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)

Beef heart mitochondrial ATPase (F1) exhibited a single binding site for Pi. The interaction with Pi was reversible, partially dependent on the presence of divalent metal ions, and characterized by a dissociation constant at pH 7.5 of 80 micronM. A variety of substances known to influence oxidative phosphorylation or the activity of the soluble ATPase (F1) also influenced Pi binding by the enzyme. Thus aurovertin, an inhibitor of oxidative phosphorylation, which was bound tightly by F1 and inhibited ATPase activity, enhanced Pi binding via a 4-fold increase in the affinity of the enzyme for Pi (KD = 20 micronM) but did not alter binding stoichiometry. Anions such as SO4(2-), SO3(2-), chromate, and 2,4-dinitrophenolate, which stimulated ATPase activity of F1, also enhanced Pi binding. Inhibitors of ATPase activity such as nickel/bathophenanthroline and the protein ATPase inhibitor of Pullman and Monroy (Pullman, M. E., and Monroy, G. C. (1963) J. Biol. Chem. 238, 3762-3769) inhibited Pi binding. The adenine nucleotides ADP, ATP, and the ATP analog adenylyl imidodiphosphate as well as the Pi analog arsenate, also inhibited Pi binding. The observations suggest that the Pi binding site was located in or near an adenine nucleotide binding site on the molecule.
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PMID:Reversible binding of Pi by beef heart mitochondrial adenosine triphosphatase. 1 6

We demonstrate the use of immobilized metal ion affinity chromatography for isolating the constituent subunits of assembled mitochondrial ATP synthase (mtATPase) wherein a single subunit of the complex has been modified to contain hexahistidine. Genes encoding subunit d or OSCP of mtATPase from Saccharomyces cerevisiae were modified each to encode a polypeptide having a C-terminal addition of six consecutive histidines. Expression of plasmid-borne modified genes, in host yeast cells lacking a functional copy of the relevant endogenous gene, generated functional mtATPase complexes as judged by growth of rescued cells on the nonfermentable substrate ethanol. Significantly, the oligomycin-sensitive ATP hydrolase activity in mitochondria from cells expressing tagged subunits was similar to that of cells expressing unmodified subunits, indicating that there had been no impairment of the functional integrity of mtATPase. Mitochondrial lysates were prepared from each strain and subjected to chromatography under nondenaturing conditions on a resin containing immobilized Ni2+. It is likely that the mtATPase complexes adsorbed by immobilized metal ion affinity chromatography are fully assembled because their subunit composition closely matches that of a preparation of assembled mtATPase conventionally isolated from mitochondrial lysates by ammonium sulfate precipitation and purification by sucrose gradient centrifugation. Furthermore, assembled mtATPase containing a tagged subunit could be adsorbed, albeit at lower yield, when the relevant modified gene was expressed in wild-type host cells. The general application of this novel isolation procedure greatly simplifies and reduces the number of steps required for the isolation of assembled multi-subunit complexes. Moreover, the approach may be used for studying subunit-subunit interactions within the mtATPase complex.
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PMID:Entrapment by immobilized metal ion affinity chromatography of assembled yeast mitochondrial ATP synthase containing individual subunits tagged with hexahistidine. 866 May 78

An engineered gamma subunit of Escherichia coli F1-ATPase with extra 14 and 20 amino acid residues at the N- and C-termini (His-tag gamma), respectively, was overproduced in E. coli and purified. Six histidines are included in the C-terminal extension. The reconstituted F1 containing alpha, beta, and His-tagged gamma exhibited sixty percent of the wild-type ATPase activity. The reconstituted alphabeta His-tag gamma complex was subjected to affinity chromatography with nickel-nitrilotriacetic acid (Ni-NTA) agarose resin. ATPase activity was eluted specifically with imidazole. These results implied that the tag sequence protruded to the surface of the complex and did not seriously impair the activity. The reconstituted alphabeta His-tag gamma complex, even after its binding to the resin, exhibited ATPase activity suggesting that the gamma subunit, when fixed to a solid phase, may rotate the alphabeta complex. This system may provide a new approach for analysis of the rotation mechanisms in F1-ATPase.
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PMID:Reconstitution of F1-ATPase activity from Escherichia coli subunits alpha, beta and subunit gamma tagged with six histidine residues at the C-terminus. 961 1

