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)

All-trans retinoic acid is a potent inhibitor of [125I]-thyroxine (T4) binding to human erythrocyte membranes and can block the activation by thyroid hormone of erythrocyte Ca(2+)-ATPase [J. Biol. Chem. (1989) 264, 687-689]. In the present studies, retinoic acid was examined for its ability to displace thyroxine from binding sites on human transthyretin (TTR). Scatchard analysis of [125I]T4 binding to purified TTR, determined by equilibrium dialysis, revealed two classes of binding sites with association constants of 3.2 x 10(9) M-1 and 8.1 x 10(6) M-1. All-trans retinoic acid also displaced [125I]T4; 40% of the specifically bound [125I]T4 was displaced at a retinoic acid concentration of 2 x 10(-5) M. Analysis of the high affinity T4 binding site suggests that the Ka for retinoic acid to that site is approx. 10(7) M-1. 8-Anilinonaphthalene-1-sulfonate (ANS), a strongly fluorescing dye, binds to the thyroxine binding sites on TTR. T4 and 3,5,3'-L-triiodothyronine (T3) shifted the fluorescence emission maximum and intensity of an ANS-TTR solution toward the spectrum obtained from uncomplexed ANS. All-trans retinoic acid caused a similar shift in the emission spectrum of ANS, but was less potent than T4. Retinol failed to quench the emission intensity of the ANS-TTR complex, while 13-cis-retinoic acid was less effective than all-trans retinoic acid.
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PMID:Retinoic acid inhibition of thyroxine binding to human transthyretin. 828 Jul 58

A number of protein reactive compounds, including the thiol reagents diamide and arsenite, are known inducers of heat shock protein (HSP) synthesis and thermotolerance. These compounds are thought to damage cellular protein, which has been proposed to serve as the signal for induction. The specific mechanism of protein damage and its relation to thermal denaturation are unknown. The Ca2+-ATPase of sarcoplasmic reticulum, a membrane protein that contains 24 cys residues, was used to determine the effect of diamide, arsenite, N-ethylmaleimide (NEM), and the cys-specific probes Br-DMC and IAEDANS, which label one or two specific cys residues, respectively, on protein conformation and stability. The Ca2+-ATPase was chosen because diamide has been shown to affect the thermal properties of a class of membrane proteins of CHO cells (Freeman et al., 1995). The labeling of one or two thiols has no effect on activity or conformation, while more extensive reaction (but with less than approximately five to eight groups titrated) results in destabilization of the Ca2+-ATPase such that it denatures thermally at 37 degrees C. Higher levels of titration result in greater destabilization such that the protein is no longer stable at room temperature, with the production of a state similar to the thermally denatured state as assayed by activity, differential scanning calorimetry, ANS binding, and light scattering. The fractional denaturation induced by these thiol reagents, determined by the decrease in the heat absorbed during thermal denaturation, is directly proportional to inactivation of ATPase activity. Thus, inactivation of the Ca2+-ATPase by thiol reagents occurs because of denaturation not through oxidation of essential thiols. These results indicate that these thiol-specific heat shock inducers function by two mechanisms: (1) destabilization of proteins such that they thermally denature at 37 degrees C and (2) direct denaturation, apparently driven by thermal processes at room temperature, following more extensive reaction which results in extreme destabilization. We suggest that these are general mechanisms by which heat shock inducers damage proteins.
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PMID:Destabilization of the Ca2+-ATPase of sarcoplasmic reticulum by thiol-specific, heat shock inducers results in thermal denaturation at 37 degrees C. 928 92

The inactivation and conformational changes of the bacterial chaperonin GroEL have been studied in SDS solutions with different concentrations. The results show that increasing the SDS concentration caused the intrinsic fluorescence emission intensity to increase and the emission peak to slightly blue-shift, indicating that increasing the SDS concentration can cause the hydrophobic surface to be slightly buried. The changes in the ANS-binding fluorescence with increasing SDS concentration also showed that the GroEL hydrophobic surface decreased. At low SDS concentrations, less than 0.3 mM, the GroEL ATPase activity increased with increasing SDS concentration. Increasing the SDS concentration beyond 0.3 mM caused the GroEL ATPase activity to quickly decrease. At high SDS concentrations, above 0.8 mM, the residual GroEL ATPase activity was less than 10% of the original activity, but the GroEL molecule maintained its native conformation (as indicated by the exposure of buried thiol groups, electrophoresis, and changes of CD spectra). The above results suggest that the conformational changes of the active site result in the inactivation of the ATPase even though the GroEL molecule does not markedly unfold at low SDS concentrations.
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PMID:SDS-induced conformational changes and inactivation of the bacterial chaperonin GroEL. 1060 40

