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

---

Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Second and fourth derivatives have been calculated from the fluorescence emission spectra of N-acetyl tryptophanamide in solvents of varying polarity. It is demonstrated that the otherwise featureless fluorescence emission spectrum can be resolved into a series of discrete bands by the use of the derivative technique. These bands appear to have their origins in the transitions of electrons from the first excited singlet state back to the various vibrational levels of the ground state. The shifting of the fluorescence emission maximum to shorter wavelengths upon decreasing the solvent polarity is shown to be due to changes in the relative contributions of each of the bands combined with smaller changes in the band positions. Derivative spectra have also been obtained from the intrinsic tryptophan fluorescence of the (Ca2+ + Mg2+)-adenosine triphosphatase of sarcoplasmic reticulum membranes. A similar pattern of bands is observed to that found in the model system and is consistent with the majority of the tryptophan residues being located in hydrophobic environments. Addition of calcium ions to the protein results in enhancement of the protein fluorescence accompanied by a small and hitherto unseen blue-shift of the spectrum. The mechanistic implications of this finding are discussed in relation to the calcium transport function of the protein.
...
PMID:Derivative spectroscopy of tryptophan fluorescence used to study conformational transitions in the (Ca2+ + Mg2+)-adenosine triphosphatase of sarcoplasmic reticulum. 294 31

Binding constants for myristoleic, palmitoleic, palmitic, oleic, and eicosanoic acids and oleyland stearylamine to lipid bilayers have been determined by using microelectrophoresis. Quenching of the fluorescence of the hydrophobic tryptophan analogue N-palmitoyl-L-tryptophan n-hexyl ester incorporated into lipid bilayers by oleic acid and oleylamine and their brominated derivatives is interpreted in terms of unlimited binding to the bilayers. The tryptophan fluorescence of the (Ca2+-Mg2+)-ATPase purified from sarcoplasmic reticulum is quenched when reconstituted into bilayers of 1,2-bis(9,10-dibromostearoyl)-phosphatidylcholine (BRPC). Addition of fatty acids, oleylamine, oleyl alcohol, and methyl oleate to the ATPase reconstituted with BRPC reduces the quenching caused by BRPC, indicating binding of these molecules at the lipid-protein interface (annular sites). The charged molecules bind more strongly at the annular sites than do the uncharged molecules. Additional quenching of BRPC-ATPase by brominated derivatives of these molecules indicates binding at sites distinct from the lipid-protein interface, with binding constants similar to those for binding at annular sites, except for oleylamine. Quenching of tryptophan fluorescence of the ATPase by fatty acids and oleylamine suggests that ca. 50% of the tryptophan residues of the ATPase are located close to the lipid-water interface of the membrane.
...
PMID:Binding of long-chain alkyl derivatives to lipid bilayers and to (Ca2+-Mg2+)-ATPase. 294 59

Enhanced fluorescence of the ATP analogue 2',3'-O-(2,4,6-trinitrocyclohexyldienylidine)adenosine 5'-triphosphate (TNP-ATP), bound to the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum, is closely related to phosphoenzyme levels (Bishop, J. E., Johnson, J. D., and Berman, M. C. (1984) J. Biol. Chem. 259, 15163-15171) and has an emission maximum consistent with decreased polarity of the TNP-ATP-binding site. The phosphoenzyme conformation responsible for increased nucleotide-binding site hydrophobicity has been studied by redistribution of phosphoenzyme intermediates following specific thiol group modification. N-Ethylmaleimide, in the presence of 50 microM Ca2+, 1 mM adenyl-5'-yl imidodiphosphate, pH 7.0, at 25 degrees C for 30 min, selectively modified the SH group essential for phosphoenzyme decomposition, which resulted in decreased ATPase activity, Ca2+ uptake, and a decrease in ATP-induced TNP-ATP fluorescence. Phosphorylated (Ca2+, Mg2+)-ATPase levels from [gamma-32P] ATP remained relatively unaffected (3.1 nmol/mg), but the ADP-insensitive fraction decreased from 56 to 15%. Phosphoenzyme levels from 32Pi were also decreased to the same extent as turnover, with equivalent loss of Pi-induced TNP-ATP fluorescence. The E1 to E2 transition, as monitored by the change in intrinsic tryptophan fluorescence, was unaffected. Modification of thiol groups of unknown function did not modify turnover-induced TNP-ATP fluorescence. It is concluded that the ADP-insensitive phosphoenzyme, E2-P, is responsible for enhanced TNP-ATP fluorescence. This suggests that the conformational transition, 2Ca2+outE1 approximately P----2Ca2+inE2-P, is associated with altered properties of the noncatalytic, or regulatory, nucleotide-binding site.
...
PMID:Phosphoenzyme conformational states and nucleotide-binding site hydrophobicity following thiol modification of the Ca2+-ATPase of sarcoplasmic reticulum from skeletal muscle. 295 14