The membrane topology of the a subunit of the F1F0 ATP synthase from Escherichia coli has been probed by surface labeling using 3-(N-maleimidylpropionyl) biocytin. Subunit a has no naturally occurring cysteine residues, allowing unique cysteines to be introduced at the following positions: 8, 24, 27, 69, 89, 128, 131, 172, 176, 196, 238, 241, and 277 (following the COOH-terminal 271 and a hexahistidine tag). None of the single mutations affected the function of the enzyme, as judged by growth on succinate minimal medium. Membrane vesicles with an exposed cytoplasmic surface were prepared using a French pressure cell. Before labeling, the membranes were incubated with or without a highly charged sulfhydryl reagent, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid. After labeling with the less polar biotin maleimide, the samples were solubilized with octyl glucoside/cholate and the subunit a was purified via the oligohistidine at its COOH terminus using immobilized nickel chromatography. The purified samples were electrophoresed and transferred to nitrocellulose for detection by avidin conjugated to alkaline phosphatase. Results indicated cytoplasmic accessibility for residues 69, 172, 176, and 277 and periplasmic accessibility for residues 8, 24, 27, and 131. On the basis of these and earlier results, a transmembrane topology for the subunit a is proposed.
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PMID:Membrane topology of subunit a of the F1F0 ATP synthase as determined by labeling of unique cysteine residues. 963 82

In the mitochondrial ATP synthase (mtATPase) of the yeast Saccharomyces cerevisiae, the stoichiometry of subunits d, oligomycin-sensitivity conferring protein (OSCP), and b is poorly defined. We have investigated the stoichiometry of these subunits by the application of hexahistidine affinity purification technology. We have previously demonstrated that intact mtATPase complexes incorporating a Hex6-tagged subunit can be isolated via Ni2+-nitrilotriacetic acid affinity chromatography (Bateson, M., Devenish, R. J., Nagley, P., and Prescott, M. (1996) Anal. Biochem. 238, 14-18). Strains were constructed in which Hex6-tagged versions of subunits d, OSCP, and b were coexpressed with the corresponding wild-type subunit. This coexpression resulted in a mixed population of mtATPase complexes containing untagged wild-type and Hex6-tagged subunits. The stoichiometry of each subunit was then assessed by determining whether or not the untagged wild-type subunit could be recovered from Ni2+-nitrilotriacetic acid purifications as an integral component of those complexes absorbed by virtue of the Hex6-tagged subunit. As only the Hex6-tagged subunit was recovered from such purifications, we demonstrate that the stoichiometry of subunits d, OSCP, and b in yeast is 1 in each case.
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PMID:Single copies of subunits d, oligomycin-sensitivity conferring protein, and b are present in the Saccharomyces cerevisiae mitochondrial ATP synthase. 1006 11

The cooCTJ gene products are coexpressed with CO-dehydrogenase (CODH) and facilitate in vivo nickel insertion into CODH. A Ni(2+) transport assay was used to monitor uptake and accumulation of (63)Ni(2+) into R. rubrum and to observe the effect of mutations in the cooC, cooT, and cooJ genes on (63)Ni(2+) transport and accumulation. Cells grown either in the presence or absence of CO transported Ni(2+) with a K(m) of 19 +/- 4 microM and a V(max) of 310 +/- 22 pmol of Ni/min/mg of total protein. Insertional mutations disrupting the reading frame of the cooCTJ genes, either individually or all three genes simultaneously, transported Ni(2+) the same as wild-type cells. The nickel specificity for transport was tested by conducting the transport assay in the presence of other divalent metal ions. At a 17-fold excess Mn(2+), Mg(2+), Ca(2+), and Zn(2+) showed no inhibition of (63)Ni(2+) transport but Co(2+), Cd(2+), and Cu(2+) inhibited transport 35, 58, and 66%, respectively. Nickel transport was inhibited by cold (50% at 4 degrees C), by protonophores (carbonyl cyanide m-chlorophenylhydrazone, 44%, and 2,4-dinitrophenol, 26%), by sodium azide (25%), and hydroxyl amine (33%). Inhibitors of ATP synthase (N, N'-dicyclohexylcarbodiimide and oligomycin) and incubation of cells in the dark stimulated Ni(2+) transport. (63)Ni accumulation after 2 h was four times greater in CO-induced cells than in cells not exposed to CO. The CO-stimulated (63)Ni(2+) accumulation coincided with the appearance of CODH activity in the culture, suggesting that the (63)Ni(2+) was accumulating in CODH. The cooC, cooT, and cooJ genes are required for the increased (63)Ni(2+) accumulation observed upon CO exposure because cells containing mutations disrupting any or all of these genes accumulated (63)Ni(2+) like cells unexposed to CO.
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PMID:Ni(2+) transport and accumulation in Rhodospirillum rubrum. 1041 53

