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)

1. The cell-membrane ATP phosphohydrolase of vegetatively grown Clostridium pasteurianum was specifically Mg2+-dependent, but demonstrated significant activity with GTP, CTP and UTP. It displayed approximate Michaelis-Menten kinetics only in the presence of certain effectors (e.g. phosphoenolpyruvate, fructose 1,6-bis-phosphate) which decreased the Km for ATP (to below 2 mM) but also V, whilst extending to pH 5.8 the effective pH range of activity of the enzyme. 2. ATP phosphohydrolase activity of the membrane ATPase (BF0F1) was inhibited by N,N'-dicyclohexylcarbodiimide, butyricin 7423, Dio-9, 4-chloro-7-nitrobenzofurazan, efrapeptin, leucinostatin and quercetin, and to a lesser degree by aurovertin and citreoviridin. The enzyme was not inhibited by oligomycin, spegazzinine, tributyl tin, triethyl tin or venturicidin. The soluble ATPase (BF1) component differed in not being inhibited by N,N'-dicyclohexylcarbodiimide, butyricin 7423 or leucinostatin. 3. The ATPase (BF0F1) complex and its soluble (BF1) component were separately purified. 4. Dodecylsulphate/polyacrylamide gel electrophoresis separated only four polypeptide components in the purified ATPase (BF0F1), with approximate molecular weights (+/- 10%) as follows: subunit a, 65 500; subunit c, 57 500; subunit da, 43 000; subunit fa, 15 000. The soluble (BF1 component contained only the three polypeptide subunits a, c and da. These were present in the BF0F1 preparation in the ratio 2 : 1 : 2; the contribution of subunit fa could not satisfactorily be quantified. 5. Subunit a was identified as the component binding 4-chloro-7-nitrobenzofurazan and subunit fa as the component binding N,N'-dicyclohexylcarbodiimide. The ATP phosphohydrolase activity of the membrane ATPase was not activated by trypsin treatment and the ATPase (BF0F1) contained no trypsin-sensitive inhibitor protein subunit. 6. Purified ATPase (BF0F1) was incorporated into artificial proteoliposomes which demonstrated ATP-dependent enhancement of 8-anilinonaphthalene-1-sulphonate fluorescence and ATP-dependent proton influx. These reactions were abolished by proton conductors (e.g. carbonylcyanide m-chlorophenylhydrazone) by valinomycin in the presence of a high external concentration of K+, or by N,N'-dicyclohexylcarbodiimide, butyricin 7423, Dio-9, 4-chloro-7-nitrobenzofurazan or leucinostatin. Oligomycin, tributyl tin, triethyl tin and venturicidin were not inhibitory. 7. When stripped of the soluble BF1 component, such ATPase-proteoliposomes demonstrated nil ATP phosphohydrolase activity and did not display ATP-dependent enhancement of 8-anilino-naphthalene-1-sulphonate fluorescence or ATP-dependent protein influx. All of these activities were restored by incubation of the BF1-depleted proteoliposomes with a purified preparation of the soluble BF1 component.
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PMID:The proton-translocating adenosine triphosphatase of the obligately anaerobic bacterium Clostridium pasteurianum. 1. ATP phosphohydrolase activity. 3 58

1. The membrane perturbations induced by the interaction of the fluorescent probe 1-anilino-8-naphthalene sulfonate (ANS) with human red blood cells were studied. 2. ANS below 0.5 mM inhibits partially (20% maximum) the ouabain-insensitive Na+ and K+ influx and efflux. Above 0.5 mM ANS increases both Na+ and K+ leak fluxes. The increased cation leaks are larger for Na+ than K+. 3. The (Na+ +K+)-ATPase and ouabain-sensitive Na+ and K+ fluxes are inhibited by ANS. Ouabain-insensitive, Mg2+-dependent ATPase activity of ghosts is stimulated by [ANS] less than 0.3 mM and inhibited by [ANS] greater than 0.3 mM. 4. ANS also inhibits the Na+-dependent, ouabain-insensitive K+ influx that is inhibited by ethacrynic acid and furosemide. 5. Red cells become crenated with [ANS] less than 1 mM and sphere at [ANS] greater than 1 mM. In the former conditions hypotonic hemolysis is decreased whereas the latter increase osmotic fragility. 6. It is suggested that ANS expands the membrane asymmetrically by binding preferentially to the external membrane surface. 7. It is concluded that ANS is a general inhibitor of ion transport, particularly of those processes thought to involve facilitated-diffusion mechanisms. The increased cation leaks observed at high ANS concentrations may be related to prehemolytic membrane disruption. 8. The membrane perturbations caused by ANS are compared to those caused by other reversible inhibitors of anion exchange in red blood cells. Their possible modes of action are discussed.
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PMID:Asymmetric membrane expansion and modification of active and passive cation permeability of human red cells by the fluorescent probe 1-anilino-8-napththalene sulfonate. 12 20

