A H+-gated urea channel: The link between Helicobacter pylori urease and gastric colonization

Science

Volumn 287, Issue 5452, 2000, Pages 482-485

  Weeks, David L ^a   Eskandari, Sepehr ^a   Scott, David R ^a   Sachs, George ^a  

^a UNIVERSITY OF CALIFORNIA   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

BACTERIAL PROTEIN; CARRIER PROTEIN; PROTEIN UREL; UNCLASSIFIED DRUG; UREA; UREASE;

ANIMAL CELL; ARTICLE; BACTERIAL COLONIZATION; CHRONIC GASTRITIS; CONTROLLED STUDY; CYTOPLASM; ENZYME ACTIVITY; HELICOBACTER PYLORI; NONHUMAN; OOCYTE; PRIORITY JOURNAL; STOMACH; STOMACH CANCER; STOMACH PH; STOMACH ULCER; UREA CYCLE; XENOPUS;

AMINO ACID SEQUENCE; ANIMALS; BACTERIAL PROTEINS; BIOLOGICAL TRANSPORT; CELL MEMBRANE; CELL MEMBRANE PERMEABILITY; CYTOPLASM; ENZYME ACTIVATION; GASTRIC ACID; GLYCOSYLATION; HELICOBACTER PYLORI; HISTIDINE; HUMANS; HYDROGEN-ION CONCENTRATION; MEMBRANE TRANSPORT PROTEINS; MOLECULAR SEQUENCE DATA; OOCYTES; RECOMBINANT PROTEINS; STOMACH; TEMPERATURE; UREA; UREASE; XENOPUS;

ANIMALIA; BACTERIA (MICROORGANISMS); HELICOBACTER PYLORI;

EID: 0034695684     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.287.5452.482     Document Type: Article

References (29)

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13. - (Stratagene, La Jolla, CA). The coding sequence of urel was replaced with the coding sequence for kanR. The ureB to -E cassette containing the urel/kanR substitution was transferred to the suicide plasmid pJM703.1 and transformed into an American Type Culture Collection (ATCC) 43504 by electroporation. A primer pair annealing to the kanR coding sequence and to ureA (outside the ureB to -E cassette) confirmed the integration into the genome.
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15. - (Invitrogen, Carlsbad, CA). The urel gene sequence was inserted upstream of the poly(A) cassette and downstream of the pcDNA3.1 T7 promoter. cRNA was prepared using the mMessage mMachine in vitro transcription system (Ambion, Austin, TX). Fifty nanoliters of cRNA (1 μg/μl ) was injected and oocytes were maintained at 18°C in Barth's solution for 3 days before use in uptake experiments.
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17. 14C-urea at pH 5.0 for a value of 1.09 ± 0.03 nmole urea per oocyte per 15 min.
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19. Periodic acid-silver staining was performed according to C. Tsai and C. Frasch, Anal. Biochem. 119, 115 (1982).
20. D. Weeks, S. Eskandari, D. Scott, G. Sachs, data not shown.
21. Membrane fractionation was performed according to J. J. Tudor and M. A. Karp, J. Bacteriol. 176, 948 (1994).
22. + -ATPase β subunit, containing five N-linked glycosylation consensus sequences. Glycosylation indicates translocation of the COOH-terminus into the microsomal membrane.
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24. Histidine replacement was performed by a technique described by B. Chen and A. E. Przybyla, Biotechniques 17, 657 (1994) and was verified by sequencing. Expression was confirmed by Western blot analysis.
25. 2, was measured as pre-viously described (5).
26. Antibody generation and affinity purification were carried out by Alpha Diagnostics International (San Antonio, TX). The epitopes UP1 (CEGAEDIAQVSHHLTNFYGPATC) and UP2 (CAILSHYSDMLDDHKVLGITEGD) (24) are within the first and second periplasmic loops. Homogenate and membranes were resolved on 10% SDS-tricine gels. Proteins were transferred to either nitrocellulose (Bio-Rad, Hercules, CA) or polyvinylidine diflouride (Millipore, Bedford, MA). After transfer, blots were blocked by incubation in a 5% solution of nonfat dry milk in phosphate-buffered saline-Tween for 1 hour. The membranes were incubated with antibodies to UP2 at a 1:2000 dilution in blocking solution. Binding was detected using a peroxidase-coupled rabbit antibody to immunoglobulin G at 1:20,000 dilution (American Qualex, San Clemente, CA) with ECL or ECL plus (Amersham, Arlington, IL).
27. 14C-urea, and was terminated with the transfer of the oocytes to an ice-cold buffer (pH 7.5). Each oocyte was individually dissolved with SDS and mixed with scintillation cocktail for counting of the labeled compounds.
28. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
29. We thank E. Wright, D. Leung, S. Hallen, P. Voland, and K. Melchers. Supported by U.S. Department of Veteran's Affairs and by NIH grants DK46917, 53462, 41301, 19567, and 17294.