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 sarcoplasmic reticulum ATPase was studied (after 3 h to 14 days) in rats treated with 2,4-dichlorophenoxyacetate to induce myotonia. It was found that ATPase decreased in the treated rats after the establishment of myotonia.
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PMID:Sarcoplasmic reticulum ATPase in tibialis anterior muscle from rats with experimental myotonia. 294 34

The calcium pump of plasma membranes is an ATPase of the E1E2 type; that is, it forms a phosphoenzyme during the reaction cycle and is inhibited by vanadate. It differs from the Ca2+-transporting ATPase of sarcoplasmic reticulum in molecular mass, immunological properties and Ca2+/ATP stoichiometry. Its affinity for calcium, which is low in the absence of calmodulin (Km, 10-20 microM), is increased by the latter (to a Km of about 0.5 microM). The effect of calmodulin is mimicked by acidic phospholipids (including the phosphorylated products of phosphatidylinositol), long-chain polyunsaturated fatty acids, and controlled treatment with a number of proteases. The ATPase has been purified to homogeneity from a number of plasma membranes using calmodulin affinity chromatography. The purified enzyme (a single polypeptide of molecular mass 138 kDa) pumps calcium into reconstituted liposomes in exchange for protons. Controlled trypsin proteolysis has shown that about one-third of the enzyme mass can be removed without impairing calcium transport. It has also indicated that the ability to bind calmodulin and to respond to it resides in a 9 kDa sequence of the enzyme molecule. The sequence contains a 4 kDa domain that binds calmodulin, and a 5 kDa domain which is essential for the stimulation.
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PMID:The calcium pump of plasma membranes. 294 87

Time-resolved filtration measurements using radioactive calcium were conducted to investigate with leaky preparations the kinetic features of the dissociation of transported calcium to the luminal side of the sarcoplasmic reticulum calcium pump, which occurs concomitantly with isomerization of the phosphorylated ATPase. At pH 6 and 20 degrees C, Ca2+ dissociation was moderately fast in the absence of potassium (3-5 s-1 at 0.05 mM ATP), implying that the dephosphorylation step (about 1.5 s-1) was the main contributor to rate limitation under these conditions. Potassium slowed down Ca2+ release but stimulated dephosphorylation, so that in its presence Ca2+-releasing isomerization did contribute to rate limitation, especially at neutral pH. At pH 6 in the absence of potassium and in the presence of magnesium, millimolar concentrations of ATP doubled the rate of Ca2+ dissociation, as also shown by dual-wavelength detection of fast changes in the absorbance of the Ca2+-sensitive dye Antipyrylazo III. Under the same conditions, low-affinity binding of ATP to phosphoenzyme was demonstrated. It is suggested that this low-affinity acceleration by ATP of the crucial step leading to dissociation of transported Ca2+ is the specific interaction responsible for the low-affinity acceleration of overall ATPase activity generally observed in the presence of potassium at neutral pH. Hydrolysis of the Ca2+-deprived phosphoenzyme was accelerated by ATP in the absence but not in the presence of Mg2+ in the dephosphorylation medium. We suggest that metal-free ATP is a more potent activator than Mg X ATP for transitions involving phosphoenzyme.
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PMID:Rapid filtration study of the phosphorylation-dependent dissociation of calcium from transport sites of purified sarcoplasmic reticulum ATPase and ATP modulation of the catalytic cycle. 294 63

Sarcoplasmic reticulum membrane vesicles from rabbit skeletal muscle were treated with iodoacetamide (IAA) at pH 7.0 and 30 degrees C. At 1.0 mM IAA, 1 mol of IAA per mol of ATPase peptide was bound in 1 h. Under these conditions, IAA was attached specifically to the B-tryptic fragment portion of the peptide. The binding of IAA did not affect the Ca2+-transporting activity of ATPase. Three fluorescent derivatives of iodoacetamide, 5-(2-acetamidoethyl)aminonaphthalene-1-sulfonate (IAEDANS), 5-iodoacetamido fluorescein (IAF), and 5-iodoacetamido eosin (IAE), were also tested for reactivity toward sarcoplasmic reticulum ATPase at 30 degrees C and pH 7.0. In 1 h at 50 microM concentration, each of these fluorescent labels modified ATPase to a labeling density of 1 mol per mol of ATPase. Neither IAEDANS nor IAF at this labeling density affected Ca2+-transporting activity, but IAE reduced it to 20% of the untreated control. The target site of IAEDANS at this labeling density was located exclusively on the B-fragment portion, as was the case with IAA, but IAF label was found on both A1 and B fragments after limited tryptic digestion. IAEDANS was used as a B-fragment portion-directed conformational probe of Ca2+-transport ATPase, and an increase in fluorescence intensity accompanying E1Ca-P formation was detected. The fluorescence enhancement was abolished when E1Ca-P X ADP beta S was formed by adding ADP beta S to preformed E1Ca-P. This suggests that the conformation of ATPase in the neighborhood of the IAEDANS binding site may be altered in response to the dissociation of ADP from the phosphorylated intermediate.
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PMID:Chemical modification and fluorescence labeling study of Ca2+,Mg2+-adenosine triphosphatase of sarcoplasmic reticulum using iodoacetamide and its N-substituted derivatives. 295 79

