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. 1980 Jun;77(6):3176–3180. doi: 10.1073/pnas.77.6.3176

REgulation of insulin binding to isolated hepatocytes: correction for bound hormone fragments linearizes Scatchard plots.

D B Donner
PMCID: PMC349577  PMID: 6997871

Abstract

Fragments of 125I-labeled insulin (125I-insulin) are rapidly produced after the initial cell binding process. After association of 125I-insulin with hepatocytes, hormone fragments remain bound to cells. At 23 degrees C, approximately 20% of the label bound at steady state was soluble in trichloroacetic acid. Correction of saturation experiments for the presence of bound trichloroacetic acid-soluble insulin fragments decreased the number and increased the affinity of 125I-insulin-binding sites. Label extracted from cell pellets recovered from saturation experiments was characterized by gel filtration; 59%, 55%, 40%, and 36% of the bound label was from intact hormone after recovery from incubation mixtures containing 0.18, 0.60, 4.6, and 7.5 nM applied 125I-insulin, respectively. At high applied 125I-insulin concentrations, the hormone predominantly interacted with lower affinity degradation systems. When binding data were corrected to assay for undegraded 125I-insulin only, curvilinear Scatchard plots were linearized. The insulin receptor is therefore not composed of heterogeneous or negatively cooperative sites. It is necessary to correct for retained fragments of 125I-insulin in order to define mechanisms through which hormone binding and cellular response may be regulated.

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Selected References

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

  1. Cuatrecasas P., Hollenberg M. D., Chang K. J., Bennett V. Hormone receptor complexes and their modulation of membrane function. Recent Prog Horm Res. 1975;31:37–94. doi: 10.1016/b978-0-12-571131-9.50006-2. [DOI] [PubMed] [Google Scholar]
  2. Cuatrecasas P. Properties of the insulin receptor isolated from liver and fat cell membranes. J Biol Chem. 1972 Apr 10;247(7):1980–1991. [PubMed] [Google Scholar]
  3. 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]
  4. Cuatrecasas P. The insulin receptor. Diabetes. 1972;21(2 Suppl):396–402. doi: 10.2337/diab.21.2.s396. [DOI] [PubMed] [Google Scholar]
  5. DeRubertis F. R., Chayoth R., Zor U., Field J. B. Evidence for persistent binding of biologically active thyrotropin to thyroid in vitro. Endocrinology. 1975 Jun;96(6):1579–1586. doi: 10.1210/endo-96-6-1579. [DOI] [PubMed] [Google Scholar]
  6. Donner D. B., Martin D. W., Sonenberg M. Accumulation of a slowly dissociable peptide hormone binding component by isolated target cells. Proc Natl Acad Sci U S A. 1978 Feb;75(2):672–676. doi: 10.1073/pnas.75.2.672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Donner D. B., Nakayama K., Tani S., Lutz U., Sonenberg M. The uptake and degradation of growth hormone fragments by isolated rat hepatocytes. J Biol Chem. 1978 Oct 10;253(19):6717–6723. [PubMed] [Google Scholar]
  8. Freychet P., Kahn R., Roth J., Neville D. M., Jr Insulin interactions with liver plasma membranes. Independence of binding of the hormone and its degradation. J Biol Chem. 1972 Jun 25;247(12):3953–3961. [PubMed] [Google Scholar]
  9. Gammeltoft S., Kristensen L. O., Sestoft L. Insulin receptors in isolated rat hepatocytes. Reassessment of binding properties and observations of the inactivation of insulin at 37 degrees C. J Biol Chem. 1978 Dec 10;253(23):8406–8413. [PubMed] [Google Scholar]
  10. Gavin J. R., 3rd, Gorden P., Roth J., Archer J. A., Buell D. N. Characteristics of the human lymphocyte insulin receptor. J Biol Chem. 1973 Mar 25;248(6):2202–2207. [PubMed] [Google Scholar]
  11. Gliemann J., Sonne O. Binding and receptor-mediated degradation of insulin in adipocytes. J Biol Chem. 1978 Nov 10;253(21):7857–7863. [PubMed] [Google Scholar]
  12. Hammond J. M., Jarett L., Mariz I. K., Daughaday W. H. Heterogeneity of insulin receptors on fat cell membranes. Biochem Biophys Res Commun. 1972 Nov 15;49(4):1122–1128. doi: 10.1016/0006-291x(72)90329-4. [DOI] [PubMed] [Google Scholar]
  13. Kahn C. R., Freychet P., Roth J., Neville D. M., Jr Quantitative aspects of the insulin-receptor interaction in liver plasma membranes. J Biol Chem. 1974 Apr 10;249(7):2249–2257. [PubMed] [Google Scholar]
  14. Knights E. B., Baylin S. B., Foster G. V. Control of polypeptide hormones by enzymatic degradation. Lancet. 1973 Sep 29;2(7831):719–723. doi: 10.1016/s0140-6736(73)92550-6. [DOI] [PubMed] [Google Scholar]
  15. Pollet R. J., Standaert M. L., Haase B. A. Insulin binding to the human lymphocyte receptor. Evaluation of the negative cooperativity model. J Biol Chem. 1977 Aug 25;252(16):5828–5834. [PubMed] [Google Scholar]
  16. Rodbell M. The problem of identifying the glucagon receptor. Fed Proc. 1973 Aug;32(8):1854–1858. [PubMed] [Google Scholar]
  17. Roth J., Kahn C. R., Lesniak M. A., Gorden P., De Meyts P., Megyesi K., Neville D. M., Jr, Gavin J. R., 3rd, Soll A. H., Freychet P. Receptors for insulin, NSILA-s, and growth hormone: applications to disease states in man. Recent Prog Horm Res. 1975;31:95–139. doi: 10.1016/b978-0-12-571131-9.50007-4. [DOI] [PubMed] [Google Scholar]
  18. Shechter Y., Hernaez L., Cuatrecasas P. Epidermal growth factor: biological activity requires persistent occupation of high-affinity cell surface receptors. Proc Natl Acad Sci U S A. 1978 Dec;75(12):5788–5791. doi: 10.1073/pnas.75.12.5788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Silhavy T. J., Szmelcman S., Boos W., Schwartz M. On the significance of the retention of ligand by protein. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2120–2124. doi: 10.1073/pnas.72.6.2120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Terris S., Steiner D. F. Binding and degradation of 125I-insulin by rat hepatocytes. J Biol Chem. 1975 Nov 10;250(21):8389–8398. [PubMed] [Google Scholar]
  21. de Meyts P., Roth J., Neville D. M., Jr, Gavin J. R., 3rd, Lesniak M. A. Insulin interactions with its receptors: experimental evidence for negative cooperativity. Biochem Biophys Res Commun. 1973 Nov 1;55(1):154–161. doi: 10.1016/s0006-291x(73)80072-5. [DOI] [PubMed] [Google Scholar]

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