1. From the intrinsic fluorescence spectral properties and fluorescence quenching experiments done with acrylamide and iodide, using native sarcoplasmic reticulum vesicles, purified ATPase and ATPase solubilized with 1% Triton X-100, it is deduced that practically all the fluorescent tryptophanyl residues of this protein belong to a single population showing similar hydrophobic microenvironments. 2. Both acrylamide and iodide seem to be able to penetrate through the sarcoplasmic reticulum membrane. 3. The intrinsic fluorescence of the Ca2+-ATPase due to tryptophan residues probably buried inside the membrane is used as a tool to follow thermotropic changes in membrane fluidity of reconstituted systems.
...
PMID:Structural properties of sarcoplasmic reticulum Ca2+-ATPase as studied by intrinsic protein fluorescence. 296 35

Chymotryptic subfragment 1 (S-1) prepared from rabbit skeletal myosin has lost its ATPase activity upon incubation at 35 degrees C for 3 h. The loss in ATPase activity was accompanied by the perturbation of the structure of the 50K domain as indicated by a dramatic increase in the tryptic susceptibility of this domain without any change in the susceptibility of the other domains of S-1. The perturbation starts at the C-terminal region of the domain as suggested by the appearance of a 29K intermediate protein band in the tryptic peptide pattern of the heat-treated S-1. The heat-treated molecule essentially retained its actin and polyphosphate binding ability, and the actin binding was still sensitive to the presence of ATP or pyrophosphate. However, as opposed to native S-1, in heat-treated S-1 the addition of ATP does not induce an increase in tryptophan fluorescence, and, in the case of the treated species, the fluorescence of 1,N6-ethenoadenosine 5'-diphosphate added to the mixture is quenchable by acrylamide. This latter observation suggests that the binding of the adenine ring of the nucleotide has been altered following the heat treatment. The results indicate that the actin and polyphosphate binding sites of S-1 are distinct and that they are relatively independent of the adenine ring binding site.
...
PMID:Effect of mild heat treatment on actin and nucleotide binding of myosin subfragment 1. 296 71

Fluorescence stopped-flow experiments were performed to elucidate the elementary steps of the ATPase mechanism of scallop heavy meromyosin in the presence and in the absence of Ca2+. ATP binding and hydrolysis, as monitored by the change in tryptophan fluorescence, appear to be Ca2+-insensitive, whereas both Pi release and ADP release are markedly suppressed in the absence of Ca2+. Rate constants for Pi release are 0.2 s-1 and 0.002 s-1 and for ADP release are 6 s-1 and 0.01 s-1 in the presence and in the absence of Ca2+ respectively. Ca2+ binding to the specific site of the regulatory domain is rapid and its release occurs at 25 s-1, consistent with the time scale of a twitch of the striated adductor muscle. Nucleotide binding is a multi-step process requiring a minimum of three states. In such a model Ca2+ controls the rate of conformational changes at the active site in both the forward and the reverse direction, leading to a large dependence of the rate of nucleotide release, but a lesser effect on the overall equilibrium position. The kinetic trapping of nucleotides and Pi at the active site, in the absence of Ca2+, appears to be a fundamental step in suppressing the interaction of the myosin head with the thin filaments in relaxed molluscan muscle.
...
PMID:Transient-kinetic studies of the adenosine triphosphatase activity of scallop heavy meromyosin. 296 25

To localize and characterize the regulatory nucleotide site of skeletal muscle sarcoplasmic reticulum Ca2+-ATPase, we have investigated the effects of ADP, ATP, and analogues of these nucleotides on the rate of dephosphorylation of both native ATPase and ATPase modified with fluorescein 5'-isothiocyanate (FITC), a reagent which hinders access of nucleotides to the ATPase catalytic site without affecting phosphorylation from Pi. Dephosphorylation of the phosphoenzyme formed from Pi was monitored by rapid filtration or stopped-flow fluorescence, mostly at 20 degrees C, pH 6.0, and in the absence of potassium. Fluorescence measurements were made possible through the use of 8-bromo-ATP, which selectively quenched certain tryptophan residues of the ATPase, thereby allowing the intrinsic fluorescence changes associated with dephosphorylation to be measured in the presence of bound nucleotide. ATP, 8-bromo-ATP, and trinitrophenyladenosine diand triphosphate, but not ADP, enhanced the rate of dephosphorylation of native ATPase 2-3-fold when added in the absence of divalent cations. Millimolar concentrations of Mg2+ eliminated the accelerating effects. Acceleration in the absence of Mg2+ was observed at relatively low concentrations of ATP and 8-bromo-ATP (0.01-0.1 mM) and binding of metal-free ATP and ADP, but not Mg.ATP, to the phosphoenzyme in this concentration range was demonstrated directly. Modification of the ATPase with FITC blocked nucleotide binding in the submillimolar concentration range and eliminated the nucleotide-induced acceleration of dephosphorylation. These results show that dephosphorylation, under these conditions, is regulated by ATP but not by Mg.ATP or ADP, and that the catalytic site is the locus of this "regulatory" ATP binding site.
...
PMID:ATP regulation of sarcoplasmic reticulum Ca2+-ATPase. Metal-free ATP and 8-bromo-ATP bind with high affinity to the catalytic site of phosphorylated ATPase and accelerate dephosphorylation. 297 Apr 58