Microcrystalline arrays of Ca2+-transporting ATPase (EC 3.6.1.38) develop in detergent-solubilized sarcoplasmic reticulum upon exposure to 10-20 mM CaCl2 at pH 6.0 for several weeks at 2 degrees C, in a crystallization medium that preserves the ATPase activity for several months. Of 48 detergents tested, optimal crystallization was obtained with Brij 36T, Brij 56, and Brij 96 at a detergent:protein weight ratio of 4:1 and with octaethylene glycol dodecyl ether at a ratio of 2:1. Similar Ca2+-induced crystalline arrays were obtained with the purified or delipidated Ca2+-ATPase of sarcoplasmic reticulum but at lower detergent:protein ratios. The crystals are stabilized by fixation with glutaraldehyde and persist even after the removal of phospholipids by treatment with phospholipases A or C and by extraction with organic solvents. The crystals obtained so far can be used only for electron microscopy, but ongoing experiments suggest that under similar conditions large ordered arrays may develop that are suitable for x-ray diffraction analysis.
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PMID:Crystallization of Ca2+-ATPase in detergent-solubilized sarcoplasmic reticulum. 295 50

The erythrocyte plasma membrane Ca2+-pumping ATPase is known to form an acyl-phosphate catalytic intermediate, but there is otherwise little structural information linking it to the other mammalian ion-pumping ATPases which also form phosphorylated intermediates (the Na+, K+-ATPase of plasma membranes, the Ca2+-ATPase of sarcoplasmic reticulum, and the H+, K+-ATPase of gastric mucosa). We show here that this enzyme possesses a fluorescein isothiocyanate-reactive region similar to that possessed by these other ATPases. Low concentrations (10 microM) of fluorescein isothiocyanate inhibit the ATPase activity of this pump, and this inhibition is prevented by 4 mM ATP. ATP also inhibits the reaction of fluorescein isothiocyanate with a single amino acid residue on the 138-kDa polypeptide chain. A tryptic fragment containing the fluorescein-conjugated residue was isolated by high pressure liquid chromatography. The sequence of this peptide was determined to be NH2-Met1-Tyr2-Ser3-Lys4-Gly5-Ala6-Ser7-Glu8++ +-Ile9-Ile10-Leu11-Arg12-COOH; fluorescein isothiocyanate reacts with the lysine residue. The identities of residues 4-8 are the same as those in a sequence common to the other ATPases mentioned above, except that serine-7 of this sequence is changed to a proline in those ATPases. This substitution, sometimes not considered a homologous one, is not expected to have a major effect on the secondary structure or polarity of this region. Outside of this 5-residue core region of the fluorescein isothiocyanate-reactive site, the homologies among the different ion-pumping ATPases are limited.
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PMID:The ATP-binding site of the erythrocyte membrane Ca2+ pump. Amino acid sequence of the fluorescein isothiocyanate-reactive region. 295 52

The effects of tricyclohexyltin hydroxide (Plictran), an organotin acaricide, on 45Ca2+ uptake and Ca2+ ATPase were studied in vitro and in vivo in rat heart ventricular membrane vesicles, primarily sarcoplasmic reticulum. There was a concentration dependent inhibition of both 45Ca2+ uptake and Ca2+ ATPase in vivo as well as in vitro. Isoproterenol, a beta-adrenergic agonist, stimulated 45Ca2+ uptake and Ca2+ ATPase of sarcoplasmic reticulum and this was also inhibited by Plictran. Since cardiac relaxation is mediated by beta-adrenergic stimulation via Ca+ uptake by sarcoplasmic reticulum, the inhibition of calcium pump activity by Plictran may result in alterations in cardiac Ca2+ fluxes leading to cardiac dysfunction.
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PMID:Inhibition of beta-adrenergic stimulated calcium pump of rat cardiac sarcoplasmic reticulum by tricyclohexyltin hydroxide. 295 2

