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)

Cultured rabbit lenses were irradiated with UV (311 nm peak; 295-340 nm) for 30 to 60 min. The entire spectrum lies in the near-UV, the major component is UVB, with a minor portion (25%) of UVA, and is henceforth referred to as near-UV(B). Posterior irradiation caused no cataract and no significant ionic imbalances compared to anterior irradiation, which caused opacification and marked changes in sodium and calcium concentrations. Anterior irradiation also resulted in reduced Na/K-ATPase activity in the epithelium. ATPase activity was not immediately inhibited; rather, only after culture was enzyme activity reduced. The concentration of reduced glutathione (GSH) decreased rapidly in the epithelium and more slowly in the underlying lens fibers. Loss of GSH was more rapid and extensive when irradiation occurred in the presence of oxygen. Irradiation under anaerobic conditions resulted in opacification but was considerably less extensive than when irradiation of lenses occurred in the presence of 7% oxygen. Near-UV(B) damage following anaerobic irradiation and 20 hrs of culture resulted in an increase in sodium levels and loss of GSH; calcium levels were not significantly elevated. Since irradiation of tryptophan solutions produced small amounts of hydrogen peroxide, the possibility of hydrogen peroxide-mediated damage was investigated but no role could be substantiated. Peroxide detoxification by the epithelium of near-UV(B) cataracts was observed, as measured by its ability to eliminate hydrogen peroxide added as a bolus.
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PMID:Mechanisms involved in cataract development following near-ultraviolet radiation of cultured lenses. 132 94

Raman analysis of Na+,K(+)-ATPase structural changes induced by cation binding reveals a slight decrease ( < 10%) of the alpha-helical content upon E1-E2 transition. Pronounced conformational changes of the enzyme are unlikely as the character of the environment of tyrosine residues remains unaltered. However, local changes can take place as evidenced by changes in tryptophan vibration at about 880 cm-1.
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PMID:Changes in Na+,K(+)-ATPase structure induced by cation binding. Approach by Raman spectroscopy. 133 Jun 84

The 26 amino acid bee venom toxin, melittin, is an amphipathic helical polypeptide which inhibits the gastric (H+ + K+)ATPase. The site of interaction with the (H+ + K+)ATPase was shown to be the alpha subunit of the (H+ + K+)ATPase in studies using [125I]azidosalicylyl melittin, a radioactive photoaffinity analog of melittin. A synthetic amphipathic polypeptide (Trp3) containing tryptophan, which exhibits a structure similar to that of melittin, also inhibited the gastric (H+ + K+)ATPase, and prevented labeling by [125I]azidosalicylyl melittin. These findings suggested that melittin and the synthetic amphipathic helical polypeptide were bound to the same or overlapping site(s). In the present studies, novel tritiated photoaffinity analogs of Trp3 containing benzoylphenylalanine (in place of tryptophan) were used to photoaffinity label the (H+ + K+)ATPase. These studies help to establish that the (H+ + K+)ATPase contains a binding site for polypeptides which exhibit an amphipathic helical motif. The precise amino acid sequence of the polypeptide appears to be of secondary importance for interaction with the (H+ + K+)ATPase as long as the alpha helical motif is present. The benzoylphenylalanine containing polypeptides are ideal for mapping the binding site on the (H+ + K+)ATPase. Using an antibody which recognizes this amphipathic helical ('melittin-like') motif, we have demonstrated that the gastric parietal cell contains a 67 kDa 'melittin-like' protein. This protein was associated with the gastric parietal cell apical membrane in the stimulated (secreting) state, but not in the resting (non-secreting) state. The binding site for the gastric 'melittin-like' protein appears to overlap with the melittin binding site on the alpha subunit of the (H+ + K+)ATPase. The potential physiological significance of the melittin binding site and the overlapping binding site for this newly identified endogenous 'melittin-like' protein on the (H+ + K+)ATPase to regulated HCl secretion by the parietal cell is presently under investigation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Interaction of polypeptides with the gastric (H+ + K+)ATPase: melittin, synthetic analogs, and a potential intracellular regulatory protein. 133 29

