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)

The catalytic sector, F1, and the membrane sector, F0, of the mitochondrial ATP synthase complex are joined together by a 45-A-long stalk. Knowledge of the composition and structure of the stalk is crucial to investigating the mechanism of conformational energy transfer between F0 and F1. This paper reports on the near neighbor relationships of the stalk subunits with one another and with the subunits of F1 and F0, as revealed by cross-linking experiments. The preparations subjected to cross-linking were bovine heart submitochondrial particles (SMP) and F1-deficient SMP. The cross-linkers were three reagents of different chemical specificities and different lengths of cross-linking from zero to 10 A. Cross-linked products were identified after gel electrophoresis of the particles and immunoblotting with subunit-specific antibodies to the individual subunits alpha, beta, gamma, delta, OSCP, F6, A6L, a (subunit 6), b, c, and d. The results suggested that the two b subunits form the principal stem of the stalk to which OSCP, d, and F6 are bound independent of one another. Subunits b, OSCP, d, and F6 cross-linked to alpha and/or beta, but not to gamma or delta. The COOH-terminal half of A6L, which is extramembranous, cross-linked to d but not to any other stalk or F1 subunit. No cross-links of subunits a and c with any stalk or F1 subunits were detected. In F1-deficient SMP, cross-linked b+b and d+F6 dimers appeared, and the extent of cross-linking between b and OSCP diminished greatly. The addition of F1 to F1-deficient particles appeared to reverse these changes. Treatment of F1-deficient particles with trypsin rapidly hydrolyzed away OSCP and F6, fragmented b to membrane-bound 18-, 12-, and 8-9-kDa antigenic fragments, which cross-linked to d and/or with one another. Trypsin also removed the COOH-terminal part of A6L, but the remainder still cross-linked to subunit d. Models showing the near neighbor relationships of the stalk subunits with one another and with the alpha and beta subunits at a level near the proximal end (bottom) of F1 and at the membrane-matrix interface are presented.
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PMID:ATP synthase complex. Proximities of subunits in bovine submitochondrial particles. 783 33

The exposure to trypsinolysis of subunits of F1F0-ATPase and of its F0 domain have been compared in everted inner membrane vesicles (submitochondrial particles) made from bovine mitochondria. Treatment of submitochondrial particles with guanidine hydrochloride removed the subunits of F1-ATPase and the oligomycin-sensitivity conferral protein (OSCP), and exposed sites that were occluded in the intact F1F0-ATPase complex. These sites were identified by purifying the subunits from the isolated F0 and F1F0-ATPase complexes before and after proteolysis of the vesicles, and by characterizing them by N-terminal sequencing and electrospray-ionization mass spectrometry. In the stripped vesicles, subunit F6 was completely digested away by either trypsin or chymotrypsin. Trypsin also cleaved subunit b, first at the bond arginine-166-glutamine-167, and then at the consecutive linkages, lysine-120-arginine-121 and arginine-121-histidine-122. Chymotrypsin-sensitive sites were observed after the adjacent methionines 164 and 165. Trypsin also removed amino acids 1-3 of subunit d, and minor cleavage sites were observed in subunit d between amino acids 24 and 25, in subunit g between amino acids 5 and 6, and after amino acid 40 in subunit e. The other subunits remained protected from proteolysis. In membrane-bound F1F0-ATPase, the N-terminus of subunit d was also accessible to trypsin, and subunit e was more susceptible to proteolysis than in F0. Otherwise the F0 subunits and the OSCP were protected. Subunits alpha and beta were cleaved by trypsin at the same sites in their N-terminal regions as in purified F1-ATPase. The trypsinized F0 was incapable of binding F1-ATPase in the presence of the OSCP. These experiments and in vitro re-assembly experiments described elsewehere, that were guided by the results of the proteolysis experiments, have helped to establish a central role for subunit b in the formation of the stalk connecting the F1 and F0 domains of the F1F0-ATPase complex.
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PMID:ATP synthase from bovine heart mitochondria: identification by proteolysis of sites in F0 exposed by removal of F1 and the oligomycin-sensitivity conferral protein. 798 Apr 27