1. A further investigation has been made of the way in which the fluorescent probes 1-anilino-naphthalene-8-sulphonate and 2-(N-methyl-anilino) naphthalene-6-sulphonate report on the energised state of bovine heart submitochondrial particles. 2. A comparison of the probe responses to energisation with ATP or to a potassium diffusion potential has been made. The fluorescence enhancements seen in these two cases have different characteristics, and in view of this it is questioned whether a substrate generated energised state of a submitochondrial particle can be equated with a trans-membrane potassium diffusion potential. 3. Substitution of ITP for ATP reduces the rate at which either of the probes respond to energisation. In contrast reducing the ATPase activity of the particles by treatment with the covalent ATPase inhibitors 4-chloro-7-nitrobenzofurazan or N,N'-dicyclohexyl-carbodiimide has no effect on this rate. This finding that the rate of the fluorescence changes is directly sensitive to events at the level of the ATPase, but not to the total ATPase activity, suggests that this rate may not be controlled by a delocalised energised state. Reduction of ATPase activity decreases the extent of the fluorescence enhancement and a relationship between the change in probe fluorescence and ATPase activity is given. 4. The results in this paper are discussed in the context of the mechanisms which have been proposed to account for the fluorescence enhancements of N-aryl naphthalene sulphonate probes upon energisation of submitochondrial particles.
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PMID:On the nature of the energised state of submitochondrial particles; investigations with N-aryl naphthalene sulphonate probes. 12 65

Fragmented sarcoplasmic reticulum was prepared from the rabbit heart. The function of vesicles was characterized by the activities of Mg++ and ATP-dependent 'Calcium-Binding' and Ca-ATPase. A fluorescence probe, 1-anilino-8-naphthalene sulfonate (ANS) was employed to examine the physicochemical changes in vesicular membranes. Chlorpromazine, dl-propranolol and lidocaine inhibited Calcium-Binding activity and increased ANS-fluorescence. There was a good correlation between the changes in two parameters. The potency of three drugs on both parameters decreased in the above-mentioned order. These drugs, however, induced no change in the ANS-fluorescence associated by serum albumin or lecithin. The drugs would act presumably at lipid-protein interfaces in vesicles to increase ANS-fluorescence. Both Ca-ATPase and Basic-ATPase were inhibited by dl-propranolol. These results suggest that the inhibitory action of dl-propranolol on the calcium transport system is ascribable to direct physicochemical effects on vesicular membranes.
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PMID:Studies on calcium transport system in cardiac sarcoplasmic vesicles and its inhibition by dl-propranolol. 13 44

Membrane fractions were isolated from Streptococcus faecalis cells of a glycolyzing microorganism, devoid of the respiratory chain, using the methods of osmotic shock of the protoplasts, ultrasonic treatment of the cells and ultrasonic treatment of the protoplasts. All fractions possessed the ATPase activity, the highest activity being observed in the fraction isolated by ultrasonication of the protoplasts. All preparations were estimated with respect to the presence of vesicles, formed by the "inside-out" and "inside-in" membranes, using ATPase as a marker of the membrane orientation. In the membrane fractions obtained by ultrasonication of the protoplasts, the "inside-out" vesicles were prevalent. ATP-dependent energization of the membranes, sensitive to the action of dicyclohexylcarbodiimide and tetrachlorotrifluoromethyl benzimidazole, was demonstrated by measuring the transport of the lipophylic anion of phenyldicarbaundecaborane and aniline naphthalene sulfonate fluorescence.
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PMID:[Energization of membrane vesicles from the cells of glycolyzing bacterium Streptococcus faecalis]. 14 4

