Solubilization and characterization of calcitonin gene-related peptide binding site from porcine spinal cord.
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.
Hiroshima
O
O
Tsukuba Research Laboratories, Eisai Co., Ltd., Ibaraki, Japan.
Sano
Y
Y
Yuzuriha
T
T
Yamato
C
C
Saito
A
A
Okamura
N
N
Uchiyama
Y
Y
Kimura
S
S
Goto
K
K
D000818:Animals; D001665:Binding Sites; D015740:Calcitonin Gene-Related Peptide; D002462:Cell Membrane / Q000378:metabolism; D002793:Cholic Acids; D002850:Chromatography, Gel; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009479:Neuropeptides / Q000378:metabolism*; D018003:Receptors, Calcitonin; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D013116:Spinal Cord / Q000378:metabolism*; D013552:Swine; D013696:Temperature
D002793:Cholic Acids; D009479:Neuropeptides; D018003:Receptors, Calcitonin; D011956:Receptors, Cell Surface; D015740:Calcitonin Gene-Related Peptide; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate