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. 1987 Sep 1;246(2):279–286. doi: 10.1042/bj2460279

Proteolytic conversion of proinsulin into insulin. Identification of a Ca2+-dependent acidic endopeptidase in isolated insulin-secretory granules.

H W Davidson 1, M Peshavaria 1, J C Hutton 1
PMCID: PMC1148274  PMID: 3318807

Abstract

The nature of the endoproteolytic activity involved in the post-translational processing of proinsulin has been investigated in rat insulinoma tissue. 125I-proinsulin was converted by lysed insulin-secretory granules into insulin via an intermediate form identified as des-dibasic-proinsulin. This activity co-localized with immunoreactive (endogenous) insulin and carboxypeptidase H upon subcellular fractionation of the tissue, indicating a secretory-granular location. Under optimized conditions, conversion was quantitative. Inhibitor studies demonstrated that processing occurred by a reaction sequence involving cleavage on the C-terminal side of the pairs of basic amino acids, with subsequent removal of the newly exposed basic residues by carboxypeptidase H. Endoproteolytic activity was abolished by EDTA and CDTA (1,2-cyclohexanediaminetetra-acetic acid), but not by 1,10-phenanthroline or by group-specific inhibitors of serine, thiol or acidic proteinases. Inhibition by EDTA and CDTA could be reversed by both Ca2+ and Zn2+, although the former appeared to be the ion of physiological importance. Addition of Ca2+ in the absence of chelators stimulated endoproteinase activity, with a maximal effect at 5 mM, a concentration consistent with the intragranular environment. Similarly the pH optimum of 5.5 coincides with the prevailing intragranular pH. Together these properties suggest that the Ca2+-dependent endopeptidase described here is involved in vivo in the proteolytic processing of proinsulin.

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Selected References

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

  1. Achstetter T., Wolf D. H. Hormone processing and membrane-bound proteinases in yeast. EMBO J. 1985 Jan;4(1):173–177. doi: 10.1002/j.1460-2075.1985.tb02333.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amory A., Foury F., Goffeau A. The purified plasma membrane ATPase of the yeast Schizosaccharomyces pombe forms a phosphorylated intermediate. J Biol Chem. 1980 Oct 10;255(19):9353–9357. [PubMed] [Google Scholar]
  3. Andersson T., Berggren P. O., Gylfe E., Hellman B. Amounts and distribution of intracellular magnesium and calcium in pancreatic beta-cells. Acta Physiol Scand. 1982 Feb;114(2):235–241. doi: 10.1111/j.1748-1716.1982.tb06977.x. [DOI] [PubMed] [Google Scholar]
  4. Ansorge S., Kirschke H., Friedrich K. Conversion of proinsulin into insulin by cathepsins B and L from rat liver lysosomes. Acta Biol Med Ger. 1977;36(11-12):1723–1727. [PubMed] [Google Scholar]
  5. 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]
  6. Chick W. L., Warren S., Chute R. N., Like A. A., Lauris V., Kitchen K. C. A transplantable insulinoma in the rat. Proc Natl Acad Sci U S A. 1977 Feb;74(2):628–632. doi: 10.1073/pnas.74.2.628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Davidson H. W., Hutton J. C. The insulin-secretory-granule carboxypeptidase H. Purification and demonstration of involvement in proinsulin processing. Biochem J. 1987 Jul 15;245(2):575–582. doi: 10.1042/bj2450575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Docherty K., Carroll R. J., Steiner D. F. Conversion of proinsulin to insulin: involvement of a 31,500 molecular weight thiol protease. Proc Natl Acad Sci U S A. 1982 Aug;79(15):4613–4617. doi: 10.1073/pnas.79.15.4613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Docherty K., Carroll R., Steiner D. F. Identification of a 31,500 molecular weight islet cell protease as cathepsin B. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3245–3249. doi: 10.1073/pnas.80.11.3245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Docherty K., Hutton J. C. Carboxypeptidase activity in the insulin secretory granule. FEBS Lett. 1983 Oct 3;162(1):137–141. doi: 10.1016/0014-5793(83)81065-5. [DOI] [PubMed] [Google Scholar]
  11. Docherty K., Hutton J. C., Steiner D. F. Cathepsin B-related proteases in the insulin secretory granule. J Biol Chem. 1984 May 25;259(10):6041–6044. [PubMed] [Google Scholar]
  12. Docherty K., Steiner D. F. Post-translational proteolysis in polypeptide hormone biosynthesis. Annu Rev Physiol. 1982;44:625–638. doi: 10.1146/annurev.ph.44.030182.003205. [DOI] [PubMed] [Google Scholar]
  13. Fletcher D. J., Quigley J. P., Bauer G. E., Noe B. D. Characterization of proinsulin- and proglucagon-converting activities in isolated islet secretory granules. J Cell Biol. 1981 Aug;90(2):312–322. doi: 10.1083/jcb.90.2.312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fricker L. D., Snyder S. H. Purification and characterization of enkephalin convertase, an enkephalin-synthesizing carboxypeptidase. J Biol Chem. 1983 Sep 25;258(18):10950–10955. [PubMed] [Google Scholar]
  15. Given B. D., Cohen R. M., Shoelson S. E., Frank B. H., Rubenstein A. H., Tager H. S. Biochemical and clinical implications of proinsulin conversion intermediates. J Clin Invest. 1985 Oct;76(4):1398–1405. doi: 10.1172/JCI112116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gold G., Gishizky M. L., Chick W. L., Grodsky G. M. Contrasting patterns of insulin biosynthesis, compartmental storage, and secretion. Rat tumor versus islet cells. Diabetes. 1984 Jun;33(6):556–561. doi: 10.2337/diab.33.6.556. [DOI] [PubMed] [Google Scholar]
  17. Habener J. F., Chang H. T., Potts J. T., Jr Enzymic processing of proparathyroid hormone by cell-free extracts of parathyroid glands. Biochemistry. 1977 Aug 23;16(17):3910–3917. doi: 10.1021/bi00636a029. [DOI] [PubMed] [Google Scholar]
  18. Hutton J. C., Davidson H. W., Grimaldi K. A., Peshavaria M. Biosynthesis of betagranin in pancreatic beta-cells. Identification of a chromogranin A-like precursor and its parallel processing with proinsulin. Biochem J. 1987 Jun 1;244(2):449–456. doi: 10.1042/bj2440449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hutton J. C., Davidson H. W., Peshavaria M. Proteolytic processing of chromogranin A in purified insulin granules. Formation of a 20 kDa N-terminal fragment (betagranin) by the concerted action of a Ca2+-dependent endopeptidase and carboxypeptidase H (EC 3.4.17.10). Biochem J. 1987 Jun 1;244(2):457–464. doi: 10.1042/bj2440457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hutton J. C., Penn E. J., Jackson P., Hales C. N. Isolation and characterization of calmodulin from an insulin-secreting tumour. Biochem J. 1981 Mar 1;193(3):875–885. doi: 10.1042/bj1930875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hutton J. C., Penn E. J., Peshavaria M. Isolation and characterisation of insulin secretory granules from a rat islet cell tumour. Diabetologia. 1982 Oct;23(4):365–373. doi: 10.1007/BF00253746. [DOI] [PubMed] [Google Scholar]
