase. The binding of F1 to the particles and the concomitant conferral of oligomycin sensitivity on the ATPase activity required the presence of cations in the incubation medium. NH4+, K+, Rb+, Na+ and Li+ promoted reconstitution maximally at 40-74 mM, guanidinium+ and Tris+ at 20-30 mM, and Ca2+ and Mg2+ at 3-5 mM. The particles exhibited a negative zeta-potential, as determined by microelectrophoresis, and this was neutralized by mono- and divalent cations in the same concentration range as that needed to promote F1 binding and reconstitution of oligomycin-sensitive ATPase. It is concluded that the cations act by neutralizing negative charges on the membrane surface, mainly negatively charged phospholipids. These results are discussed in relation to earlier findings reported in the literature with F1-depleted thylakoid membranes and with submitochondrial particles depleted of both F1 and the coupling proteins F6 and oligomycin sensitivity-conferring protein.
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PMID:Cation requirement for the reconstitution of oligomycin-sensitive ATPase by means of soluble F1-ATPase and F1-depleted submitochondrial particles. 621 97

A sodium dodecyl sulfate (SDS)-urea polyacrylamide gel system was used to investigate certain properties of the subunits of the beef heart mitochondrial ATPase, (native F1, nF1). By examining the affects of urea concentration and acrylamide concentration upon the electrophoretic mobilities of the polypeptides comprising the nF1 enzyme, we have obtained conditions under which all five subunits are simultaneously resolved when the discontinuous buffer system of Laemmli is used (U. K. Laemmli (1970) Nature (London) 277, 680-685). The determination of the apparent molecular weights by analysis of Ferguson plots (K. A. Ferguson (1964) Metabolism 13, 985-1002) revealed that the addition of urea to the SDS gels resulted in a decrease in the apparent molecular weight of the beta subunit. A dramatic increase in the apparent molecular weight of the delta subunit was also brought about by the presence of urea in the SDS gels. In addition, the apparent molecular weight of both the alpha and the beta subunits was dependent upon the acrylamide concentration used, indicating that these subunits contain either areas highly resistant to denaturation by the combined action of urea and SDS, or covalent modifications leading to anomalous electrophoretic mobility. The results of experiments in which urea analogs were used indicate that the interactions of urea with the beta subunit involve the formation of hydrogen bonds between urea and regions of this subunit. On the other hand, the interactions of urea with the delta subunit are primarily of a hydrophobic nature, suggesting that these interactions could involve domains of the delta subunit required for binding of the coupling factor to the mitochondrial membrane.
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PMID:Hydrophobic and ionic effects upon the electrophoretic mobilities of the subunits of coupling factor 1 from mitochondria. 623 67

A rapid method for the preparative purification of the subunits of oligomeric proteins like chloroplast and Rhodospirillum rubrum coupling factors is presented. It involves the dissociation of the protein in urea and the separation of its subunits by isoelectric focusing in flat-beds of Sepharose CL-4B or Sephadex G-75 superfine, in the presence of urea and in an overnight run. Using this procedure in the pH range 5-7, we have purified to homogeneity the alpha, beta and delta subunits of chloroplast coupling factor, as well as the alpha and beta subunits of Rhodospirillum rubrum coupling factor. The full separation of the gamma and epsilon subunits of chloroplast coupling factor, which focused at the same pH, was achieved by gel filtration high-performance liquid chromatography.
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PMID:Preparative purification of the subunits of chloroplast and Rhodospirillum rubrum coupling factors by flat-bed electrofocusing in granulated gels. 624 May 6

An N-ethylmaleimide-sensitive phosphate transport protein has been isolated from rat liver mitochondria, substantially purified, and reconstituted into phospholipid vesicles. Purified inner mitochondrial membrane vesicles depleted of F1-ATPase by urea treatment proved to be the most satisfactory starting material. Treatment of these membrane vesicles with Triton X-100 resulted in solubilization of the phosphate transport protein. Further purification was achieved using hydroxylapatite powder. Polyacrylamide gel electrophoresis of the purified fraction in sodium dodecyl sulfate indicated the presence of two Coomassie blue-staining bands with apparent Mr's of 30,000 and 35,000. Labeling of the 35,000 Mr band by the Pi transport inhibitor diazobenzene sulfonate was reduced markedly by prior treatment of the mitochondria with the inhibitor N-ethylmaleimide. The purified fraction containing both proteins could be reconstituted into liposomes prepared from purified asolectin. Phosphate efflux from these vesicles was inhibited by N-ethylmaleimide, by the impermeant mercurial agent, p-chloromercuribenzoate, and by diazobenzene sulfonate. Treatment of the purified fraction with N-ethylmaleimide prior to incorporation into liposomes resulted in a reconstituted system incapable of catalyzing Pi efflux. These studies summarize the first detailed attempt to purify the Pi/H+ transport system from rat liver mitochondria and emphasize the need to commence the purification with purified inner membrane vesicles depleted of F1-ATPase. In addition, these studies show that the final fraction contains a reconstitutively active transport system which when incorporated into phospholipid vesicles has its essential sulfhydryl groups oriented outward. Finally, it is shown that the purified fraction also contains a 30,000 Mr component.
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PMID:Isolation and reconstitution of an N-ethylmaleimide-sensitive phosphate transport protein from rat liver mitochondria. 630 81

