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
. 1976 Jun;73(6):1964–1968. doi: 10.1073/pnas.73.6.1964

Cell-free synthesis of rat preproinsulins: characterization and partial amino acid sequence determination.

S J Chan, P Keim, D F Steiner
PMCID: PMC430428  PMID: 778852

Abstract

Whole nucleic acid fractions of isolated rat islets of Langerhans greatly stimulate incorporation of radioactive amino acids into protein in a wheat germ ribosomal system. Approximately 30% of the synthetic product is precipitated with antisera to insulin or proinsulin. Characterization of this material by gel chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates a molecular mass of 11,500 daltons. Trypsin digestion releases intact A chain as well as tryptic fragments of the C-peptides and B chains of the two rat proinsulins. Automated sequence determination of labeled cell-free product purified by immunoprecipitation discloses the presence of 23 additional amino acids NH2-terminal to the B chain sequence of proinsulin. The partial amino acid sequence of this extension is as follows: NH2-X-Leu (Lys) Met-x-Phe-Leu-Phe-Leu-Leu (Lys) Leu-Leu-x-leu-X-X-X-X-X-X-X-X-proinsulin. On the basis of the above evidence we have designated this peptide preproinsulin.

Full text

PDF
1968

Selected References

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

  1. Blobel G., Dobberstein B. Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J Cell Biol. 1975 Dec;67(3):835–851. doi: 10.1083/jcb.67.3.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blobel G., Dobberstein B. Transfer of proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components. J Cell Biol. 1975 Dec;67(3):852–862. doi: 10.1083/jcb.67.3.852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boime I., Boguslawski S., Caine J. The translation of a human placental lactogen mRNA fraction in heterologous cell-free systems: the synthesis of a possible precursor. Biochem Biophys Res Commun. 1975 Jan 6;62(1):103–109. doi: 10.1016/s0006-291x(75)80411-6. [DOI] [PubMed] [Google Scholar]
  4. Boime I., McWilliams D., Szczesna E., Camel M. Synthesis of human placental lactogen messenger RNA as a function of gestation. J Biol Chem. 1976 Feb 10;251(3):820–825. [PubMed] [Google Scholar]
  5. Clark J. L., Steiner D. F. Insulin biosynthesis in the rat: demonstration of two proinsulins. Proc Natl Acad Sci U S A. 1969 Jan;62(1):278–285. doi: 10.1073/pnas.62.1.278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Devillers-Thiery A., Kindt T., Scheele G., Blobel G. Homology in amino-terminal sequence of precursors to pancreatic secretory proteins. Proc Natl Acad Sci U S A. 1975 Dec;72(12):5016–5020. doi: 10.1073/pnas.72.12.5016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gozes I., Schmitt H., Littauer U. Z. Translation in vitro of rat brain messenger RNA coding for tubulin and actin. Proc Natl Acad Sci U S A. 1975 Feb;72(2):701–705. doi: 10.1073/pnas.72.2.701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kemper B., Habener J. F., Ernst M. D., Potts J. T., Jr, Rich A. Pre-proparathyroid hormone: analysis of radioactive tryptic peptides and amino acid sequence. Biochemistry. 1976 Jan 13;15(1):15–19. doi: 10.1021/bi00646a003. [DOI] [PubMed] [Google Scholar]
  9. Kemper B., Habener J. F., Mulligan R. C., Potts J. T., Jr, Rich A. Pre-proparathyroid hormone: a direct translation product of parathyroid messenger RNA. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3731–3735. doi: 10.1073/pnas.71.9.3731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Lundquist R. E., Lazar J. M., Klein W. H., Clark J. M., Jr Translation of satellite tobacco necrosis virus ribonucleic acid. II. Initiation of in vitro translation in procaryotic and eucaryotic systems. Biochemistry. 1972 May 23;11(11):2014–2019. doi: 10.1021/bi00761a004. [DOI] [PubMed] [Google Scholar]
  12. Milstein C., Brownlee G. G., Harrison T. M., Mathews M. B. A possible precursor of immunoglobulin light chains. Nat New Biol. 1972 Sep 27;239(91):117–120. doi: 10.1038/newbio239117a0. [DOI] [PubMed] [Google Scholar]
  13. Roberts B. E., Paterson B. M. Efficient translation of tobacco mosaic virus RNA and rabbit globin 9S RNA in a cell-free system from commercial wheat germ. Proc Natl Acad Sci U S A. 1973 Aug;70(8):2330–2334. doi: 10.1073/pnas.70.8.2330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Rosen J. M., Woo S. L., Holder J. W., Means A. R., O'Malley B. W. Preparation and preliminary characterization of purified ovalbumin messenger RNA from the hen oviduct. Biochemistry. 1975 Jan 14;14(1):69–78. doi: 10.1021/bi00672a012. [DOI] [PubMed] [Google Scholar]
  15. Schechter I., Burstein Y. Partial sequence of the precursors of immunoglobulin light-chains of different subgroups: evidence that the immunoglobulin variable-region gene is larger than hitherto known. Biochem Biophys Res Commun. 1976 Jan 26;68(2):489–496. doi: 10.1016/0006-291x(76)91172-4. [DOI] [PubMed] [Google Scholar]
  16. Shih D. S., Kaesberg P. Translation of brome mosaic viral ribonucleic acid in a cell-free system derived from wheat embryo. Proc Natl Acad Sci U S A. 1973 Jun;70(6):1799–1803. doi: 10.1073/pnas.70.6.1799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Steiner D. F., Cunningham D., Spigelman L., Aten B. Insulin biosynthesis: evidence for a precursor. Science. 1967 Aug 11;157(3789):697–700. doi: 10.1126/science.157.3789.697. [DOI] [PubMed] [Google Scholar]
  18. Steiner D. F., Kemmler W., Tager H. S., Peterson J. D. Proteolytic processing in the biosynthesis of insulin and other proteins. Fed Proc. 1974 Oct;33(10):2105–2115. [PubMed] [Google Scholar]
  19. Suchanek G., Kindås-Mügge I., Kreil G. Translation of honeybee promelittin messenger RNA. Formation of a larger product in a mammalian cell-free system. Eur J Biochem. 1975 Dec 1;60(1):309–315. doi: 10.1111/j.1432-1033.1975.tb21005.x. [DOI] [PubMed] [Google Scholar]
  20. Sussman P. M., Tushinski R. J., Bancroft F. C. Pregrowth hormone: product of the translation in vitro of messenger RNA coding for growth hormone. Proc Natl Acad Sci U S A. 1976 Jan;73(1):29–33. doi: 10.1073/pnas.73.1.29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Tager H. S., Steiner D. F. Primary structures of the proinsulin connecting peptides of the rat and the horse. J Biol Chem. 1972 Dec 25;247(24):7936–7940. [PubMed] [Google Scholar]
  22. Yip C. C., Hew C. L., Hsu H. Translation of messenger ribonucleic acid from isolated pancreatic islets and human insulinomas. Proc Natl Acad Sci U S A. 1975 Dec;72(12):4777–4779. doi: 10.1073/pnas.72.12.4777. [DOI] [PMC free article] [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