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 role of physicochemical factors, electronic and hydrophobic, and a hydrogen donor index in the inhibition of gastric (H+/K+)-ATPase by 4-phenyl-2-guanidinothiazoles and the 4-indolyl-2-guanidinothiazoles has been quantitatively analysed. For the first congeneric series, the resonance effect of the ortho- and para-substituents and hydrogen donor property of the meta-substituent in the phenyl ring play crucial role, whereas for 4-indolyl analogues, the hydrophobicity and electron withdrawing effect of X-substituents in the indolyl ring are shown to be important decisive factors. Also the substitution of the guanidine moiety, e.g. by benzyl, raises the activity of proton pump inhibitors. The substitution at 5-position of thiazole ring does not enhance the potency.
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PMID:Structure-activity relationship studies of 4-substituted-2-guanidinothiazoles: reversible inhibitors of gastric (H+/K+)-ATPase. 827 27

We reported previously that poliovirus infection induces alkalinization in HeLa cells and that an alkaline intracellular pH (pHi) promoted viral replication. Additional experiments were carried out to understand the underlying mechanism. Virus-infected or control monolayer cultures were incubated with nominally bicarbonate-free Eagle's minimal essential medium (MEM) buffered with N-2-hydroxyethylpiperazine-N-3-ethanesulfonic acid (HEPES), and immediately following preincubations, changes in pHi were monitored via benzoic acid uptake around 2 h postinfection. The absence of pH increase in cells infected with ultraviolet light-inactivated virus (UV-virus) indicated that viral gene expression was required for this effect. On the other hand, lack of effect of 3 mM guanidine, an inhibitor of poliovirus-specific RNA but not protein synthesis, suggested that translation of input viral genome RNA is sufficient for the pH increase. Activation of Na+/H+ exchange, Cl(-)HCO3- exchange, or H(+)-ATPase was considered as possible mechanisms by which alkalinization occurs in virus-infected cells. Na+/H+ exchange was excluded because the pH effect occurred in a Na+/H+ exchange deficient HeLa cell mutant. Similarly, Cl-/HCO3- exchange was excluded because virus-specific alkalinization was evident in the presence of Cl- or bicarbonate deficient medium and was not associated with an increase in HCO3- uptake or a decrease in Cl- uptake. Lack of dependence on Na+, abrogation by 10 microM 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl), and resistance to 1 mM vandate suggested that this effect was due to the activation of a vacuolar-type (V) proton ATPase. Studies using protein kinase inhibitors indicated that activation of the ATPase in virus-infected cells probably involved protein kinase C-mediated phosphorylation.
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PMID:Poliovirus-induced intracellular alkalinization involves a proton ATPase and protein phosphorylation. 838 3

Chemical modification of mitochondrial F1-ATPase from Schizosaccharomyces pombe by the tryptophan-specific reagent N-bromosuccinimide (NBS) at pH 5.0 in the presence of 20% glycerol produced a characteristic lowering in both enzyme absorbance at 280 nm and intrinsic fluorescence at 332 nm that varied with NBS/F1 molar ratio up to a value of 130. Fluorometric titration of tryptophans and correlation to residual ATPase activity showed that modification of three reactive residues among the seven present on alpha- and epsilon-subunits did not markedly modify the enzyme activity but efficiently released endogenous ATP and abolished the fluorescence quenching related to GDP or ATP binding to the catalytic site. Additional modification of one, less reactive, tryptophan altered both negative cooperativity of ATP hydrolysis and sensitivity to azide inhibition and produced a nearly complete inactivation at high NBS/F1 molar ratio. NBS-induced inactivation of F1 was favored by catalytic-site saturation with GDP or low ATP concentration and on the contrary was prevented by noncatalytic-site saturation with ADP or high ATP concentration. When reactive tryptophans were selectively modified by NBS in the presence of ADP, and subunits were isolated after guanidine hydrochloride dissociation by one-step purification on reversed-phase HPLC, the absorbance of alpha-subunit at 280 nm was decreased, whereas that of epsilon-subunit was unchanged. Cyanogen bromide cleavage of alpha-subunit and fragments separation by reversed-phase HPLC showed that one peptide of 3 kDa apparent molecular mass had decreased absorbance. N-Terminal sequencing allowed its identification to fragment 255-282 that contains tryptophan257.
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PMID:Chemical modification of alpha-subunit tryptophan residues in Schizosaccharomyces pombe mitochondrial F1 adenosine 5'-triphosphatase: differential reactivity and role in activity. 842 30