The addition of a His6 tag to the N terminus of subunit a of the F0 complex of the Escherichia coli ATP synthase allowed the purification of an ab2 subcomplex after solubilization of membranes with n-dodecyl-beta-d-maltoside and subsequent nickel-nitrilotriacetic acid affinity chromatography. After co-reconstitution of the ab2 subcomplex with purified subunit c, passive proton translocation rates as well as coupled ATPase activities after binding of F1 were measured that were comparable with those of wild type F0. The interaction between subunits a and b, which has been shown to be stoichiometric and functional, is not triggered by any cross-linking reagent and therefore reflects subunit interactions occurring within the F0 complex in vivo.
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PMID:Direct interaction of subunits a and b of the F0 complex of Escherichia coli ATP synthase by forming an ab2 subcomplex. 1272 21

In Escherichia coli the peripheral stalk of F1F0-ATP synthase consists of a parallel dimer of identical b subunits. However, the length of the two b subunits need not be fixed. This led us to ask whether it is possible for two b subunits of unequal length to dimerize in a functional enzyme complex. A two-plasmid expression system has been developed that directs production of b subunits of unequal lengths in the same cell. Two b subunits differing in length have been expressed with either a histidine or V5 epitope tag to facilitate nickel-affinity resin purification (Ni-resin) and Western blot analysis. The epitope tags did not materially affect enzyme function. The system allowed us to determine whether the different b subunits segregate to form homodimers or, conversely, whether a heterodimer consisting of both the shortened and lengthened b subunits can occur in an intact enzyme complex. Experiments expressing different b subunits lengthened and shortened by up to 7 amino acids were detected in the same enzyme complex. The V5-tagged b subunit shortened by 7 amino acids (b Delta 7-V5) was detected in Ni-resin-purified membrane preparations only when coexpressed with a histidine-tagged b subunit in the same cell. The results demonstrate that the enzyme complex can tolerate a size difference between the two b subunits of up to 14 amino acids. Moreover, the experiments demonstrated the feasibility of constructing enzyme complexes with non-identical b subunits that will be valuable for research requiring specific chemical modification of a single b subunit.
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PMID:Integration of b subunits of unequal lengths into F1F0-ATP synthase. 1284 3

The green alga Chlamydomonas reinhardtii is a model organism for the study of photosynthesis. The chloroplast ATP synthase is responsible for the synthesis of ATP during photosynthesis. Using genetic engineering and biolistic transformation, a string of eight histidine residues has been inserted into the amino-terminal end of the beta subunit of this enzyme in C. reinhardtii. The incorporation of these amino acids did not impact the function of the ATP synthase either in vivo or in vitro and the resulting strain of C. reinhardtii showed normal growth. The addition of these amino acids can be seen through altered gel mobility of the beta subunit and the binding of a polyhistidine-specific dye to the subunit. The purified his-tagged CF1 has normal Mg(2+)-ATPase activity, which can be stimulated by alcohol and detergents and the enzyme remains active while bound to a nickel-coated surface. Potential uses for this tagged enzyme as a biochemical tool are discussed.
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PMID:Expression by Chlamydomonas reinhardtii of a chloroplast ATP synthase with polyhistidine-tagged beta subunits. 1746 33

The effects of 10 microM cadmium, copper and nickel on the activities of vacuolar membrane and plasma membrane (PM) ATP-dependent proton pumps was investigated in Cucumis sativus L. root cells. It was demonstrated that vacuolar H+-ATPase (EC 3.6.3.14) and PM H+-ATPase (EC 3.6.3.6) differed in sensitivity to heavy metals. Exposure of cucumber seedlings to Cd, Cu and Ni had no significant effect on the activity of the vacuolar proton pump and, in the case of Ni, also on the activity of the PM proton pump. In contrast, Cd and Cu ions diminished both ATP hydrolysis and proton transport in plasma membranes. Transcript levels of genes encoding PM enzyme as well as the subunit A of tonoplast enzyme in roots stressed with heavy metals were similar to the control. Cd, Cu and Ni were accumulated in cucumber roots with similar efficiency, but their relative distribution between the symplast and apoplast differed. To explain the mechanism of heavy metal action on the plasma membranes of cucumber roots, the MDA content, as a lipid peroxidation product, and fatty acid composition were analyzed. It was shown that exposure of plants to Cd, Cu and Ni did not enhance the lipid peroxidation in the PM fraction. However, all metals caused an increase in the saturation of PM fatty acids and a decrease in the length of the fatty acid chain.
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PMID:Comparison of heavy metal effect on the proton pumps of plasma membrane and tonoplast in cucumber root cells. 1765 57


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