FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2023488999> ?p ?o ?g. }

• W2023488999 endingPage "14027" @default.
• W2023488999 startingPage "14018" @default.
• W2023488999 abstract "Subunit KtrA of the bacterial Na+-dependent K+-translocating KtrAB systems belongs to the KTN/RCK family of regulatory proteins and protein domains. They are located at the cytoplasmic side of the cell membrane. By binding ligands they regulate the activity of a number of K+ transporters and K+ channels. To investigate the function of KtrA from the bacterium Vibrio alginolyticus (VaKtrA), the protein was overproduced in His-tagged form (His10-VaKtrA) and isolated by affinity chromatography. VaKtrA contains a G-rich, ADP-moiety binding β-α-β-fold (“Rossman fold”). Photocross-linking and flow dialysis were used to determine the binding of [32P]ATP and [32P]NAD+ to His10-VaKtrA. Binding of other nucleotides was estimated from the competition by these compounds of the binding of the 32P-labeled nucleotides to the protein. [γ-32P]ATP bound with high affinity to His10-VaKtrA (KD of 9 μm). All other nucleotides tested exhibited KD (Ki) values of 30 μm or higher. Limited proteolysis with trypsin showed that ATP was the only nucleotide that changed the conformation of VaKtrA. ATP specifically promoted complex formation of VaKtrA with the His-tagged form of its K+-translocating partner, VaKtrB-His6, as detected both in an overlay experiment and in an experiment in which VaKtrA was added to VaKtrB-His6 bound to Ni2+-agarose. In intact cells of Escherichia coli both a high of membrane potential and a high cytoplasmic ATP concentration were required for VaKtrAB activity. C-terminal deletions in VaKtrA showed that for in vivo activity at least 169 N-terminal amino acid residues of its total of 220 are required and that its 40 C-terminal residues are dispensable. Subunit KtrA of the bacterial Na+-dependent K+-translocating KtrAB systems belongs to the KTN/RCK family of regulatory proteins and protein domains. They are located at the cytoplasmic side of the cell membrane. By binding ligands they regulate the activity of a number of K+ transporters and K+ channels. To investigate the function of KtrA from the bacterium Vibrio alginolyticus (VaKtrA), the protein was overproduced in His-tagged form (His10-VaKtrA) and isolated by affinity chromatography. VaKtrA contains a G-rich, ADP-moiety binding β-α-β-fold (“Rossman fold”). Photocross-linking and flow dialysis were used to determine the binding of [32P]ATP and [32P]NAD+ to His10-VaKtrA. Binding of other nucleotides was estimated from the competition by these compounds of the binding of the 32P-labeled nucleotides to the protein. [γ-32P]ATP bound with high affinity to His10-VaKtrA (KD of 9 μm). All other nucleotides tested exhibited KD (Ki) values of 30 μm or higher. Limited proteolysis with trypsin showed that ATP was the only nucleotide that changed the conformation of VaKtrA. ATP specifically promoted complex formation of VaKtrA with the His-tagged form of its K+-translocating partner, VaKtrB-His6, as detected both in an overlay experiment and in an experiment in which VaKtrA was added to VaKtrB-His6 bound to Ni2+-agarose. In intact cells of Escherichia coli both a high of membrane potential and a high cytoplasmic ATP concentration were required for VaKtrAB activity. C-terminal deletions in VaKtrA showed that for in vivo activity at least 169 N-terminal amino acid residues of its total of 220 are required and that its 40 C-terminal residues are dispensable. The major solute in the cytoplasm of all cells, K+, plays an important role in osmo-regulation. Many bacteria react to hyperosmotic shock by rapidly accumulating K+ from the medium, thereby restoring their turgor pressure (1Dinnbier U. Limpinsel E. Schmid R. Bakker E.P. Arch. Microbiol. 1988; 150: 348-357Crossref PubMed Scopus (298) Google Scholar, 2Stumpe S. Schloösser A. Schleyer M. Bakker E.P. Konings W.N. Kaback H.R. Lolkema J.S. Handbook of Biological Physics, Volume 2: Transport Processes in Eukaryotic and Prokaryotic Organelles. 1996: 474-499Google Scholar, 3Record T.M. Courtenay E.S. Cayley D.S. Guttman H.J. Trends Biochem. Sci. 1998; 23: 143-1484Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). For this function, bacteria possess at least three types of K+-uptake systems, Kdp, Trk, and Ktr (2Stumpe S. Schloösser A. Schleyer M. Bakker E.P. Konings W.N. Kaback H.R. Lolkema J.S. Handbook of Biological Physics, Volume 2: Transport Processes in Eukaryotic and Prokaryotic Organelles. 