Steady-state ATPase activity, calcium binding, formation of phosphorylated enzyme intermediate with ATP in the presence of Ca2+, or with Pi in the absence of Ca2+, and association of ATPase molecules into bidimensional crystals, were studied using vesicular fragments of sarcoplasmic reticulum. The vesicles were exposed to increasing concentrations of urea in order to produce stepwise perturbations of protein structure and to test the effect of such perturbations on the partial reactions and crystallization pattern of sarcoplasmic reticulum ATPase. It was found that low concentrations of urea produce specific inhibition of Pi binding and enzyme phosphorylation with Pi (but not with ATP). Intermediate concentrations of urea reduce calcium binding affinity and cooperativity, while the ability of the enzyme to be phosphorylated with ATP and to form dimeric arrays is retained. These observations demonstrate that the sarcoplasmic reticulum ATPase is sensitive to physical perturbations producing specific and reversible changes in the Pi and calcium binding domains. These changes interfere with enzyme turnover, indicating that conformational effects related to binding and dissociation of Pi and calcium are tightly coupled to catalysis and energy transduction. Higher concentrations of urea produce irreversible denaturation, accompanied by total inhibition of calcium binding, enzyme phosphorylation with ATP, and association of ATPase chains in bidimensional crystals. Under these conditions, protein unfolding is manifested by a sharp reduction in the fluorescence of intrinsic tryptophan residues and of a covalently bound probe. These observations suggest that dimeric association and a tendency to form bidimensional crystals correspond to a basic property of the enzyme, which is linked to its native structure and whose character may change in the presence of ligands and/or during the catalytic cycle. On the other hand, the decavanadate-induced crystallization pattern cannot be interpreted in terms of a mechanistic relationship of ATPase dimerization with one of the intermediate states of the catalytic cycle.
...
PMID:Effect of urea on the partial reactions and crystallization pattern of sarcoplasmic reticulum adenosine triphosphatase. 297 Aug 23

The Ca2+ binding site region of the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum was labeled with several fluorescent analogs of dicyclohexylcarbodiimide. As has been shown by Chadwick and Thomas, in the absence of Ca2+ in the medium, labeling with the naphthyl carbodiimide results in the inhibition of enzyme activity. Further, Ca2+ occupancy of the high affinity sites of the enzyme protects against incorporation into the site(s). The fluorescent carbodiimide has been used to determine the depth of the site of label incorporation relative to the aqueous-bilayer interfaces by quenching studies using spin-labeled fatty acid derivatives. The series of quenchers used have their spin-label moiety located at different positions along the fatty acid chain. It was found that after suitable correction for differences in partitioning of the various derivatives, the order of quenching efficiency was 16- greater than 12- greater than 10- greater than 7- greater than 5-NS, indicating that the naphthyl moiety is near the center of the bilayer. In contrast, quenching with the aqueous-restricted I- indicated that the label is accessible from the external milieu, likewise for a presumed aqueous quencher, acrylamide. The aqueous quenchers accessibilities were altered upon Ca2+ binding to the ATPase. Quenching of the intrinsic fluorescence with the x-NS derivatives indicates that the ATPase tryptophan residues are primarily localized at the aqueous-membrane interfaces, with the order of quenching being 5- greater than 7- greater than 10- greater than 12- greater than 16-NS. The trp residue(s) which changes its fluorescence upon Ca2+ binding is shown to be near the membrane surface.
...
PMID:Molecular topography of the Ca2+-ATPase of sarcoplasmic reticulum. 297 13

Phosphorescence of protein tryptophan was analyzed in sarcoplasmic reticulum vesicles, and in the purified Ca2+ transport ATPase in deoxygenated aqueous solutions at room temperature. Upon excitation with light of 295 nm wavelength, the emission maxima of fluorescence and phosphorescence were at 330 nm and at 445 nm, respectively. The phosphorescence decay was multiexponential; the lifetime of the long-lived component of phosphorescence was approximately equal to 22 ms. ATP and vandate significantly reduced the phosphorescence in the presence of either Ca2+ or EGTA; ADP was less effective, while AMP was without effect. The quenching by ATP showed saturation consistent with the idea that the ATP-enzyme complex had a lower phosphorescence yield. Upon exhaustion of ATP, the phosphorescence returned to starting level. Significant quenching of phosphorescence with a decrease in phosphorescence lifetime was also caused by NaNO2, methylvinyl ketone and trichloroacetate, without effect on ATPase activity; this quenching did not show saturation and was therefore probably collisional in nature.
...
PMID:Tryptophan phosphorescence of the Ca2+-ATPase of sarcoplasmic reticulum. 297 55


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---