  22. Hutton J. C., Penn E. J., Peshavaria M. Low-molecular-weight constituents of isolated insulin-secretory granules. Bivalent cations, adenine nucleotides and inorganic phosphate. Biochem J. 1983 Feb 15;210(2):297–305. doi: 10.1042/bj2100297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hutton J. C., Peshavaria M. Proton-translocating Mg2+-dependent ATPase activity in insulin-secretory granules. Biochem J. 1982 Apr 15;204(1):161–170. doi: 10.1042/bj2040161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hutton J. C. The internal pH and membrane potential of the insulin-secretory granule. Biochem J. 1982 Apr 15;204(1):171–178. doi: 10.1042/bj2040171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Julius D., Brake A., Blair L., Kunisawa R., Thorner J. Isolation of the putative structural gene for the lysine-arginine-cleaving endopeptidase required for processing of yeast prepro-alpha-factor. Cell. 1984 Jul;37(3):1075–1089. doi: 10.1016/0092-8674(84)90442-2. [DOI] [PubMed] [Google Scholar]
  26. Kemmler W., Peterson J. D., Steiner D. F. Studies on the conversion of proinsulin to insulin. I. Conversion in vitro with trypsin and carboxypeptidase B. J Biol Chem. 1971 Nov 25;246(22):6786–6791. [PubMed] [Google Scholar]
  27. Kemmler W., Steiner D. F., Borg J. Studies on the conversion of proinsulin to insulin. 3. Studies in vitro with a crude secretion granule fraction isolated from rat islets of Langerhans. J Biol Chem. 1973 Jul 10;248(13):4544–4551. [PubMed] [Google Scholar]
  28. Kemmler W., Steiner D. F. Conversion of proinsulin to insulin in a subcellular fraction from rat islets. Biochem Biophys Res Commun. 1970 Dec 9;41(5):1223–1230. doi: 10.1016/0006-291x(70)90217-2. [DOI] [PubMed] [Google Scholar]
  29. Kitahara A., Ohtsuki H., Kirihata Y., Yamagata Y., Takano E., Kannagi R., Murachi T. Selective localization of calpain I (the low-Ca2+-requiring form of Ca2+-dependent cysteine proteinase) in B-cells of human pancreatic islets. FEBS Lett. 1985 May 6;184(1):120–124. doi: 10.1016/0014-5793(85)80666-9. [DOI] [PubMed] [Google Scholar]
  30. Loh Y. P., Brownstein M. J., Gainer H. Proteolysis in neuropeptide processing and other neural functions. Annu Rev Neurosci. 1984;7:189–222. doi: 10.1146/annurev.ne.07.030184.001201. [DOI] [PubMed] [Google Scholar]
  31. Loh Y. P., Parish D. C., Tuteja R. Purification and characterization of a paired basic residue-specific pro-opiomelanocortin converting enzyme from bovine pituitary intermediate lobe secretory vesicles. J Biol Chem. 1985 Jun 25;260(12):7194–7205. [PubMed] [Google Scholar]
  32. Nolan C., Margoliash E., Peterson J. D., Steiner D. F. The structure of bovine proinsulin. J Biol Chem. 1971 May 10;246(9):2780–2795. [PubMed] [Google Scholar]
  33. Orci L., Ravazzola M., Amherdt M., Madsen O., Perrelet A., Vassalli J. D., Anderson R. G. Conversion of proinsulin to insulin occurs coordinately with acidification of maturing secretory vesicles. J Cell Biol. 1986 Dec;103(6 Pt 1):2273–2281. doi: 10.1083/jcb.103.6.2273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Orci L., Ravazzola M., Amherdt M., Madsen O., Vassalli J. D., Perrelet A. Direct identification of prohormone conversion site in insulin-secreting cells. Cell. 1985 Sep;42(2):671–681. doi: 10.1016/0092-8674(85)90124-2. [DOI] [PubMed] [Google Scholar]
  35. Panyim S., Chalkley R. High resolution acrylamide gel electrophoresis of histones. Arch Biochem Biophys. 1969 Mar;130(1):337–346. doi: 10.1016/0003-9861(69)90042-3. [DOI] [PubMed] [Google Scholar]
  36. Parish D. C., Tuteja R., Altstein M., Gainer H., Loh Y. P. Purification and characterization of a paired basic residue-specific prohormone-converting enzyme from bovine pituitary neural lobe secretory vesicles. J Biol Chem. 1986 Nov 5;261(31):14392–14397. [PubMed] [Google Scholar]
  37. Perrie W. T., Perry S. V. An electrophoretic study of the low-molecular-weight components of myosin. Biochem J. 1970 Aug;119(1):31–38. doi: 10.1042/bj1190031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Robbins D. C., Blix P. M., Rubenstein A. H., Kanazawa Y., Kosaka K., Tager H. S. A human proinsulin variant at arginine 65. Nature. 1981 Jun 25;291(5817):679–681. doi: 10.1038/291679a0. [DOI] [PubMed] [Google Scholar]
  39. Sorenson R. L., Shank R. D., Lindall A. W. Effect of pH on conversion of proinsulin to insulin by a subcellular fraction of rat islets. Proc Soc Exp Biol Med. 1972 Feb;139(2):652–655. doi: 10.3181/00379727-139-36207. [DOI] [PubMed] [Google Scholar]
  40. Sorenson R. L., Steffes M. W., Lindall A. W. Subcellular localization of proinsulin to insulin conversion in isolated rat islets. Endocrinology. 1970 Jan;86(1):88–96. doi: 10.1210/endo-86-1-88. [DOI] [PubMed] [Google Scholar]
  41. Storer A. C., Cornish-Bowden A. Concentration of MgATP2- and other ions in solution. Calculation of the true concentrations of species present in mixtures of associating ions. Biochem J. 1976 Oct 1;159(1):1–5. doi: 10.1042/bj1590001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Virji M. A., Vassalli J. D., Estensen R. D., Reich E. Plasminogen activator of islets of Langerhans: modulation by glucose and correlation with insulin production. Proc Natl Acad Sci U S A. 1980 Feb;77(2):875–879. doi: 10.1073/pnas.77.2.875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Yamaji K., Tada K., Trakatellis A. C. On the biosynthesis of insulin in anglerfish islets. J Biol Chem. 1972 Jun 25;247(12):4080–4088. [PubMed] [Google Scholar]
  44. Yip C. C. A bovine pancreatic enzyme catalyzing the conversion of proinsulin to insulin. Proc Natl Acad Sci U S A. 1971 Jun;68(6):1312–1315. doi: 10.1073/pnas.68.6.1312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Yoi O. O., Seldin D. C., Spragg J., Pinkus G. S., Austen K. F. Sequential cleavage of proinsulin by human pancreatic kallikrein and a human pancreatic kininase. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3612–3616. doi: 10.1073/pnas.76.8.3612. [DOI] [PMC free article] [PubMed] [Google Scholar]

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