1996: 474-499Google Scholar, 4Nakamura T. Yuda R. Unemoto T. Bakker E.P. J. Bacteriol. 1998; 180: 3491-3494Crossref PubMed Google Scholar, 5Holtmann G. Bakker E.P. Uozumi N. Bremer E. J. Bacteriol. 2003; 185: 1289-1298Crossref PubMed Scopus (139) Google Scholar). These systems are all composed of several types of subunits and mediate a tightly controlled mode of K+ transport. The mechanism by which they accomplish this differs from system to system. The activities of the K+-translocating P-type ATPase Kdp and of the K+-transporter Trk are directly influenced by the magnitude of the cell turgor pressure (6Rhoads D.B. Epstein W. J. Gen. Physiol. 1978; 72: 283-295Crossref PubMed Scopus (75) Google Scholar). Trk consists of three types of subunits: TrkE/SapD that binds ATP (7Parra-Lopez C. Baer M.T. Groisman E.A. EMBO J. 1993; 12: 4053-4062Crossref PubMed Scopus (163) Google Scholar, 8Harms C. Domoto Y. Celik C. Rahe E. Stumpe S. Schmid R. Nakamura T. Bakker E.P. Microbiology. 2001; 147: 2991-3003Crossref PubMed Scopus (46) Google Scholar), TrkA that binds NAD(H) (9Schloösser A. Hamann A. Bossemeyer D. Schneider E. Bakker E.P. Mol. Microbiol. 1993; 9: 533-543Crossref PubMed Scopus (113) Google Scholar), and TrkG/TrkH which translocates K+ across the membrane (2Stumpe S. Schloösser A. Schleyer M. Bakker E.P. Konings W.N. Kaback H.R. Lolkema J.S. Handbook of Biological Physics, Volume 2: Transport Processes in Eukaryotic and Prokaryotic Organelles. 1996: 474-499Google Scholar). The third regulated bacterial K+-uptake system is Ktr (4Nakamura T. Yuda R. Unemoto T. Bakker E.P. J. Bacteriol. 1998; 180: 3491-3494Crossref PubMed Google Scholar, 10Murata T. Takase K. Yamato I. Igarashi K. Kakinuma Y. J. Biol. Chem. 1996; 271: 10042-10047Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 11Kawano M. Igarashi K. Kakinuma Y. FEMS Microbiol. Lett. 1999; 176: 449-453Crossref Google Scholar). Its activity depends on Na+ ions (12Tholema N. Bakker E.P. Suzuki A. Nakamura T. FEBS Lett. 1999; 450: 217-220Crossref PubMed Scopus (75) Google Scholar). With only two types of subunits, the KtrAB system from Vibrio alginolyticus (VaKtrAB) 4The abbreviations used are: Va, V. alginolyticus; ΔΨ, membrane potential (internally negative); ATPγS, adenosine 5′-O-(thiotriphosphate). has a relatively simple composition. The K+-translocating subunit KtrB is a member of the SKT proteins, which are believed to have evolved from simple K+ channels by multiple gene duplications and gene fusions (13Durell S.R. Hao Y. Nakamura T. Bakker E.P. Guy H.R. Biophys. J. 1999; 77: 775-789Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 14Durell S.R. Bakker E.P. Guy H.R. Biophys. J. 2000; 78: 188-199Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). KtrA is the regulatory subunit. It is a membrane surface protein. KtrA confers velocity, ion specificity, and ion coupling to the Ktr system (15Tholema N. Vor der Bruöggen M. Maöser P. Nakamura T. Schroeder J.I. Kobayashi H. Uozumi N. Bakker E.P. J. Biol. Chem. 2005; 280: 41146-41154Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Structural work on a NAD+ binding N-terminal KtrA fragment from Bacillus subtilis compared with that of an NADH binding N-terminal TrkA fragment from Methanocaldococcus jannaschii has led Roosild et al. (16Roosild T.P. Miller S. Booth I.R. Choe S. Cell. 2002; 109: 781-791Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) to propose that KtrB activity is activated by a conformational switch brought about by the replacement of NAD+ by NADH as the ligand bound to KtrA. However, recent data show that ATP rather than nicotinamide nucleotides binds with the highest affinity to the KtrA fragment (KD about 600 nm (17Albright R.A. Vasquez-Ibar J-L. Kim C.U. Gruner S.M. Morais-Cabral J.H. Cell. 2006; 126: 1147-1159Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar)). KtrA belongs to the family of KTN proteins, which are closely related to cytoplasmic RCK domains of several types of K+ channels (Refs. 18Jiang Y. Pico A. Cadene M. Chait B.T. MacKinnon R. Neuron. 