The H+-translocating ATPase from rat liver mitochondria can be disaggregated selectively to yield two distinct, stable complexes of the rutamycin-insensitive ATPase. The two ATPase complexes can be purified to homogeneity by zone sedimentation in a glycerol gradient. Based on their electrophoretic mobility in 5% polyacrylamide gels, the aggregates have been designated as type I (Rf = 0.49) ATPase and type II (Rf = 0.56) ATPase. These two complexes of the ATPase differ in ATP hydrolytic activity, in stability, in mobility on 5% polyacrylamide gel electrophoresis, in subunit composition, and in ability to reassociate with submitochondrial particles which are highly depleted in ATPase activity. The type II ATPase is similar to the F1-ATPase, but the type I ATPase contains a 26.5-kilodalton subunit not present in the type II enzyme. This 26.5-kilodalton subunit is equimolar with the gamma subunit of the ATPase (based on Coomassie blue dye binding); its presence seems to be correlated to the altered properties of the type I ATPase. Type I ATPase reconstitutes rutamycin-sensitive ATPase activity in submitochondrial particles treated with trypsin, urea, ammonia, and 1.5% silicotungstic acid. The type II ATPase does not reconstitute rutamycin-sensitive ATPase activity in these ATPase-depleted submitochondrial particles unless it is supplemented with the 26.5-kilodalton subunit isolated from the type I ATPase. The 26.5-kilodalton protein has thus been functionally identified as important for the binding of the ATPase to the membrane by providing a direct link to the membrane or by binding to the ATPase putting it in an appropriate conformation for binding.
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PMID:Selective disaggregation of the H+-translocating ATPase. Isolation of two discrete complexes of the rutamycin-insensitive ATPase differing in mitochondrial membrane-binding properties. 645 Feb 7

1. The subunit stoicheiometry of mitochondrial F1-ATPase from yeast (Saccharomyces carlsbergensis), grown in the presence of [3H]leucine and uniformly labelled [14C]glucose, has been determined. 2. The stoicheiometry on the basis of radioactivity is : alpha: beta: gamma: epsilon = 3 : 3 : 1 : 1. The amount of the smallest subunit, epsilon, could not be measured by this method. 3. The molecular weights of the subunits, determined by urea-SDS gel electrophoresis, are 53 000, 50 000, 33 000, 12 500 and 6500, respectively. The calculated molecular weight of the ATPase is 360 000, assuming the presence of one epsilon subunit per F1. 4. The amino acid composition of the total ATPase and of the individual subunits has been determined. 5. The aurovertin-binding properties of F1 are discussed in relation to the subunit stoicheiometry.
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PMID:Subunit composition of mitochondrial F1-ATPase isolated from Saccharomyces carlsbergensis. 645 Dec 40

To better understand the early events of the mitochondrial protein import process, we purified the precursor of the F1-ATPase beta subunit (pre-F1 beta) and examined its import into isolated mitochondria. Import of purified urea-denatured pre-F1 beta did not require cytosolic factors. However, the period of productive import was prolonged by the addition of reticulocyte lysate, suggesting that cytosolic factors such as molecular chaperones were acting to extend the period of import competence of pre-F1 beta. Purified pre-F1 beta bound extensively to both cardiolipin-containing liposomes and to intact mitochondria, indicating that a direct interaction between mitochondrial precursors and the mitochondrial outer membrane surface can occur. The ability to chase this surface-bound pre-F1 beta into mitochondria suggests that precursors bound to the mitochondrial surface can be maintained in an import competent conformation. Finally, our defined mitochondrial import system was used to characterize the ATP requirements of pre-F1 beta import in the absence of cytosol. We found a strong requirement for ATP on both sides of the mitochondrial inner membrane, suggesting that one or more previously undetected mitochondrial proteins outside the inner membrane utilize ATP to promote efficient pre-F1 beta import.
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PMID:Characterization of the mitochondrial binding and import properties of purified yeast F1-ATPase beta subunit precursor. Import requires external ATP. 812 31

