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

---

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

The characteristics of the H+ pump in isolated rat renal endocytotic vesicles were studied by the delta pH-sensitive dye acridine orange, the voltage-sensitive dye 3,3'-dipropylthiadicarbocyanine iodide, and by a coupled optical ATPase assay. Intravesicular acidification depended on ATP and Mg2+ concentrations with half-maximal activations at 73 and 77 microM, respectively. CTP, GTP, UTP, and ITP partially supported acidification, but ADP and AMP did not. Ouabain, ethoxzolamide, levamisole, and vanadate did not inhibit H+ uptake into endocytotic vesicles. Oligomycin inhibited partially. Depending on concentration and preincubation time, Dio-9, filipin, N-ethylmaleimide (NEM), and dicyclohexylcarbodiimide (DCCD) inhibited H+ uptake completely. Filipin and, partially, DCCD acted nonspecifically by dissipating pH gradients. A specific cation was not required for the H+ pump; Zn2+ inhibited. Compared with mannitol, ATP-driven H+ uptake was stimulated by SCN- greater than Cl- greater than Br- greater than I- much greater than HPO4(2-) = gluconate = HCO3- = F-, but not by SO4(2-), NO3-, CH3COO-, S2O3(2-), and S4O6(2-). Chloride stimulated H+ uptake from the outside of the vesicles with an apparent Km of 27 mM. In the absence of Cl-, ATP-driven proton uptake was increased by intravesicular K+ and valinomycin, suggesting that the pump is electrogenic. The electrogenicity, however, could not be demonstrated with voltage-sensitive dyes. The vesicle membrane contains no significant K+ and Cl- conductances; only a conductance for H+ was found. The vesicles exhibited an ouabain-, oligomycin-, and vanadate-insensitive ATPase activity that was inhibited by DCCD and NEM. Our data indicate the presence of an electrogenic H+ pump in endocytotic vesicles from rat renal proximal tubules with similar characteristics as H+ pumps present in various intracellular (nonmitochondrial) membranes.
...
PMID:Characteristics of the proton pump in rat renal cortical endocytotic vesicles. 242 58

Regulation of steady-state free Ca2+ concentration at rest and at stimulation have been studied in isolated permeabilized pancreatic acinar cells by measuring the free Ca2+ concentration of the surrounding incubation medium with a Ca2+-specific electrode. Ca2+ transport mechanisms have been further characterized in subcellular membrane fractions by measuring 45Ca2+ uptake into membrane vesicles and protein phosphorylation using polyacrylamide gel electrophoresis. (a) In permeabilized isolated acinar cells from exocrine glands, inositol-1,4,5-trisphosphate (IP3) releases Ca2+ from endoplasmic reticulum. (b) Secretagogue-induced Ca2+ release from permeabilized cells is accompanied by increased production of IP3. At rest, steady-state free Ca2+ concentration is regulated at 4 X 10(-7) mol/L by the rough endoplasmic reticulum (RER). Ca2+ uptake into this pool is promoted by a (Ca2+ + Mg2+)-ATPase, and is dependent on cations and anions in the incubation medium in the order K+ greater than Na+ greater than Li+ greater than choline+ and Cl- greater than Br- greater than SO4(2-) = NO3- greater than I- greater than cyclamate- greater than SCN-, respectively. Similarly, Ca2+-stimulated 32P incorporation from [gamma 32P]ATP into a 130 kD protein intermediate of (Ca2+ + Mg2+)-ATPase, as well as 32P liberation, indicating (Ca2+ + Mg2+)-ATPase activity, are cation dependent. While 32P incorporation is highest in the presence of choline, 32P liberation is higher with K+, as compared with Na+ or choline, indicating that K+ ions facilitate dephosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Intracellular messengers in stimulus-secretion coupling of pancreatic acinar cells. 243 35

