Skip to main content
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
. 1987 Mar;84(6):1521–1525. doi: 10.1073/pnas.84.6.1521

Purification and subunit composition of atrial natriuretic peptide receptor.

D B Schenk, M N Phelps, J G Porter, F Fuller, B Cordell, J A Lewicki
PMCID: PMC304466  PMID: 2882506

Abstract

A receptor for atrial natriuretic peptide (ANP) was purified 2700-fold, to apparent homogeneity, from cultured bovine aortic smooth muscle cells by affinity chromatography. The native ANP receptor has a molecular weight of 125,000 as determined by both metrizamide gradient centrifugation and nonreducing NaDodSO4/polyacrylamide gel electrophoresis. With 125I-labeled ANP as ligand, the purified receptor bound a maximum of 5.70 nmol of ligand per mg of protein and the dissociation constant was 4.0 X 10(-10)M. Upon treatment with 10 mM dithiothreitol, the purified receptor migrated as a single band at Mr 60,500 in NaDodSO4/polyacrylamide gel electrophoresis. These findings show that the holoreceptor for ANP in vascular tissue is composed of two subunits of identical apparent molecular weight, presumably linked by a disulfide bridge(s).

Full text

PDF

Images in this article

Selected References

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

  1. Atlas S. A., Kleinert H. D., Camargo M. J., Januszewicz A., Sealey J. E., Laragh J. H., Schilling J. W., Lewicki J. A., Johnson L. K., Maack T. Purification, sequencing and synthesis of natriuretic and vasoactive rat atrial peptide. Nature. 1984 Jun 21;309(5970):717–719. doi: 10.1038/309717a0. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Clarke S. The size and detergent binding of membrane proteins. J Biol Chem. 1975 Jul 25;250(14):5459–5469. [PubMed] [Google Scholar]
  4. Currie M. G., Geller D. M., Cole B. R., Boylan J. G., YuSheng W., Holmberg S. W., Needleman P. Bioactive cardiac substances: potent vasorelaxant activity in mammalian atria. Science. 1983 Jul 1;221(4605):71–73. doi: 10.1126/science.6857267. [DOI] [PubMed] [Google Scholar]
  5. De Léan A., Gutkowska J., McNicoll N., Schiller P. W., Cantin M., Genest J. Characterization of specific receptors for atrial natriuretic factor in bovine adrenal zona glomerulosa. Life Sci. 1984 Dec 3;35(23):2311–2318. doi: 10.1016/0024-3205(84)90522-8. [DOI] [PubMed] [Google Scholar]
  6. Hirata Y., Tomita M., Takada S., Yoshimi H. Vascular receptor binding activities and cyclic GMP responses by synthetic human and rat atrial natriuretic peptides (ANP) and receptor down-regulation by ANP. Biochem Biophys Res Commun. 1985 Apr 30;128(2):538–546. doi: 10.1016/0006-291x(85)90080-4. [DOI] [PubMed] [Google Scholar]
  7. Hirata Y., Tomita M., Yoshimi H., Ikeda M. Specific receptors for atrial natriuretic factor (ANF) in cultured vascular smooth muscle cells of rat aorta. Biochem Biophys Res Commun. 1984 Dec 14;125(2):562–568. doi: 10.1016/0006-291x(84)90576-x. [DOI] [PubMed] [Google Scholar]
  8. Hirose S., Akiyama F., Shinjo M., Ohno H., Murakami K. Solubilization and molecular weight estimation of atrial natriuretic factor receptor from bovine adrenal cortex. Biochem Biophys Res Commun. 1985 Jul 31;130(2):574–579. doi: 10.1016/0006-291x(85)90455-3. [DOI] [PubMed] [Google Scholar]
  9. Inui K., Saito H., Matsukawa Y., Nakao K., Morii N., Imura H., Shimokura M., Kiso Y., Hori R. Specific binding activities and cyclic GMP responses by atrial natriuretic polypeptide in kidney epithelial cell line (LLC-PK1). Biochem Biophys Res Commun. 1985 Oct 15;132(1):253–260. doi: 10.1016/0006-291x(85)91015-0. [DOI] [PubMed] [Google Scholar]
  10. Ishido M., Fujita T., Hagiwara H., Shimonaka M., Saheki T., Hirata Y., Hirose S. Physical and functional association of the atrial natriuretic peptide receptor with particulate guanylate cyclase as demonstrated using detergent extracts of bovine lung membranes. Biochem Biophys Res Commun. 1986 Oct 15;140(1):101–106. doi: 10.1016/0006-291x(86)91063-6. [DOI] [PubMed] [Google Scholar]
  11. Koseki C., Hayashi Y., Ohnuma N., Imai M. Difference in molecular size of receptors for alpha-rat atrial natriuretic polypeptide among the kidney, aorta, and adrenal gland as identified by direct UV-photoaffinity labeling. Biochem Biophys Res Commun. 1986 Apr 14;136(1):200–207. doi: 10.1016/0006-291x(86)90895-8. [DOI] [PubMed] [Google Scholar]
  12. Kudo T., Baird A. Inhibition of aldosterone production in the adrenal glomerulosa by atrial natriuretic factor. Nature. 1984 Dec 20;312(5996):756–757. doi: 10.1038/312756a0. [DOI] [PubMed] [Google Scholar]