Ca2+-Transporting ATPase of rabbit skeletal muscle sarcoplasmic reticulum contains several SH groups which are reactive with N-ethylmaleimide (MalNEt) at pH 7.0. The location of the one which is most reactive with MalNEt (SHN, Kawakita et al. J. Biochem. 87, 609 (1980)) was identified on the amino acid sequence of the ATPase. SHN was labeled by reacting sarcoplasmic reticulum membranes with [14C] MalNEt to a labeling density of 1 mol/mol ATPase. [14C]MalNEt-labeled membranes were digested with thermolysin and 14C-labeled SHN peptides were fractionated by Sephadex LH-20 chromatography to give two major peaks of radioactivity. [14C]-MalNEt-labeled peptides were further purified to homogeneity by C18-reversed phase HPLC. Two radioactive peptides containing modified cysteine (Cys), Leu-Gly-Cys-Thr-Ser and Val-Cys-Lys-Met, were finally obtained in roughly equal amounts and in reasonable recovery. Both of these sequences were found in the amino acid sequence of Ca2+-transporting ATPase (Brandl et al. Cell 44, 597 (1986)), and Cys344 and Cys364 were identified as the targets of MalNEt-modification. Thus, 0.5 mol/mol ATPase of each Cys residue actually reacted rapidly with MalNEt under the conditions leading to SHN-modification. Modification of either one with MalNEt may negatively affect the reactivity of the other. Both of the highly reactive SH groups are located in the neighborhood of Asp351, the phosphorylation site of ATPase.
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PMID:Reactive sulfhydryl groups of sarcoplasmic reticulum ATPase. I. Location of a group which is most reactive with N-ethylmaleimide. 295 11

The Syrian cardiomyopathic hamster has a hereditary disease characterized by a progressive myocyte necrosis and intracellular calcium overload. Several systems in the heart sarcolemma that regulate the rate of Ca2+ entry or efflux were examined. There is a selective decrease of Ca2+-pumping ATPase activity in the heart sarcolemma of 40-day-old myopathic hamsters, while the Na+-Ca2+ exchange system and the ouabain-sensitive (Na+ + K+)-ATPase activity remain intact. This age-dependent decrease in Ca2+-ATPase activity closely parallels the time course of lesion development. Both the affinity for Ca2+ (Km) and the maximal velocity (Vmax) of the Ca2+-dependent ATP hydrolysis are altered. In addition, there is also an increased number of calcium channel receptor binding sites. Thus the data suggest that the imbalance in Ca2+ fluxes across the cardiac plasma membrane may be involved in the pathogenesis of this cardiomyopathy.
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PMID:Defective Ca2+-pumping ATPase of heart sarcolemma from cardiomyopathic hamster. 295 83

The coupling of Ca2+ movements and phosphate fluxes as well as the time-dependent occurrence of sequential reaction intermediates in the forward mode of the Ca,Mg-dependent ATPase reaction have been investigated using leaky vesicles (A23187) in the presence of varying Ca2+, Mg2+, and K+ concentrations. The employed ATP concentration of 2 microM does not allow more than one reaction cycle to occur. The respective fractions of ADP-sensitive and ADP-insensitive phosphoenzyme have been determined. The chosen experimental conditions (0-1 degree C, pH 6.0, absence of solubilizers) allow a prolonged time of observation and exclude interfering alterations of coupling and binding parameters, respectively. It is shown that under the experimental conditions K+ interacts with at least four different reaction steps (phosphoenzyme formation, E1P----E2P transition, E2P hydrolysis, and E2----E1 transformation). Mg2+ represents the sole ionic co-factor for the formation of the substrate MgATP if it is present in high concentrations (5 mM). Additional Ca2+ is bound to the substrate as well as to unspecific sites otherwise occupied by Mg2+ if Mg2+ is reduced to 0.1 mM. In this case the E1P----E2P transition rate (including Ca2+ translocation and Ca2+ release from low-affinity sites) is little diminished. If, in the absence of K+, both Mg2+ and Ca2+ are deficient E2P hydrolysis is vastly retarded. We find Ca2+ release to occur time-coincidently with E1P formation and not concomitantly with the comparably slow appearance of E2P; the molar amount of Ca2+ released, however, rather agreed with that of E2P formed. This suggests that under the prevailing conditions of a high proton concentration, phosphoenzyme states containing occluded Ca2+ or Ca2+ bound to low-affinity sites are transitional and not detectable. Preliminary findings on this subject have been published by us and colleagues from this laboratory [Hasselbach, W., Agostini, B., Medda, P., Migala, A. & Waas, W. (1985) in The sarcoplasmic reticulum calcium pump: Early and recent developments critically overviewed (Fleischer, S. & Tonomura, Y., eds) pp. 19-49, Academic Press, Orlando].
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PMID:The effect of monovalent and divalent cations on the ATP-dependent Ca2+-binding and phosphorylation during the reaction cycle of the sarcoplasmic reticulum Ca2+-transport ATPase. 295 19


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