We have studied the effect of Ruthenium red on the sarcoplasmic reticulum Ca(2+)-ATPase. Ruthenium red does not modify the Ca2+ pumping activity of the enzyme, despite its interaction with cationic binding sites on sarcoplasmic reticulum vesicles. Two pools of binding sites were distinguished. One pool (10 nmol/mg) is dependent upon the presence of micromolar Ca2+ and may therefore represent the high-affinity Ca2+ transport sites of the Ca(2+)-ATPase. However, Ruthenium red only slightly competes with Ca2+ on these sites. The other pool (15-17 nmol/mg) is characterized as low-affinity cation binding sites of sarcoplasmic reticulum, distinct from the Mg2+ site involved in the ATP binding to the Ca(2+)-ATPase. The interaction of Ruthenium red with these low-affinity cation binding sites, which may be located either on the Ca(2+)-ATPase or on surrounding lipids, decreases tryptophan fluorescence level of the protein. As much as 25% of the tryptophan fluorescence of the Ca(2+)-ATPase is quenched by Ruthenium red (with a dissociation constant of 100 nM), tryptophan residues located near the bilayer being preferentially affected.
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PMID:Ruthenium red affects the intrinsic fluorescence of the calcium-ATPase of skeletal sarcoplasmic reticulum. 137 28

Molecular changes underlying the partial inactivation of the sarcoplasmic-reticulum (SR) Ca(2+-) ATPase in low-frequency-stimulated fast-twitch muscle were investigated in the present study. The specific Ca(2+)-ATPase activity, as well as the ATP- and acetyl phosphate-driven Ca2+ uptakes by the SR, were reduced by approx. 30% in 4-day-stimulated muscle. Phosphoprotein formation of the enzyme in the presence of ATP or Pi was also decreased to the same extent. Measurements of ATP binding revealed a 30% decrease in binding to the enzyme. These changes were accompanied by similar decreases in the ligand-induced (ATP, ADP, Pi) intrinsic tryptophan fluorescence. A decreased binding of fluorescein isothiocyanate (FITC) corresponded to the lower ATP binding and phosphorylation of the enzyme. Moreover, Pi-induced changes in fluorescence of the FITC-labelled enzyme did not differ between SR from stimulated and contralateral muscles, indicating that Ca(2+)- ATPase molecules which did not bind FITC were responsible for the decreased Pi-dependent phosphorylation, and therefore represented the inactive form of the enzyme. No differences existed between the Ca(2+)-induced changes in the intrinsic fluorescence of SR from stimulated and contralateral muscles which fit their similar Ca(2+)-binding characteristics. Taking the proposed architecture of the Ca2(+)-ATPase into consideration, our results suggest that the inactivation relates to a circumscribed structural alteration of the enzyme in sections of the active site consisting of the nucleotide-binding and phosphorylation domains.
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PMID:Inactivation of sarcoplasmic-reticulum Ca(2+)-ATPase in low-frequency-stimulated muscle results from a modification of the active site. 138 17

Beryllium and aluminum fluorides are good phosphate analogues. These compounds, like orthovanadate, form stable complexes with myosin subfragment 1 (S1) in the presence of MgADP. The formation of the stable S1-nucleotide complexes is characterized by the loss of ATPase activity. For the complete loss of ATPase activity there was necessary a higher concentration of aluminum than of beryllium or vanadate. In the presence of MgATP the onset of the inhibition is delayed, which indicates that stable complexes cannot form when a specific site is occupied by the gamma-phosphate of ATP or by P(i) derived from the gamma-phosphate. The half-lives of the S1-MgADP-(BeF3-), S1-MgADP-(AlF4-), and S1-MgADP-Vi complexes at 0 degrees C are 7, 2, and 4 days, respectively. In the presence of actin the rate of decomposition of all of the complexes is significantly enhanced; however, the order of decomposition is reversed, the fastest rate being observed with beryllium and the slowest with aluminum. The formation of the S1-MgADP-(BeF3-) and S1-MgADP-(AlF4-) complexes is accompanied by an increase in tryptophan fluorescence similar to that observed upon addition of MgATP to S1. The fluorescence increase develops rather slowly, by suggesting that the rate-limiting step in the formation of the stable complex is an isomerization. The rate of the fluorescence change accompanying the formation of the Be complex is faster than that for the Al complex. Addition of vanadate to S1 causes a static quenching of the tryptophan fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of stable beryllium fluoride, aluminum fluoride, and vanadate containing myosin subfragment 1-nucleotide complexes. 138 27