ATP-driven acidification visualized by the delta pH indicator acridine orange was used as marker for isolation of endocytic vesicles from rat liver. By differential and Percoll density gradient centrifugation, a vesicle fraction was obtained with an approx. 80-fold enriched H+-pump activity. The preparation contained vesicles that had taken up fluorescein isothiocyanate-labeled dextran or horseradish peroxidase injected into rats in vivo, proving the presence of endosomes. The H+-pump in these vesicles showed: (a) strict preference for ATP; (b) stimulation by Mg2+ and Mn2+, but not by monovalent cations; (c) stimulation by Cl-, I- and Br-; (d) electrogenicity; (e) insensitivity to vanadate, slight inhibition by oligomycin and strong inhibition by N-ethylmaleimide (NEM) and N,N'-dicyclohexylcarbodimide (DCCD). The vesicles exhibited an ouabain-, oligomycin- and levamisole-resistant ATPase activity, which was slightly stimulated by Cl-, unaffected by vanadate and inhibited by NEM and DCCD. Thus, a simple and efficient high-speed centrifugation method is available for isolation of endocytic vesicles from mammalian liver.
...
PMID:Isolation of rat liver endocytic vesicles using the proton pump as a marker. 246 Jan 36

In microsomal vesicles, as isolated from exocrine pancreas cells, MgATP-driven H+ transport was evaluated by measuring H+-dependent accumulation of acridine orange (AO). Active H+ uptake showed an absolute requirement for ATP with simple Michaelis-Menten kinetics (Km for ATP 0.43 mmol/liter) with a Hill coefficient of 0.99. H+ transport was maximal at an external pH of 6.7, generating an intravesicular pH of 4.8. MgATP-dependent H+ accumulation was abolished by protonophores, such as nigericin (10(-6) mol/liter) or CCCP (10(-5) mol/liter), and by inhibitors of nonmitochondrial H+ ATPases, such as NEM or NBD-Cl, at a concentration of 10(-5) mol/liter. Inhibitors of both mitochondrial and nonmitochondrial H+ pumps, such as DCCD (10(-5) mol/liter) or Dio 9 (0.25 mg/ml), reduced microsomal H+ transport by about 90%. Vanadate (2 x 10(-3) mol/liter), a blocker of those ATPases, which form a phosphorylated intermediate, did not inhibit H+ transport. The stilbene derivative DIDS (10(-4) mol/liter), which inhibits anion transport systems, abolished H+ transport completely. MgATP-dependent H+ transport was found to be anion dependent in the sequence Cl- greater than Br- greater than gluconate-; in the presence of SO2-4, CH3COO- or No-3, no H+ transport was observed. MgATP-dependent H+ accumulation was also cation dependent in the sequence K+ greater than Li+ greater than Na+ = choline+. As shown by dissipation experiments in the presence of different ion gradients and ionophores, both a Cl- and a K+ conductance, as well as a small H+ conductance, were found in the microsomal membranes. When membranes containing the H+ pump were further purified by Percoll gradient centrifugation (ninefold enrichment compared to homogenate), no correlation with markers for endoplasmic reticulum, mitochondria, plasma membranes, zymogen granules or Golgi membranes was found. The present data indicate that the H+ pump located in microsomes from rat exocrine pancreas is a vacuolar- or "V" -type H+ ATPase and has most similarities to that described in endoplasmic reticulum, Golgi apparatus or endosomes.
...
PMID:Characterization of MgATP-driven H+ uptake into a microsomal vesicle fraction from rat pancreatic acinar cells. 246 2

Immunological studies were designed to study the structure of the oligomycin sensitivity conferring protein (OSCP) integrated in the mitochondrial ATPase-ATPsynthase complex. The monoclonal antibody 2B1B1 used in this study could bind as well to purified or membrane bound OSCP as shown previously by Protein A-gold immunocytochemistry and by competitive immunotitration. In this paper, it is shown that 2B1B1 can also immunoprecipitate the F0F1 complex from a Triton X-100 extract. This means that not only, 2B1B1 binds to the surface of OSCP but also that the binding of 2B1B1 did not destroy the interactions between F0 and F1 and further demonstrates the external location of the 2B1B1 binding site in the ATPase-ATPsynthase complex. This antigenic site was located on the N-terminal sequence of OSCP, between residues 1 and 72, as demonstrated after chemical cleavage of OSCP with formic acid, hydroxylamine and partial cleavage with cyanogen bromide. The proximity of Tyr and Arg to the epitope was suggested by the lack of 2B1B1 binding to iodinated OSCP and by the susceptibility of this binding to trypsin or to endoproteinase Arg-C treatments of OSCP, respectively. A more precise location of the epitope has been attempted by using the method of synthesis of overlapping octapeptides on solid support. It was found that 2 groups of octapeptides could bind 2B1B1. The first group contained in common the sequence Pro7-Pro8-Val9-Gln10-Ile11-Tyr12- and the second group of peptides contained the sequence Arg62-Ser63-Val64-Lys65. Another monoclonal antibody, AF4H7, which competes with 2B1B1, also recognized the first group of peptides. The possible involvement of these 2 fragments in the epitope localized at the surface of OSCP is discussed. In addition, secondary structure theoretical analysis predicts that these 2 domains should be in a beta-strand configuration.
...
PMID:Epitope of OSCP oligomycin sensitivity conferring protein exposed at the surface of the mitochondrial ATPase-ATPsynthase complex. 247 97

