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)

It was shown that DCCD-sensitive ATPase activity of isolated membranes and preparations of F1F0 only from anaerobically grown E. coli depended on K+ activity. F1F0 include two additional proteins which correspond to the Trk system. The data improve the possibility to form supercomplex (F1F0-Trk) functioning as the H(+)-K(+)-pump.
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PMID:[K+-dependent enzyme activity of H+-ATPase in the E. coli membrane]. 183 Feb 24

To understand the molecular structure of the vacuolar H(+)-translocating ATPase from plants, cDNAs encoding the N,N'-dicyclohexylcarbodiimide-binding 16-kDa proteolipid from oat (Avena sativa L. var. Lang) have been obtained. A synthetic oligonucleotide corresponding to a region of the bovine proteolipid cDNA (Mandel, M., Moriyama, Y., Hulmes, J.D., Pan, Y.-C.E., Nelson, H., and Nelson, N. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5521-5524) was used to screen an oat cDNA library constructed in lambda gt11. The nucleotide sequences of several positive clones (VATP-P1, clones 12, 54, 93) demonstrated the presence of a small multigene family. The four clones showed extensive divergence in their codon usage and their 3'-untranslated regions; however, the deduced amino acid sequences of the proteins were 97-99% identical. These clones encoded the proteolipid subunit as one of them (clone 12) expressed a fusion protein that reacted with an antibody to the 16-kDa proteolipid. The open reading frame of one cDNA clone (VATP-P1) predicted a polypeptide of 165 amino acids with a molecular mass of 16,641. Based on hydropathy plots, a molecule with four membrane-spanning domains was predicted, in which domain IV was especially conserved among different species. This domain showed 80% identity in nucleotide or amino acid sequences between the oat and the bovine proteolipids and contained a glutamate residue that is the putative N,N'-dicyclohexylcarbodiimide-binding residue. The presence of a small multigene family of the 16-kDa proteolipid was confirmed by Southern blot analysis showing that several distinct restriction fragments of oat nuclear DNA hybridized with the VATP-P1 cDNA.
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PMID:Molecular cloning and sequencing of cDNAs encoding the proteolipid subunit of the vacuolar H(+)-ATPase from a higher plant. 183 53

Brush-border membranes from rat kidney cortex are transiently exposed to cholate to reorient ATP-driven H+ pumps to the outside of the vesicles. The carboxyl group reagent, N,N'-dicyclohexylcarbodiimide (DCCD), inhibits ATP-driven H+ uptake into cholate-pretreated vesicles irreversibly. Complete inhibition requires treatment of vesicles with 0.2 mM DCCD for greater than or equal to 15 min. ATP and ADP do not protect the H+ pump from inactivation suggesting that DCCD modifies pump subunits involved in H+ translocation, but not those related to ATP hydrolysis. With [14C]DCCD a 16 kDa protein is strongly labeled in brush-border and endosomal membranes, but not in basolateral membranes. Molecular mass of this protein and distribution similar to H(+)-ATPases suggest a role as H(+)-conducting subunit of the H+ pumps. The SH-group reagent, N-ethylmaleimide (NEM), also inhibits ATP-driven H+ uptake irreversibly. As opposed to DCCD, ATP and ADP protect the pump from irreversible inhibition indicating that NEM modifies SH-groups in the proximity of ATP hydrolysis sites. Finally, 15 nM of a potent inhibitor of vacuolar ATPases, bafilomycin B1, abolishes ATP-driven H+ uptake. Inactivation by DCCD and NEM, labeling of 16 kDa subunits by [14C]DCCD, and high sensitivity to bafilomycin indicate that the H+ pump (H(+)-ATPase) in rat renal brush-border membranes belongs to the class of vacuolar ATPases. Bafilomycin may prove a valuable tool for specific inhibition of the renal H(+)-ATPase in future studies.
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PMID:Biochemical aspects of H(+)-ATPase in renal proximal tubules: inhibition by N,N'-dicyclohexylcarbodiimide, N-ethylmaleimide, and bafilomycin. 183 71

Dicyclohexylcarbodiimide (DCCD) inhibits the activity of the F1F0-H+ ATP synthase of Escherichia coli by reacting with aspartyl 61 in subunit c of the FO sector to form a stable N-acylurea. The segment of chromosomal DNA which codes the subunits of the FO was cloned from four independently isolated DCCD-resistant mutants, and the sequence of the subunit c gene (uncE) was determined. An Ala24 to serine (A24S) substitution was found in the subunit c gene of each mutant. The A24S uncE gene was cloned into the BamHI site of a mutant derivative of plasmid pBR322. The A24S subunit c conferred DCCD resistance to a variety of recipient E. coli strains when it was overexpressed from this plasmid. A 7-base pair deletion beginning at position 132 of the plasmid vector was responsible for the observed overexpression. Hoppe et al. (Hoppe, J., Schairer, H. U., and Sebald, W. (1980) Eur. J. Biochem. 112, 17-24) had previously shown that mutation of subunit c Ile28 to threonine or valine resulted in DCCD resistance. The DCCD sensitivities of the membrane ATPase of these mutants and the A24S mutant were compared. DCCD sensitivity decreased in the order: wild-type much greater than I27V greater than I28T = A24S. The venturicidin sensitivities of wild-type and mutant membranes were also examined. The membrane ATPase of the I28T and I28V mutants was venturicidin resistant whereas the A24S substitution resulted in a hypersensitivity to inhibition by venturicidin. These results support a model in which subunit c folds in the membrane like a hairpin, where the region of residues 24-28 in transmembrane helix-1 is close to that of aspartyl 61 in transmembrane helix-2.
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PMID:Mutation of alanine 24 to serine in subunit c of the Escherichia coli F1F0-ATP synthase reduces reactivity of aspartyl 61 with dicyclohexylcarbodiimide. 183 53

1. Peroxisomes were isolated from bovine and rat liver by use of differential and density gradient centrifugations. 2. In the final density gradient (Nycodenz) a distinct peak of ATPase activity codistributed with the peroxisome marker catalase and was well separated from the bulk of the ATPase activity and from markers for other subcellular organelles. 3. The peroxisome-associated ATPase had a pH optimum of 7.5 and was inhibited by N-ethylmaleimide, by N,N'-dicyclohexylcarbodiimide and by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, but was unaffected by up to 30 microM n-tributyltin chloride. 4. Prolonged incubation with oligomycin at high concentrations indicated that 50% of peroxisomal ATPase was resistant to this inhibitor. The oligomycin-sensitive ATPase activity required at least a four-fold higher ratio of inhibitor to protein for inhibition than mitochondrial ATPase did. It was concluded that oligomycin-sensitive and oligomycin-resistant ATPase may be associated with liver peroxisomes.
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PMID:Properties of ATPase activity associated with peroxisomes of rat and bovine liver. 183 59

Ouabain- and furosemide-insensitive and ATP-dependent Cl- uptake was demonstrated in rat renal membrane vesicles. Such a Cl- uptake activity was prominent in cortical plasma membrane fractions with high activities of Cl(-)-ATPase and Na+, K(+)-ATPase. The membrane vesicles accumulated Cl- in an osmotically reactive manner with the following sequence of nucleotide specificity: ATP greater than ITP greater than UTP greater than GTP greater than CTP., beta, gamma-Methylene ATP, ADP and AMP had no effect. ATP-dependent Cl- uptake was markedly inhibited by a Cl(-)-ATPase inhibitor, ethacrynic acid (0.3 mM), but not affected by an H(+)-ATPase inhibitor, N,N'-dicyclohexylcarbodiimide (0.1 mM). These findings suggest that an ethacrynic acid-sensitive and ATP-driven
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PMID:Ethacrynic acid-sensitive and ATP-dependent Cl- transport in the rat kidney. 183 37

We have studied the mechanism by which liver Golgi apparatus maintains the acidity of its contents, using a subcellular fraction from rat liver highly enriched in Golgi marker enzymes. Proton accumulation (measured by quenching of acridine-orange fluorescence) and anion-dependent ATPase were characterized and compared. Maximal ATPase and proton accumulation required ATP; GTP and other nucleotides gave 10% to 30% of maximal activity. Among anions, Cl- and Br- approximately doubled the activities; others were much less effective. Half-maximal increase of ATPase and H+ uptake required 55 mmol/L and 27 mmol/L Cl-, respectively. In predominantly chloride media, SCN- and NO3- markedly inhibited H+ uptake. Nitrate competitively inhibited both the chloride-dependent ATPase (apparent Ki 6 mmol/L) and proton uptake (apparent Ki 2 mmol/L). Nitrate and SCN- also inhibited uptake of 36Cl. Replacing K+ with Na+ had no effect on the initial rate of proton uptake but somewhat reduced the steady state attained. Replacement of K+ with NH4+ and choline reduced proton uptake without affecting ATPase. The ATPase and H+ uptake were supported equally well by Mg2+ or Mn2+. The ATPase was competitively inhibited by 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid (apparent Ki 39 mumol/L). Other agents inhibiting both H+ uptake and ATPase were N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide, chlorpromazine, diethylstilbestrol, Zn2+, Co2+ and Cu2+. In the Cl- medium, accumulated protons were released by ionophores at the relative rates, monensin = nigericin greater than valinomycin greater than carbonyl cyanide mchlorophenylhydrazone; the last of these also reduced ATPase activity. In the absence of Cl-, monensin and valinomycin both stimulated the ATPase. These results show a close association between ATPase activity and acidification of liver Golgi vesicles. They support a role for Cl- that depends on its uptake as a counter ion for H+ and suggest that it may also stimulate proton transport by a more direct effect on a component of the transport system.
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PMID:Proton accumulation and ATPase activity in Golgi apparatus-enriched vesicles from rat liver. 184 95

