Site-directed mutagenesis of a predicted cation binding site of Na, K-ATPase.


:Chemical modification and proteolytic digestion studies have identified a transmembrane glutamic acid residue (E953) of the alpha subunit of the pig kidney Na, K-ATPase as a possible cation binding site [Goldshleger et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6911-6915]. In addition, an adjacent glutamate (E954) is conserved in all species and isoforms and may also be involved in cation binding. To further explore the role of these residues in ion transport, we have utilized a mutagenesis-expression strategy. This approach avoids the introduction of a large chemical moiety into the protein and allows specific amino acid substitutions to be introduced. Glutamic acid residues 955 and 956 of the rat alpha-1 subunit (corresponding to glutamates 953 and 954 of the pig kidney Na, K-ATPase) were replaced separately and together using site-directed mutagenesis of the rat alpha-1 cDNA. The mutant cDNAs were expressed in ouabain-sensitive HeLa cells. This system makes it possible to rapidly identify amino acid substitutions which significantly impair enzyme function, as substitutions which do not affect enzyme activity will yield colonies in the presence of ouabain, while substitutions which severely impair function will prevent or limit growth of the ouabain-sensitive HeLa cells. The amino acid replacements (E955Q, E956Q, E955Q-E956Q, E955D-E956D) all resulted in the growth of ouabain-sensitive cells, demonstrating that the modified Na, K-ATPase in each case was functional. To further study the altered enzymes, ouabain-resistant colonies were isolated and expanded into stable cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)






Van Huysse JW,Jewell EA,Lingrel JB




Has Abstract


1993-01-26 00:00:00












  • Structure of the conserved HAMP domain in an intact, membrane-bound chemoreceptor: a disulfide mapping study.

    abstract::The HAMP domain is a conserved motif widely distributed in prokaryotic and lower eukaryotic organisms, where it is often found in transmembrane receptors that regulate two-component signaling pathways. The motif links receptor input and output modules and is essential to receptor structure and signal transduction. Rec...


    pub_type: 杂志文章


    authors: Swain KE,Falke JJ

    更新日期:2007-12-04 00:00:00

  • Structural and Biochemical Investigation of PglF from Campylobacter jejuni Reveals a New Mechanism for a Member of the Short Chain Dehydrogenase/Reductase Superfamily.

    abstract::Within recent years it has become apparent that protein glycosylation is not limited to eukaryotes. Indeed, in Campylobacter jejuni, a Gram-negative bacterium, more than 60 of its proteins are known to be glycosylated. One of the sugars found in such glycosylated proteins is 2,4-diacetamido-2,4,6-trideoxy-α-d-glucopyr...


    pub_type: 杂志文章


    authors: Riegert AS,Thoden JB,Schoenhofen IC,Watson DC,Young NM,Tipton PA,Holden HM

    更新日期:2017-11-14 00:00:00

  • Molecular exchange at the lipid-rhodopsin interface: spin-label electron spin resonance studies of rhodopsin-dimyristoylphosphatidylcholine recombinants.

    abstract::The photoreceptor protein rhodopsin has been reconstituted with a single phospholipid species, dimyristoylphosphatidylcholine, at a range of different lipid/protein ratios, and the exchange rate at the lipid-protein interface has been determined from the electron spin resonance spectra of spin-labeled phosphatidylchol...


    pub_type: 杂志文章


    authors: Ryba NJ,Horváth LI,Watts A,Marsh D

    更新日期:1987-06-02 00:00:00

  • Identification of c-di-GMP derivatives resistant to an EAL domain phosphodiesterase.

    abstract::The bacterial second messenger signaling molecule bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) controls important biological processes such as biofilm formation, virulence response, and motility. This second messenger is sensed by macromolecular targets inside the cell, both protein and RNA, which ind...


    pub_type: 杂志文章


    authors: Shanahan CA,Gaffney BL,Jones RA,Strobel SA

    更新日期:2013-01-15 00:00:00

  • Structure-activity analysis of a Conus peptide blocker of N-type neuronal calcium channels.

    abstract::The synthetic peptide SNX-111 corresponding to the sequence of the omega-conopeptide MVIIA from the venom of the marine snail Conus magus is a highly potent and selective antagonist of N-type calcium channels. We have synthesized and characterized a large number of analogs of SNX-111 in order to elucidate the structur...


