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. 1979 Feb;76(2):635–639. doi: 10.1073/pnas.76.2.635

Initial site of insulin cleavage by insulin protease.

W C Duckworth, F B Stentz, M Heinemann, A E Kitabchi
PMCID: PMC383003  PMID: 284388

Abstract

Exposure of insulin to insulin protease (insulinase, EC 3.4.22.11), a degradative enzyme with considerable specificity toward insulin, results in alterations in the properties of the insulin molecule. Limited degradation by the enzyme results in a decrease in the ability of insulin to bind to membrane receptors with less change in the immunoprecipitability or trichloracetic acid precipitability of the hormone. Limited degradation by insulin protease also alters insulin so that the molecule becomes susceptible to attack by nonspecific endopeptidases which have no effect on unaltered insulin. These data demonstrate the production of an intermediate in the proteolytic degradation of insulin. By labeling with [14C]dansyl chloride, an insulin intermediate with three amino-terminal residues, glycine, phenylalanine, and leucine, was identified. Analysis of this intermediate demonstrated that it was composed of an intact A chain and a B chain cleaved between residues B16 and B17, with the three peptide chains held together by disulfide bonds. Based on these findings, we hypothesize that a stepwise degradation of insulin occurs in vivo and that an early step in the process is the cleavage between B16 and B17 that renders the molecule sucseptible to further degradation by nonspecific proteases.