2001; 29: 593-601Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 19Jiang Y. Lee A. Chen J. Cadene M. Chait B.T. MacKinnon R. Nature. 2002; 417: 515-522Crossref PubMed Scopus (1205) Google Scholar, 20Roosild T.P. Le K.T. Choe S. Trends Biochem. Sci. 2004; 29: 39-45Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 21Kuo M.M.C. Haynes W.J. Loukin S.H. Kung C. Saimi Y. FEMS Microbiol. Rev. 2005; 29: 961-985Crossref PubMed Scopus (89) Google Scholar; see supplemental Fig. S1 for an alignment of VaKtrA with that of KTN/RCK domains of known structure). The N-terminal part of these proteins (i.e. βA to βE; supplemental Fig. S1) possesses a Rossmann-fold type of β-α protein structure, closely similar to that of the NAD+ binding domains of NAD+-dependent dehydrogenases (2Stumpe S. Schloösser A. Schleyer M. Bakker E.P. Konings W.N. Kaback H.R. Lolkema J.S. Handbook of Biological Physics, Volume 2: Transport Processes in Eukaryotic and Prokaryotic Organelles. 1996: 474-499Google Scholar, 9Schloösser A. Hamann A. Bossemeyer D. Schneider E. Bakker E.P. Mol. Microbiol. 1993; 9: 533-543Crossref PubMed Scopus (113) Google Scholar, 22Parra-Lopez C. Lin R. Aspedon A. Groisman E.A. EMBO J. 1994; 13: 3964-3972Crossref PubMed Scopus (101) Google Scholar, 23Wierenga R.K. Terpstra P. Hol W.G.J. J. Mol. Biol. 1986; 187: 101-107Crossref PubMed Scopus (996) Google Scholar, 24Branden C. Tooze J. Introduction to Protein Structure. 1991; (Garland Publishing, Inc., pp. , New York and London): 144-152Google Scholar). In the N-terminal β-α domain, the glycine-rich sequence (GXGXXG...(D/E)) is important for the binding of the adenosine moiety of NAD(H). However, in several RCK domains glycine residues from this fold are replaced by other amino acids (supplemental Fig. S1). The K+ channel Mth from Methanobacterium thermoautotrophicum is one of these proteins. It binds Ca2+, and its activity depends on the presence of this divalent cation (19Jiang Y. Lee A. Chen J. Cadene M. Chait B.T. MacKinnon R. Nature. 2002; 417: 515-522Crossref PubMed Scopus (1205) Google Scholar, 25Parfenova L.V. Crane B.M. Rothberg B.S. J. Biol. Chem. 2006; 281: 21131-21138Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). For a second K+ channel, Kch from Escherichia coli, it is not known which ligand binds to its RCK domain (18Jiang Y. Pico A. Cadene M. Chait B.T. MacKinnon R. Neuron. 2001; 29: 593-601Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). Because of these uncertainties about ligand binding to KTN/RCK domains and their role in the regulation of both K+ transport and K+ channel activity, we examined nucleotide binding to the isolated full-length KtrA protein from the bacterium V. alginolyticus. We show that ATP binds with higher affinity than does NAD+ or NADH, that ATP promotes complex formation between KtrA and KtrB, and that in vivo VaKtrAB-expressing E. coli cells require ATP and the membrane potential for activity. Strains, Plasmids, Growth Conditions—The strains and plasmids used in this study are listed in supplemental Table S1. Plasmid pEL305 contains VaKtrA cloned into the NdeI and BamHI sites of plasmid pET16b (Novagen, Schwalbach, Ts, Germany). It encodes KtrA with the 21-amino acid N-terminal extension MGHHHHHHHHHHSSGHIEGRH (His10-KtrA). Plasmid pKT84 (4Nakamura T. Yuda R. Unemoto T. Bakker E.P. J. Bacteriol. 1998; 180: 3491-3494Crossref PubMed Google Scholar) contains VaKtrAB cloned into vector pHG165 (26Stewart G.S.A.B. Lubinsky-Mink S. Jackson C.G. Cassel A. Kuhn J. Plasmid. 1986; 15: 172-181Crossref PubMed Scopus (142) Google Scholar). Plasmids pMW130stop to pMW180stop were generated by PCR using the QuikChange site-directed mutagenesis kit from Stratagene, La Jolla, CA. They contain a stop codon at positions 130 to 180 of KtrA in plasmid pKT84, respectively. Plasmid pEL903 encodes VaKtrB-His6 under the control of the para promoter of plasmid pBAD18 (27Guzman L-M. Belin D. Carson M.J. Beckwith J. J. Bacteriol. 1994; 177: 4121-4130Crossref Google Scholar). Plasmid-containing cells of strains LB2003 and LB692 were grown at 37 °C under aerobic conditions in minimal medium K30 (28Epstein W. Kim B.S. J. Bacteriol. 