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)

A new continuous coupled uv-spectrophotometric assay is described for two phosphate-releasing enzymes, aspartate transcarbamylase and ATPase of herpes simplex virus (HSV). Phosphate release is coupled to the phosphorolysis of the nucleoside analog 7-methylinosine (m7Ino) catalyzed by purine nucleoside phosphorylase. When this reaction is monitored at 291 nm, the coupled assay can readily detect 10 nmol Pi released/min. Our method offers advantages over a recently reported continuous assay devised for measuring aspartate transcarbamylase activity using the nucleoside analog methylthioguanosine (MESG) as the linking substrate. In contrast to MESG, m7Ino is easily and inexpensively synthesized and is also commercially available. The spectrophotometric signal at 291 nm, produced by the difference in the extinction coefficients between nucleoside substrate and the base product, is significant over a much wider pH range than the signal difference between MESG and its phosphorolysis product at 360 nm. Saturation curves for aspartate and carbamyl phosphate and pH rate profiles have been reproduced using the purine nucleoside phosphorylase/m7Ino coupled assay. Initial velocity patterns constructed over micromolar to millimolar concentrations of aspartate and carbamyl phosphate yielded four kinetic parameters simultaneously. To further illustrate the application of this coupled assay, kinetic parameters were determined for the DNA-dependent ATPase reaction of HSV helicase-primase.
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PMID:A continuous spectrophotometric assay for aspartate transcarbamylase and ATPases. 905 87

The Acanthamoeba myosin-IA and myosin-IB molecular motors bind to membranes, so they may produce the force to move organelles and membranes along actin filaments. We have determined the rate constants for the actin-activated myosin-I ATPase by pre-steady state kinetic analysis. ATP binds rapidly to myosin-I and dissociates the enzyme from actin filaments at a rate > 500 s-1. Myosin-I hydrolyzes ATP to ADP and inorganic phosphate (Pi) at 20-50 s-1. Phosphate dissociation is the rate limiting step in the ATPase cycle, 0.01 s-1 for myosin-I alone and at 10 s-1 when myosin-I is bound to actin filaments. ADP dissociation is rapid. Phosphorylation controls the ATPase cycle by increasing the rate of phosphate release from myosin-I bound to actin. At steady state the major species are myosin-ATP and myosin-ADP-Pi, which rapidly bind to and dissociate from actin filaments. During the ATPase cycle myosin-I binds so weakly to actin filaments that it cannot support processive movement like kinesin, unless several motors cluster together on a membrane or actin filament. These properties of the enzyme emphasize the importance of characterizing mechanisms that promote the self-association of myosin-I isoforms at specific binding sites in cells.
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PMID:The chemical mechanism of myosin-I: implications for actin-based motility and the evolution of the myosin family of motor proteins. 911 40

Peroxynitrite anion, the reaction product of superoxide and nitric oxide, is a potent biological oxidant, which inactivates mammalian heart mitochondrial NADH-coenzyme Q reductase (complex I), succinate dehydrogenase (complex II), and ATPase, without affecting cytochrome c oxidase (complex IV). In this paper, we evaluated the effect of peroxynitrite on mitochondrial membrane integrity and permeability under low calcium concentration. Phosphate buffer was used in most of our experiments since Hepes, Tris, mannitol, and sucrose were found to inhibit the oxidative chemistry of peroxynitrite. Peroxynitrite (0.1-1.0 mM) caused a dose-dependent decrease in the ability of mitochondria to build up a membrane potential when N,N,N',N'-tetramethyl-p-phenylenediamine/ascorbate were used as substrate. Elimination of the membrane potential was accompanied by penetration of the osmotic support (KCl/NaCl) into the matrix as judged by the parallel occurrence of mitochondrial swelling. This swelling was partially inhibited by dithiothreitol (DTT) or butylated hydroxytoluene (BHT) and was insensitive to ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, ADP, and cyclosporin A. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane proteins indicated that alterations in membrane permeability were associated with the production of protein aggregates due to membrane protein thiol cross-linking. The protective effect of DTT on both mitochondrial swelling and protein polymerization suggests the involvement of disulfide bonds in the membrane permeabilization process. In addition, the increase in thiobarbituric acid-reactive substances and the partial inhibitory effect of BHT indicate the occurrence of lipid peroxidation. These results support the idea that under our experimental conditions peroxynitrite causes mitochondrial structural and functional alterations by Ca2+-independent mechanisms through lipid peroxidation and protein sulfhydryl oxidation.
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PMID:Ca2+-independent permeabilization of the inner mitochondrial membrane by peroxynitrite is mediated by membrane protein thiol cross-linking and lipid peroxidation. 930 96