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Selected References

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  1. Baskin F. K., Duckworth W. C., Kitabchi A. E. Sites of cleavage of glucagon by insulin-glucagon protease. Biochem Biophys Res Commun. 1975 Nov 3;67(1):163–169. doi: 10.1016/0006-291x(75)90297-1. [DOI] [PubMed] [Google Scholar]
  2. Baskin F. K., Kitabchi A. E. Substrate studies for insulin-specific protease. Eur J Biochem. 1973 Sep 3;37(3):489–496. doi: 10.1111/j.1432-1033.1973.tb03010.x. [DOI] [PubMed] [Google Scholar]
  3. Brush J. S. Purification and characterizatoion of a protease with specificity for insulin from rat muscle. Diabetes. 1971 Mar;20(3):140–145. [PubMed] [Google Scholar]
  4. Crofford O. B., Rogers N. L., Russell W. G. The effect of insulin on fat cells. An insulin degrading system extracted from plasma membranes of insulin responsive cells. Diabetes. 1972;21(2 Suppl):403–413. doi: 10.2337/diab.21.2.s403. [DOI] [PubMed] [Google Scholar]
  5. Cuatrecasas P. Membrane receptors. Annu Rev Biochem. 1974;43(0):169–214. doi: 10.1146/annurev.bi.43.070174.001125. [DOI] [PubMed] [Google Scholar]
  6. Dial L. K., Miyamoto S., Arquilla E. R. Modulation of 125I-insulin degradation by receptors in liver plasma membranes. Biochem Biophys Res Commun. 1977 Jan 24;74(2):545–552. doi: 10.1016/0006-291x(77)90338-2. [DOI] [PubMed] [Google Scholar]
  7. Duckworth W. C., Heinemann M. A., Kitabchi A. E. Purification of insulin-specific protease by affinity chromatography. Proc Natl Acad Sci U S A. 1972 Dec;69(12):3698–3702. doi: 10.1073/pnas.69.12.3698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Duckworth W. C., Heinemann M., Kitabchi A. E. Proteolytic degradation of insulin and glucagon. Biochim Biophys Acta. 1975 Feb 19;377(2):421–430. doi: 10.1016/0005-2744(75)90322-8. [DOI] [PubMed] [Google Scholar]
  9. Duckworth W. C. Insulin and glucagon degradation by the kidney. I. Subcellular distribution under different assay condition. Biochim Biophys Acta. 1976 Jul 21;437(2):518–530. doi: 10.1016/0304-4165(76)90020-9. [DOI] [PubMed] [Google Scholar]
  10. Duckworth W. C. Insulin and glucagon degradation by the kidney. II. Characterization of the mechanisms at neutral pH. Biochim Biophys Acta. 1976 Jul 21;437(2):531–542. doi: 10.1016/0304-4165(76)90021-0. [DOI] [PubMed] [Google Scholar]
  11. Duckworth W. C., Kitabchi A. E. Insulin and glucagon degradation by the same enzyme. Diabetes. 1974 Jun;23(6):536–543. doi: 10.2337/diab.23.6.536. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Fujino M., Wakimasu M., Taketomi S., Iwatsuka H. Insulin-like activities and insulin-potentiating actions of a modified insulin B21-26 fragment: beta-Ala-Arg-Gly-Phe-Phe-Tyr-NH2. Endocrinology. 1977 Aug;101(2):360–364. doi: 10.1210/endo-101-2-360. [DOI] [PubMed] [Google Scholar]
  14. Hammond J. M., Jarett L. Insulin degradation by isolated fat cells and their subcellular fractions. Diabetes. 1975 Nov;24(11):1011–1019. doi: 10.2337/diab.24.11.1011. [DOI] [PubMed] [Google Scholar]
  15. Izzo J. L., Bartlett J. W., Roncone A., Izzo M. J., Bale W. F. Physiological processes and dynamics in the disposition of small and large doses of biologically active and inactive 131-I-insulins in the rat. J Biol Chem. 1967 May 25;242(10):2343–2355. [PubMed] [Google Scholar]
  16. KATZEN H. M., STETTEN D., Jr Hepatic glutathione-insulin transhydrogenase. Diabetes. 1962 Jul-Aug;11:271–280. [PubMed] [Google Scholar]
  17. Kerr M. A., Kenny A. J. The molecular weight and properties of a neutral metallo-endopeptidase from rabbit kidney brush border. Biochem J. 1974 Mar;137(3):489–495. doi: 10.1042/bj1370489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kitabchi A. E. Proinsulin and C-peptide: a review. Metabolism. 1977 May;26(5):547–587. doi: 10.1016/0026-0495(77)90099-3. [DOI] [PubMed] [Google Scholar]
  19. Kitabchi A. E., Stentz F. B. Degradation of insulin and proinsulin by various organ homogenates of rat. Diabetes. 1972 Nov;21(11):1091–1101. doi: 10.2337/diab.21.11.1091. [DOI] [PubMed] [Google Scholar]
  20. Phelps B. H., Varandani P. T. Stimulation by insulin of cyclic AMP phosphodiesterase. Role of glutathione-insulin transhydrogenase. Biochem Biophys Res Commun. 1977 Mar 21;75(2):302–310. doi: 10.1016/0006-291x(77)91043-9. [DOI] [PubMed] [Google Scholar]
  21. Pullen R. A., Lindsay D. G., Wood S. P., Tickle I. J., Blundell T. L., Wollmer A., Krail G., Brandenburg D., Zahn H., Gliemann J. Receptor-binding region of insulin. Nature. 1976 Feb 5;259(5542):369–373. doi: 10.1038/259369a0. [DOI] [PubMed] [Google Scholar]
  22. Roth J. Peptide hormone binding to receptors: a review of direct studies in vitro. Metabolism. 1973 Aug;22(8):1059–1073. doi: 10.1016/0026-0495(73)90225-4. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Varandani P. T., Shroyer L. A., Nafz M. A. Sequential degradation of insulin by rat liver homogenates. Proc Natl Acad Sci U S A. 1972 Jul;69(7):1681–1684. doi: 10.1073/pnas.69.7.1681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Weitzel G., Eisele K., Stock W. Structure and activity of insulin, XIII. Specificity of the arginine-guanidino group in biologically active tetrapeptides of the insulin sequence B 22-25 (Arg-Gly-Phe-Phe). Hoppe Seylers Z Physiol Chem. 1975 May;356(5):583–590. doi: 10.1515/bchm2.1975.356.1.583. [DOI] [PubMed] [Google Scholar]

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