1971; 108: 639-644Crossref PubMed Google Scholar) containing in addition 1 mg/liter thiamine, 40 mg/liter methionine (only strain LB2003), 100 mg/liter carbenicillin, and 10 mm glucose. For overproduction of His10-KtrA, plasmid pEL305-containing cells of strain BL21(DE3)pLysS (29Studier F.W. J. Mol. Biol. 1991; 219: 37-41Crossref PubMed Scopus (681) Google Scholar) were grown at 30 °C under aerobic conditions in KML medium containing 10 g/liter KCl, 10 g/liter Tryptone, 5 g/liter yeast extract, 100 mg/liter ampicillin, and 34 mg/liter chloramphenicol. For production of KtrB-His6, cells of strain Rosetta™2(DE3)/pEL903 were grown as described in Tholema et al. (15Tholema N. Vor der Bruöggen M. Maöser P. Nakamura T. Schroeder J.I. Kobayashi H. Uozumi N. Bakker E.P. J. Biol. Chem. 2005; 280: 41146-41154Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Complementation of K+ Transport by Growth Measurements; Net K+-uptake Activity—These assays were done as described previously (12Tholema N. Bakker E.P. Suzuki A. Nakamura T. FEBS Lett. 1999; 450: 217-220Crossref PubMed Scopus (75) Google Scholar, 15Tholema N. Vor der Bruöggen M. Maöser P. Nakamura T. Schroeder J.I. Kobayashi H. Uozumi N. Bakker E.P. J. Biol. Chem. 2005; 280: 41146-41154Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 28Epstein W. Kim B.S. J. Bacteriol. 1971; 108: 639-644Crossref PubMed Google Scholar). Detection of C-terminal-truncated KtrA in a Minicell System—Plasmid pMWstop-encoded, C-terminal-truncated KtrA proteins labeled with [35S]methionine were detected in minicells of E. coli DK6 (30Klionsky D.J. Brusilow W.S.A. Simoni R.D. J. Bacteriol. 1984; 160: 1055-1060Crossref PubMed Google Scholar) as described in Nakamura et al. (31Nakamura T. Yamamuro Unemoto T. Stumpe S. Bakker E.P. Microbiology. 1998; 144: 2281-2289Crossref PubMed Scopus (39) Google Scholar). Experiments on Energy Coupling of VaKtr—Cells of strain LB692/pKT84 were depleted of ATP and K+ by incubation with 2,4-dinitrophenol as described in Harms et al. (8Harms C. Domoto Y. Celik C. Rahe E. Stumpe S. Schmid R. Nakamura T. Bakker E.P. Microbiology. 2001; 147: 2991-3003Crossref PubMed Scopus (46) Google Scholar). For the transport assays cells were preincubated for 30 min at 20 °C with 10 mm glucose, 20 mm disodium succinate or without substrate. Transport was initiated by the addition at t = 0 of 1 mm KCl, 2 μm [14C]glutamine (40 nCi/ml), or 4 μm [14C]proline (100 nCi/ml) to the cell suspension. If present, 2,4-dinitrophenyl was added at t =–5 min. Uptake of K+ or radioactive compounds by the cells was measured as a function of time, as described in Harms et al. (8Harms C. Domoto Y. Celik C. Rahe E. Stumpe S. Schmid R. Nakamura T. Bakker E.P. Microbiology. 2001; 147: 2991-3003Crossref PubMed Scopus (46) Google Scholar). The ATP content of these cells was determined by the method of Kimmich et al. (32Kimmich G.A. Randles J. Brand J.S. Anal. Biochem. 1975; 69: 187-206Crossref PubMed Scopus (309) Google Scholar). Overproduction and Purification of His10-KtrA—Overnight grown cells of E. coli BL21 (DE3)pLysS/pEL305 were transferred at an OD578 value of about 0.05 to 4 1-liter portions of fresh KML medium and grown at 30 °C. 1 mm isopropyl 1-thio-β-d-galactopyranoside was added at OD578 of 0.25–0.3. After a further 20 min the cultures received 0.1 g/liter rifampicin. The cultures were shaken for an additional 2 h. Subsequently the cells were harvested by centrifugation at 10,000 × g. The cell pellets from 1 liter of cell culture were suspended in 18 ml of buffer A, subsequently frozen with liquid nitrogen, and stored at –80 °C. Buffer A contained 300 mm NaCl, 1 mm EDTA, and 50 mm Tris-HCl, pH 8.0. For cell fractionation 18 ml of cell suspension in buffer A plus 180 μl of protease inhibitor mixture P8849 (Sigma/Aldrich) were sonicated 5 times for 30 s each (50% duty cycle) with maximal output from the large tip of a Branson Sonifer (Branson, Heusenstamm, Germany). Cell debris and the membrane fraction were removed by subsequent centrifugation steps at 28,000 × g for 15 min and 350,000 × g for 30 min, respectively. The supernatant of the latter (cell fraction of soluble proteins) was stored in liquid nitrogen. For affinity purification of recombinant His10-VaKtrA, prepacked protino®Ni 2000 column (Machery-Nagel GmbH & Co. KG, Duören Germany) preincubated with the supplied buffer plus 5 mm imidazole, 200 mm NaCl, and 30 mm 2-mercaptoethanol was used. After His10-VaKtrA binding at 5 mm imidazole, the column