The hydrolytic activity of the chicken liver F1-ATPase is strongly inhibited by detergents. The inhibition is concentration dependent and the highest value is obtained at the critical micelle concentration. Cationic and anionic detergents are good inhibitor of the enzyme, however the inhibition is correlated rather with the micelle size than the charge of the detergent as demonstrated by the inhibition observed with the non-ionic beta-D-alkyl glucosides. The effect of detergents upon subunits interactions was studied by trypsin limited proteolysis. Incubation with cholate results in the proteolytic cleavage of the alpha and gamma subunits whereas in the presence of DTAB also the beta subunit is digested. The small subunits delta and epsilon instead are not accessible when the enzyme is treated with both detergents. The results may suggest that hydrophobic areas of defined size located at the interface among alpha, beta and gamma subunit are crucial for the catalytic activity of the enzyme.
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PMID:Effect of detergents on the chicken liver F1-ATPase: functional and structural aspects. 801 84

Mutant strain AN1518 or AN2387 (Gly48-->Asp in epsilon-subunit) and partial revertant strain AN2540 (Gly48-->Asp, Pro47-->Ser in epsilon-subunit) of E. coli were used in a kinetic study of membrane-bound H(+)-ATPase. It was found that at pH 9.0 mutant strain AN1518 or AN2387 and partial revertant strain AN2540 gave a low initial rate, which increased with time until linearity was reached after 1-2 min. This phenomenon was prominent in mutant strains, but was not so obvious in wild-type AN346 of E. coli; this property is similar to F1-ATPase reported by Cox [1]. The mechanism of the slow activation of membrane-bound H(+)-ATPase was further investigated in this paper. The experimental results indicated that the hydrolytic rate of E. coli F1F0-ATPase that increased with time was membrane protein concentration- and pH-dependent, and that the product ADP produced during ATP hydrolysis is the factor causing the slow activation. Preincubation of the hydrolytic product ADP with a concentration comparable to that produced in the assay (20 microM) caused initial activation of ATP hydrolysis and abolished the slow activation. On the other hand, with the removal of ADP during the progress of the hydrolytic reaction it could be seen that the slow activation was abolished as well. In order to test the relationship between the epsilon-subunit and ADP involved in the slow activation, trypsin treatment was carried out on the membrane-bound H(+)-ATPase of various strains. The activation observed after trypsin treatment was on the order of AN1518 > AN2540 > AN346. The activation effects of ADP and trypsin were not found to be additive. This implies that ADP acted in a similar way to trypsin, i.e., to cause removal of the epsilon-subunit. A tentative mechanism of the slow activation was proposed that ADP, a product of ATP hydrolysis, could induce conformational changes of F1F0 at alkaline pH 9.0, thus weakening the binding strength between the epsilon-subunit and other subunits of F1F0, and resulting in removal or partial removal of the epsilon-subunit. This further impaired the coupling of F1 and F0 in the mutant strains; as a consequence the rate of ATP hydrolysis was increased.
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PMID:Product-activation of Escherichia coli membrane-bound H(+)-ATPase (F1F0-ATPase) connected with epsilon-subunit at alkaline pH. 814 15

Certain forms of ceroid lipofuscinosis, a hereditary degenerative disease, are characterized by accumulation of large amounts of subunit c of mitochondrial ATP synthase in lysosomal storage bodies of numerous tissues. The subunit c protein appears to constitute a major fraction of the total storage body protein. In previous studies it was demonstrated that hydrolysates of total storage body protein from affected humans and sheep contain significant amounts of epsilon-N-trimethyllysine (TML). This finding suggested that one or both of the two lysine residues of subunit c might be methylated in the stored form of the protein. The normal subunit c protein from mitochondria does not appear to be methylated. Using a putative canine model for the juvenile form of ceroid lipofuscinosis, analyses were conducted to determine whether lysosomal storage of subunit c was accompanied by lysine methylation of this protein. In affected dogs, as in humans and sheep with hereditary ceroid lipofuscinosis, the storage bodies were found to contain large amounts of subunit c protein, as indicated by polyacrylamide gel electrophoresis and partial amino acid sequence analysis. The subunit c protein partially purified from isolated storage bodies was found to contain lysine and TML in an almost equimolar ratio. Normal subunit c contains 2 lysine residues, one at position 7 and the other at position 43. Removal of the first 7 residues of the partially purified protein through sequential Edman degradation resulted in a dramatic increase in the TML to lysine ratio in the residual protein. This suggests that lysine residue 43 is methylated. Confirmation that residue 43 of the stored protein is TML was obtained by amino acid sequence analysis after cleavage of the protein with trypsin. This finding strongly suggests that specific methylation of lysine residue 43 of mitochondrial ATP synthase plays a central role in the lysosomal storage of this protein.
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PMID:Lysine methylation of mitochondrial ATP synthase subunit c stored in tissues of dogs with hereditary ceroid lipofuscinosis. 814 84

