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:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The uncB402 mutation in Escherichia coli results in formation of an H+-ATPase complex that is defective in energy-transducing capacity. The mutation, originally described by Butlin et al. (Butlin, J.D., Cox, G.B., and Gibson, F. (1973) Biochim. Biophys. Acta 292, 366-375), alters the F0 sector of the H+-ATPase complex. Here, we show that uncB402 is an amber-suppressible, chain-terminating mutation that results in loss of the chi subunit from F0. This was demonstrated in crude membrane fractions after overproduction of the ATPase complex by heat induction of a lambda transducing phage carrying the unc operon of uncB402. The lambda-uncB402 DNA was used as a template in an in vitro transcription-translation system. A synthesis product that may correspond to the truncated form of the chi subunit was observed. Despite the absence of chi, the F1-ATPase was still bound to the membrane, although more weakly than in wild type. The omega subunit of F0 ("dicyclohexylcarbodiimide-binding protein") shows normal reactivity with dicyclohexylcarbodiimide, indicating that at least this portion of F0 integrates properly in the membrane in the absence of the chi subunit. The F0 of uncB402 was not functional in H+ translocation activity. This was shown by direct H+ flux measurements with crude membrane vesicles that were treated with guanidine to disrupt the binding of F1 to F0. Secondly, a method was developed for isolation of F0 from F1-depleted membranes. The F0 from uncB402 was shown to have less than 5% the proton-translocase activity of wild type F0 when reconstituted into liposomes. Although the uncB402 mutant shows these defects, the question of whether the chi subunit plays a direct role in F1-binding or H+ translocation remains open, since the loss of chi may lead to subtle changes in the assembly of the other F0 subunits. Analysis of other mutants should permit a more definitive assignment of function.
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PMID:H+-ATPase of Escherichia coli uncB402 mutation leads to loss of chi subunit of subunit of F0 sector. 621 6

The physiochemical properties of the isolated alpha subunit of the Escherichia coli coupling factor ATPase, and changes resulting from the interaction of alpha with ATP, were studied. Amino acid analysis of alpha revealed 42% polar residues, 4 cysteine residues, and a single tryptophan residue. The partial specific volume, v, of alpha was 0.74 cm3 g-1. Molecular weight value of alpha, determined by sedimentation equilibrium, of 55,000 to 59,000 were observed in guanidine hydrochloride, or in nondenaturing buffer in either the presence of absence of ATP, which alpha binds with high affinity. Sedimentation velocity experiments gave a value of s20,w0-3.52 S for alpha. In the presence of ATP, this value increased to 4.00 S, indicating a large conformational change of alpha when ATP is bound. A slow dissociation rate of alpha x ATP was suggested by the finding that a substantial portion of [2-3H]ATP mixed with alpha remained bound to the protein during native polyacrylamide gel electrophoresis, causing alpha to migrate with a higher relative mobility. A dissociation rate constant, koff, of 0.21 min-1 at 22 degrees C was measured by following the rate at which unlabeled ATP displaced [2-3H]ATP from the protein. The properties of the interaction of alpha with ATP suggest that this subunit may be the site of the "tightly bound" nucleotides of the coupling factor ATPase.
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PMID:ATP causes a large change in the conformation of the isolated alpha subunit of Escherichia coli F1 ATPase. 644 13

The hydrophobic sector of the mitochondrial ATPase complex was purified by sequential extraction with cholate and octylglucoside, by further differential solubilization with guanidine and cholate in the presence of phosphatidylcholine, and by fractionation with ammonium sulfate. A polypeptide with a mass of 28,000 dalton was present in the purified hydrophobic section which was cleaved by trypsin, resulting in loss of reconstitution activity. In contrast, dicyclohexylcarbodiimide-binding proteolipid remained unimpaired after exposure to trypsin. The 32Pi-ATP exchange activity of the reconstituted ATPase complex was inhibited by p-hydroxymercuribenzoate, which reacted primarily with the 28,000-dalton protein, as monitored by acrylamide gel electrophoresis with 14C-labeled inhibitor. The function of a 22,000-dalton polypeptide and of some minor components in the region of the proteolipid remains unknown. An examination of the phospholipid requirements for reconstitution of an active complex revealed an unexpected discrepancy. With an excess of phosphatidylethanolamine, optimal reconstitution of 32Pi-ATP exchange and ATP synthesis in the presence of bacteriorhodopsin and light was achieved: at a high phosphatidylcholine:phosphatidylethanolamine ratio, the rate of ATP synthesis remained high, but the rate of 32Pi-ATP exchange dropped precipitously. A new procedure is described for the reconstitution of the ATPase complex with purified phospholipids which is stable for at least 15 days.
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PMID:Isolation, characterization, and reconstitution of a solubilized fraction containing the hydrophobic sector of the mitochondrial proton pump. 646 Jul 56

