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Volumn 289, Issue 5479, 2000, Pages 625-628

Soluble adenylyl cyclase as an evolutionarily conserved bicarbonate sensor

Author keywords

[No Author keywords available]

Indexed keywords

ADENYLATE CYCLASE; BICARBONATE; CYCLIC AMP;

EID: 0034725748     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.289.5479.625     Document Type: Article
Times cited : (728)

References (25)
  • 8
    • 0343842808 scopus 로고    scopus 로고
    • note
    • Rabbit polyclonal antisera were raised against the catalytic regions of sAC (C1 and C2; amino acids 1 through 469), which were expressed in bacteria and purified from inclusion bodies by excision from SDS-polyacrylamide gel electrophoresis. Antisera were affinity-purified, and specificity was confirmed with heterologously expressed proteins (Fig. 1A). Tissues for Western blotting were purchased from Pel-Freez Biologicals (Rogers, AR). Goat anti-rabbit horseradish peroxidase was used as secondary antibody, and detection was done with enhanced chemiluminescence (Pierce).
  • 10
    • 0342971600 scopus 로고    scopus 로고
    • note
    • Immunoprecipitations were performed on rat testis cytosol prepared by 100,000g centrifugation. Cytosol was incubated with 50 μl of preimmune or anti-sAC antisera for 2 hours at 4°C. After a preclearing spin (for 10 min in a microcentrifuge at full speed), antigen-antibody complexes were harvested by incubation with 60 μl of protein A-Sepharose beads for 1 hour at 4°C. Immunecomplexes were washed twice with 50 mM tris-HCl (pH 7.5) and 100 mM NaCl and were assayed as described (4).
  • 12
    • 85176997515 scopus 로고    scopus 로고
    • note
    • 2) for 24 hours. In vitro AC assays were performed at pH 7.5 in tris-buffered solutions, except where indicated. Kinetic parameters were determined with KinetAsyst II (IntelliKinetics, State College, PA).
  • 14
    • 0342971599 scopus 로고    scopus 로고
    • unpublished data
    • M. J. Cann, unpublished data.
    • Cann, M.J.1
  • 16
    • 0343406849 scopus 로고    scopus 로고
    • note
    • 2+-NTA Sepharose Resin (Qiagen).
  • 22
    • 0343406845 scopus 로고
    • Department of Genetics, University of Washington, Seattle
    • J. Felsenstein (Department of Genetics, University of Washington, Seattle, 1993).
    • (1993)
    • Felsenstein, J.1
  • 25
    • 0342971598 scopus 로고    scopus 로고
    • note
    • We thank R. B. Silver for assistance with in vivo pH measurement; R. Duvoisin, M. C. Gershengorn, C. Nathan, and M. R. Reidenberg for suggestions; J. Qiu for technical assistance; and M. Ohmori for the kind gift of Spirulina platensis CyaC in bacterial expression vector. Supported by Weill Medical College or Cornell University Research Associates, the Irma T. Hirschl Trust, The L. W. Frohlich Charitable Trust, Edith C. Blum Foundation, and NIH.


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.