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 kinetics of Ca2+ dissociation from and binding to the Ca2(+)-ATPase and the coupled tryptophan fluorescence changes were compared in the heavy and the light sarcoplasmic reticulum (SR) fractions. It was found that in the light SR both the dissociation of Ca2+ from the Ca2(+)-ATPase and the coupled tryptophan fluorescence change took place in a biphasic fashion, but they were monophasic in the heavy SR. On the other hand, the time courses of both the Ca2+ binding and the coupled tryptophan fluorescence increase were biphasic, and virtually indistinguishable between the heavy and the light SR fractions. Submicromolar ruthenium red altered the kinetics of both the Ca2+ dissociation and the coupled fluorescence change in heavy, but not in light SR; the monophasic time course characteristic of the heavy fraction became biphasic, similar to that found for light SR in the absence of ruthenium red. Extraction of non-ATPase proteins from the heavy SR vesicles also changed the kinetics of the Ca2+ dissociation-coupled fluorescence decrease in the heavy SR from monophasic to biphasic. These results suggest that the difference between the heavy and light SR Ca2(+)-ATPase in the Ca2+ dissociation kinetics is most probably produced by a ruthenium-red-sensitive interaction of the Ca2(+)-ATPase with additional protein(s), which is/are present exclusively in the heavy SR, rather than being due to a difference inherent to the Ca2(+)-ATPase polypeptide itself.
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PMID:Non-identical behavior of the Ca2(+)-ATPase in the terminal cisternae and the longitudinal tubules fractions of sarcoplasmic reticulum. 248 84

The solubilization and delipidation of sarcoplasmic reticulum Ca2+-ATPase by different nonionic detergents were measured from changes in turbidity and recovery of intrinsic fluorescence of reconstituted ATPase in which tryptophan residues had been quenched by replacement of endogenous phospholipids with brominated phospholipids. It was found that incorporation of C12E8 or dodecyl maltoside (DM) at low concentrations in the membrane, resulting in membrane "perturbation" without solubilization, displaced a few of the phospholipids in contact with the protein; perturbation was evidenced by a parallel drop in ATPase activity. As a result of further detergent addition leading to solubilization, the tendency toward delipidation of the immediate environment of the protein was stopped, and recovery of enzyme activity was observed, suggesting reorganization of phospholipid and detergent molecules in the solubilized ternary complex, as compared to the perturbed membrane. After further additions of C12E8 or DM to the already solubilized membrane, the protein again experienced progressive delipidation which was only completed at a detergent concentration about 100-fold higher than that necessary for solubilization. Delipidation was correlated with a decrease in enzyme activity toward a level similar to that observed during perturbation. On the other hand, Tween 80, Tween 20, and Lubrol WX failed to solubilize SR membranes and to induce further ATPase delipidation when added after preliminary SR solubilization by C12E8 or dodecyl maltoside. For Tween 80, this can be related to an inability to solubilize pure lipid membrane; in contrast, Tween 20 and Lubrol WX were able to solubilize liposomes but not efficiently to solubilize SR membranes. In all three cases, insertion of the detergent in SR membranes is, however, demonstrated by perturbation of enzyme activity. Correlation between detergent structure and ability to solubilize and delipidate the ATPase suggests that one parameter impeding ATPase solubilization might be the presence of a bulky detergent polar headgroup, which could not fit close to the protein surface. We also conclude that in the active protein/detergent/lipid ternary complexes, solubilized by C12E8 or dodecyl maltoside, most phospholipids remain closely associated with the ATPase hydrophobic surface as in the membranous form. Binding of only a few detergent molecules on this hydrophobic surface may be sufficient for inhibition of ATPase activity observed at high ATP concentration, both during perturbation and in the completely delipidated, solubilized protein.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Membrane solubilization by detergent: use of brominated phospholipids to evaluate the detergent-induced changes in Ca2+-ATPase/lipid interaction. 252 49