It has been demonstrated that palytoxin binds to and forms a channel within the Na+/K+-ATPase. To investigate whether palytoxin-induced channel formation within the sodium pump can occur independently of ATP hydrolysis and phosphorylation of the enzyme, an Asp369-->Ala mutant of the alpha1 subunit of the sheep sodium pump was produced and coexpressed with beta subunits in the yeast Saccharomyces cerevisiae. This aspartic acid residue, which during ion transport becomes phosphorylated from ATP, is essential for the function of the sodium pump. Therefore, as expected, microsomes isolated from yeast expressing the mutant sodium pump do not exhibit any ouabain sensitive ATPase activity, whereas in microsomes from yeast expressing the wild-type sodium pump, 60% of the total ATPase activity is ouabain-sensitive. Ouabain binds to yeast membranes containing either wild-type or mutant sodium pumps with similar Bmax (1.45+/-0.05 versus 1.37+/-0.02 pmol/mg) and Kd values (27.7+/-0.91 versus 29.57+/-0.93 nM), thus indicating that the mutant sodium pumps are expressed in the yeast and that the mutation does not considerably affect the conformation of the enzyme. In the presence of phosphate ouabain binds to microsomes containing the wild-type sodium pump with a Kd of 3.62+/-0.34 nM, showing that, although not necessary, phosphoenzyme formation enhances binding of the steroid. Phosphate or ATP, however, inhibit binding of ouabain to microsomes containing the mutant sodium pump with IC50 values of 78+/-3 microM and 3.0+/-0.4 microM, respectively. Despite these radical changes in the interactions of the mutant enzyme with ouabain, the interactions with palytoxin are not affected by the mutation. Palytoxin causes K+ efflux from yeast cells expressing the wild-type or mutant sodium pumps with EC50 values of 3.5+/-0.4 nM and 6.2+/-0.9 nM, respectively. Palytoxin-induced efflux from cells expressing wild-type or mutant sodium pumps occurs with similar t1/2 values of 20.3+/-2.1 min and 22.2+/-3.1 min, respectively. Ouabain inhibits K+ efflux from both cell types with IC50 values of 28+/-2 microM and 210+/-15 microM, respectively. Cells expressing the Asp369-->Ala mutants have an IC50 7.5-fold higher than that obtained with cells expressing the wild-type sodium pumps, possibly because ATP or phosphate present in the cytosol of the yeast cells influence and decrease ouabain binding to the mutant sodium pump. Thus, while ouabain binding and the associated inhibition of ion fluxes is promoted by phosphorylation of the wild-type enzyme by phosphate or ATP, palytoxin-induced channel formation is independent of phosphorylation and can be separated from the ATPase function of the sodium pump. Since ion fluxes through the sodium pump protein do not depend on ATP hydrolysis, the results suggest that the ionophores of pumps and ion channels might share common structural features.
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PMID:Palytoxin-induced channel formation within the Na+/K+-ATPase does not require a catalytically active enzyme. 934 22

