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)

Fluorescein-labeled heavy meromyosin subfragment-1 (F-S-1) has been purified by ion exchange chromatography and characterized in terms of its ability to bind specifically to actin. F-S-1 activates the Mg++-adenosine triphosphatase activity of rabbit skeletal muscle actin and decorates actin as shown by negative stains and thin sections of rabbit actin and rat embryo cell microfilament bundles, respectively. Binding of F-S-1 to cellular structures is prevented by pyrophosphate and by competition with excess unlabeled S-1. The F-S-1 is used in light microscope studies to determine the distribution of actin-containing structures in wnterphase and mitotic rat embryo and rat kangaroo cells. Interphase cells display the familiar pattern of fluorescent stress fibers. Chromosome-to-pole fibers are fluorescent in mitotic cells. The glycerol extraction procedures employed provide an opportunity to examine cells prepared in an identical manner by light and electron microscopy. The latter technique reveals that actin-like microfilaments are identifiable in spindles of glycerinated cells before and after addition of S-1 or HMM. In some cases, microfilaments appear to be closely associated with spindle microtubles. Comparison of the light and electron microscope results aids in the evaluation of the fluorescent myosin fragment technique and provides further evidence for possible structural and functional roles of actin in the mitotic apparatus.
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PMID:Myosin subfragment binding for the localization of actin-like microfilaments in cultured cells. A light and electron microscope study. 7 3

Fluorescein 5'-isothiocyanate (FITC) was used to modify the lysine residue in the active site of tonoplast H(+)-ATPase from etiolated mung-bean (Vigna radiata L.) seedlings. FITC caused marked inactivation of the enzyme activities of both membrane-bound and soluble ATPase and its associated H+ translocation. The SDS/PAGE pattern revealed that the FITC-binding site was in the large (A) subunit of ATPase. Inhibition could be substantially prevented by its physiological substrate ATP, pyrophosphate and nucleotides in the decreasing order: ATP greater than pyrophosphate greater than ADP greater than AMP greater than GTP greater than CTP greater than UTP. The mode of inhibition by FITC was competitive with respect to ATP. Loss of ATPase activity followed pseudo-first-order kinetics with a Ki of 0.33 mM, a minimum inactivation half-time of 110 s, and a first-order rate constant of 0.244 s-1. A double-logarithmic plot of apparent rate constant versus FITC concentration gave a slope of 0.913, indicating that inactivation results from reaction of at least one lysine residue at the catalytic site of the large subunit. Labelling studies indicated that the incorporation of approx. 1 mol of FITC/mol of ATPase is sufficient to inhibit ATPase completely. The enhancement and blue shift of emission maxima of FITC after modification of ATPase indicated that the labelled lysine residue was located in a relatively hydrophobic domain.
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PMID:Inhibition of tonoplast ATPase from etiolated mung bean seedlings by fluorescein 5'-isothiocyanate. 138 33

The objective of this study was to determine the relationship between cytosolic pH and vesicular pH during ATP depletion. Using digitized video microscopy and single, cultured rat hepatocytes, cytosolic pH and vesicular pH were quantitated by ratio imaging of BCECF (2', 7' biscarboxyethyl-5,6-carboxyfluorescein) fluorescence and fluorescein-dextran fluorescence, respectively. Basal value for cytosolic pH was 7.26 and basal value for vesicular pH was 4.86. During ATP depletion by metabolic inhibition with KCN plus iodoacetic acid or antimycin A, cytosolic pH decreased 0.71 units to 6.55. In separate experiments under identical conditions, vesicular pH increased 1.59 units to 6.45, suggesting that protons were leaking from acidic vesicles during ATP depletion. Fluorescein-dextran fluorescence remained punctate, indicating that the rise in vesicular pH was due to an efflux of protons from vesicles and not loss of vesicle integrity. To determine whether efflux of protons from acidic vesicles can acidify cytosolic pH, we used two maneuvers that result in leakage of protons from acidic vesicles without significantly decreasing cellular ATP: (a) hypotonic stress in K(+)-free media and (b) exposure of the cells to the H(+)-ATPase inhibitor NBD-Cl. Both hypotonic stress and NBD-Cl decreased cytosolic pH 0.4 units to 6.86 and increased vesicular pH 2.0 units to 6.76, resulting in near-equilibration of cytosolic pH and vesicular pH. Thus an efflux of protons from intracellular compartments will acidify cytosolic pH of hepatocytes (pH 6.86), but not to the same degree as ATP depletion (pH 6.55).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Efflux of protons from acidic vesicles contributes to cytosolic acidification of hepatocytes during ATP depletion. 171 57

Fluorescein 5'-isothiocyanate (FITC) covalently modifies the Lys-501 residue of the catalytic (alpha) subunit of Na+,K(+)-ATPase and resides at a conformation-sensitive site in or near the ATP binding site. In these studies, FITC-directed antibodies which quench this hapten's fluorescence were used to infer the solvent accessibility of the enzyme-bound probe. These antibodies identified two FITC labeling populations. An antibody-accessible population, representing 20-50% of the bound FITC fluorescence, was essentially (95%) quenched by the antibody. The second population was irreversibly labeled, was inaccessible to antibody, and was the fraction of probe whose fluorescence intensity is sensitive to the enzyme's conformation. The anti-FITC antibodies therefore permitted the selective investigation of FITC at this active site. Distinct differences between the two labeling sites were then demonstrated. Shifts in the absorption spectrum suggested that the active-site-bound probe resides in a hydrophobic environment, while polarization values indicated a rigid, rotationally restricted location. These two properties were not altered by ligand additions. Iodide quenching studies, however, showed that in the E1Na+ conformation there was a 50% decrease in solvent access to the active-site-bound probe as compared to free probe while the E1Na(+)----E2K+ transition decreased this accessibility an additional 50%. Similarly, there was a significant decrease in the relative quantum yield of FITC linked at this site that was reduced further by the E1Na(+)----E2K+ transition. In contrast, frequency domain spectroscopy showed no significant differences in the lifetimes of fluorescence decay for the two different labeling populations nor for the high (E1Na+) and low (E2K+) fluorescence intensity conformations. We have found that static (lifetime independent) quenching rather than collisional processes or protonation changes accounts for the fluorescence intensity changes undergone by FITC bound at the ATP-protectable site.
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PMID:Immunochemical and spectroscopic characterization of two fluorescein 5'-isothiocyanate labeling sites on Na+,K(+)-ATPase. 184 73

The binding and conformational properties of the divalent cation site required for H+,K(+)-ATPase catalysis have been explored by using Ca2+ as a substitute for Mg2+. 45Ca2+ binding was measured with either a filtration assay or by passage over Dowex cation exchange columns on ice. In the absence of ATP, Ca2+ was bound in a saturating fashion with a stoichiometry of 0.9 mol of Ca2+ per active site and an apparent Kd for free Ca2+ of 332 +/- 39 microM. At ATP concentrations sufficient for maximal phosphorylation (10 microM), 1.2 mol of Ca2+ was bound per active site with an apparent Kd for free Ca2+ of 110 +/- 22 microM. At ATP concentrations greater than or equal to 100 microM, 2.2 mol of Ca2+ were bound per active site, suggesting that an additional mole of Ca2+ bound in association with low affinity nucleotide binding. At concentrations sufficient for maximal phosphorylation by ATP (less than or equal to 10 microM), APD, ADP + Pi, beta,gamma-methylene-ATP, CTP, and GTP were unable to substitute for ATP. Active site ligands such as acetyl phosphate, phosphate, and p-nitrophenyl phosphate were also ineffective at increasing the Ca2+ affinity. However, vanadate, a transition state analog of the phosphoenzyme, gave a binding capacity of 1.0 mol/active site and the apparent Kd for free Ca2+ was less than or equal to 18 microM. Mg2+ displaced bound Ca2+ in the absence and presence of ATP but Ca2+ was bound about 10-20 times more tightly than Mg2+. The free Mg2+ affinity, like Ca2+, increased in the presence of ATP. Monovalent cations had no effect on Ca2+ binding in the absence of ATP but dit reduce Ca2+ binding in the presence of ATP (K+ = Rb+ = NH4 + greater than Na+ greater than Li+ greater than Cs+ greater than TMA+, where TMA is tetramethylammonium chloride) by reducing phosphorylation. These results indicate that the Ca2+ and Mg2+ bound more tightly to the phosphoenzyme conformation. Eosin fluorescence changes showed that both Ca2+ and Mg2+ stabilized E1 conformations (i.e. cytosolic conformations of the monovalent cation site(s)) (Ca.E1 and Mg.E1). Addition of the substrate acetyl phosphate to either Ca.E1 or Mg.E1 produced identical eosin fluorescence showing that Ca2+ and Mg2+ gave similar E2 (extracytosolic) conformations at the eosin (nucleotide) site. In the presence of acetyl phosphate and K+, the conformations with Ca2+ or Mg2+ were also similar. Comparison of the kinetics of the phosphoenzyme and Ca2+ binding showed that Ca2+ bound prior to phosphorylation and dissociated after dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Calcium binding to the H+,K(+)-ATPase. Evidence for a divalent cation site that is occupied during the catalytic cycle. 216 18