We examined the tryptophan decay kinetics of sarcoplasmic reticulum Ca2+-ATPase using frequency-domain fluorescence. Consistent with earlier reports on steady-state fluorescence intensity, our intensity decays reveal a reproducible and statistically significant 2% increase in the mean decay time due to calcium binding to specific sites involved in enzyme activation. This Ca2+ effect could not be eliminated with acrylamide quenching, which suggests a global effect of calcium on the Ca2+-ATPase, as opposed to a specific effect on a single water-accessible tryptophan residue. The tryptophan anisotropy decays indicate substantial rapid loss of anisotropy, which can be the result of either intramolecular energy transfer or a change in segmental flexibility of the ATPase protein. Energy transfer from tryptophan to TNP-ATP in the nucleotide binding domain, or to IEADANS on Cys-670 and -674, indicates that most tryptophan residues are 30 A or further away from these sites and that this distance is not decreased by Ca2+. In light of known structural features of the Ca2+-ATPase, the tryptophan fluorescence changes are attributed to stabilization of clustered transmembrane helices resulting from calcium binding.
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PMID:Characterization of the tryptophan fluorescence from sarcoplasmic reticulum adenosinetriphosphatase by frequency-domain fluorescence spectroscopy. 252 24

The 23-kDa N-terminal tryptic fragment was isolated from the heavy chain of rabbit skeletal myosin subfragment 1 (S-1). The heavy-chain fragments were dissociated by guanidine hydrochloride following limited trypsinolysis, and the 23-kDa fragment was isolated by gel filtration and ion-exchange chromatography. Finally, the fragment was renatured by removing the denaturants. The CD spectrum of the renatured fragment shows the presence of ordered structure. The tryptophan fluorescence emission spectrum of the fragment is considerably shifted to the red upon adding guanidine hydrochloride which indicates that the tryptophans are located in relatively hydrophobic environments. The two 23-kDa tryptophans, unlike the rest of the S-1 tryptophans, are fully accessible to acrylamide as indicated by fluorescence quenching. The isolated 23-kDa fragment cosediments with F-actin in the ultracentrifuge and significantly increases the light scattering of actin in solution which indicates actin binding. The binding is rather tight (Kd = 0.1 microM) and ionic strength dependent (decreasing with increasing ionic strength). ATP, pyrophosphate, and ADP dissociate the 23-kDa-actin complex with decreasing effectiveness. The isolated 23-kDa fragment does not have ATPase activity; however, it inhibits the actin-activated ATPase activity of S-1 by competing presumably with S-1 for binding sites on actin. F-Actin binds to the 23-kDa fragment immobilized on the nitrocellulose membrane. The fragment was further cleaved, and one of the resulting peptides, containing the 130-204 stretch of residues, was found to bind actin on the nitrocellulose membrane, indicating that this region of the 23-kDa fragment participates in forming an actin binding site.
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PMID:Isolation and characterization of the N-terminal 23-kilodalton fragment of myosin subfragment 1. 252 54

Multifrequency phase-modulation lifetime data were acquired for sarcoplasmic reticulum Ca2+-ATPase. The intrinsic tryptophan fluorescence decay was complex and was fitted either with three exponentials or with bimodal Lorentzian distributions of lifetimes. Ca2+ binding to the high affinity sites in the ATPase produced an increase of 11% in the center of the main component of the bimodal distribution, shifting the lifetime from 4.04 to 4.50 ns. The effects of solvent on the ATPase were studied with the enzyme dissolved in reverse micelles of detergent bis-(2-ethylhexyl)sulfosuccinate in hexane. Increasing amounts of water up to a water/bis-(2-ethylhexyl)sulfosuccinate molar ratio of 4 produced marked changes in the fluorescence emission of the protein. Comparison of data obtained for micellar solutions of tryptophan or ATPase indicated that the tryptophan residues in the protein are protected from exposure to water. Correlation of water effects on emission intensity and lifetimes suggested that interaction with solvent may result in structural changes that cause a mixture of dynamic and static quenching of ATPase intrinsic fluorescence. Evidence for an effect of hydration on the structure of the active site was obtained by measurements of the fluorescence properties of fluorescein isothiocianate-labeled ATPase in reverse micelles.
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PMID:Fluorescence decay of sarcoplasmic reticulum ATPase. Ligand binding and hydration effects. 252 52