The fluorescence decay of the plasma membrane calmodulin-activated Ca2(+)-ATPase from the erythrocyte was measured for the first time. The availability of a novel procedure for on-line blank subtraction in frequency-domain lifetime data acquisition (G.G. Reinhart, B. Feddersen, D. Jameson and E. Gratton, Biophys. J. 57 (1990) 189a) permitted the elimination of background interference from detergent-solubilized purified plasma membrane ATPase samples. The fluorescence decay of the erythrocyte Ca2(+)-ATPase was measured in the absence of Ca2+, or in the presence of Ca2+ or Ca2+ plus calmodulin. In the three different experimental conditions the fluorescence decay was very heterogeneous and could be best described by Lorentzian distributions of lifetime values. In the absence of Ca2+ the decay was described by a broad lifetime distribution centered at 4.4 ns with a width of 3.2 ns, indicating heterogeneity of tryptophan microenvironments in the ATPase. Calcium ion binding promoted an 11% increase in the center and a 27% decrease in the width of the distribution. By contrast, addition of calmodulin in the presence of Ca2+ caused a 15% decrease in the center of the distribution, revealing structural difference between calmodulin-activated and Ca2(+)-activated states of the ATPase. These results indicate the usefulness of on-line blank subtraction in frequency-domain lifetime measurements to investigate conformational changes in detergent-solubilized membrane protein samples.
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PMID:Time-resolved fluorescence of erythrocyte plasma membrane Ca2(+)-ATPase in different functional states. 153 98

Inhibition of the myosin subfragment 1 (S-1) ATPase activity by beryllium fluoride was studied directly in the presence of MgATP and following preincubation of samples with MgADP. In both cases, the rates of inhibition were very slow, with kapp = 0.5 and 58 M-1 s-1, respectively, in analogy to the rates of inhibition of myosin ATPase by vanadate [Goodno, C. C. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2620-2624]. The very different rates of inhibition in the presence of MgATP and on preincubation with MgADP suggested that beryllium fluoride binds to the M.ADP state of myosin. The slow inhibition rates and the nonlinear dependence of the observed rates on beryllium fluoride concentration were consistent with a two-step inhibition process involving a rapid binding equilibrium to yield a collisional complex, M.ADP.BeF3-, and its slow isomerization into M++.ADP.BeF3-. A third, much slower, step was required to account for the conversion of the stable M++.ADP.BeF3- to a virtually irreversibly inhibited complex. Kinetic description of the inhibition pathway was derived from the observed rates of inhibition of myosin ATPase, information on the binding of beryllium fluoride to M.ADP, and measurements of epsilon ADP chase from M++.epsilon ADP.BeF3-. The isomerization rate and equilibrium constants were 1.4 x 10(-2) s-1 and 50, respectively, and the overall binding constant of beryllium fluoride to M.ADP was 5 x 10(5) M-1. The inhibitory complex showed a 16% enhancement to tryptophan fluorescence of S-1 and a reduced quenching of epsilon ADP by acrylamide. It is concluded that M++.ADP.BeF3- is analogous to the M++.ADP.Vi and M**.ADP.Pi states of myosin.
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PMID:Inhibition of myosin ATPase by beryllium fluoride. 153 58

We have quantitated the interactions of two rabbit skeletal troponin C fragments with troponin I and the troponin I inhibitory peptide. The calcium binding properties of the fragments and the ability of the fragments to exert control in the regulated actomyosin ATPase assay have also been studied. The N- and C-terminal divalent metal binding domains of rabbit skeletal troponin C, residues 1-97 and residues 98-159, respectively, were prepared by specific cleavage at cysteine-98 and separation by gel exclusion chromatography. Both of the troponin C fragments bind calcium. The calcium affinity of the weak sites within the N-terminal fragment is about an order of magnitude greater than is reported for these sites in troponin C, suggesting interaction between the calcium-saturated strong sites and the weak sites. Stoichiometric binding (1:1) of the troponin I inhibitory peptide to each fragment and to troponin C increased the calcium affinities of the fragments and troponin C. Complex formation was detected by fluorescence quenching or enhancement using dansyl-labeled troponin C (and fragments) or tryptophan-labeled troponin I inhibitory peptide. The troponin C fragments bind to troponin I with 1:1 stoichiometry and approximately equal affinities (1.6 x 10(6) M-1) which are decreased 4-fold in the presence of magnesium versus calcium. These calcium effects are much smaller than is observed for troponin C. The summed free energies for the binding of the troponin C fragments to troponin I are much larger than the free energy of binding troponin C. This suggests a large positive interaction free energy for troponin C binding to troponin I relative to the fragments.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Interaction of troponin C and troponin C fragments with troponin I and the troponin I inhibitory peptide. 155 24

1. o-Iodosobenzoic acid (IOB) caused the formation of a disulfide bridge between SH1 and SH2 groups of myosin SF1 rendering inactive its ATPase activity. 2. IOB at high concentrations provoked fragmentation of SF1 at its tryptophan residues. 3. The main fragmentation point was located at 15 K from the amino terminus of the myosin heavy chain. 4. Actin was not fragmented by IOB. It protected SF1 tryptophans from IOB attack. 5. These results suggest a possible use of IOB as a reagent to study protein tryptophan under nondenaturing conditions.
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PMID:o-Iodosobenzoic oxidation and cleavage of myosin subfragment 1. 158 26


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