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. 1972 May;69(5):1277–1281. doi: 10.1073/pnas.69.5.1277

Affinity Chromatography and Purification of the Insulin Receptor of Liver Cell Membranes

Pedro Cuatrecasas 1
PMCID: PMC426681  PMID: 4504339

Abstract

Relatively simple and rapid procedures are described for the large-scale preparation of liver membranes that contain virtually all of the high affinity insulin-binding activity of liver homogenates. The presumed insulin recepotr, which is extracted from these membranes in soluble form with Triton X-100, can be further purified by ammonium sulfate fractionation (3-fold purification) or by diethylaminoethyl-cellulose chromatography (60-fold purification). Several insulin-agarose derivatives have been synthesized that can efficiently extract the insulin-binding protein from the detergent extracts of the membranes. The receptor macro-molecule can be eluted from the affinity columns in high (50-80%) yield by use of urea-containing buffers of moderately low pH. The receptor, thus purified by small-scale affinity chromatography experiments, approaches theoretical purity on the basis of its specific activity. This protein is purified about 250,000-fold from the liver homogenate by detergent extraction and affinity chromatography.

Keywords: insulin-agarose, detergent extraction

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Selected References

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

  1. Blatt L. M., Kim K. H. Regulation of hepatic glycogen synthetase. Stimulation of glycogen synthetase in an in vitro liver system by insulin bound to sepharose. J Biol Chem. 1971 Aug 25;246(16):4895–4898. [PubMed] [Google Scholar]
  2. Cuatrecasas P. Affinity chromatography. Annu Rev Biochem. 1971;40:259–278. doi: 10.1146/annurev.bi.40.070171.001355. [DOI] [PubMed] [Google Scholar]
  3. Cuatrecasas P., Desbuquois B., Krug F. Insulin-receptor interactions in liver cell membranes. Biochem Biophys Res Commun. 1971 Jul 16;44(2):333–339. doi: 10.1016/0006-291x(71)90604-8. [DOI] [PubMed] [Google Scholar]
  4. Cuatrecasas P., Illiano G. Membrane sialic acid and the mechanism of insulin action in adipose tissue cells. Effects of digestion with neuraminidase. J Biol Chem. 1971 Aug 25;246(16):4938–4946. [PubMed] [Google Scholar]
  5. Cuatrecasas P. Insulin--receptor interactions in adipose tissue cells: direct measurement and properties. Proc Natl Acad Sci U S A. 1971 Jun;68(6):1264–1268. doi: 10.1073/pnas.68.6.1264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cuatrecasas P. Interaction of insulin with the cell membrane: the primary action of insulin. Proc Natl Acad Sci U S A. 1969 Jun;63(2):450–457. doi: 10.1073/pnas.63.2.450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cuatrecasas P. Isolation of the insulin receptor of liver and fat-cell membranes (detergent-solubilized-( 125 I)insulin-polyethylene glycol precipitation-sephadex). Proc Natl Acad Sci U S A. 1972 Feb;69(2):318–322. doi: 10.1073/pnas.69.2.318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cuatrecasas P. Properties of the insulin receptor of isolated fat cell membranes. J Biol Chem. 1971 Dec 10;246(23):7265–7274. [PubMed] [Google Scholar]
  9. Cuatrecasas P. Protein purification by affinity chromatography. Derivatizations of agarose and polyacrylamide beads. J Biol Chem. 1970 Jun;245(12):3059–3065. [PubMed] [Google Scholar]
  10. Cuatrecasas P. Unmasking of insulin receptors in fat cells and fat cell membranes. Perturbation of membrane lipids. J Biol Chem. 1971 Nov;246(21):6532–6542. [PubMed] [Google Scholar]
  11. Cuatrecasas P., Wilchek M., Anfinsen C. B. Selective enzyme purification by affinity chromatography. Proc Natl Acad Sci U S A. 1968 Oct;61(2):636–643. doi: 10.1073/pnas.61.2.636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cutrecasas P. Perturbation of the insulin receptor of isolated fat cells with proteolytic enzymes. Direct measurement of insulin-receptor interactions. J Biol Chem. 1971 Nov;246(21):6522–6531. [PubMed] [Google Scholar]
  13. Freychet P., Roth J., Neville D. M., Jr Insulin receptors in the liver: specific binding of ( 125 I)insulin to the plasma membrane and its relation to insulin bioactivity. Proc Natl Acad Sci U S A. 1971 Aug;68(8):1833–1837. doi: 10.1073/pnas.68.8.1833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Freychet P., Roth J., Neville D. M., Jr Monoiodoinsulin: demonstration of its biological activity and binding to fat cells and liver membranes. Biochem Biophys Res Commun. 1971 Apr 16;43(2):400–408. doi: 10.1016/0006-291x(71)90767-4. [DOI] [PubMed] [Google Scholar]
  15. House P. D., Weidemann M. J. Characterization of an [125 I]-insulin binding plasma membrane fraction from rat liver. Biochem Biophys Res Commun. 1970 Nov 9;41(3):541–548. doi: 10.1016/0006-291x(70)90046-x. [DOI] [PubMed] [Google Scholar]
  16. Kono T., Barham F. W. The relationship between the insulin-binding capacity of fat cells and the cellular response to insulin. Studies with intact and trypsin-treated fat cells. J Biol Chem. 1971 Oct 25;246(20):6210–6216. [PubMed] [Google Scholar]
  17. Krug F., Desbuquois B., Cuatrecasas P. Glucagon affinity absorbents: selective binding of receptors of liver cell membranes. Nat New Biol. 1971 Dec 29;234(52):268–270. doi: 10.1038/newbio234268a0. [DOI] [PubMed] [Google Scholar]
  18. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  19. Oka T., Topper Y. J. Insulin-sepharose and the dynamics of insulin action. Proc Natl Acad Sci U S A. 1971 Sep;68(9):2066–2068. doi: 10.1073/pnas.68.9.2066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Turkington R. W. Stimulation of RNA synthesis in isolated mammary cells by insulin and prolactin bound to sepharose. Biochem Biophys Res Commun. 1970 Dec 9;41(5):1362–1367. doi: 10.1016/0006-291x(70)90239-1. [DOI] [PubMed] [Google Scholar]

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