was washed with 50 ml of buffer containing 5 mm imidazole. His10-VaKtrA was eluted from the column in four steps by the successive addition of 800 μl of buffer containing 250 mm imidazole to the column. Imidazole was removed from the purified His10-VaKtrA fractions by buffer exchange against 0.2 m NaCl, 20 mm NaH2PO4, 2 mm 2-mercaptoethanol, 1 mm EDTA, pH 7.0 (buffer B) on NAP-5 columns (Amersham Biosciences). Purified His10-VaKtrA was stored at 4 °C, at which it remained in solution for several days. Removal of the His Tag from His10-VaKtrA—The tag was removed by digestion for 16 h at 20 °C with 50 units of protease Factor Xa (Novagen)/mg in buffer B containing 3–5 mg/ml His10-VaKtrA protein. Subsequently, the split-off His tag was removed on a NAP-5 column. The product (VaKtrA) contains a histidine residue as the N-terminal extension. Trypsin Digestion of His10-VaKtrA—About 60 μg of His10-VaKtrA in 300 μl of buffer B containing either no nucleotides, 1 mm NAD+, 1 mm NADH, or 1 mm ATP were incubated with 1.2 μg of trypsin. 50-μl samples were removed at different times and mixed immediately with an equivalent amount of soybean trypsin inhibitor. The protein pattern of the samples was analyzed by SDS-PAGE. Some fragments were identified by N-terminal sequencing. Antibodies—Rabbit polyclonal antibodies against purified VaKtrA were produced by Charles Rivers, Kisslegg, Germany. Monoclonal anti-penta-His antibody was purchased from Qiagen, Hilden, Germany. Overlay Experiment—VaKtrB-His6-containing membranes from strain Rosetta2(DE3)/pEL903 were prepared as described in Tholema et al. (15Tholema N. Vor der Bruöggen M. Maöser P. Nakamura T. Schroeder J.I. Kobayashi H. Uozumi N. Bakker E.P. J. Biol. Chem. 2005; 280: 41146-41154Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Proteins from this fraction were separated by SDSPAGE and transferred to a nitrocellulose sheet where they were stained with Ponceau Red. Separate protein lanes were cut out from the sheet. After removal of the stain and treatment with 3% of the blocking agent bovine serum albumin, these lanes were incubated with purified VaKtrA in the absence or presence of nucleotides at the concentration specified in the legend to Fig. 5. KtrA was detected with its specific antibody used at a 1:300,000 dilution followed by horseradish peroxidase-conjugated secondary antibodies (1:1000 dilution) and visualization with 4-nitro-blue-tetrazolium chloride/5-bromo4-chloro-3-indolylphosphate according to the recommendations of the supplier (Roche Diagnostics). In parallel, VaKtrB-His6 in the protein lane was detected with the monoclonal anti-penta-His antibody. Binding of Solubilized VaKtrB-His6 to Ni2+-agarose—VaKtrB-His6 was solubilized from membranes of strain Rosetta2(DE3)/pEL903 as described in Tholema et al. (15Tholema N. Vor der Bruöggen M. Maöser P. Nakamura T. Schroeder J.I. Kobayashi H. Uozumi N. Bakker E.P. J. Biol. Chem. 2005; 280: 41146-41154Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), except that membranes were present at 20 mg/ml and that the detergent n-dodecyl β-d-maltoside was present at 1.3% (w/v). Solubilized His-tagged protein was bound to Ni2+-agarose (Qiagen) in the presence of 5 mm imidazole. Accompanying, non-His-tagged proteins were eluted by washing the column with 50 and 8 bed volumes of buffer containing 30 and 50 mm imidazole, respectively. Complex Formation between VaKtrA and VaKtrB-His6 Bound to Ni2+-agarose; Elution of the Complex—An amount of 1 ml of column material containing about 0.2 mg of bound VaKtrB-His6 was incubated with 0.5 ml of buffer B without EDTA containing 0.5 mg of VaKtrA and either 100 μm ATP or no nucleotide. Subsequently the column was washed again with 50 and 8 bed volumes of buffer containing 30 and 50 mm imidazole, respectively. The KtrAB complex was eluted with buffer containing 250 mm imidazole. Detection of KtrA or KtrB-His6 was done with the specific antibodies as described above for the overlay experiment. Photochemical Binding of Adenine-(di)-nucleotides—Photochemical binding of [32P]NAD+ or [γ-32P]ATP (Amersham Biosciences; Ref. 33Carrol S.F. Lory S. Collier R.J. J. Biol. Chem. 1980; 255: 12020-12024Abstract Full Text PDF PubMed Google Scholar) to His10-VaKtrA was done as in Schloösser et al. (9Schloösser A. Hamann A. Bossemeyer D. Schneider E. Bakker E.P. Mol. Microbiol. 