Fluorescein isothiocyanate-conjugated dextran (FITC-dextran) is internalized by endocytosis into the lysosome-like vacuoles of Saccharomyces cerevisiae (Makarow, M., 1985, EMBO (Eur. Mol. Biol. Organ.) J. 4:1861-1866). Here we show that under energy depletion conditions FITC-dextran accumulated in a cytoplasmic compartment, from which it could be chased to the vacuole when the energy block was removed. The internal pH of the intermediate compartment under energy depletion was determined by fluorometry to be 5.8. The pH could be raised by the lysosomotropic agent ammonium chloride, the protonophore carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (CCCP) and the ATPase inhibitors dicyclohexylcarbodiimide (DCCD) and sodium vanadate. The pH of the vacuole was found to be 6.5. It was raised by ammonium chloride, CCCP, and DCCD, but not with sodium vanadate. Efrapeptin had no effect on the internal pH of either compartment. By dissecting the endocytic pathway, two portions of the route leading to the vacuole could be studied separately. The internalization of FITC-dextran from the extracellular fluid to the intermediate compartment followed linear kinetics, was independent of energy, and occurred at temperatures of between 15 degrees and 37 degrees C. Transfer of the marker from the intermediate compartment to the vacuole required energy, took place at temperatures between 19 degrees and 37 degrees C, and had a half-time of 7 min at 37 degrees C. Transport of the marker from the exterior of the cell to the vacuole did not require acidic pH values in the intermediate compartment or the vacuole. We suggest that the cytoplasmic compartment revealed by FITC-dextran, under energy depletion, represents the equivalent of the endosomes of mammalian cells.
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PMID:Transport of a fluorescent macromolecule via endosomes to the vacuole in Saccharomyces cerevisiae. 243 74

The density of Langerhans cells (LC) is significantly decreased in skin sites exposed to ultraviolet radiation (UVR). This results in reduced antigen-presenting cell (APC) activity in UVR-exposed skin. We have previously reported that the recovery in the density of ATPase/Ia+ epidermal cells (EC) parallels the restoration of APC function in the UVR-exposed skin of mice. The present study was designed to determine whether the spleen might serve as a source of ATPase/Ia+ cells to restore APC function to the skin after UVR exposure. ATPase/Ia+ EC densities were calculated from skin biopsies taken from BALB/c mice at various time points after low dose UVR treatment (2000 J/m2 protracted over 4 days). The recovery rate of ATPase/Ia+ EC in splenectomized mice after UVR exposure was similar, although delayed, compared to that of sham-operated mice. For example, 3 days after UVR exposure the density of ATPase/Ia+ EC in sham controls was 90% of normal and exceeded normal values by 5 days. In contrast, ATPase/Ia+ EC density in splenectomized mice was 60% of normal at 3 days and did not exceed normal values until 7 days after UVR exposure. Normal recovery of ATPase/Ia+ EC was restored to splenectomized mice given an adoptive transfer of syngeneic spleen cells immediately after UVR exposure. Fluorescein-labeled spleen cells used for adoptive transfer were observed within the epidermis of splenectomized mice 1 and 3 days after UVR exposure. Indirect immunofluorescent staining employing phycoerythrin-labeled reagents revealed that the fluorescein-labeled splenic EC expressed Ia, MAC-1, CLA, and Fc-receptor molecules. These results indicate that a portion of the ATPase/Ia+ EC that recovery after UVR exposure originate from the spleen. These cells may be distinct from LC, but appear to restore APC function to the skin following LC depletion by UVR exposure.
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PMID:Splenic contribution to the recovery of ATPase/Ia+ epidermal cells in the skin of mice after exposure to ultraviolet radiation. 253 51

