Solubilization and Characterization of Calcitonin Gene-Related Peptide Binding Site from Porcine Spinal Cord |
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Authors: | Osamu Hiroshima Yoshihisa Sano Teruaki Yuzuriha Chiyuki Yamato Akira Saito Naomichi Okamura† Yasuo Uchiyama† Sadao Kimura † Katsutoshi Goto |
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Affiliation: | Tsukuba Research Laboratories, Eisai Co., Ltd., Ibaraki, Japan. |
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Abstract: | The binding site for calcitonin gene-related peptide (CGRP) was solubilized with 3-(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) in an active form from porcine spinal cord. 125I-labeled human alpha-CGRP (125I-CGRP) binding to the solubilized protein was determined by filtration using a GF/B glass filter. The maximal binding activity (approximately 60% of the crude membrane fraction) was obtained with 5 mM CHAPS. 125I-CGRP binding to the solubilized protein was of high affinity, saturability, and high specificity, having KD and Bmax values of 3.69 pM and 338 fmol/mg of protein, respectively. The binding activity was eluted in a single peak with a molecular mass of 400,000 daltons by gel filtration on TSK gel G4000SW. These results suggest that the solubilized protein may be responsible for the specific binding site. |
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Keywords: | Solubilization Calcitonin gene-related peptide Binding site Spinal cord 3-[(3-Cholamidopropyl) dimethylammonio]-l-propane sulfonate |
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