Terbium ions and terbium formycin triphosphate have been used to investigate the interactions between the cation and nucleotide binding sites of the sarcoplasmic reticulum Ca2+-ATPase. Three classes of Tb3+-binding sites have been found: a first class of low-affinity (Kd = 10 microM) corresponds to magnesium binding sites, located near a tryptophan residue of the protein; a second class of much higher affinity (less than 0.1 microM) corresponds to the calcium transport sites, their occupancy by terbium induces the E1 to E2 conformational change of the Ca2+-ATPase; a third class of sites is revealed by following the fluorescence transfer from formycin triphosphate (FTP) to terbium, evidencing that terbium ions can also bind into the nucleotide binding site at the same time as FTP. Substitution of H2O by D2O shows that Tb-FTP binding to the enzyme nucleotide site is associated with an important dehydration of the terbium ions associated with FTP. Two terbium ions, at least, bind to the Ca2+-ATPase in the close vicinity of FTP when this nucleotide is bound to the ATPase nucleotide site. Addition of calcium quenches the fluorescence signal of the terbium-FTP complex bound to the enzyme. Calcium concentration dependence shows that this effect is associated with the replacement of terbium by calcium in the transport sites, inducing the E2----E1 transconformation when calcium is bound. One interpretation of this fluorescence quenching is that the E1----E2 transition induces an important structural change in the nucleotide site. Another interpretation is that the high-affinity calcium sites are located very close to the Tb-FTP complex bound to the nucleotide site.
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PMID:Evidence of a calcium-induced structural change in the ATP-binding site of the sarcoplasmic-reticulum Ca2+-ATPase using terbium formycin triphosphate as an analogue of Mg-ATP. 252 52

Phenotypic revertants have been selected from mutants of the yeast Schizosaccharomyces pombe devoid of either alpha or beta subunits of mitochondrial ATPase-ATPsynthase. In contrast to parental mutants, phenotypic revertants are able to grow on glycerol respiratory medium and show immunodetectable alpha and beta subunits. However, growth and cellular respiration are only partially restored as compared to the wild strain, indicating that the recovered subunits are mutated. ATPase activity of revertant submitochondrial particles shows markedly different parameters: more acidic optimal pH, absence of bicarbonate activation and decreased sensitivity to azide inhibition in the alpha subunit-modified R3.51. Opposite differences are observed in the beta subunit-modified R4.3: more alkaline optimal pH, much higher bicarbonate activation, and increased sensitivity to azide. The ITPase activity of R4.3 submitochondrial particles is also more sensitive to azide as compared to the wild strain. ATPase activity of purified F1 also exhibits marked differences: loss of bicarbonate-sensitive negative cooperativity, decreased sensitivity to both ADP and azide inhibitions in the R3.51 revertant. On the contrary, increased negative cooperativity and increased sensitivity to both ADP and azide inhibitions are observed for the R4.3 revertant enzyme which in addition exhibits a much lower maximal rate. The beta subunit-mutation of R4.3 also increases the sensitivity of ITPase activity to tripolyphosphate inhibition, whereas the alpha subunit-mutation of R3.51 is without any effect. Soluble F1 with beta subunit-mutation is very sensitive to high ammonium sulfate concentrations required for enzyme precipitation and concentration and known to partially deplete the enzyme from its endogenous nucleotides. On the contrary, poly(ethylene)glycol is very efficient for preparing from any strain a pure and very stable enzyme retain-ing high amounts of endogenous nucleotides. The R4.3 revertant F1 retains even more nucleotides than the wild-strain F1 and is much less sensitive to high iodide concentrations which favor enzyme dissociation and precipitation. The tryptophan intrinsic fluorescence of F1 is modified by both mutations that increase the maximal emission intensity. The most important effect is produced by beta subunit-mutation which decreases the quenchable fraction, one-third to one-half tryptophans being no longer accessible to iodide. The overall results suggest that both mutations modify enzyme-nucleotide interactions: the alpha subunit-mutation of R3.51 would favor ADP release by lowering interactions with the adenine moiety, whereas the beta subunit-mutation of R4.3 would lower ADP release by strengthening interactions with the phosphate chain moiety.
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PMID:Structure-function relationships of mitochondrial ATPase-ATPsynthase using Schizosaccharomyces pombe yeast mutants with altered F1 subunits. 252 9