The structure of the isolated alpha subunit of F1-ATPase from the thermophilic Bacillus strain PS3 was probed using limited proteolysis by four different proteases, and the following results were obtained. 1) Distribution of 21 protease-cleaved sites is similar to that of the beta subunit of F1-ATPase (Tozawa, K., Odaka, M., Date, T., and Yoshida, M. (1992) J. Biol. Chem. 267, 16484-16490), thus providing experimental evidence for similar folding topology of the two subunits, and the locations of 11 water-exposed loop regions in the tertiary structure are predicted. 2) Most proteolytic peptides remain associated to maintain the gross structure of the alpha subunit and can reassociate each other after denaturing urea treatment. 3) However, the carboxyl-terminal peptides comprising approximately 80 residues (C1 peptides) are released from other peptide(s) during proteolysis, and those comprising approximately 105 residues (C2 peptides) are released during native polyacrylamide gel electrophoresis after proteolysis. 4) Inclusion of Mg-ATP in the native electrophoretic system prevents the release of the C2 peptide. Addition of Mg-ATP to the proteolysis mixtures results in an increase of the C2 peptide population and a decrease of the C1 peptide population. Thus, Mg-ATP induces a conformational change at the regions of C1 and C2 peptides of the alpha subunit. 5) Except for the trypsin-treated one, protease-treated alpha subunits are reconstitutable with the native beta subunit into the form of alpha 3 beta 3 complexes, which show significantly higher ATPase activities than the intact alpha 3 beta 3 complex. This activation is attributable to the cleavage of a peptide bond that produces C2 peptides. The carboxyl-terminal region of the alpha subunit is likely to be involved in the regulation of ATPase activity in F1-ATPase.
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PMID:Structure of the alpha subunit of F1-ATPase probed by limited proteolysis. 836 Jan 91

Synthesis of [14C]dequalinium, 1,1'-(1,10-[1,10-14C]decanediyl)bis[4-amino-2-methylquinolinium ], is described, which photoinactivates the bovine heart mitochondrial F1-ATPase (MF1). Maximal photoinactivation occurs on incorporation of about 1.5 mol of [14C]dequalinium/mol of MF1. Three radioactive species were resolved when photoinactivated enzyme was submitted to polyacrylamide gel electrophoresis at pH 4.0 in the presence of tetradecyltrimethylammonium bromide, which correspond to the alpha and beta subunits and a cross-linked species with an M(r) of 116,000. Fractionation of a tryptic digest of photoinactivated enzyme by high-performance liquid chromatography led to isolation of a radioactive peptide which contains residues 399-420 of a alpha subunit. Two fragments containing equal amounts of radioactivity were obtained on fractionation of an endoproteinase Asp-N digest of the isolated radioactive tryptic peptide by high-performance liquid chromatography. Amino acid sequence analysis showed that both fragments contained residues 399-408 of the alpha subunit, but one was missing Phe-alpha 403 and the other was lacking Phe-alpha 406. Fractionation of a cyanogen bromide digest of photoinactivated enzyme followed by trypsin digestion of partially purified cyanogen bromide fragments and fractionation of the resulting radioactive tryptic fragments yielded several radioactive species comprised of residues 399-420 of the alpha subunit cross-linked to residues 440-459 of the beta subunit and a radioactive fragment containing residues 399-420 of the alpha subunit. Partial sequence analyses of the cross-linked fragments suggest that Phe-alpha 403 and Phe-alpha 406 participate in cross-links, whereas no information was obtained on the site or sites of cross-linking in the beta subunit fragment.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Photoinactivation of the bovine heart mitochondrial F1-ATPase by [14C]dequalinium cross-links phenylalanine-403 or phenylalanine-406 of an alpha subunit to a site or sites contained within residues 440-459 of a beta subunit. 844 63

