Solubilization and Characterization of Calcitonin Gene‐Related Peptide Binding Site from Porcine Spinal Cord

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.

Calcitonin gene-related peptide and its receptors: molecular genetics, physiology, pathophysiology, and therapeutic potentials

“…Specific CGRP receptors have been found in porcine coronary arteries (M r 90,000 and 70,000) and in the atria (318). In the porcine spinal cord, only the 70,000 M r form was identified (318)(319)(320)(321). The reported M r of the CGRP-binding protein measured by autoradiography after cross-linking with bifunctional reagents varies from 13,700 to 400,000 (296, 292-301, 315-322).…”

Calcitonin Gene‐Related Peptide‐Binding Sites of Porcine Cardiac Muscles and Coronary Arteries: Solubilization and Characterization

“…We previously found that CHAPS solubilized the binding sites retaining the specific binding activity to CGRP from the spinal cord but not from coronary arteries, atria, or ventricles (Hiroshima et al, 1988). In the present study, among the various types of detergents so far examined, only the nonionic detergent I922 Y. SANO ET AL.…”

“…The binding assay of the crude membrane as well a:< the solubilized fraction was carried out according to the method described previously (Hiroshima et al, 1988). In brief, the crude membrane or solubilized fractions (100 p l ) were mixed with 900 pl of I2'1-human a-CGRP (18-23 pM) in buffer A containing 1% digitonin in the presence or absence of 1.0 pLM unlabeled human a-CGRP.…”

“…CGRP stimulates the activity of adenylate cyclase and subsequently the synthesis of cyclic adenosine monophosphate in cardiac muscles and blood vessels (Edvinsson et al, 1985;Kubota et al, 1985;Sigrist et al, 1986;Ishikawa et al, 1987;Shoji et a]., 1987), but not in the spinal cord (Goltzman and Mitchell, 1985). Recently, CGRPbinding sites were solubilized from the porcine spinal cord using 3-[(3-cholamidopropyI)-dimethylammoniol-1 -propanesulfonate (CHAPS), an ionic detergent (Hiroshima et al, 1988). However, the same detergent did not show a satisfactory result in terms of solubilization of specific CGRP-binding sites in tissues of the cardiovascular system (unpublished observations).…”

Electrophysiological effects of calcitonin gene‐related peptide in bull‐frog and guinea‐pig atrial myocytes.

“…The presence of a specific receptor (Foord & Graig, 1987;Hiroshima et al 1988) and the ability of CGRP to activate adenylate cyclase suggest that the stimulatory effect of CGRP on ICa may be mediated by a GTP-binding protein (Neer & Clapham, 1988;Birnbaumer et al 1989). We have obtained evidence for involvement of GTP-binding protein (s) in these CGRP effects.…”