The addition of bacteriophage T5 to anaerobic, fermenting cells of Escherichia coli B or K-12 in the presence of 8-anilino-1-naphthalene sulfonate (ANS), N-phenylnaphthyl-1-amine (NPN), or dansyl ethylamine causes the fluorescence of these probes to rise in two steps, the first occurring immediately upon addition, the second delayed by 6 min. The conditions necessary for observing this phenomenon are defined (cell density, probe concentration, substrate, absence of an electron acceptor, multiplicity of infection, growth, and harvesting conditions). The magnitudes of the first and second steps in fluorescence are dependent upon the multiplicity of infection; the timing of the steps is not. The first step correlates with a breakdown in the potassium or rubidium permeability barrier of the cells, and it occurs either aerobically or anaerobically, with fermentable or nonfermentable substrates. The second step occurs only with cells that are without an available electron acceptor, are fermenting, and which have a functional membrane-bound, Ca2+-dependent adenosine triphosphatase (ATPase). The results are consistent with disturbance of energization of the cell membrane by the membrane-bound ATPase at the time of the second step in fluorescence. No changes in the intracellular level of adenosine 5'-triphosphate (ATP) was seen, whereas the extracellular level increased sharply, starting 3--6 min after phage addition. The quantity of ATP found in the medium by 30 min after infection amounted to about four times the amount present inside the cells at the time of infection. The quantity and rate of efflux of ATP was similar under aerobic and anaerobic conditions.
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PMID:Relationship between steps in 8-anilino-1-naphthalene sulfonate (ANS) fluorescence and changes in the energized membrane state and in intracellular and extracellular adenosine 5'-triphosphate (ATP) levels following bacteriophage T5 infection of Escherichia coli. 15 81

The phospholipid composition of the electron transport particles and coupling factor-depleted electron transport particles of Mycobacterium phlei are the same, but they differ in contents. The accessibility of partially purified phospholipase A to these membrane phospholipids was found to be different. Treatment of membranes of Mycobacterium phlei with phospholipase A impairs the rate of oxidation as well as phosphorylation. The inhibition of phosphorylation can be reversed by washing the membranes with defatted bovine serum albumin. The reconstitution of membrane-bound coupling factor-latent ATPase activity to phospholipase A-treated depleted electron transport particles and their capacity to couple phosphorylation to oxidation of substrates remained unaffected after phospholipase A treatment. However, the pH gradient as measured by bromthymol blue was not restored after reconstitution of phospholipase A-treated depleted electron transport particles with membrane-bound coupling factor-latent ATPase. These findings show that the phosphorylation coupled to the oxidation of substrates can take place without a pronounced pH gradient in these membrane vesicles. The dye 1-anilino-8-naphthalene sulfonic acid (ANS) exhibited low levels of energized and nonenergized fluorescence in phospholipase A-treated membranes. This decrease in the level of ANS fluorescence in phospholipase A-treated membranes was found to be directly related to the amount of phospholipids cleaved. The decrease in the energy-dependent ANS response in phospholipase A-treated electron transport particles, as compared with untreated electron transport particles, was shown to be a result of a change in the apparent K-d of the dye-membrane complex, and of a decrease in the number of irreversible or slowly reversible binding sites, with no change in the relative quantum efficiency of the dye. The decrease in ANS fluorescence in phospholipase A-treated particles appears to be due to a decrease in the hydrophobicity of the membranes.
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PMID:Effect of phospholipase A on the structure and functions of membrane vesicles from Mycobacterium phlei. 23 99

1. Evidence is presented which indicates that inactivation of the mitochondrial ATPase from bovine heart by the reagent 4-chloro-7-nitrobenzofurazan results from modification of one tyrosine residue per enzyme molecule. Activity can be restored by a variety of sulphydryl reagents. 2. In sodium dodecyl sulphate, the nitrogenzofurazan group on tyrosine is transfered to newly exposed sulphydryl groups on the enzyme. 3. The rate of transfer of the nitrobenzofurazan moiety from theenzyme to sulphydryl compounds is compared with that for transfer from the model compound N-acetyl-tyrosine-0(7-nitrobenzo-furazan) ethyl ester, the synthesis and properties of which are also described. 4. The ligands ATP and ADP exert a protective effect on the rate of reaction between the mitochondrial ATPase and 4-chloro-7-nitrobenzofurazan. The variation in rate of this reaction with change in pH has also been examined and a pKa of 9.5 estimated for the tyrosine residue. 5. The modification does not prevent substrate binding as judged by changes in the fluorescence of aurovertin, an antibiotic with specific affinity for mitochondiral ATPases. 6. When the ATPase activity of submitochondrial particles is inhibited by 4-chloro-7-nitrobenzo-furazan, there is a parallel decrease in the extent of the energy-linked fluorescence enhancement of 1-anilino-naphthalene-8-sulphonate induced by ATP hydrolysis. Both ATPase activity and the fluorescence enhancement are restored by sluphydryl reagents.
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PMID:The mitochondrial ATPase. Evidence for a single essential tyrosine residue. 23 39