  13. Kuno T., Andresen J. W., Kamisaki Y., Waldman S. A., Chang L. Y., Saheki S., Leitman D. C., Nakane M., Murad F. Co-purification of an atrial natriuretic factor receptor and particulate guanylate cyclase from rat lung. J Biol Chem. 1986 May 5;261(13):5817–5823. [PubMed] [Google Scholar]
  14. 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]
  15. Lang R. E., Thölken H., Ganten D., Luft F. C., Ruskoaho H., Unger T. Atrial natriuretic factor--a circulating hormone stimulated by volume loading. Nature. 1985 Mar 21;314(6008):264–266. doi: 10.1038/314264a0. [DOI] [PubMed] [Google Scholar]
  16. Longenecker J. P., Kilty L. A., Johnson L. K. Glucocorticoid influence on growth of vascular wall cells in culture. J Cell Physiol. 1982 Nov;113(2):197–202. doi: 10.1002/jcp.1041130203. [DOI] [PubMed] [Google Scholar]
  17. MERRIFIELD R. B. SOLID-PHASE PEPTIDE SYNTHESIS. 3. AN IMPROVED SYNTHESIS OF BRADYKININ. Biochemistry. 1964 Sep;3:1385–1390. doi: 10.1021/bi00897a032. [DOI] [PubMed] [Google Scholar]
  18. Meloche S., Ong H., Cantin M., De Léan A. Affinity cross-linking of atrial natriuretic factor to its receptor in bovine adrenal zona glomerulosa. J Biol Chem. 1986 Feb 5;261(4):1525–1528. [PubMed] [Google Scholar]
  19. Misono K. S., Grammer R. T., Rigby J. W., Inagami T. Photoaffinity labeling of atrial natriuretic factor receptor in bovine and rat adrenal cortical membranes. Biochem Biophys Res Commun. 1985 Aug 15;130(3):994–1001. doi: 10.1016/0006-291x(85)91713-9. [DOI] [PubMed] [Google Scholar]
  20. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  21. Napier M. A., Vandlen R. L., Albers-Schönberg G., Nutt R. F., Brady S., Lyle T., Winquist R., Faison E. P., Heinel L. A., Blaine E. H. Specific membrane receptors for atrial natriuretic factor in renal and vascular tissues. Proc Natl Acad Sci U S A. 1984 Oct;81(19):5946–5950. doi: 10.1073/pnas.81.19.5946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Petruzzelli L., Herrera R., Rosen O. M. Insulin receptor is an insulin-dependent tyrosine protein kinase: copurification of insulin-binding activity and protein kinase activity to homogeneity from human placenta. Proc Natl Acad Sci U S A. 1984 Jun;81(11):3327–3331. doi: 10.1073/pnas.81.11.3327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Scarborough R. M., Schenk D. B., McEnroe G. A., Arfsten A., Kang L. L., Schwartz K., Lewicki J. A. Truncated atrial natriuretic peptide analogs. Comparison between receptor binding and stimulation of cyclic GMP accumulation in cultured vascular smooth muscle cells. J Biol Chem. 1986 Oct 5;261(28):12960–12964. [PubMed] [Google Scholar]
  24. Schenk D. B., Hubert J. J., Leffert H. L. Use of a monoclonal antibody to quantify (Na+,K+)-ATPase activity and sites in normal and regenerating rat liver. J Biol Chem. 1984 Dec 10;259(23):14941–14951. [PubMed] [Google Scholar]
  25. Schenk D. B., Johnson L. K., Schwartz K., Sista H., Scarborough R. M., Lewicki J. A. Distinct atrial natriuretic factor receptor sites on cultured bovine aortic smooth muscle and endothelial cells. Biochem Biophys Res Commun. 1985 Mar 15;127(2):433–442. doi: 10.1016/s0006-291x(85)80179-0. [DOI] [PubMed] [Google Scholar]
  26. Schenk D. B., Phelps M. N., Porter J. G., Scarborough R. M., McEnroe G. A., Lewicki J. A. Identification of the receptor for atrial natriuretic factor on cultured vascular cells. J Biol Chem. 1985 Dec 5;260(28):14887–14890. [PubMed] [Google Scholar]
  27. Schneider W. J., Goldstein J. L., Brown M. S. Partial purification and characterization of the low density lipoprotein receptor from bovine adrenal cortex. J Biol Chem. 1980 Dec 10;255(23):11442–11447. [PubMed] [Google Scholar]
  28. Vandlen R. L., Arcuri K. E., Napier M. A. Identification of a receptor for atrial natriuretic factor in rabbit aorta membranes by affinity cross-linking. J Biol Chem. 1985 Sep 15;260(20):10889–10892. [PubMed] [Google Scholar]
  29. Wimalasena J., Moore P., Wiebe J. P., Abel J., Jr, Chen T. T. The porcine LH/hCG receptor. Characterization and purification. J Biol Chem. 1985 Sep 5;260(19):10689–10697. [PubMed] [Google Scholar]
  30. Yip C. C., Laing L. P., Flynn T. G. Photoaffinity labeling of atrial natriuretic factor receptors of rat kidney cortex plasma membranes. J Biol Chem. 1985 Jul 15;260(14):8229–8232. [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