The interaction faces of the gamma and epsilon subunits in the Escherichia coli F1-ATPase have been explored by a combination of cross-linking and chemical modification experiments using several mutant epsilon subunits as follows: epsilonS10C, epsilonH38C, epsilonT43C, epsilonS65C, epsilonS108C, and epsilonM138C, along with a mutant of the gamma subunit, gammaT106C. The replacement of Ser-10 by a Cys or Met-138 by a Cys reduced the inhibition of ECF1 by the epsilon subunit, while the mutation S65C increased this inhibitory effect. Modification of the Cys at position 10 with N-ethylmaleimide or fluoroscein maleimide further reduced the binding affinity of, and the maximal inhibition by, the epsilon subunit. Similar chemical modification of the Cys at position 43 of the epsilon subunit (in the mutant epsilonT43C) and a Cys at position 106 of the gamma subunit (gammaT106C) also affected the inhibition of ECF1 by the epsilon subunit. The various epsilon subunit mutants were reacted with TFPAM3, and the site(s) of cross-linking within the ECF1 complex was determined. Previous studies have shown cross-linking from the Cys at positions 10 and 38 with the gamma subunit and from a Cys at position 108 to an alpha subunit (Aggeler, R., Chicas-Cruz, K., Cai, S. X., Keana, J. F. W., and Capaldi, R. A. (1992) Biochemistry 31, 2956-2961; Aggeler, R., Weinreich, F., and Capaldi, R. A. (1995) Biochim. Biophys. Acta 1230, 62-68). Here, cross-linking was found from a Cys at position 43 to the gamma subunit and from the Cys at position 138 to a beta subunit. The site of cross-linking from Cys-10 of epsilon to the gamma subunit was localized by peptide mapping to a region of the gamma subunit between residues 222 and 242. Cross-linking from a Cys at position 38 and at position 43 was with the C-terminal part of the gamma subunit, between residues 202 and 286. ECF1 treated with trypsin at pH 7.0 still binds purified epsilon subunit, while enzyme treated with the protease at pH 8.0 does not. This identifies sites around residue 70 and/or between 202 and 212 of the gamma subunit as involved in epsilon subunit binding.
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PMID:Characterization of the interface between gamma and epsilon subunits of Escherichia coli F1-ATPase. 862 95

At least part of the gamma subunit of the catalytic portion of the chloroplast ATP synthase (CF1) is present in the middle of the alpha3beta3 heterohexamer. Interactions of the alpha/beta subunits with the gamma subunit stabilize the hexameric structure. Surprisingly, neither reduction of the gamma disulfide nor selective proteolysis of alpha, beta and gamma affects the thermal stability of EDTA-treated CF1 preparations, as determined by differential scanning calorimetry. Dissociation of the enzyme in the cold may be monitored by loss of the ATPase activity of CF1 subunit depleted of its epsilon subunit [CF1(-epsilon)]. The rate of cold inactivation of ATPase activity of reduced and alkylated CF1(-epsilon) treated with trypsin in solution was much faster than that CF1(-epsilon)(8.1 versus 38.7 min, respectively, for 50% loss of activity). The increased cold liability of the trypsin-treated enzyme was not a consequence of the cleavage of the gamma. CF1 incubated with trypsin under conditions in which gamma is not cleaved was as cold labile as CF1 with cleaved gamma. Instead, loss of the delta subunit and a few residues from the C-terminal of the beta subunits were responsible for the increased cold liability of the enzyme.
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PMID:Structural stability of chloroplast coupling factor 1 determined by differential scanning calorimetry and cold inactivation. 866 76

The treatment of the soluble F1-ATPase with the Fe2+-ascorbate oxidative system has resulted in the inactivation and fragmentation of the enzyme. Up to 10 polypeptide fragments could be readily observed on the SDS-PAGE. Addition of free Mg2+ or EDTA effectively prevented inactivation and fragmentation. Both alpha and beta subunits of the F1-ATPase were cleaved, with predominant cleavage sites being identified on alpha. Oxidative fragmentation of the F1-ATPase showed nucleotide dependence. Removal of nucleotides from the F1-ATPase as well as their excess in the medium dramatically affected the fragmentation pattern. On the basis of the M(r) of the fragments, their immunorecognition with the antibodies against subunits of the F1-ATPase, and the results of the mild proteolysis of the F1-ATPase with trypsin, cleavage sites are suggested to be located in the nucleotide-binding domain of both alpha and beta subunits. Finally, it is hypothesized that similar structural damage of the F1-ATPase may occur in mitochondrion in vivo under oxidative stress conditions.
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PMID:Mitochondrial ATP synthase: Fe2+-catalyzed fragmentation of the soluble F1-ATPase. 891 43


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