We have isolated a chaperonin from the hyperthermophilic archaeon Sulfolobus solfataricus based on its ability to inhibit the spontaneous refolding at 50 degrees C of dimeric S. solfataricus malic enzyme. The chaperonin, a 920-kDa oligomer of 57-kDa subunits, displays a potassium-dependent ATPase activity with an optimum temperature at 80 degrees C. S. solfataricus chaperonin promotes correct refoldings of several guanidine hydrochloride-denatured enzymes from thermophilic and mesophilic sources. At a molar ratio of chaperonin oligomer to single polypeptide chain of 1:1, S. solfataricus chaperonin completely inhibits spontaneous refoldings and suppresses aggregation upon dilution of the denaturant; refoldings resume upon ATP hydrolysis, with yields of active molecules and rates of folding notably higher than in spontaneous processes. S. solfataricus chaperonin prevents the irreversible inactivations at 90 degrees C of several thermophilic enzymes by the binding of the denaturation intermediate; the time-courses of inactivations are unaffected and most activity is regained upon hydrolysis of ATP. S. solfataricus chaperonin completely prevents the formation of aggregates during thermal inactivation of chicken egg white lysozyme at 70 degrees C, without affecting the rate of activity loss; ATP hydrolysis results in the recovery of most lytic activity. Tryptophan fluorescence measurements provide evidence that S. solfataricus chaperonin undergoes a dramatic conformational rearrangement in the presence of ATP/Mg, and that the hydrolysis of ATP is not required for the conformational change. The ATP/Mg-induced conformation of the chaperonin is fully unable to bind the protein substrates, probably due to disappearance or modification of the substrate binding sites. This is the first archaeal chaperonin whose involvement in protein folding has been demonstrated.
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PMID:The chaperonin from the archaeon Sulfolobus solfataricus promotes correct refolding and prevents thermal denaturation in vitro. 783 6

We have examined the intrinsic fluorescence properties of a highly purified chloroplast H(+)-ATPase (CF0F1) preparation [R. D. Kirch and P. Graber (1992) Acta Physiol. Scand. 746, 9-12). Unlike the catalytic CF1 portion alone, CF0F1 fluorescence was dominated by tryptophan fluorescence both at 277-nm excitation, favoring tyrosine excitation, and at 295-nm excitation, favoring tryptophan excitation. A broad tryptophan fluorescence peak was observed with a maximum at around 335 nm and a broad shoulder around 350 nm. Denaturation of the enzyme complex with guanidine-HCl resulted in a significant increase (approximately 40%) in tyrosine fluorescence. The fluorescence spectrum (lambda ex = 295 nm) of the inhibitory epsilon subunit isolated from CF1 resembled that of CF1, indicating the presence of two tryptophan species located in different environments. Fluorescence quenching by potassium iodide indicated a substantial increase in the solvent accessibility of one of the two tryptophans following isolation of epsilon from CF1. Thus, when epsilon binds to CF1, a tryptophan residue becomes partially buried, probably at an interface between epsilon and another (possibly gamma) CF1 subunit. Removal of the epsilon subunit from CF1 leads to an increase in tyrosine fluorescence of a magnitude similar to that obtained upon denaturation of the CF0F1 complex. The results suggest that the reversible association of the epsilon subunit with CF0F1 or with isolated CF1 may be monitored by following changes in the intrinsic fluorescence of the enzyme complex.
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PMID:Intrinsic fluorescence of the chloroplast H(+)-ATPase. 787

The effects of the anti-acid secretory agents, cimetidine (N-cyano-N'-methyl-N"-(2-([(5-methyl-1H-imidazol-4-yl)methyl]thio)ethyl) guanidine), ranitidine (N-(2-(((-5-[(dimethylamino)methyl]-2-furanyl)methyl)thio)ethyl)-N'-meth yl- 2-nitro-1,1-ethene-diamine), roxatidine (2-acetoxy-N-(3-[m-(1-piperidinylmethyl)phenoxy]-propyl) acetamide hydrochloride), FRG-8813 (2-(furfurylsulfinyl)-N-(4-[4-(piperidinomethyl)-2-pyridyl]o xy-(z)-2- butenyl)acetamide), omeprazole (5-methoxy-2-([(4-methoxy-3,5-dimethylpyridinyl)methyl]sulfinyl)- 1H-benzimidazole), and NC-1300-O-3 (2-([2-(isobutylmethylamino)benzyl]sulfinyl)-1H- benzimidazole), on mucin biosynthesis were studied in rat gastric mucosa by using an organ culture technique. [3H]Glucosamine incorporation was stimulated in the corpus region by the histamine H2 receptor antagonists which have a six-membered aromatic ring, roxatidine and FRG-8813, and the new H+,K(+)-ATPase inhibitor, NC-1300-O-3. Thus, these drugs not only inhibit acid secretion but may also promote gastric mucosal protective actions. The present observations also demonstrate that the determination of mucin biosynthesis may be a useful tool for evaluation of mucosal protective activity.
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PMID:Effects of acid-inhibitory antiulcer drugs on mucin biosynthesis in the rat stomach. 790 83

