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Cys⁵⁷⁷ is a conformationally mobile residue in the atp-binding domain of the Na,K-atpase α-subunit

JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第35期274卷 24995-25003页

作者： Gatto, C Thornewell, SJ Holden, JP Kaplan, JH Oregon Hlth & Sci Univ Dept Biochem & Mol Biol Portland OR 97201 USA

2-[4'-Maleimidylanilino]naphthalene 6-sulfonic acid (MIANS) irreversibly inactivates Na,K-atpase in a time- and concentration-dependent manner. Inactivation is prevented by 3 mM atp or low K+ (<1 mM);the protective effect K+ is reversed at higher concentrations, This biphasic effect was also observed with K+ congeners, In contrast, Na+ ions did not protect. MIANS inactivation disrupted high affinity atp binding. Tryptic fragments of MIANS-labeled protein were analyzed by reversed phase high performance liquid chromatography, atp clearly protected one major labeled peptide peak. This observation was confirmed by separation of tryptic peptides in SDS-polyacrylamide gel electrophoresis revealing a single fluorescently-labeled peptide of similar to 5 kDa, N-terminal. amino acid sequencing identified the peptide (V(545)LGFCH...), This hydrophobic peptide contains only two Cys residues in all sodium pump alpha-subunit sequences and is found in the major cytoplasmic loop between M4 and M5, a region previously associated with atp binding. Subsequent digestion of the tryptic peptide with V8 protease and N-terminal amino acid sequencing identified the modified residue as Cys(577). The cation-dependent change in reactivity of Cys(577) implies structural alterations in the atp-binding domain following cation binding and occlusion in the intramembrane domain of Na,K-atpase and expands our knowledge of the extent to which cation binding and occlusion are sensed in the atp hydrolysis domain.

关键词：腺苷三磷酸/代谢苯胺基萘磺酸类/药理学结合部位阳离子/药理学色谱法高压液相半胱氨酸/化学电泳聚丙烯酰氨凝胶酶抑制剂/药理学动力学肽碎片/化学蛋白质结合序列分析钠钾交换atp酶/化学胰蛋白酶

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The β-subunits of Na⁺,K⁺-atpase and gastric H⁺,K⁺-atpase have a high preference for their own α-subunit and affect the K⁺ affinity of these enzymes

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第17期274卷 11604-11610页

作者： Koenderink, JB Swarts, HGP Hermsen, HPH De Pont, JJHHM Catholic Univ Nijmegen Dept Biochem Inst Cellular Signaling NL-6500 HB Nijmegen Netherlands

The alpha- and beta-subunits of Na+,K+-atpase and H+,K+ atpase were expressed in Sf9 cells in different combinations. Immunoprecipitation of the alpha-subunits resulted in coprecipitation of the accompanying beta-subunit independent of the type of beta-subunit, This indicates cross-assembly of the subunits of the different atpases, The hybrid atpase with the catalytic subunit of Na+,K+- atpase and the beta-subunit of H+,K+-atpase (NaK alpha HK beta) showed an atpase activity, which was only 12 +/- 4% of the activity of the Na+,K+-atpase with its own beta-subunit. Likewise, the complementary hybrid atpase with the catalytic subunit of H+,K+-atpase and the beta-subunit of Na+,K+-atpase (HK alpha NaK beta) showed an atpase activity which was 9 +/- 2% of that of the recombinant H+,K+ atpase, In addition, the apparent K+ affinity of hybrid NaK alpha HK beta was decreased, while the apparent K+ affinity of the opposite hybrid HK alpha NaK beta was increased. The hybrid NaK alpha HK beta could be phosphorylated by atp to a level of 21 +/- 7% of that of Na+,K+-atpase, These values, together with the atpase activity gave turnover numbers for NaK alpha beta and NaK alpha HK beta of 8800 +/- 310 min(-1) and 4800 +/- 160 min(-1), respectively. Measurements of phosphorylation of the HK alpha NaK beta and HK alpha beta enzymes are consistent with a higher turnover of the former. These findings suggest a role of the beta-subunit in the catalytic turnover. In conclusion, although both Na+,K+-atpase and H+,K+-atpase have a high preference for their own beta-subunit, they can function with the beta-subunit of the other enzyme, in which case the K+ affinity and turnover number are modified.

