Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1982 Jun;79(12):3739–3743. doi: 10.1073/pnas.79.12.3739

Covalent crosslinking of angiotensin II to its binding sites in rat adrenal membranes.

S Paglin, J D Jamieson
PMCID: PMC346502  PMID: 6285362

Abstract

To identify the molecular components of the angiotensin II (AII) receptor in the adrenal cortex, we have covalently linked 125I-labeled AII (125I-AII) to its binding sites by using the chemical crosslinker disuccinimidyl suberate. Membrane fractions from rat adrenal tissue were incubated with 125I-AII, washed, and treated with disuccinimidyl suberate. NaDodSO4/polyacrylamide gel electrophoresis carried out under reducing conditions followed by autoradiography showed that a major polypeptide(s) with an average Mr of 116,000 was covalently tagged with 125I-AII. Proteins with Mr of 54,000 and 45,000 were also lightly labeled. Labeling of the Mr 116,000 species was specific in that it could be abolished by prior incubation of the membranes with [Sar1,Leu8]AII or unlabeled AII. Labeling of the smaller proteins was less affected by prior incubation with [Sar1,Leu8]AII. Experiments in which crosslinking was carried out in the presence of a variety of protease inhibitors indicated that the Mr 116,000 species is not an enzyme involved in AII degradation. Gel electrophoresis under nonreducing conditions showed that this component is not derived from larger disulfide-linked entities. We suggest that the Mr 116,000 moiety is a part of the receptor system for AII. The relationship, if any, of the Mr 54,000 and 45,000 peptides to the AII receptor remains unknown.

Full text

PDF
3739

Images in this article

3740Image
on p.3740
3741Image
on p.3741
3741Image
on p.3741
3741Image
on p.3741

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Capponi A. M., Catt K. J. Angiotensin II receptors in adrenal cortex and uterus. Binding and activation properties of angiotensin analogues. J Biol Chem. 1979 Jun 25;254(12):5120–5127. [PubMed] [Google Scholar]
  2. Capponi A. M., Catt K. J. Solubilization and characterization of adrenal and uterine angiotensin II receptors after photoaffinity labeling. J Biol Chem. 1980 Dec 25;255(24):12081–12086. [PubMed] [Google Scholar]
  3. Castle J. D., Jamieson J. D., Palade G. E. Secretion granules of the rabbit parotid gland. Isolation, subfractionation, and characterization of the membrane and content subfractions. J Cell Biol. 1975 Jan;64(1):182–210. doi: 10.1083/jcb.64.1.182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chiu A. T., Ryan J. W., Stewart J. M., Dorer F. E. Formation of angiotensin III by angiotensin-converting enzyme. Biochem J. 1976 Apr 1;155(1):189–192. doi: 10.1042/bj1550189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Desbuquois B., Krug F., Cuatrecasas P. Inhibitors of glucagon inactivation. Effect on glucagon--receptor interactions and glucagon-stimulated adenylate cyclase activity in liver cell membranes. Biochim Biophys Acta. 1974 Mar 20;343(1):101–120. doi: 10.1016/0304-4165(74)90242-6. [DOI] [PubMed] [Google Scholar]
  6. Douglas J., Aguilera G., Kondo T., Catt K. Angiotensin II receptors and aldosterone production in rat adrenal glomerulosa cells. Endocrinology. 1978 Mar;102(3):685–696. doi: 10.1210/endo-102-3-685. [DOI] [PubMed] [Google Scholar]
  7. Farruggia P., Sachs S., Palaic D. Effect of angiotensinase inhibitors on angiotensin receptors in rabbit aorta. Mol Pharmacol. 1979 May;15(3):525–530. [PubMed] [Google Scholar]
  8. Fredlund P., Saltman S., Catt K. J. Aldosterone production by isolated adrenal glomerulosa cells: stimulation by physiological concentrations of angiotensin II. Endocrinology. 1975 Dec;97(6):1577–1586. doi: 10.1210/endo-97-6-1577. [DOI] [PubMed] [Google Scholar]
  9. Glossmann H., Baukal A. J., Catt K. J. Properties of angiotensin II receptors in the bovine and rat adrenal cortex. J Biol Chem. 1974 Feb 10;249(3):825–834. [PubMed] [Google Scholar]
  10. Kasuga M., Van Obberghen E., Nissley S. P., Rechler M. M. Demonstration of two subtypes of insulin-like growth factor receptors by affinity cross-linking. J Biol Chem. 1981 Jun 10;256(11):5305–5308. [PubMed] [Google Scholar]
  11. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  12. Lin S. Y., Goodfriend T. L. Angiotensin receptors. Am J Physiol. 1970 May;218(5):1319–1328. doi: 10.1152/ajplegacy.1970.218.5.1319. [DOI] [PubMed] [Google Scholar]
  13. Massague J., Guillette B. J., Czech M. P. Affinity labeling of multiplication stimulating activity receptors in membranes from rat and human tissues. J Biol Chem. 1981 Mar 10;256(5):2122–2125. [PubMed] [Google Scholar]
  14. Nussdorfer G. G., Robba C., Mazzochi G., Rebuffat P. Effects of angiotensin II on the zona fasciculata of the rat adrenal cortex: an ultrastructural stereologic study. J Anat. 1981 Mar;132(Pt 2):235–242. [PMC free article] [PubMed] [Google Scholar]
  15. Ondetti M. A., Rubin B., Cushman D. W. Design of specific inhibitors of angiotensin-converting enzyme: new class of orally active antihypertensive agents. Science. 1977 Apr 22;196(4288):441–444. doi: 10.1126/science.191908. [DOI] [PubMed] [Google Scholar]
  16. Pilch P. F., Czech M. P. Interaction of cross-linking agents with the insulin effector system of isolated fat cells. Covalent linkage of 125I-insulin to a plasma membrane receptor protein of 140,000 daltons. J Biol Chem. 1979 May 10;254(9):3375–3381. [PubMed] [Google Scholar]
  17. Regoli D., Park W. K., Rioux F. Pharmacology of angiotensin. Pharmacol Rev. 1974 Jun;26(2):69–123. [PubMed] [Google Scholar]
  18. Umezawa H. Structures and activities of protease inhibitors of microbial origin. Methods Enzymol. 1976;45:678–695. doi: 10.1016/s0076-6879(76)45058-9. [DOI] [PubMed] [Google Scholar]
  19. Vallotton M. B., Capponi A. M., Grillet C., Knupfer A. L., Hepp R., Khosla M. C., Bumpus F. M. Characterization of angiotensin receptors on bovine adrenal fasciculata cells. Proc Natl Acad Sci U S A. 1981 Jan;78(1):592–596. doi: 10.1073/pnas.78.1.592. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES