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Solubilization and characterization of covalently labeled angiotensin II receptors in cultured mouse spinal cord cells

Authors
Journal
Neuropeptides
0143-4179
Publisher
Elsevier
Publication Date
Volume
9
Issue
4
Identifiers
DOI: 10.1016/0143-4179(87)90008-4
Disciplines
  • Chemistry

Abstract

Abstract To investigate the physicochemical characteristics of angiotensin II receptors in nervous tissue, [ 125I]-labeled AII was covalently linked to its binding sites in cultured mouse spinal cord cells, using the chemical crosslinking agent disuccinimidyl suberate. The liganded complexes were analyzed by SDS/polyacrylamide gel electrophoresis. Autoradiograms revealed a single band which corresponding to a component with a calculated Mr value of 68,000 ± 5,000. The same radioactive band was observed under reducing or non-reducing conditions, although in the presence of β-mercaptoethanol the band was less intense. This suggests that the AII binding unit may contain essential disulfide bonds close to the AII binding site. After solubilization with 1% Triton X-100, extracts were analyzed by HPLC gel filtration. Three radioactive species were identified with apparent molecular weights of 65,000; 115,000 and 185,000. Results obtained in the presence or absence of 10 nM unlabeled AII showed that these three units were common to both the high and the low affinity binding sites. The results also demonstrate that AII receptors in nervous tissue have similar physicochemical properties to those from other tissues, and that the 68,000 subunit probably contains the AII binding site.

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