Systems for overexpression and purification of active alpha, beta and gamma subunits of Escherichia coli H(+)-ATPase were established. The alpha and beta subunits recovered as soluble form were purified by hydroxyapatite column chromatography. Since the gamma subunit was overexpressed as the insoluble form, this subunit was purified by polyacrylamide gel-electrophoresis containing sodium dodecyl sulfate. By subsequent denaturation of this subunit with guanidine hydrochloride and renaturation, the active gamma subunit for reconstitution of the F1-ATPase activity with the purified alpha and beta subunit was obtained. The delta and epsilon subunits which were fused to the carboxy terminus of glutathione S-transferase (GST) were overproduced and purified by affinity chromatography. These fused proteins (delta-GST and epsilon-GST) were incubated with the purified alpha, beta and gamma subunits and applied to affinity chromatography. The alpha beta gamma delta-GST and alpha beta gamma epsilon-GST complex were eluted specifically by addition of glutathione and exhibited high and low ATPase activity, respectively, with a subunit stoichiometry similar to that in the native F1-ATPase, indicating that active complexes could be reconstituted with the fused proteins. These results suggested that the amino-terminal ends of the delta and epsilon subunits are not involved in formation of the active complex. The fused epsilon-GST bound the gamma subunit strongly, and the alpha subunit weakly. The delta-GST bound the gamma subunit significantly, and the alpha and beta subunits very weakly.
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PMID:Reconstitution of the F1-ATPase activity from purified alpha, beta, gamma and delta or epsilon subunits with glutathione S-transferase fused at their amino termini. 857 96

We report that protein 2C, the putative nucleoside triphosphatase/helicase protein of poliovirus, is required for the initiation of negative-strand RNA synthesis. Preinitiation RNA replication complexes formed upon the translation of poliovirion RNA in HeLa S10 extracts containing 2 mM guanidine HCI, a reversible inhibitor of viral protein 2C. Upon incubation in reactions lacking guanidine, preinitiation RNA replication complexes synchronously initiated and elongated negative-strand RNA molecules, followed by the synchronous initiation and elongation of positive-strand RNA molecules. The immediate and exclusive synthesis of negative-strand RNA upon the removal of guanidine demonstrates that guanidine specifically blocks the initiation of negative-strand RNA synthesis. Readdition of guanidine HCl to reactions synchronously elongating nascent negative-strand RNA molecules did not prevent their continued elongation and completion. In fact, readdition of guanidine HCl to reactions containing preinitiation complexes elongating nascent negative-strand RNA molecules had no effect on subsequent positive-strand RNA synthesis initiation or elongation. Thus, the guanidine-inhibited function of viral protein 2C was not required for the elongation of negative-strand RNA molecules, the initiation of positive-strand RNA molecules, or the elongation of positive-strand RNA molecules. The guanidine-inhibited function of viral protein 2C is required only immediately before or during the initiation of negative-strand RNA synthesis. We suggest that guanidine may block an irreversible structural maturation of protein 2C and/or RNA replication complexes necessary for the initiation of RNA replication.
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PMID:Synchronous replication of poliovirus RNA: initiation of negative-strand RNA synthesis requires the guanidine-inhibited activity of protein 2C. 934 5

Vacuolar-type (V) ATPases are thought to be the main determinant of phagosomal acidification. In phagosomes containing mycobacteria, which ostensibly impair the delivery of V-ATPases to the phagosomal membrane, the pH would be expected to be near neutral. This prediction was tested by microfluorescence ratio imaging using macrophages from mice susceptible to mycobacterial infection. Although less acidic than their counterparts containing dead bacteria, phagosomes containing live Mycobacteria bovis were nearly 1 pH unit more acidic than the cytosol, suggesting the existence of alternate H+ transport mechanisms. We therefore investigated whether Na+/H+ exchange (NHE) contributes to phagosomal acidification. Immunoblotting, reverse transcriptase-polymerase chain reaction, and pharmacological studies indicated that NHE1 is the predominant isoform of the exchanger in macrophages. Fractionation revealed that NHE1 is incorporated into the phagosomal membrane, and measurements of pH indicated that it is functional in this location. Nevertheless, acidification of the lumen of phagosomes containing either latex beads or live M. bovis was insensitive to (3-methylsulfonyl-4-piperidinobenzoyl)-guanidine methanesulfonate, a potent inhibitor of NHE1. This may have been due to the absence of an appropriate lumen to cytosol Na+ gradient, because the phagosomal membrane was found to be devoid of Na+/K+ pumps. Unexpectedly, the acidification of M. bovis phagosomes was fully reversed by specific inhibitors of the vacuolar H+-ATPase, suggesting that ATPases are present only transiently or in reduced quantities in the phagosomal membrane. Alternatively, acid equivalents accumulated in endosomes by V-ATPases may be delivered to the mycobacterial phagosome by carrier vesicles devoid of ATPases.
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PMID:Regulation of phagosomal acidification. Differential targeting of Na+/H+ exchangers, Na+/K+-ATPases, and vacuolar-type H+-atpases. 936 53