1993; 9: 533-543Crossref PubMed Scopus (113) Google Scholar). Samples of 20 μl of buffer B containing 10–40 μg of protein, 0.5–1 μCi of 32P-labeled compound (specific activity 500–1000 Ci/mmol), and varying concentrations of nonradioactive (di)-nucleotides were irradiated with ultraviolet light for 20–40 min on ice. Subsequently each sample received 20 μl of 2×-concentrated sample solubilization buffer (8% SDS, 24% glycerol, 100 mm Tris-HCl, 4% 2-mercaptoethanol, 0.02% Serva blue, pH 6.8), and the mixture was incubated for 30 min at 40 °C. Proteins from 20-μl samples were separated by SDS-PAGE. Radioactivity on the dried gel was detected and quantified by phosphorimaging using an Amersham Biosciences Storm 820 apparatus. Flow Dialysis for Determining Nucleotide Binding to His10-VaKtrA—Binding of nucleotides to His10-VaKtrA was determined at room temperature by flow dialysis (34Colowick S.P. Womack F.C. J. Biol. Chem. 1969; 244: 774-777Abstract Full Text PDF PubMed Google Scholar). The upper chamber was filled with 1 ml of buffer D containing 2 mm β-mercaptoethanol, 0.2 m NaCl, 1 mm EDTA, 20 mm sodium phosphate, pH 7.0, and radioactivity (1 μCi (about 1–5 pmol) of [32P]NAD+ or [γ-32P]ATP) in the absence or presence of 1–2 mg of His10-VaKtrA. The lower spiral chamber was separated from the upper chamber by a Visking type 36 dialysis membrane (Biomol, Hamburg, Germany). Buffer D was passed through the lower chamber at a rate of about 1 ml/min. 0.8-ml samples of the dialysate were collected and mixed with 4 ml of scintillation fluid Ecolume (ICN, Heidelberg, Germany), and their radioactivity was determined in a Tricarb 2300 TR liquid scintillation counter (PerkinElmer Life Sciences). Binding of radioactivity to His10-VaKtrA was calculated from the difference between radioactivity in the dialysate in the absence and presence of His10-VaKtrA. The KD-value for ATP binding and the amount of ATP maximally bound to His10-KtrA on a molar basis were determined according to the method of Scatchard (35Fersht A. Enzyme Structure and Mechanism, 2nd Ed., W.H. Freeman and Co., New York. 1985; Google Scholar). For this calculation the molar concentration of His10-VaKtrA was determined spectroscopically using a ɛ280 value of 5,600 m–1·cm–1, calculated from the contributions at that wavelength of the one tryptophan and five tyrosine residues per VaKtrA molecule (4Nakamura T. Yuda R. Unemoto T. Bakker E.P. J. Bacteriol. 1998; 180: 3491-3494Crossref PubMed Google Scholar). Binding constants for nucleotides other than ATP were determined by first binding a small amount of radioactive ATP to His10-VaKtrA and then gradually releasing the radioactivity by adding increasing concentrations of the second nucleotide. For this calculation it was assumed that the KD value for ATP was 9 μm and that KtrA possesses a single nucleotide binding site. ATPase Activity of His10-KtrA—This activity was measured in a flow system as described in Arnold et al. (36Arnold A. Wolf H.U. Ackermann B.P. Anal. Biochem. 1976; 71: 209-213Crossref PubMed Scopus (77) Google Scholar). Overproduction and Purification—VaktrA was cloned into the NdeI and BamHI sites of the expression plasmid pET16b, giving plasmid pEL305. This plasmid encodes VaKtrA with a 21-residue N-terminal extension of 10 histidine residues followed by a factor Xa proteolytic cleavage site (His10-VaKtrA). Overproduction of His10-VaKtrA in strain BL21(DE3)pLysS/pEL305 gave good yields of the protein in both the cell membrane and soluble protein fractions. From the latter, His10-VaKtrA was isolated by affinity chromatography on nickel-agarose (Fig. 1A). The yield was about 250 mg of His10-VaKtrA/l cell culture, and the preparation had a purity of at least 95% (Fig. 1A, elution fractions 1–4), which was sufficient for the intended experiments. His10-VaKtrA ran as a single peak with an apparent Mr of about 280,000 on a Sepharose 200 column (data not shown), suggesting that His10-VaKtrA is a decamer. Because this type of Mr determination is inaccurate, our data are compatible with KtrA being an octamer (17Albright R.A. Vasquez-Ibar J-L. Kim C.U. Gruner S.M. Morais-Cabral J.H. Cell. 2006; 126: 1147-1159Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Photochemical Binding of [32P]NAD+ to His10-VaKtrA—Because the first two KTN protein fragments for which a structure was known were complexed with NAD+ and NADH, respectively (16Roosild T.P. Miller S. Booth I.R. Choe S. Cell. 2002; 109: 781-791Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), we started our nucleotide binding studies with [32P]NAD+. Previously we have shown that [32P]NAD+ can be photochemically bound to the KTN protein TrkA from E. coli (9Schloösser A. Hamann A. Bossemeyer D. Schneider E. Bakker E.P. Mol. Microbiol. 