Biochemical analysis of solubilized angiotensin II receptors from murine neuroblastoma N1E-115 cells by covalent cross-linking and affinity purification.

Abstract

Angiotensin II (Ang-II) receptors were solubilized from differentiated N1E-115 neuroblastoma cell membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), whereas other detergents, such as digitonin, sodium cholate, and Triton X-100, were much less effective. Binding of 125I-Ang-II or the antagonist 125I-Sar1,Ile8-Ang-II to 1% CHAPS-solubilized membranes was saturable and of high affinity. Moreover, these solubilized receptors retained the pharmacological specificity characteristic of particulate receptors. Covalent cross-linking of 125I-Ang-II to either particulate or solubilized membrane fractions, with the homobifunctional cross-linker disuccinimidyl suberate, followed by size exclusion chromatography or sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, resulted in the identification of the same two distinct 125I-Ang-II binding entities, with approximate molecular masses of 111 kDa and 68 kDa. The estimated molecular weights of the Ang-II binding sites in differentiated N1E-115 cells are in good agreement with the molecular weights obtained previously from solubilized rat brain membranes, suggesting that the N1E-115 Ang-II receptors are similar to those present in the brain. Finally, solubilized N1E-115 membranes could be purified by Ang-II affinity chromatography, resulting in only a single protein (66 kDa), which retained its ability to specifically bind 125I-Ang-II.

• Authors: I R Siemens, H J Adler, K Addya, S J Mah, S J Fluharty
• Journal: Molecular pharmacology (Mol Pharmacol) Vol. 40 Issue 5 Pg. 717-26 (Nov 1991) ISSN: 0026-895X [Print] United States
• PMID: 1944241 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Membrane Proteins
• Receptors, Angiotensin
• Angiotensin II

Topics

• Angiotensin II (metabolism)
• Animals
• Binding Sites
• Binding, Competitive
• Chromatography, Affinity
• Chromatography, Gel
• Electrophoresis, Polyacrylamide Gel
• Membrane Proteins (isolation & purification)
• Mice
• Neuroblastoma (chemistry)
• Receptors, Angiotensin (isolation & purification)
• Solubility
• Tumor Cells, Cultured