We present a new method to specifically and stably label proteins by attaching extrinsic probes to amino acids that are thiophosphorylated by protein kinases and ATP gamma S. The method was demonstrated for labeling of a thiophosphorylatable serine of the isolated regulatory light chain of smooth muscle myosin. We stoichiometrically blocked the single thiol (Cys-108) either by forming a reversible intermolecular disulfide bond or by reacting with iodoacetic acid. The protein was stoichiometrically thiophosphorylated at Ser-19 by myosin light chain kinase and ATP gamma S. The nucleophilic sulfur of the protein phosphorothioate was coupled at pH 7.9 and 25 degrees C to the fluorescent haloacetate [3H]-5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1- sulfonic acid ([3H]IAEDANS) by displacement of the iodide. Typical labeling efficiencies were 70-100%. The labeling was specific for the thiophosphorylated Ser-19, as determined from the sequences of two labeled peptides isolated from a tryptic digest of the labeled protein. [3H]IAEDANS attached to the thiophosphorylated Ser-19 was stable at pH 3-10 at 25 degrees C, and to boiling in high concentrations of reductant. The labeled light chains were efficiently exchanged for unlabeled regulatory light chains of the whole myosin molecule. The resulting labeled myosin had normal ATPase activities in the absence of actin, indicating that the modification of Ser-19 and the exchange of the labeled light chain into myosin did not significantly disrupt the protein. The labeled myosin partially retained the elevated actin-activated Mg(2+)-ATPase activity which is characteristic of thiophosphorylated myosin. This indicates that labeling of the thiophosphate group with [3H]IAEDANS did not completely disrupt the functional properties of the thiophosphorylated protein in the presence of actin.
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PMID:A new method to specifically label thiophosphorylatable proteins with extrinsic probes. Labeling of serine-19 of the regulatory light chain of smooth muscle myosin. 142 Apr 39

The naphthalene-induced cataract in rats has been studied for many years as a possible model of human aging-related cataract. While the molecular mechanism of this cataract is unclear, it has recently been demonstrated that the aldose reductase inhibitor ALO1576 can prevent lens opacification in this system. The present study was undertaken to investigate the molecular basis for the effects of naphthalene on the lens and the role of pigmentation in the cataractogenic mechanism. Cataracts were induced in five strains of rats (two pigmented, three albino) by oral administration of naphthalene. Initial lens changes were observed after 1 week by slit-lamp; by 3 weeks a distinct shell-like opacity was present in the deep cortex. Little difference in the course of opacification was found between the pigmented and albino strains. Major biochemical effects were a decrease of 20-30% in glutathione (GSH) by 1 week of feeding, disulfide cross-linking of lens proteins present by 3 weeks, and a nearly 20-fold increase in the content of protein-GSH mixed disulfide. No effect was seen in the ability of the affected lenses to accumulate activity [3H]choline or 86Rb from the medium in organ culture nor in the activity of the Na+/K(+)-ATPase. ALO1576 (10 mg kg-1 day-1) completely prevented all morphological and biochemical changes in the lenses of the naphthalene-fed rats in both pigmented and non-pigmented strains. These results indicate that pigmentation is not required for induction of naphthalene cataract in rats. Naphthalene dihydrodiol was found in the aqueous humor and lens of naphthalene-fed rats. It is proposed that naphthalene dihydrodiol produced in the liver reaches the aqueous humor and penetrates the lens where it is further metabolized ultimately to form the toxic species, naphthoquinone.
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PMID:The possible mechanism of naphthalene cataract in rat and its prevention by an aldose reductase inhibitor (ALO1576). 154 42


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