Examination of organelle- and membrane-specific processes such as signal transduction necessitates the use of plasma membrane vesicles with cytoplasmic side-in orientation. We are interested in the structural identity and subcellular localization of in vivo [32P]phosphoric acid ([32Pi])-labeled phosphoinositides, including the recently discovered phosphatidyl-scyllo-inositol, for signal transduction studies. In the first part of this investigation, plasma membrane vesicles from barley aleurone cells were isolated employing the aqueous polymer (Dextran and polyethylene glycol) two-phase partition method. The membrane vesicles that partitioned into the upper and lower phases of the aqueous polymer two-phase system were characterized and the purity of the vesicles ascertained by assaying for two marker enzymes, K+-stimulated, Mg2+-dependent adenosine triphosphatase (EC 3.6.1.3, ATPase), localized in the plasma membranes, and cytochrome c oxidase, localized in the mitochondria. Inhibitors for ATPases such as azide, molybdate, and vanadate were used to distinguish between plasma membrane-associated and intracellular membrane-associated ATPases. These inhibitor studies suggest that the plasma membrane preparation contained about 7% of intracellular membrane vesicles and the intracellular membrane fraction contained about 6% of plasma membrane vesicles. Orientation of the plasma membrane vesicles was ascertained by measuring the latent ATPase activity. These latency studies suggest that about 95% of the plasma membrane vesicles were of cytoplasmic side-in orientation. In the second part of this investigation, intracellular distribution and in vivo [32Pi] labeling of phosphoinositides in the plasma membranes and intracellular membranes were investigated. Preferential accumulation of [32Pi]-labeled phosphatidyl-myo-inositol monophosphate (myo-PIP) and phosphatidyl-myo-inositol bisphosphate (myo-PIP2) was observed in the plasma membrane. However, scyllo-phosphatidylinositol (scyllo-PI) was detected in both the plasma membrane and the intracellular membranes. The cellular concentration of myo-phosphoinositides was determined, and, after 24 h of labeling with [32Pi], the ratio of radiolabel in myo-PI, PIP, and PIP2 paralleled the relative concentrations in aleurone cells.
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PMID:The isolation and characterization of right-side-out plasma membrane vesicles from barley aleurone cells. 1018

Trinitrophenylation of the reactive lysine (Lys84) in skeletal myosin subfragment 1 (S1) introduces a chiral probe (TNP) into an interface of the catalytic and lever arm domains of S1 [Muhlrad (1977) Biochim. Biophys. Acta 493, 154-166]. Characteristics of the TNP absorption and circular dichroism (CD) spectra in TNP-modified S1 (TNP-Lys84-S1), and the Lys84 trinitrophenylation rate in native S1, indicate a one-to-one correspondence between ATPase transients and trapped phosphate analogues. Phosphate analogue-induced structures of TNP-Lys84-S1 were modeled using the crystallographic coordinates of S1 [Rayment et al. (1993) Science 261, 50-58] with swivels at Gly699 and Gly710 to approximate conformational changes during ATPase. The CD and absorption spectral characteristics of the model structures were compared to those observed for analogue-induced structures. The model calculations, first tested on a trinitrophenylated hexapeptide with known structure, were applied to TNP-Lys84-S1. They showed that ATP binding initiates swiveling at Gly699 and that swiveling at both Gly710 and Gly699 accompanied ATP splitting just prior to product release. The computed lever arm trajectory during ATPase suggests (i) a plausible mechanism for the nucleotide-induced inhibition of Lys84 trinitrophenylation, and (ii) trinitrophenylation-induced changes in S1 Mg2+- and K+-EDTA ATPase are from collision of the lever arm with TNP at Lys84. TNP is a site-specific structural perturbant of S1 and a chiral reporter group for the effect of Lys84 modification on dynamic S1 structure. As such, TNP-Lys84-S1 is equivalent to a genetically engineered mutant with intrinsic sensitivity to structure local to the modified residue.
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PMID:Trinitrophenylated reactive lysine residue in myosin detects lever arm movement during the consecutive steps of ATP hydrolysis. 1035 Apr 61

Transport of inorganic orthophosphate (Pi) across the tonoplast membrane was studied using intact vacuoles isolated from suspension-cultured cells of Catharanthus roseus. Orthophosphate uptake was strongly stimulated in the presence of Mg-ATP and Mg-pyrophosphate and inhibited by bafilomycin and concanamycin which are potent inhibitors of the vacuolar H+-ATPase. These results indicated that the build-up of an electrochemical gradient by the H - pumps was essential for the uptake of Pi. Potassium thiocyanate, which dissipates the membrane potential across the tonoplast, strongly inhibited the Mg-ATP-stimulated uptake of Pi, while only a weak inhibition was observed in the presence of NH4Cl, which dissipates the pH gradient. These results indicate that, as observed for other anions like malate or chloride, the electrical component is the driving force of Pi uptake, whereas the deltapH plays only a minor role. Possible competitive inhibitors of Pi, MoO4(2-) , VO4(3-) and CrO4(2-) were tested. Among them, CrO4(2-) strongly inhibited Pi uptake into the vacuoles. Various inhibitors of anion transport were also tested. Only 4,4-diisothiocyanostilbene-2,2'-disulfonic acid strongly inhibited Pi uptake into the vacuoles. The function of the vacuolar Pi transporters for cytoplasmic Pi homeostasis is discussed.
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PMID:Phosphate uptake across the tonoplast of intact vacuoles isolated from suspension-cultured cells of Catharanthus roseus (L.) G. Don. 1098 58