The total Ca-ATPase activity in the sarcoplasmic reticulum (SR) membrane fraction isolated from skeletal muscles of winter hibernating ground squirrel Spermophilus undulatus is approximately 2.2-fold lower than in preparations obtained from summer active animals. This is connected in part with approximately 10% decrease of the content of Ca-ATPase protein in SR membranes. However, the enzyme specific activity calculated with correction for its content in SR preparations is still approximately 2-fold lower in hibernating animals. Analysis of the protein composition of SR membranes has shown that in addition to the decrease in Ca-ATPase content in hibernating animals, the amount of SR Ca-release channel (ryanodine receptor) is decreased approximately 2-fold, content of Ca-binding proteins calsequestrin, sarcalumenin, and histidine-rich Ca-binding protein is decreased approximately 3-4-fold, and the amount of proteins with molecular masses 55, 30, and 22 kD is significantly increased. Using the cross-linking agent cupric-phenanthroline, it was shown that in SR membranes of hibernating ground squirrels Ca-ATPase is present in a more aggregated state. The affinity of SR membranes to the hydrophilic fluorescent probe ANS is higher and the degree of excimerization of the hydrophobic probe pyrene is lower (especially for annular lipids) in preparations from hibernating than from summer active animals. The latter indicates an increase in the microviscosity of the lipid environment of Ca-ATPase during hibernation. We suggest that protein aggregation as well as the changes in protein composition and/or in properties of lipid bilayer SR membranes can result in the decrease of enzyme activity during hibernation.
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PMID:Characteristics of sarcoplasmic reticulum membrane preparations isolated from skeletal muscles of active and hibernating ground squirrel Spermophilus undulatus. 1156 64

Calmodulin (CaM) and troponin C (TnC) are the most similar members of EF-hand family and show few differences in the primary structure. Here, we use mutants of troponin that mimic calmodulin and changes in temperature to investigate the factors that determine their specificity as regulatory proteins. Using a double mutant of troponin that resembles calmodulin in lacking both the N-terminal helix and KGK(91-93) we observe a small difference from troponin in binding to the erythrocyte Ca(2+)-ATPase, and an improvement in enzyme activation. A triple mutant, where in addition, the residues 88-90 are replaced with the corresponding sequence from calmodulin is equivalent to calmodulin in maximal activation, and it restores protein ability to increase Ca(2+) affinity for the enzyme. However, this mutant also binds less tightly (1/100) than calmodulin. Remarkably, a decrease in temperature has a more marked effect in protein binding than either mutation, reducing the difference in affinities to 18-fold, but without any improvement in their ability to increase Ca(2+) affinity for the enzyme. Spectroscopic analysis of hydrophobic domain exposure in EF-hand proteins was carried out using 8-anilino-1-naphthalenesulfonic acid (ANS). The probe shows a much higher fluorescence when bound to the complex Ca(4)-calmodulin than to Ca(4)-troponin. Decreasing the temperature exposes additional hydrophobic regions of troponin. Changing the Mg(2+) concentration does not affect their bindings to the enzyme. It is suggested that the requirements for troponin to mimic calmodulin in binding to the target enzyme, and those for activating it, are met by different regions of the protein.
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PMID:Converting troponin C into calmodulin: effects of mutations in the central helix and of changes in temperature. 1194 96

