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Volumn 20, Issue 20, 2010, Pages 6040-6044

Elucidation of the active conformation of the amino terminus of receptor-bound secretin using intramolecular disulfide bond constraints

Author keywords

Active peptide conformation; Family B G protein coupled receptor; Ligand binding; Secretin; Secretin receptor

Indexed keywords

CYSTEINE; SECRETIN;

EID: 77956898278     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2010.08.062     Document Type: Article
Times cited : (6)

References (31)
  • 18
    • 0024009303 scopus 로고    scopus 로고
    • note
    • 3, and was diluted with water to yield a peptide concentration of 0.5-1 mg/ml. While stirring and exposed to the air, dimethyl sulfoxide was added to a final concentration of 10% (v/v). The oxidation process was monitored for completion using reversed-phase HPLC, typically taking between 2 and 5 h. All linear and disulfide-bonded cyclic peptides were purified to homogeneity by reversed-phase HPLC on an octadecylsilane column running a 10-60% acetonitrile gradient with a background of 0.1% trifluoroacetic acid. Expected molecular masses of the synthetic products were verified by matrix-assisted laser desorption/ionization- time of flight mass spectrometry.
  • 19
    • 77956938654 scopus 로고    scopus 로고
    • note
    • 125I and exposure to the IODO-BEAD solid-phase oxidant (Pierce Chemical Co, Rockford, IL) for 15 s in 0.1 M borate buffer (pH 9). This was purified using reversed-phase HPLC to yield a specific radioactivity of approximately 2000 Ci/mmol (Powers, S. P.; Pinon, D. I.; Miller, L. J. Int. J. Pept. Protein Res. 1988, 31, 429). In brief, approximately 5-10 μg of CHO-SecR membranes were incubated with a constant amount of secretin radioligand (approximately 20,000 rpm) and increasing concentrations of secretin or one of its analogs (from 0 to 1 μM) in KRH medium containing 0.01% soybean trypsin inhibitor, 1 mM phenylmethylsulfonyl fluoride and 0.2% bovine serum albumin for 1 h at room temperature (reaction volume, 500 μl). Separation of bound from free radioligand was performed by centrifugation at 14,000 rpm at 4 °C, and washing twice with ice-cold KRH medium. Bound radioactivity was quantified with a γ-spectrometer. Non-saturable binding was determined in the presence of 1 μM secretin and represented <15% of total radioligand bound. Binding kinetics were determined by analysis with the LIGAND program of Munson and Rodbard (Munson, P. J.; Rodbard, D. Anal. Biochem. 1980, 107, 220).
  • 20
    • 77956927806 scopus 로고    scopus 로고
    • note
    • 3. The cAMP levels in the lysates were assayed in a 384-well white Optiplate using a LANCE kit from PerkinElmer (Boston, MA).


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.