    pub_type: 杂志文章


    authors: Nadasdi L,Yamashiro D,Chung D,Tarczy-Hornoch K,Adriaenssens P,Ramachandran J

    更新日期:1995-06-27 00:00:00

  • Stereospecific Radical-Mediated B12-Dependent Methyl Transfer by the Fosfomycin Biosynthesis Enzyme Fom3.

    abstract::Fom3, the antepenultimate enzyme in the fosfomycin biosynthetic pathway in Streptomyces spp., is a class B cobalamin-dependent radical SAM methyltransferase that catalyzes methylation of (5'-cytidylyl)-2-hydroxyethylphosphonate (2-HEP-CMP) to form (5'-cytidylyl)-2-hydroxypropylphosphonate (2-HPP-CMP). Previously, the ...


    pub_type: 杂志文章


    authors: McLaughlin MI,van der Donk WA

    更新日期:2018-08-21 00:00:00

  • Histone-dependent reconstitution and nucleosomal localization of a nonhistone chromosomal protein: the H2A-specific protease.

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    pub_type: 杂志文章


    authors: Watson DK,Moudrianakis EN

    更新日期:1982-01-19 00:00:00

  • Generality of the NUX rule: kinetic analysis of the results of systematic mutations in the trinucleotide at the cleavage site of hammerhead ribozymes.

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    pub_type: 杂志文章


    authors: Shimayama T,Nishikawa S,Taira K

    更新日期:1995-03-21 00:00:00

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    pub_type: 杂志文章


    authors: Bossi RT,Negri A,Tedeschi G,Mattevi A

    更新日期:2002-03-05 00:00:00

  • Effect of insulin on ATP-citrate lyase phosphorylation: regulation of peptide A and peptide B phosphorylations.

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    pub_type: 杂志文章


    authors: Ramakrishna S,Murthy KS,Benjamin WB

    更新日期:1989-01-24 00:00:00

  • Structural basis for hydration dynamics in radical stabilization of bilin reductase mutants.

    abstract::Heme-derived linear tetrapyrroles (phytobilins) in phycobiliproteins and phytochromes perform critical light-harvesting and light-sensing roles in oxygenic photosynthetic organisms. A key enzyme in their biogenesis, phycocyanobilin:ferredoxin oxidoreductase (PcyA), catalyzes the overall four-electron reduction of bili...


    pub_type: 杂志文章


    authors: Kohler AC,Gae DD,Richley MA,Stoll S,Gunn A,Lim S,Martin SS,Doukov TI,Britt RD,Ames JB,Lagarias JC,Fisher AJ

    更新日期:2010-07-27 00:00:00

  • Nuclear Magnetic Resonance Solution Structure of DNA Featuring Clustered 2'-Deoxyribonolactone and 8-Oxoguanine Lesions.

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    pub_type: 杂志文章


    authors: Zálešák J,Constant JF,Jourdan M

    更新日期:2016-07-19 00:00:00

  • Regulatory mechanisms of tumor suppressor P16(INK4A) and their relevance to cancer.

    abstract::P16(INK4A) (also known as P16 and MTS1), a protein consisting exclusively of four ankyrin repeats, is recognized as a tumor suppressor mainly because of the prevalence of genetic inactivation of the p16(INK4A) (or CDKN2A) gene in virtually all types of human cancers. However, it has also been shown that an elevated le...


    pub_type: 杂志文章,评审


    authors: Li J,Poi MJ,Tsai MD

    更新日期:2011-06-28 00:00:00

  • Structure-function relationships of the Escherichia coli ATP synthase probed by trypsin digestion.

    abstract::Trypsin cleavage has been used to probe structure-function relationships of the Escherichia coli ATP synthase (ECF1F0). Trypsin cleaved all five subunits, alpha, beta, gamma, delta, and epsilon, in isolated ECF1. Cleavage of the alpha subunit involved the removal of the N-terminal 15 residues, the beta subunit was cle...


    pub_type: 杂志文章


    authors: Gavilanes-Ruiz M,Tommasino M,Capaldi RA

    更新日期:1988-01-26 00:00:00

  • Identification of a region of the ileal-type sodium/bile acid cotransporter interacting with a competitive bile acid transport inhibitor.