Phosphate uptake by the phosphate-accumulating denitrifier Pseudomonas sp. JR12 was examined with different combinations of electron and carbon donors and electron acceptors. Phosphate uptake in acetate-supplemented cells took place with either oxygen or nitrate but did not take place when nitrite served as the final electron acceptor. Furthermore, nitrite reduction rates by this denitrifier were shown to be significantly reduced in the presence of phosphate. Phosphate uptake assays in the presence of the H(+)-ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD), in the presence of the uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP), or with osmotic shock-treated cells indicated that phosphate transport over the cytoplasmic membrane of this bacterium was mediated by primary and secondary transport systems. By examining the redox transitions of whole cells at 553 nm we found that phosphate addition caused a significant oxidation of a c-type cytochrome. Based on these findings, we propose that this c-type cytochrome serves as an intermediate in the electron transfer to both nitrite reductase and the site responsible for active phosphate transport. In previous studies with this bacterium we found that the oxidation state of this c-type cytochrome was significantly higher in acetate-supplemented, nitrite-respiring cells (incapable of phosphate uptake) than in phosphate-accumulating cells incubated with different combinations of electron donors and acceptors. Based on the latter finding and results obtained in the present study it is suggested that phosphate uptake in this bacterium is subjected to a redox control of the active phosphate transport site. By means of this mechanism an explanation is provided for the observed absence of phosphate uptake in the presence of nitrite and inhibition of nitrite reduction by phosphate in this organism. The implications of these findings regarding denitrifying, phosphate removal wastewater plants is discussed.
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PMID:Relationship between nitrite reduction and active phosphate uptake in the phosphate-accumulating denitrifier Pseudomonas sp. strain JR 12. 1109 96

The interaction between sulfite, an efficient Mg2+-dependent F1-ATPase activator, and chloroplast CF1-ATPase was studied. The sulfite anion was shown to inhibit ADP and ATP binding to the noncatalytic sites of CF1. The stimulating activity of sulfite persists when all noncatalytic sites are nucleotide-occupied. Phosphate, a competing candidate for binding to CF1 catalytic sites, suppresses this activity. These results support the suggestion that the stimulation of Mg2+-dependent ATPase activity of CF1 is caused by sulfite binding to its catalytic sites.
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PMID:Interaction of sulfite with the noncatalytic and catalytic sites of chloroplast coupling factor cf1. 1140 48

The establishment of the arbuscular mycorrhizal symbiosis results in a modification of the gene expression pattern in both plant and fungus to accomplish the morphological and physiological changes necessary for the bidirectional transfer of nutrients between symbionts. H(+)-ATPase enzymes play a key role establishing the electrochemical gradient required for the transfer of nutrients across the plasma membrane in both fungi and plants. Molecular analysis of the genetic changes in arbuscular mycorrhizal fungi during symbiosis allowed us to isolate a fungal cDNA clone encoding a H(+)-ATPase, GmPMA1, from Glomus mosseae (BEG12). Despite the high conservation of the catalytic domain from H(+)-ATPases, detailed analyses showed that GmPMA1 was strongly related only to a previously identified G. mosseae ATPase gene, GmHA5, and not to the other four ATPase genes known from this fungus. A developmentally regulated expression pattern could be shown for both genes, GmPMA1 and GmHA5. GmPMA1 was highly expressed during asymbiotic development, and its expression did not change when entering into symbiosis, whereas the GmHA5 transcript was induced upon plant recognition at the appressorium stage. Both genes maintained high levels of expression during intraradical development, but their expression was reduced in the extraradical mycelium. Phosphate, a key nutrient to the symbiosis, also induced the expression of GmHA5 during asymbiotic growth, whereas sucrose had a negative effect. Our results indicate that different fungal H(+)-ATPases isoforms might be recruited at different developmental stages possibly responding to the different requirements of the life in symbiosis.
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PMID:Symbiotic status, phosphate, and sucrose regulate the expression of two plasma membrane H+-ATPase genes from the mycorrhizal fungus Glomus mosseae. 1285 34


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