The effects of anisodamine on the Ca(2+)-ATPsae of sarcoplasmic reticulum (SR) were investigated by using differential scanning calorimetry to measure the ability of anisodamine to denature the transmembrane domain and the cytoplasmic domain. Anisodamine significantly altered the thermotropic phase behaviors of the transmembrane domain of purified Ca(2+)-ATPase. Specifically, the melting temperature of the transmembrane domain moved toward lower temperatures with the concentrations of anisodamine increasing and the thermotropic phase peak was abolished at 10 mM, indicating that the stabilized structure of the transmembrane domain in the presence of Ca2+ could be destabilized by anisodamine. Decreases of the intrinsic fluorescence and increases of the extrinsic fluorescence of ANS, a fluorescent probe, showed the exposure of tryptophan and hydrophobic region, respectively, suggesting again that anisodamine caused a less compact conformation in the transmembrane domain. A marked inhibition of the Ca2+ uptake activity of SR Ca(2+)-ATPase was observed when the addition of anisodamine. The drug did not affect the cytoplasmic domain of the enzyme and only slightly decreased the ATPase activity of the enzyme at concentrations up to 10 mM. This was likely due to the destabilized protein transmembrane domain. To sum up, our results revealed that anisodamine interacted specifically with the transmembrane domain of SR Ca(2+)-ATPase and inhibited the Ca2+ uptake activity of the enzyme.
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PMID:Anisodamine causes the changes of structure and function in the transmembrane domain of the Ca(2+)-ATPase from sarcoplasmic reticulum. 1474 74

alpha-Tropomyosin (Tm) is a two-stranded alpha-helical coiled-coil protein, which participates in the regulation of muscle contraction. Unlike Tm purified from vertebrate muscle, recombinant Tm expressed in Escherichia coli is not acetylated at the N-terminal residue and loses the capacity to undergo head-to-tail polymerization, to bind actin and to inhibit actomyosin ATPase activity. These functions are restored by fusion of an N-terminal Ala-Ser (AS) dipeptide tail to recombinant Tm. Here, we have employed chemical (guanidine hydrochloride and urea) and physical (elevated hydrostatic pressures and low temperatures) denaturing agents to compare the structural stabilities of polymeric alanine-serine-tropomyosin (ASTm, containing the AS dipeptide) and dimeric "non-fusion" Tm (nfTm, i.e., not containing the AS dipeptide). Binding of the hydrophobic fluorescent dye bis-ANS, circular dichroism and size-exclusion chromatography were used to monitor the stabilities and state of association of both proteins under different solution conditions. Bis-ANS binding was markedly decreased at low concentrations (<1M) of GdnHCl or urea, whereas the secondary structures of both ASTm and nfTm were essentially unaffected in the same range of denaturant concentrations. These results suggest local unfolding of bis-ANS binding domains prior to global unfolding of Tm. In contrast, increased bis-ANS binding was observed when Tm was submitted to high pressures or to low temperatures, implying increased exposure of hydrophobic domains in the protein. Taken together, the different sensitivities of ASTm and nfTm to different denaturing agents support the notion that, at close to physiological conditions, head-to-tail interactions in polymerized ASTm are predominantly stabilized by electrostatic interactions between adjacent Tm dimers, whereas non-polar interactions appear to play a major role in the stability of the coiled-coil structure of individual Tm dimers.
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PMID:Folding and stability of a coiled-coil investigated using chemical and physical denaturing agents: comparative analysis of polymerized and non-polymerized forms of alpha-tropomyosin. 1583 71

Polygodial is a naturally occurring sesquiterpene dialdehyde that exhibits several pharmacologically interesting activities. Among them, its antifungal properties have been more thoroughly studied. The mitochondrial ATPase has been suggested as one of the possible targets for polygodial action. However, its mechanism of action is not well defined yet. The effect of polygodial on the mitochondrial energy metabolism is described in this paper. Polygodial inhibited ATP synthesis coupled to succinate oxidation in beef-heart submitochondrial particles at concentrations (IC(50)=2.4+/-0.1 microM) which marginally affected electron transport and ATPase activity (IC(50)=97+/-4 microM). A transitory stimulation of the electron transport in intact rat liver mitochondria in state 4 was also obtained at low polygodial concentrations (EC(50)=20+/-4 microM). These results suggest that polygodial uncouples ATP synthesis from electron transport at low concentrations. Similar concentrations of polygodial partially abolished the ANS fluorescence enhancement (IC(50)=2.2+/-0.4 microM) induced by succinate oxidation in submitochondrial particles but did not collapse the DeltapH. We postulate that polygodial uncouples mitochondrial ATP synthesis by affecting the electrical properties of the membrane surface and consequently collapsing the membrane potential (Deltapsi) and/or the localized transmembrane pH difference (DeltapH(S)) without affecting the DeltapH between the two bulk aqueous phases (DeltapH(B)). The relevance of these findings for the understanding of the biochemical basis of the antifungal activity of polygodial and the evaluation of its potentiality as a therapeutic agent are discussed.
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PMID:Inhibition of the mitochondrial ATP synthesis by polygodial, a naturally occurring dialdehyde unsaturated sesquiterpene. 1589 93