    abstract::Drug intervention that prevents reabsorption of circulating bile acids by the apical (ileal) sodium/bile acid cotransporter (ASBT) may be a promising new therapy for lowering of plasma cholesterol. 2164U90 is a benzothiazepine-based competitive inhibitor of bile acid transport with K(i) values of approximately 10 and ...


    pub_type: 杂志文章


    authors: Hallén S,Björquist A,Ostlund-Lindqvist AM,Sachs G

    更新日期:2002-12-17 00:00:00

  • Adenine protonation in domain B of the hairpin ribozyme.

    abstract::Protein enzymes often use ionizable side chains, such as histidine, for general acid-base catalysis because the imidazole pK(a) is near neutral pH. RNA enzymes, on the other hand, are comprised of nucleotides which do not have apparent pK(a) values near neutral pH. Nevertheless, it has been recently shown that cytidin...


    pub_type: 杂志文章


    authors: Ravindranathan S,Butcher SE,Feigon J

    更新日期:2000-12-26 00:00:00

  • Three novel oligosaccharides with the sialyl-Lea structure in human milk: isolation by immunoaffinity chromatography.

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    pub_type: 杂志文章


    authors: Kitagawa H,Nakada H,Kurosaka A,Hiraiwa N,Numata Y,Fukui S,Funakoshi I,Kawasaki T,Yamashina I,Shimada I

    更新日期:1989-10-31 00:00:00

  • Combined N- and C-terminal truncation of human apolipoprotein A-I yields a folded, functional central domain.

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    pub_type: 杂志文章


    authors: Beckstead JA,Block BL,Bielicki JK,Kay CM,Oda MN,Ryan RO

    更新日期:2005-03-22 00:00:00

  • Retardation of Folding Rates of Substrate Proteins in the Nanocage of GroEL.

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    pub_type: 杂志文章


    authors: Koculi E,Thirumalai D

    更新日期:2021-01-19 00:00:00

  • Akt-dependent antiapoptotic action of insulin is sensitive to farnesyltransferase inhibitor.

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    pub_type: 杂志文章


    authors: Park D,Pandey SK,Maksimova E,Kole S,Bernier M

    更新日期:2000-10-17 00:00:00

  • Interaction of troponin and tropomyosin: spectroscopic and calorimetric studies.

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    pub_type: 杂志文章


    authors: Ingraham RH,Swenson CA

    更新日期:1985-09-10 00:00:00

  • Zinc is associated with the beta subunit of DNA-dependent RNA polymerase of Bacillus subtilis.

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    pub_type: 杂志文章


    authors: Halling SM,Sanchez-Anzaldo FJ,Fukuda R,Doi RH,Meares CF

    更新日期:1977-06-28 00:00:00

  • Generation of a calmodulin-based EPR calcium indicator.

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    pub_type: 杂志文章


    authors: Shao J,Cieslak J,Gross A

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    pub_type: 杂志文章


    authors: Goto Y,Hagihara Y

    更新日期:1992-01-28 00:00:00

  • N1-(5'-phosphoribosyl)adenosine-5'-monophosphate cyclohydrolase: purification and characterization of a unique metalloenzyme.

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    pub_type: 杂志文章


    authors: D'Ordine RL,Klem TJ,Davisson VJ

    更新日期:1999-02-02 00:00:00

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    pub_type: 杂志文章


    authors: Masada N,Schaks S,Jackson SE,Sinz A,Cooper DM

    更新日期:2012-10-09 00:00:00

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    pub_type: 杂志文章


    authors: Rathore D,McCutchan TF,Garboczi DN,Toida T,Hernáiz MJ,LeBrun LA,Lang SC,Linhardt RJ

    更新日期:2001-09-25 00:00:00

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    pub_type: 杂志文章


    authors: Aagaard A,Gilderson G,Mills DA,Ferguson-Miller S,Brzezinski P

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    pub_type: 杂志文章


    authors: Dan-Cohen H,Sofer Y,Schwartzman ML,Natarajan RD,Nadler JL,Naor Z

    更新日期:1992-06-23 00:00:00

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    pub_type: 杂志文章


    authors: deCastro MJ,Ho CH,Stewart RJ

    更新日期:1999-04-20 00:00:00