The aziridinium of purified quinacrine mustard at 50 microM inactivates the bovine heart mitochondrial F1-ATPase with a pseudo-first order rate constant of 0.07 min-1 at pH 7.0 and 23 degrees C. An apparent Kd of 27 microM for the enzyme-reagent complex was estimated from the dependence of the rate of inactivation on the concentration of quinacrine mustard. The pH inactivation profile revealed that deprotonation of a group with a pKa of about 6.7 is necessary for inactivation. The amount of reagent incorporated into the protein increased linearly with the extent of inactivation. Complete inactivation was estimated to occur when 3 mol of reagent were incorporated/mol of F1. Enzyme, in which steady state ATPase was inactivated by 98% by quinacrine mustard, hydrolyzed substoichiometric ATP with zero order kinetics suggesting that residual activity is catalyzed by F1 in which at least one beta subunit is modified. By exploiting the reactivity of the aziridinium of covalently attached reagent with [3H] aniline, sites modified by quinacrine mustard were labeled with 3H. Isolation of radioactive cyanogen bromide peptides derived from F1 inactivated with the reagent in the presence of [3H]aniline which were identified by sequence analysis and sequence analyses of radioactive tryptic fragments arising from them have revealed the following. About two thirds of the radioactivity incorporated into the enzyme during inactivation is apparently esterified to one or more of the carboxylic acid side chains in a CNBr-tryptic fragment of the beta subunit with the sequence: 394DELSEEDK401. The remainder of the radioactivity is associated with at least two sites within the cyanogen bromide peptide containing residues 293-358 of the beta subunit. From these results it is concluded that inactivation of F1 by the aziridinium of quinacrine mustard is due, at least in part, to modification of one or more of the carboxylic acid side chains in the DELSEED segment of the beta subunit and possibly also to modification of unspecified amino acid side chains between residues 302-356 of the beta subunit.
...
PMID:Localization of sites modified during inactivation of the bovine heart mitochondrial F1-ATPase by quinacrine mustard using [3H]aniline as a probe. 252 84

We have examined the relationship of the ubiquitous 68-70-kDa cytoskeletal-associated protein beta-internexin (Napolitano, E. W., Pachter, J. S., Chin, S. S. M., and Liem, R. K. H. (1985) J. Cell Biol. 101, 1323-1331) to heat-shock cognate 70 (hsc70), the major constitutive member of the mammalian heat-shock protein 70 (hsp70) family of stress proteins. We purify beta-internexin from rat brain microtubules and confirm its identity with hsc70 and the clathrin-uncoating ATPase by the following criteria: 1) The partial sequence of a cyanogen bromide-derived peptide from beta-internexin matches the inferred amino acid sequence of the cDNA clone pRC62 encoding hsc70 from rat brain (O'Malley, K., Mauron, A., Barchas, J. D., and Kedes, L. (1985) Mol. Cell. Biol. 5, 3476-3483). 2) Mixing experiments followed by two-dimensional gel analyses reveal the precise co-migration of beta-internexin, the clathrin-uncoating ATPase, and the in vitro translation product of cDNA clone pHSP-4 encoding rat brain hsc70. 3) beta-Internexin is recognized by a monoclonal antibody reactive against the class of hsp70 proteins. 4) beta-Internexin purified from a microtubule-associated protein-enriched fraction of rat brain by virtue of high affinity binding to ATP-agarose possesses clathrin cage-specific ATPase activity.
...
PMID:Beta-internexin is a microtubule-associated protein identical to the 70-kDa heat-shock cognate protein and the clathrin uncoating ATPase. 252 48