The catalytic behavior and structural features of Ca2+-ATPase in the vesicles of longitudinal tubules and terminal cisternae of the sarcoplasmic reticulum isolated from rabbit skeletal muscles was analysed. pH measurements have shown under optimal conditions Ca2+-ATPase has similar catalytic behavior both in the fractions of longitudinal tubules and terminal cisternae. Under non-optimal conditions, the behavior similarity was not observed. The specific activity of the ATPase enzyme under optimal conditions was shown to be much higher in the fraction of longitudinal tubules than in the fraction of terminal cisternae. Caffeine added to both fractions had no effect on the catalytic behavior of Ca2+-ATPase. As judged from fluorescence analysis, the structure of Ca2+-ATPase of longitudinal tubules differs from that structure of terminal cisternae. In sarcoplasmic reticulum membrane, at least half of the tryptophan residues of Ca2+-ATPase was shown to be buried in the lipid bilayer. Our findings suggest that in terminal cisternae some of the Ca2+-ATPase molecules exist as an oligomeric protein and do not participate in ATP hydrolysis (named "silent" Ca2+-ATPase).
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PMID:[Comparison of the catalytic and structural properties of Ca2+-ATPase in longitudinal tubules and terminal cisternae of the sarcoplasmic reticulum]. 253 Dec 74

Two spectroscopic techniques, circular dichroism and steady-state fluorescence, were employed in order to study conformational changes of the purified, detergent-solubilized (Ca2+-Mg2+)-ATPase of porcine erythrocyte ghost membranes. Circular dichroism (CD) spectra in the peptide region were obtained from the purified (Ca2+-Mg2+)-ATPase of porcine erythrocyte ghost membranes with the aim to investigate the secondary structure of the enzyme in the presence of calmodulin (CaM) or phosphatidylserine (PS), as well as in the E1 and E2 states. The E1 conformation was stabilized by 10 microM free Ca2+, while the E2 conformation was stabilized by 0.1 mM ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). It was found that the E1 and E2 states of the enzyme strikingly differed in their secondary structure (66% and 46% of calculated alpha-helix content, respectively). In the presence of Ca2+, PS decreased the helical content of the ATPase to 61%, while CaM to 55%. Quenching of intrinsic fluorescence of (Ca2+-Mg2+)-ATPase by acrylamide, performed in the presence of Ca2+, gave evidence for a single class of tryptophan residues with Stern-Volmer constant (KSV) of 10 M-1. Accessibility of tryptophan residues varied depending on the conformational status of the enzyme. Addition of PS and CaM decreased the KSV value to 7.6 M-1 and 8.5 M-1, respectively. In the absence of Ca2+, KSV was 7.0 M-1. KI and CsCl were less effective as quenchers. The fluorescence energy transfer between (Ca2+-Mg2+)-ATPase tryptophan residues and dansyl derivative of covalently labeled CaM occurred in the presence of EGTA, but was further promoted by Ca2+. It is concluded that the interaction of CaM and PS with (Ca2+-Mg2+)-ATPase results in different conformational states of the enzyme. CD and fluorescence spectroscopy allowed to distinguish these states from the E1 and E2 conformational forms of the ATPase.
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PMID:Conformational changes of (Ca2+-Mg2+)-ATPase of erythrocyte plasma membrane caused by calmodulin and phosphatidylserine as revealed by circular dichroism and fluorescence studies. 253 12

The effect of vitamin A deficiency on the intestinal absorption of nutrients and the activities of brush border enzymes were studied in albino rats. Intestinal uptakes of D-glucose, L-methionine, L-tryptophan and L-histidine were significantly greater in vitamin A-deficient animals than in controls. The specific activities of total adenosine triphosphatase (ATPase), ouabain-sensitive ATPase, maltase and sucrase in the intestinal mucosa of vitamin A-deprived rats were 121, 124, 131 and 134 per cent respectively, of the corresponding values in control animals. The DNA content of the small intestine in vitamin A-deficient rats was 36.5 per cent lower than in control rats. The stimulation in digestive and absorptive capacity appears to be an adaptive change in vitamin A-deficiency which decreases the intestinal cell population.
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PMID:Effect of vitamin A deficiency on rat intestinal digestive & absorptive functions. 253 19


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