The involvement of ATP synthase in the imbalance between the photoactivities of PS I and PS II under light-limiting conditions, was examined in broken lettuce chloroplasts using modulated fluorimetry. The imbalance, in favor of PS II, was minimal and roughly constant between pH 6.5-7.3 (ratio of PS II/PS I activities about 1.1), and maximal at pH 8.5 (ratio of PS II/PS I activities about 1.4). This increase was strongly inhibited by a treatment of the chloroplasts with the CF0 ATP synthase inhibitor DCCD, but unaffected by the CF1 ATPase inhibitor, tentoxin. However, tentoxin plus ADP-P1 did inhibit the high pH-induced increased imbalance. These results, when considered with the previous results on the effect of high pH on proton flux through the ATP synthase, suggest that the rate of such proton flow controls the imbalance between the two photo-systems. It is possible that there is an in vivo fine-tuning regulating mechanism of the photosystems imbalance via the opening and closing of proton gradient dissipation through the ATP synthase. This mechanism may help alleviate photoinhibitory damage.
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PMID:Proton flow through the ATP synthase in chloroplasts regulates the distribution of light energy between PS I and PS II. 184 95

Glucose transport in the bloodstream form of the protozoan parasite Trypanosoma brucei was characterized by enzymatically measuring the D-glucose uptake. Uptake kinetics showed a concentration-dependent saturable process, typical for a carrier-mediated transport system, with an apparent Km = 0.49 +/- 0.14 mM and Vmax = 252 +/- 43 nmol.min-1.mg cell protein-1 (equal to 2.25 x 10(8) trypanosomes). The specificity of glucose transport was investigated by inhibitor studies. Glucose uptake was shown to be sodium independent; neither the Na+/K(+)-ATPase inhibitor ouabain (1 mM) nor the ionophor monensin (1 microM) inhibited uptake. Transport was also unaffected by the H(+)-ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD; 20 microM) and the uncoupler carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP; 1 microM). However, highly significant inhibition was obtained with both phloretin (82% at 0.13 mM; Ki = 64 microM) and cytochalasin B (77% at 0.3 mM; Ki = 0.44 mM), and partial inhibition with phlorizin (14% at 0.5 mM; Ki = 3.0 mM). In each case, inhibition was noncompetitive, partially reversible (45%) for phloretin and completely reversible for cytochalasin B and phlorizin. Measurement of the temperature-dependent glucose uptake between 25 degrees C and 37 degrees C resulted in a temperature quotient of Q10 = 1.97 +/- 0.02 and an activation energy of Ea = 52.12 +/- 1.00 kJ/mol for glucose uptake. We conclude that glucose uptake in T. brucei bloodstream forms occurs via a facilitated diffusion system, clearly distinguished from the human erythrocyte-type glucose transporter with about a 10-fold higher affinity for glucose and about a 1000-fold decreased sensitivity to the inhibitor cytochalasin B.
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PMID:Specificity of glucose transport in Trypanosoma brucei. Effective inhibition by phloretin and cytochalasin B. 193 76

Strain F, a recently isolated ruminal bacterium, grew rapidly with glutamate or glutamine as an energy source in the presence but not the absence of Na. Monensin, a Na+/H+ antiporter, completely inhibited bacterial growth and significantly reduced ammonia production (85%), but 3,3',4',5-tetrachlorosalicylanide (a protonophore) and valinomycin had little effect on growth or ammonia production. Dicyclohexylcarbodiimide, a H(+)-ATPase, inhibitor had no effect. The kinetics of glutamate and glutamine transport were biphasic, showing unusually high rates at high substrate concentrations. On the basis of low substrate concentrations (less than 100 microM), the Km values for glutamate and glutamine were 4 and 11 microM, respectively. Strain F had separate carriers for glutamate and glutamine which could be driven by a chemical gradient of Na. An artificial delta psi was unable to drive transport even when Na was present. The glutamate carrier had a single binding site for Na with a Km of 21 mM; the glutamine carrier appeared to have more than one binding site, and the Km was 2.8 mM. Neither carrier could use Li instead of Na. Histidine and serine were also rapidly transported by Na-dependent systems, but serine alone did not allow growth even when Na was present. Because exponentially growing cells at pH 6.9 had little delta psi (-3 mV) and a slightly reversed Z delta pH (+17 mV), it appeared that the membrane bioenergetics of strain F were solely dependent on Na circulation.
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PMID:Transport and deamination of amino acids by a gram-positive, monensin-sensitive ruminal bacterium. 197 63


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