The rapid removal of Ca2+ ions from the cytosol, necessary for the efficient relaxation of cardiac muscle cells, is performed by the Ca2+-pumping ATPase of the sarcoplasmic reticulum. The calcium pump is activated by cyclic AMP- and calmodulin-dependent phosphorylation of phospholamban, an integral membrane protein of the sarcoplasmic reticulum. Using a heterobifunctional crosslinking agent which can be cleaved and photoactivated, we provide evidence for a direct interaction between the two proteins. Only the non-phosphorylated form of phospholamban interacts with the ATPase, demonstrating that phospholamban is an endogenous inhibitor that is removed from the ATPase by phosphorylation. Non-phosphorylated phospholamban interacts only with the calcium-free conformation of the ATPase and is released when it is converted to the calcium-bound state. We localized the site of interaction to a single peptide isolated after cyanogen bromide cleavage of the ATPase. The peptide derives from a domain just C-terminal to the aspartyl phosphate of the active site. This domain is unique to ATPases of the sarcoplasmic reticulum in that it has no homology with any other phosphorylation-type ion pump. The domain occurs in both slow- and fast-twitch isoforms of the ATPase, even though phospholamban is not expressed in fast-twitch muscles.
...
PMID:Nature and site of phospholamban regulation of the Ca2+ pump of sarcoplasmic reticulum. 253 Apr 54

We have examined the effect of second messengers on ATP-driven H+ transport in an H+ ATPase-bearing endosomal fraction isolated from rabbit renal cortex. cAMP (0.1 mM) had no effect on H+ transport. Acridine orange fluorescence in the presence of 0.5 mM Ca2+ (+1 mM EGTA) was 19 +/- 6% of control. Inhibition of ATP-driven H+ transport by Ca2+ was concentration dependent; 0.25 and 0.5 mM Ca2+ (+1 mM EGTA) inhibited acridine orange fluorescence by approximately 50 and approximately 80%, respectively. Ca2+ also produced a concentration-dependent increase in the rate of pH-gradient dissipation. Ca2+ did not affect ATP hydrolysis. ATP-dependent Br- uptake was virtually unchanged in the presence of 0.5 mM Ca2+ (+1 mM EGTA). These vesicles were also shown to transport Ca2+ in an ATP-dependent mode. Inositol 1,4,5-trisphosphate had no effect on ATP-dependent Ca2+ uptake. These results are consistent with the co-existence of an H+ ATPase and an H+/Ca2+ exchanger on these endosomes, the latter transport system using the H+ gradient to energize Ca2+ uptake. Attempts to demonstrate an H+/Ca2+ antiporter in the absence of ATP have been unsuccessful. Yet, when a pH gradient was established by preincubation with ATP and residual ATP was subsequently removed by hexokinase + glucose, stimulation of Ca2+ uptake could be demonstrated. A Ca2(+)-dependent increase in H+ permeability and an ATP-dependent Ca2+ uptake might have important implications for the regulation of vacuolar H+ ATPase activity as well as the homeostasis of cytosolic Ca2+ concentration.
...
PMID:H+/Ca2+ exchange in rabbit renal cortical endosomes. 253 22

Clathrin-coated vesicle acidification is mediated by an endomembrane proton translocating ATPase. This pump is electrogenic, and significant pH gradient formation requires the parallel movement of chloride through a chloride transporter in order to maintain net electroneutrality. We have solubilized, isolated and achieved 270-fold purification of this chloride transporter by means of selective detergent solubilization with cholate and polyoxyethelene 9-lauryl ether (C12E9), hydroxylapatite chromatography, and glycerol gradient centrifugation. Stabilization of the solubilized transporter requires 5 mM dithiothreitol. The partially purified transporter was co-reconstituted with the purified clathrin-coated vesicle proton translocating complex to yield preparations of proteoliposomes capable of valinomycin-independent proton pumping, as assessed by ATP-generated acridine orange quenching. In addition, the chloride transporter was independently reconstituted and was shown to catalyze diisothiocyano-disulfonic acid stilbene-sensitive 36Cl uptake. The anionic conductive selectivity of the reconstituted transporter (chloride = bromide greater than nitrate) exactly matched that of the transporter of native clathrin-coated vesicles. These studies demonstrate that the chloride transporter of vacuolar acidification systems is structurally and functionally dissociable from co-existing proton pumps and allow for investigations of pump-transporter interactions in a resolved system.
...
PMID:Isolation and reconstitution of the chloride transporter of clathrin-coated vesicles. 257 98


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---