1993; 9: 533-543Crossref PubMed Scopus (113) Google Scholar). Hence we applied the same technique to His10-VaKtrA (Fig. 2). After strong illumination of His10-VaKtrA with light of 260 nm in the presence of [32P]NAD+, the protein became radioactively labeled. This process was prevented by increasing concentrations of nonradioactive NAD+ (Fig. 2A, lanes 2–4). Quantification of the amount of radioactive NAD+ bound to His10-VaKtrA showed that under the assay conditions of Fig. 2, only a few percent of VaKtrA had bound the radioactive nucleotide. The photochemical binding assay was used to determine how other nucleotides competed with [32P]NAD+ binding to His10-VaKtrA. ATP and NADH were more effective in reducing [32P]NAD+ binding than did NAD+ itself (Fig. 2, A, lanes 10–13, lanes 6–9, and lanes 2–5, and D, closed circles, closed squares, and closed triangles, respectively). By the same criterion, NADP+ and NADPH bound with lower affinity than did NAD+ (Fig. 2, B, lanes 1–4, lanes 5–8, and A, lanes 2–5; D, crosses, closed diamonds, and closed diamonds, respectively). Moreover, ADP and AMP also bound strongly to the protein (Fig. 2C). The observation that ATP was most effective in inhibiting [32P]NAD+ binding to His10-VaKtrA led us to do the reverse experiment in which [γ-32P]ATP was photoreacted to His10-VaKtrA. NAD+ was a weak inhibitor of this cross-linking (Fig. 2B, lanes 9–12), confirming that the affinity with which ATP binds to His10-VaKtrA is higher than that of NAD+. From these data we conclude that the order of affinity for binding of nucleotides is ATP > NADH > NAD+ > NADP+ ≅ NADPH. Flow Dialysis—Because photocross-linking experiments are not suitable for determining ligand binding constants accurately, we resorted for these determinations to the technique of flow dialysis (34Colowick S.P. Womack F.C. J. Biol. Chem. 1969; 244: 774-777Abstract Full Text PDF PubMed Google Scholar). It allows accurate KD determinations under the condition that this constant is less than or equal to that of the protein concentration, which was 30–50 μm for His10-VaKtrA. Fig. 3A shows an experiment in which the binding of [γ-32P]ATP to His10-KtrA was measured. Compared with the situation without protein (open triangles), in the presence of His10-VaKtrA only about 20% of the added 30 μm of [γ-32P]ATP appeared in the dialysate (closed triangles), indicating strong binding of the nucleotide to the protein. Subsequent additions of nonradioactive ATP gradually released the radioactivity from the protein (Fig. 3A). From these data a KD value of 7 μm was calculated for the binding of ATP to His10-VaKtrA. Repetition of the experiment gave a KD value of 9 μm ± 0.8 for three different His10-VaKtrA preparations (Fig. 3B and Table 1). A similar value was obtained with VaKtrA from which its His tag had been removed previously. In addition, the presence of 1 mm concentrations of any of the divalent cations Mg2+, Ca2+, Fe2+, Cu2+, Mn2+, Ni2+, or Co2+ did not influence ATP binding to His10-KtrA (results not shown).TABLE 1Dissociation constants (KD values) for the binding of nucleotides to VaKtrANucleotideKDNumber of determinationsμmATP9 ± 0.83ADP27 ± 32AMP65 ± 72CTP73 ± 112GTP≥7002NAD+≥7003NADH103 ± 112FAD33 ± 82 Open table in a new tab A similar flow dialysis experiment with [32P]NAD+ gave a completely different result. The amount of radioactivity in the dialysate was only slightly lower" @default.
• W2023488999 created "2016-06-24" @default.
• W2023488999 creator A5002940746 @default.
• W2023488999 creator A5039680714 @default.
• W2023488999 creator A5046017194 @default.
• W2023488999 creator A5058650042 @default.
• W2023488999 creator A5072361885 @default.
• W2023488999 creator A5091008532 @default.
• W2023488999 date "2007-05-01" @default.
• W2023488999 modified "2023-10-15" @default.