关键词：杆状病毒科/遗传学催化作用 H(+)K(+)交换atp酶/化学 H(+)K(+)交换atp酶/代谢钾/代谢重组蛋白质类/化学重组蛋白质类/代谢钠钾交换atp酶/化学钠钾交换atp酶/代谢斜纹夜蛾属胃/酶学动物大鼠

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Functional immobilization of biomembrane fragments on planar waveguides for the investigation of side-directed ligand binding by surface-confined fluorescence

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FARADAY DISCUSSIONS 1998年第111期111卷 273-288页

作者： Pawlak, M Grell, E Schick, E Anselmetti, D Ehrat, M Novartis Pharma AG DMPK Preclin Safety CH-4002 Basel Switzerland Max Planck Inst Biophys D-65096 Frankfurt Germany Novartis Serv AG Sci Serv CH-4002 Basel Switzerland

A method for the functional immobilization of Na,K-atpase-rich membrane fragments on planar metal oxide waveguides has been developed. A novel optical technique based on the highly sensitive detection of surface-confined fluorescence in the evanescent held of the waveguide allowed us to investigate the interactions of the immobilized protein with cations and ligands. For specific binding studies, a FITC-Na,K-atpase was used, which had been labelled covalently within the atp-binding domain of the protein. Fluorophore labels of the surface-bound enzyme can be selectively excited in the evanescent field. A preserved functional activity of the immobilized enzyme was only found when a phospholipid monolayer was preassembled onto the hydrophobic chip surface to form a gentle, biocompatible interface. In situ atomic force microscopy (AFM) was used to examine and optimize the conditions for the lipid and membrane fragment assembly and the quality of the formed layers. The enzyme's functional activity was tested by selective K+ cation binding, interaction with anti-fluorescein antibody 4-4-20, phosphorylation of the protein and binding of inhibitory ligand ouabain. The comparison with corresponding fluorescence intensity changes found in bulk solution provides information about the side-directed surface binding of the Na,K-atpase membrane fragments. The affinity constants of K+ ions to the Na,K-atpase was the same for the immobilized and the non-immobilized enzyme, providing evidence for the highly native environment on the surface. The method for the functional immobilization of membrane fragments on waveguide surfaces will be the basis for future applications in pharmaceutical research where advanced methods for exploring the molecular mechanisms of membrane receptor targets and drug screening are required.

关键词：结合部位细胞膜/化学荧光染料配体膜人工钠钾交换atp酶/化学表面特性动物

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17β-O-aminoalkyloximes of 5β-androstane-3β,14β-diol with digitalis-like activity:: Synthesis, cardiotonic activity, structure-activity relationships, and molecular modeling of the Na⁺,K⁺-atpase receptor

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JOURNAL OF MEDICINAL CHEMISTRY 2000年第12期43卷 2332-2349页

作者： Cerri, A Almirante, N Barassi, P Benicchio, A Fedrizzi, G Ferrari, P Micheletti, R Quadri, L Ragg, E Rossi, R Santagostino, M Schiavone, A Serra, F Zappavigna, MP Melloni, P Prassis Ist Ric Sigma Tau Dept Med Chem I-20019 Settimo Milanese MI Italy Prassis Ist Ric Sigma Tau Dept Cardiovasc Pharmacol I-20019 Settimo Milanese MI Italy Univ Milan Fac Agr Chem Sect Dept Agr & Food Mol Sci I-20133 Milan Italy

A series of digitalis-like compounds with a 17-aminoalkoxyiminoaIkyl or -alkenyl substituent was synthesized and evaluated for inhibition of Na+,K+-atpase and for inotropic activity. The highest inhibition was found with compounds having the substituent in configuration 17 beta and the amino group at a distance of 6 or 7 bonds from C(17) of the digitoxigenin skeleton. The presence of the oxime function strengthens the interaction with the receptor, more if alpha,beta-unsaturated, thus mimicking the electronic situation of the unsaturated lactone in natural digitalis compounds. The most active compounds showed Na+,K+-atpase inhibitory potencies (IC50) 17-25 times higher than the standards digitoxigenin and digoxin and 3-11 times higher inotropic potencies (EC50) in isolated guinea pig left atria. These features are supported by a molecular model suggesting the possible interactions of the groups described above with particular amino acid residues in the H1-H2 domains of Na+,K+-atpase. Some interactions are the classical ones already described in the literature;a new, very strong interaction of the basic group with the Cys138 was found and adds new possibilities to design compounds interacting with this region of the receptor. The most interesting compounds were also studied in vivo in the anesthetized guinea pig for evaluating their inotropic effect versus the lethal dose. Compounds 9 and 12 showed a slightly higher safety ratio than digoxin and deserve further evaluation.