The soluble ATPase purified from an aerobic thermophilic eubacterium, Thermus thermophilus, was not a usual F1-ATPase but a V1-ATPase, a peripheral section of plasma membrane V-type ATPase (Yokoyama, K., Oshima, T., and Yoshida, M. (1990) J. Biol. Chem. 265, 21946-21950). Here, we report the purification of V-type ATPase from the same bacterium (V0V1-ATPase) which consists of V1-ATPase and a membrane-integrated section, V0. The V0V1-ATPase, either in the Triton X-100-solubilized membrane fraction or in the purified state, migrates as a single band in a non-denaturing polyacrylamide gel electrophoresis for membrane protein complexes, and eight kinds of polypeptides are found when this band is developed in a second dimension denaturing gel electrophoresis in the presence of sodium dodecyl sulfate. The 66-, 56-, 30-, and 12-kDa polypeptides are the subunits of V1-ATPase and the 100-, 38-, 24-, and 13-kDa polypeptides are candidates for V0 (or V0-associated) subunits. The amino-terminal amino acid sequences of the 38- and 24-kDa subunits do not show obvious similarity to any subunits of eukaryotic V0V1-ATPases. The kinetic properties of the purified V0V1-ATPase are very similar to those of V1-ATPase: a very low ATPase activity, stimulation by bisulfite, inhibition by nitrate, and resistance against inhibitors of eukaryotic V-type ATPases, Bafilomycin A1 and N-ethylmaleimide. The V0 vesicles prepared from reconstituted V0V1-ATPase vesicles by 6 M urea treatment show the H+ channel activity. This H+ channel activity is abolished either by treatment of vesicles with dicyclohexylcarbodiimide or by the back addition of V1-ATPase. These results indicate that the coupling ion of this V0V1-ATPase is H+.
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PMID:Isolation of prokaryotic V0V1-ATPase from a thermophilic eubacterium Thermus thermophilus. 816 30

We compare translocation into inside-out plasma membrane vesicles (INV) of the in vitro synthesized outer membrane proteins LamB and OmpA and the periplasmic protein Skp of Escherichia coli and demonstrate a precursor-specific dependence on the export factors SecA, SecB, and the proton-motive force (delta mu H+). A partial reduction in soluble SecA caused a 50% decrease in translocation of preLamB. In contrast, removal of INV-bound SecA by urea extraction was required to see a decrease in translocation of preOmpA and preSkp, with 8% of preSkp still being translocated into urea-treated INV. Translocation of the three precursors into INV showed a corresponding differential sensitivity toward dissipation of delta mu H+ following removal of the F1-ATPase from the INV. While depletion of both F1 and SecA or simply lowering of the reaction temperature resulted in an inhibition of complete transmembrane translocation, it interfered less severely with signal sequence cleavage, indicating the formation of translocation intermediates under these conditions. The relative amounts of intermediate obtained were also different for the three preproteins correlating a low requirement for SecA and delta mu H+ with a facilitated initiation of translocation. Whereas preSkp was translocated independently of SecB, preLamB was not even targeted to the INV in its absence. Functional targeting of preOmpA required the presence of SecB during incubation of the precursor with INV and not during its synthesis. SecB, exogenously added during the period of synthesis, did not prevent the formation of translocation-incompetent preLamB. The latter results are consistent with an important targeting function of SecB, which so far has mostly been described as a molecular chaperone. The findings are discussed with respect to current models of bacterial protein export usually derived from the analysis of a single precursor.
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PMID:Precursor-specific requirements for SecA, SecB, and delta muH+ during protein export of Escherichia coli. 817 98

The yeast ATP synthase subunit d was over-expressed in E. coli and formed inclusion bodies. It was purified by solubilization in urea and slow removal of the urea by stepwise dialysis in the presence of a non-ionic detergent. The resulting soluble subunit d was used to prepare polyclonal antibodies. Blots of yeast mitochondrial proteins were probed with these antibodies. The strain disrupted in ATP4 gene encoding the subunit 4 displayed only 8% of the wild type subunit d. Antibodies against subunit d did not inhibit the wild type ATPase activity.
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PMID:Over-expression of the yeast ATP synthase subunit D in Escherichia coli: use of polyclonal antibodies directed against recombinant subunit D. 817 22


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