• W2023488999 title "ATP Binding to the KTN/RCK Subunit KtrA from the K+-uptake System KtrAB of Vibrio alginolyticus" @default.
• W2023488999 cites W1513843435 @default.
• W2023488999 cites W1517364723 @default.
• W2023488999 cites W1524836192 @default.
• W2023488999 cites W1553379514 @default.
• W2023488999 cites W1574972316 @default.
• W2023488999 cites W1608918896 @default.
• W2023488999 cites W1625553880 @default.
• W2023488999 cites W1665555949 @default.
• W2023488999 cites W1764225276 @default.
• W2023488999 cites W1896452697 @default.
• W2023488999 cites W1969381663 @default.
• W2023488999 cites W1973352749 @default.
• W2023488999 cites W1984065377 @default.
• W2023488999 cites W1987469737 @default.
• W2023488999 cites W1992838345 @default.
• W2023488999 cites W1998322821 @default.
• W2023488999 cites W2006670481 @default.
• W2023488999 cites W2008000048 @default.
• W2023488999 cites W2018746058 @default.
• W2023488999 cites W2024332597 @default.
• W2023488999 cites W2025205859 @default.
• W2023488999 cites W2031267910 @default.
• W2023488999 cites W2038645762 @default.
• W2023488999 cites W2039037981 @default.
• W2023488999 cites W2061487060 @default.
• W2023488999 cites W2063480230 @default.
• W2023488999 cites W2065329484 @default.
• W2023488999 cites W2076999300 @default.
• W2023488999 cites W2082362903 @default.
• W2023488999 cites W2088144279 @default.
• W2023488999 cites W2088986479 @default.
• W2023488999 cites W2089954238 @default.
• W2023488999 cites W2094832359 @default.
• W2023488999 cites W2101204196 @default.
• W2023488999 cites W2104215185 @default.
• W2023488999 cites W2120961946 @default.
• W2023488999 cites W2126007769 @default.
• W2023488999 cites W2152619379 @default.
• W2023488999 cites W2153269862 @default.
• W2023488999 cites W2154577330 @default.
• W2023488999 cites W2160084442 @default.
• W2023488999 cites W2176634422 @default.
• W2023488999 cites W290094515 @default.
• W2023488999 cites W299200106 @default.
• W2023488999 cites W2068990198 @default.
• W2023488999 doi "https://doi.org/10.1074/jbc.m609084200" @default.
• W2023488999 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17344221" @default.
• W2023488999 hasPublicationYear "2007" @default.
• W2023488999 type Work @default.
• W2023488999 sameAs 2023488999 @default.
• W2023488999 citedByCount "38" @default.
• W2023488999 countsByYear W20234889992012 @default.
• W2023488999 countsByYear W20234889992013 @default.
• W2023488999 countsByYear W20234889992014 @default.
• W2023488999 countsByYear W20234889992015 @default.
• W2023488999 countsByYear W20234889992016 @default.
• W2023488999 countsByYear W20234889992017 @default.
• W2023488999 countsByYear W20234889992019 @default.
• W2023488999 countsByYear W20234889992020 @default.
• W2023488999 countsByYear W20234889992021 @default.
• W2023488999 countsByYear W20234889992022 @default.
• W2023488999 crossrefType "journal-article" @default.
• W2023488999 hasAuthorship W2023488999A5002940746 @default.
• W2023488999 hasAuthorship W2023488999A5039680714 @default.
• W2023488999 hasAuthorship W2023488999A5046017194 @default.
• W2023488999 hasAuthorship W2023488999A5058650042 @default.
• W2023488999 hasAuthorship W2023488999A5072361885 @default.
• W2023488999 hasAuthorship W2023488999A5091008532 @default.
• W2023488999 hasBestOaLocation W20234889991 @default.
• W2023488999 hasConcept C104292427 @default.
• W2023488999 hasConcept C104317684 @default.
• W2023488999 hasConcept C107824862 @default.
• W2023488999 hasConcept C185592680 @default.
• W2023488999 hasConcept C2776154503 @default.
• W2023488999 hasConcept C2778968793 @default.
• W2023488999 hasConcept C523546767 @default.
• W2023488999 hasConcept C54355233 @default.
• W2023488999 hasConcept C55493867 @default.
• W2023488999 hasConcept C86803240 @default.
• W2023488999 hasConcept C89423630 @default.
• W2023488999 hasConceptScore W2023488999C104292427 @default.
• W2023488999 hasConceptScore W2023488999C104317684 @default.
• W2023488999 hasConceptScore W2023488999C107824862 @default.
• W2023488999 hasConceptScore W2023488999C185592680 @default.
• W2023488999 hasConceptScore W2023488999C2776154503 @default.
• W2023488999 hasConceptScore W2023488999C2778968793 @default.
• W2023488999 hasConceptScore W2023488999C523546767 @default.

• next