关键词：雄甾烷类/化学合成雄甾烷类/化学雄甾烷类/药理学雄甾烷醇类/化学合成雄甾烷醇类/化学雄甾烷醇类/药理学结合部位强心药/化学合成强心药/化学强心药/药理学洋地黄糖甙类/药理学酶抑制剂/化学合成酶抑制剂/化学酶抑制剂/药理学心房/药物作用肾/酶学模型分子心肌收缩/药物作用哇巴因/化学哇巴因/代谢肟类/化学合成肟类/化学肟类/药理学钠钾交换atp酶/拮抗剂和抑制剂钠钾交换atp酶/化学钠钾交换atp酶/代谢构效关系动物狗豚鼠男(雄)性

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Cloning, expression, purification and crystallization of the N-domain from the α₂ subunit of the membrane-spanning Na,K-atpase protein

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ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY 2003年第7期59卷 1259-1261页

作者： Haue, L Pedersen, PA Jorgensen, PL Håkansson, KO Univ Copenhagen August Krogh Inst Biomembrane Ctr DK-2100 Copenhagen OE Denmark

The nucleotide-binding domain of the Na,K-atpase ion pump was expressed with a His tag in Escherichia coli and purified. The soluble 24 kDa derivative consists of 214 amino-acid residues and was crystallized in the presence of NiCl2. The crystals belong to space group F23, with unit-cell parameters a = b = c = 147.5 Angstrom, and diffract to 3.1 Angstrom. Complete data sets could be collected from native and thimerosal-treated crystals frozen in 50% sucrose. Five mercury positions were found and initial SIR phases calculated.

关键词：氨基酸序列结合部位克隆分子结晶/方法膜蛋白质类/化学镍蛋白质结构三级蛋白质亚单位/化学钠钾交换atp酶/化学猪硫柳汞 X线衍射/方法动物

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CONSTRAINTS ON MODELS FOR THE FOLDING OF THE NA,K-atpASE

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ANNALS OF THE NEW YORK ACADEMY OF SCIENCES 1992年第1期671卷 217-227页

作者： SWEADNER, KJ ARYSTARKHOVA, E HARVARD UNIV SCH MEDDEPT CELLULAR & MOLEC PHYSIOLBOSTONMA 02115

We have attempted to bring together in graphic fashion the available evidence on the structure of the Na,K-atpase and the H,K-atpase. There appears to be much room for modification of the existing models for transmembrane folding. More sites on each side of the membrane need to be identified. Whether these will be antibody epitopes, sites of covalent modification, or tags inserted by mutagenesis is less important than that there be many of them and that each be verified by alternative approaches. If any single principle has emerged from the study of the topography of membrane proteins, it is that it is easy to reach conclusions too soon.

关键词：氨基酸序列结合部位抗体钙转运atp酶类/化学细胞膜/酶学细胞膜/超微结构表位/分析 H(+)K(+)交换atp酶/化学模型结构分子序列数据蛋白质折叠序列同源性氨基酸钠钾交换atp酶/化学动物大鼠

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Combined allosteric and competitive interaction between extracellular Na⁺ and K⁺ during ion transport by the α₁, α₂, and α₃ isoforms of the Na, K-atpase

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BIOPHYSICAL JOURNAL 2000年第2期79卷 853-862页

作者： Balshaw, DM Millette, LA Tepperman, K Wallick, ET Univ Cincinnati McMicken Coll Arts & Sci Dept Biol Sci Cincinnati OH 45267 USA Univ Cincinnati Coll Med Dept Pharmacol & Cell Biophys Cincinnati OH 45267 USA

A combined allosteric and competitive model describes the interaction between extracellular Na+ and Rb+ during ion transport mediated by the Na, K-atpase. The model was developed from experiments based on Rb-86 uptake by whole cells transfected with rat isoforms of the enzyme. In the absence of Na+, only a single transport site for extracellular Rb+ exists. After the occupation of the Na+-specific allosteric site, the Rb+ transport pocket opens to allow occupation by an additional Rb+ and the subsequent transport of the two Rb+ ions into the cells. Na+ can also directly compete with Rb+ for binding to at least one of the transport sites. While the model derived here applies to each of the three rat isoforms of the Na, K-atpase expressed in HeLa cells, subtle differences exist among the isoforms. The alpha(3)* isoform has an increased intrinsic affinity for Rb+ and a lower affinity for the allosteric Na+ site than alpha(1) or alpha(2)*. The stimulation of uptake observed according to the best-fit model is due to the displacement by Rb+ of inhibitory Na+ bound to the transport site.

关键词：别构调节别构部位结合竞争性 HeLa细胞同工酶类/化学同工酶类/代谢动力学模型化学钾/代谢重组蛋白质类/化学重组蛋白质类/代谢铷/药代动力学钠/代谢钠钾交换atp酶/化学钠钾交换atp酶/代谢转染动物人类大鼠

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