It has been shown that many proteins, when converted into partially unfolded structures, interact strongly with a lipid bilayer. SecA protein of Escherichia coli is an unusual water-soluble protein which, in the native form, can readily penetrate the membrane and lipid bilayer. In order to see whether the native SecA exhibits partially unfolded characteristics, the stability and solvent accessibility of SecA were studied using various spectroscopic and hydrogen-exchange methods. The results are compared with the reported data for native and molten globule forms of alpha-lactalbumin (alpha-LA), as well as those for apocytochrome c. The exposure of hydrophobic residues of SecA, as monitored by 8-anilinonaphthalene-1-sulfonic acid (ANS) binding, and the extent of amide hydrogen exchange were comparable to those of native alpha-LA. On the other hand, equilibrium unfolding experiments showed that SecA is less stable than native alpha-LA. The results of tryptic digestion and the change of endogenous ATPase activity induced by guanidine hydrochloride were suggestive of the C-terminal half of SecA being more flexible than the rest of the protein. The overall conclusion is that the SecA as a whole has a somewhat open structure due to a relatively unstable C-domain which may facilitate its penetration into a lipid bilayer.
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PMID:Stability and solvent accessibility of SecA protein of Escherichia coli. 944 18

The highly conserved non-structural protein 2C of picornaviruses is involved in viral genome replication and encapsidation and in the rearrangement of intracellular structures. 2C binds RNA, has nucleoside triphosphatase activity, and shares three motifs with superfamily III helicases. Motifs "A" and "B" are involved in nucleotide triphosphate (NTP) binding and hydrolysis, whereas a function for motif "C" has not yet been demonstrated. Poliovirus RNA replication is inhibited by millimolar concentrations of guanidine hydrochloride (GdnHCl). Resistance and dependence to GdnHCl map to 2C. To characterize the nucleoside triphosphatase activity of 2C, we purified poliovirus recombinant 2C fused to glutathione S-transferase (GST-2C) from Escherichia coli. GST-2C hydrolyzed ATP with a Km of 0.7 mM. Other NTPs, including GTP, competed with ATP for binding to 2C but were poor substrates for hydrolysis. Mutation of conserved residues in motif A and B abolished ATPase activity, as did mutation of the conserved asparagine residue in motif C, an observation indicating the involvement of this motif in ATP hydrolysis. GdnHCl at millimolar concentrations inhibited ATP hydrolysis. Mutations in 2C that confer poliovirus resistant to or dependent on GdnHCl increased the tolerance to GdnHCl up to 100-fold.
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PMID:Characterization of the nucleoside triphosphatase activity of poliovirus protein 2C reveals a mechanism by which guanidine inhibits poliovirus replication. 1006 53

Cardiac intracellular Na+and Ca2+homeostasis is regulated by the concerted action of ion channels, pumps and exchangers. The Na+, K+-ATPase produces the electrochemical concentration gradient for Na+, which is the driving force for Ca2+removal from the cytosol via the Na+/Ca2+exchange. Reduction of this gradient by increased intracellular Na+concentration leads to cellular Ca2+overload resulting in arrhythmias and contractile dysfunction. Na+and Ca2+overload-associated arrhythmias can be produced experimentally by inhibition of Na+efflux (digitalis-induced intoxication) and by abnormal Na+influx via modulated Na+channels (veratridine, DPI 201-106; hypoxia) or via the Na+, H+exchanger. Theoretically, blockers of Na+and Ca2+channels, inhibitors of abnormal oscillatory release of Ca2+from internal stores or modulators of the Na+, Ca2+and Na+, H+exchanger activities could protect against cellular Na+and Ca2+overload. Three exemplary drugs that prevent Na+and Ca2+overload, i.e. the benzothiazolamine R56865, the methylenephenoxydioxy-derivative CP-060S, and the benzoyl-guanidine Hoe 642, a Na+, H+exchange blocker, are briefly reviewed with respect to their efficacy on digitalis-, veratridine- and ischaemia/reperfusion-induced arrhythmias.
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PMID:Drugs preventing Na+ and Ca2+ overload. 1009 40

The purpose of this study was to investigate the mechanism by which the H2-antagonists ranitidine and famotidine interacted with the paracellular space during their transport across Caco-2 cell monolayers. Transport experiments with ranitidine and famotidine across Caco-2 cell monolayers were performed to determine the apical-to-basolateral flux at various concentrations. Kinetic analysis of the transport data showed that ranitidine and famotidine were transported by both saturable and nonsaturable processes. Na+, K+-ATPase inhibitor ouabain and metabolic inhibitors sodium azide + 2-deoxy-D-glucose did not affect ranitidine transport, suggesting that the active transport was not involved. Famotidine and some other guanidine-containing compounds, e.g., guanethidine, Arg-Gly, L-arginine methyl ester, and L-argininamide, inhibited the transport of ranitidine, whereas other guanidine-containing compounds with an additional negative charge, e.g., L-arginine, did not. 2,4, 6-Triaminopyrimidine (TAP), an inhibitor of paracelluar cationic conductance, also inhibited the transport of both ranitidine and famotidine. On the basis of these results, it is proposed that the saturable transport of ranitidine and famotidine across Caco-2 cell monolayers appears to be via a facilitated diffusion process mediated by the paracellular anionic sites. This mechanism is consistent with the observation that ranitidine and famotidine caused a concentration-dependent increase in transepithelial electrical resistance (TEER) across Caco-2 cell monolayers, presumably by blocking the paracellular anionic sites and thus inhibiting the flux of cations (e.g., Na+).
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PMID:Saturable transport of H2-antagonists ranitidine and famotidine across Caco-2 cell monolayers. 1039 65


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