Here, we provide functional and direct structural evidence that alphaB-crystallin, a member of the small heat-shock protein family, suppresses thermal unfolding and aggregation of the myosin II molecular motor. Chicken skeletal muscle myosin was thermally unfolded at heat-shock temperature (43 degrees C) in the absence and in the presence of alphaB-crystallin. The ATPase activity of myosin at 25 degrees C was used as a parameter to monitor its unfolding. Myosin retained only 65% and 8% of its ATPase activity when incubated at heat-shock temperature for 15 min and 30 min, respectively. However, 84% and 58% of the myosin ATPase activity was maintained when it was incubated with alphaB-crystallin under the same conditions. Furthermore, actin-stimulated ATPase activity of myosin was reduced by approximately 90%, when myosin was thermally unfolded at 43 degrees C for 30 min, but was reduced by only approximately 42% when it was incubated with alphaB-crystallin under the same conditions. Light-scattering assays and bound thioflavin T fluorescence indicated that myosin aggregates when incubated at 43 degrees C for 30 min, while alphaB-crystallin suppressed this thermal aggregation. Photo-labeled bis-ANS alphaB-crystallin fluorescence studies confirmed the transient interaction of alphaB-crystallin with myosin. These findings were further supported by electron microscopy of rotary shadowed molecules. This revealed that approximately 94% of myosin molecules formed inter and intra-molecular aggregates when incubated at 43 degrees C for 30 min. alphaB-Crystallin, however, protected approximately 48% of the myosin molecules from thermal aggregation, with protected myosin appearing identical to unheated molecules. These results are the first to show that alphaB-crystallin maintains myosin enzymatic activity and prevents the aggregation of the motor under heat-shock conditions. Thus, alphaB-crystallin may be critical for nascent myosin folding, promoting myofibrillogenesis, maintaining cytoskeletal integrity and sustaining muscle performance, since heat-shock temperatures can be produced during multiple stress conditions or vigorous exercise.
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PMID:alphaB-crystallin maintains skeletal muscle myosin enzymatic activity and prevents its aggregation under heat-shock stress. 1654 10

The mechanism of inhibition of Ca2+-transport activity of rabbit sarcoplasmic reticulum Ca 2+-ATPase (SERCA) by anisodamine (a drug isolated from a medicinal herb Hyoscyamuns niger L) was investigated by using ANS (1-anilino-8-naphthalenesulfonate) fluorescence probe, intrinsic fluorescence quenching and Ca 2+-transport activity assays. The number of ANS binding sites for apo Ca2+-ATPase was determined as 8, using a multiple-identical binding site model. Both anisodamine and Ca2+ at millimolar level enhanced the ANS binding fluorescence intensities. Only anisodamine increased the number of ANS molecules bound by SERCA from 8 to 14. The dissociation constants of ANS to the enzyme without any ligand, with 30 mM anisodamine and with 15 mM Ca 2 were found to be 53.0 microM, 85.0 microM and 50.1 microM, respectively. Both anisodamine and Ca2+ enhanced the ANS binding fluorescenc with apparent dissociation constants of 7.6 mM and 2.3 mM, respectively, at a constant concentration of the enzyme. Binding of anisodamine significantly decreased the binding capacity of Ca2+ with the dissociation constant of 9.5 mM, but binding of Ca2+ had no obvious effect on binding of anisodamine. Intrinsic fluorescence quenching and Ca2+-transport activity assays gave the dissociation constants of anisodamine to SERCA as 9.7 and 5.4 mM, respectively, which were consistent with those obtained from ANS-binding fluorescence changes during titration of SERCA with anisodamine and anisodamine + 15 mM Ca2+, respectively. The results suggest that anisodamine regulates Ca2+-transport activity of the enzyme, by stabilizing the trans-membrane domain in an expanded, inactive conformation, at least at its annular ring region.
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PMID:Mechanism of inhibition of Ca2+-transport activity of sarcoplasmic reticulum Ca2+-ATPase by anisodamine. 1728 99


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