Chaperonin 60 and chaperonin 10 (GroEL and GroES homologues, respectively) have been isolated from extracts of the anaerobic thermophile Thermoanaerobacter brockii. A simple and rapid purification for chaperonin 60 made use of hydrophobic and anion-exchange chromatographies, and could be readily scaled up; approximately 2 mg pure chaperonin 60 was obtained/g cells. In contrast with all other prokaryotic chaperonin 60 proteins that have been studied, which are tetradecamers, including those from Thermus sp., the T. brockii protein is a heptamer, and as isolated was not in association with chaperonin 10. The preparation is readily crystallized using 2-propanol or poly(ethylene glycol) with MgCl2. The N-terminal amino acid sequence of this preparation is similar to other thermophilic chaperonin 60 proteins. Chaperonin 10 was purified from the flow-through of the first hydrophobic column (which bound chaperonin 60) using a more hydrophobic adsorbent to remove contaminating proteins, followed by anion-exchange chromatography. Chaperonin 10 was obtained with a yield of approximately 10% that of chaperonin 60. The subunit molecular mass of chaperonin 10 determined by electrospray mass spectrometry is 10254 +/- 0.4 Da, which is very similar to the molecular mass of Escherichia coli GroES. Similarly, the subunit size of chaperonin 60 determined by mass spectrometry is very similar to that of GroEL, at 57949 +/- 10 Da. T. brockii chaperonin 60 has an ATPase activity that is suppressed by chaperonin 10, and the two proteins together are active in protein-folding assays. Mitochondrial malate dehydrogenase was successfully refolded at 37 degrees C after denaturation in guanidine hydrochloride, using T. brockii chaperonin 60 and chaperonin 10, or chaperonin 60 and E. coli GroES. The denatured enzyme was protected from aggregation by association with chaperonin 60. Guanidine-hydrochloride-denatured preparations of isocitrate dehydrogenase and secondary alcohol dehydrogenase isolated from T. brockii were also refolded at 60-65 degrees C. In each case, refolding required chaperonin 60, chaperonin 10 and ATP, giving up to 80% regeneration of control activity.
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PMID:Purification and characterization of chaperonin 60 and chaperonin 10 from the anaerobic thermophile Thermoanaerobacter brockii. 791 71

In this study, cimetidine uptake and its regulation by LLCPK1 monolayers were investigated. Uptake was temperature dependent with kinetic and specificity characteristics typical of a carrier-mediated mechanism. With cimetidine uptake in the presence of an excess concentration of the potent inhibitor quinidine as a measure of nonspecific transport, the estimated kinetic parameters for cimetidine uptake at 37 degrees C under steady-state conditions are Km = 32.3 +/- 6.4 microM and Vmax = 20.2 +/- 2.1 pmol/mg per minute. Amiloride, quinidine, and quinine inhibited cimetidine uptake, whereas N1-methylnicotinamide, tetraethylammonium, and guanidine did not. The uptake of cimetidine was increased in the presence of a cell-->lumen H+ gradient, consistent with the behavior of a cimetidine-H+ antiport system. Furthermore, the activity of both the Na(+)-H+ exchanger and H(+)-ATPase acted to dissipate the cell-->lumen H+ gradient, thereby decreasing net cimetidine transport. These results suggest that there is a cimetidine-H+ exchange system in LLCPK1 cells and that the net secretion of organic base in vivo may be regulated by luminal acidification mechanisms.
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PMID:Characterization of cimetidine transport in LLCPK1 cells. 794 86

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

SecA, the peripheral ATPase domain of the Escherichia coli precursor protein translocase, was denatured in 6 M guanidine hydrochloride. Circular dichroism and intrinsic tryptophan fluorescence spectra revealed that the protein is transformed into a random-coil configuration. Upon dilution of the chaotropic agent, SecA refolds into its native, functional conformation as a homodimer. As structural criteria, the native dimeric state was assayed by size-exclusion chromatography, chemical cross-linking, tryptophan fluorescence, and circular dichroism. Functional SecA heterodimers were formed of which the individual subunits were tagged with fluorescent dyes to allow measurements of the association state of the monomers by resonance energy transfer using steady-state and time-resolved fluorescence spectroscopy. SecA retained its dimeric structure during translocation, while energy transfer was abolished only by denaturation. The "half-of-the-sites activity" was investigated by constructing heterodimers formed from native and 8-azido-ATP-inactivated SecA. Heterodimers have lost the ability to support translocation of the precursor protein proOmpA in an in vitro translocation system. It is concluded that the dimeric structure is maintained during translocation and required for functionality.
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PMID:SecA, the peripheral subunit of the Escherichia coli precursor protein translocase, is functional as a dimer. 824 Nov 73


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