Techniques are described for using blocking agents to distinguish between enzymes which are functional monomers and oligomers. To achieve this distinction the blocking agent must react exclusively at the active site with a stoichiometry of one mole of site per mole enzyme. The effect of the blocking agent on enzymatic activity in oligomers of n = 2 and 4 are described and the optimal degree of blocking is considered for tests of enzyme activity at saturating and less than saturating substrate concentrations. For saturating concentrations and a dimer the distinction between dimer and monomer is best observed with 50 per cent of sites blocked. For a tetramer the distinction is best made at higher degrees of blockade. The use of saturating substrate concentrations is thus limited to small oligomers. If nonsaturating substrate concentrations are used and normalized double reciprocal plots of the dependence of enzyme activity on substrate concentrations are made then the distinction between monomer and oligomer can readily be made for dimers, tetramers, and higher n-mers. The principles developed to distinguished monomeric from oligomeric enzymes are applied to published data obtained with the Ca Mg-ATPase of sarcoplasmic reticulum. Fluorescein isothiocyanate is the blocking agent. Plots of the published data support both the monomeric and tetrameric models for allosteric regulation with the preponderance of the data supporting the monomeric model.
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PMID:Distinguishing between functional monomeric and oligomeric complexes of the Ca,Mg-ATPase in sarcoplasmic reticulum. 294 43

Competitive binding and fluorescence energy transfer experiments were used to examine the binding of 2'[3']-O-(2,4,6-trinitrophenyl) adenosine-5'-diphosphate (TNP-ADP) to the catalytic site of Ca ATPase. Fluorescein isothiocyanate (FITC), which is known to covalently bind near the catalytic site (13), was shown to exclude all TNP-ADP binding. TNP-ADP, in turn, will protect against FITC labeling of the Ca ATPase in its native state and in both phosphoenzyme forms. The competitive nature of these probes indicates that TNP-ADP binds to the catalytic site exclusively. Fluorescence energy transfer studies using TNP-ADP as an energy acceptor and 1,N6-ethenoadenosine-5'-diphosphate (epsilon-ADP) as an energy donor were used to estimate the distance between nucleotide binding sites in the enzyme complex. A lower limit for the distance measured was 44 A. This is interpreted to be the distance between catalytic sites on adjacent monomers of a dimer unit. The results of this work are consistent with a single nucleotide site per ATPase monomer.
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PMID:A fluorescence investigation of the nucleotide binding sites of the Ca ATPase. 296 4

The mechanisms of allosteric regulation of the Ca-ATPases of cardiac and skeletal sarcoplasmic reticulum by ATP have been compared. Although both enzymes showed stimulation of ATPase activity by ATP, the cardiac enzyme did not show the plateau in ATPase activity at 10-100 microM ATP seen with the skeletal enzyme. Likewise the phosphoenzyme (EP) levels did not plateau with the cardiac enzyme as they did with the skeletal enzyme. The apparent negative cooperatively which was seen in the kinetics of ATP hydrolysis at low ATP concentrations was not due to negative cooperatively in substrate binding to either enzyme. The cardiac enzyme did show, however, much higher affinity for the ATP analog, AMPPCP, which helps explain how AMPPCP blocks ATPase activity in the cardiac enzyme and stimulates ATPase activity in the skeletal enzyme. Fluorescein isothiocyanate was used to determine if allosteric regulation takes place through site-site interactions in oligomers. The 1 to 1 ratio between AMPPCP binding sites and FITC binding sites eliminated allosteric regulation by effector sites in both enzymes. The allosteric mechanism which remained was one in which the active-site becomes an effector-site by the early departure of ADP in the reaction mechanism. The step stimulated by the binding of ATP at the active-site turned effector-site was a nonphosphorylated form of the enzyme in cardiac sarcoplasmic reticulum and a phosphorylated form in skeletal sarcoplasmic reticulum.
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PMID:Allosteric regulation of cardiac sarcoplasmic reticulum Ca-ATPase: a comparative study. 297 11


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