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. 1972 Feb;69(2):505–509. doi: 10.1073/pnas.69.2.505

Insulin Biosynthesis: Studies of Islet Polyribosomes*

M A Permutt 1, D M Kipnis 1
PMCID: PMC426490  PMID: 4551147

Abstract

A method is described for separation of polyribosomes from as few as 25 isolated Islets of Langerhans, representing about 250 μg of pancreatic tissue. Islets are labeled with [3H]leucine and polysomes are isolated with liver polyribosomes, which serve as carrier and inhibitor of ribonuclease activity. Islets incubated at 37°C for 45 min in 15.5 mM glucose, then pulsed with [3H]leucine, incorporated about 2-3 times more label into nascent peptides on islet polysomes than islets incubated in 2.8 mM glucose. Sucrose gradient analysis of the labeled polysomes indicated that raising the glucose concentration preferentially stimulated synthesis of peptides on trisomes and larger polyribosomes. Islets incubated with [3H]leucine for 15 min incorporated two-thirds of the label into proteins on membrane-bound polysomes. At least 85% of the proinsulin synthesis during this time occurs on membrane-bound polysomes.

Keywords: nascent peptides, sucrose gradient analysis, gel filtration

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Selected References

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

  1. Blobel G., Potter V. R. Ribosomes in rat liver: an estimate of the percentage of free and membrane-bound ribosomes interacting with messenger RNA in vivo. J Mol Biol. 1967 Sep 28;28(3):539–542. doi: 10.1016/s0022-2836(67)80103-7. [DOI] [PubMed] [Google Scholar]
  2. Blobel G., Potter V. R. Studies on free and membrane-bound ribosomes in rat liver. I. Distribution as related to total cellular RNA. J Mol Biol. 1967 Jun 14;26(2):279–292. doi: 10.1016/0022-2836(67)90297-5. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. DAVOREN P. R. The isolation of insulin from a single cat pancreas. Biochim Biophys Acta. 1962 Sep 10;63:150–153. doi: 10.1016/0006-3002(62)90347-5. [DOI] [PubMed] [Google Scholar]
  5. Fan H., Penman S. Regulation of protein synthesis in mammalian cells. II. Inhibition of protein synthesis at the level of initiation during mitosis. J Mol Biol. 1970 Jun 28;50(3):655–670. doi: 10.1016/0022-2836(70)90091-4. [DOI] [PubMed] [Google Scholar]
  6. Ganoza M. C., Williams C. A. In vitro synthesis of different categories of specific protein by membrane-bound and free ribosomes. Proc Natl Acad Sci U S A. 1969 Aug;63(4):1370–1376. doi: 10.1073/pnas.63.4.1370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gelehrter T. D., Tomkins G. M. Posttranscriptional control of tyrosine aminotransferase synthesis by insulin. Proc Natl Acad Sci U S A. 1970 Jun;66(2):390–397. doi: 10.1073/pnas.66.2.390. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Henshaw E. C., Hirsch C. A., Morton B. E., Hiatt H. H. Control of protein synthesis in mammalian tissues through changes in ribosome activity. J Biol Chem. 1971 Jan 25;246(2):436–446. [PubMed] [Google Scholar]
  9. Hicks S. J., Drysdale J. W., Munro H. N. Preferential synthesis of ferritin and albumin by different populations of liver polysomes. Science. 1969 May 2;164(3879):584–585. doi: 10.1126/science.164.3879.584. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Lacy P. E., Kostianovsky M. Method for the isolation of intact islets of Langerhans from the rat pancreas. Diabetes. 1967 Jan;16(1):35–39. doi: 10.2337/diab.16.1.35. [DOI] [PubMed] [Google Scholar]
  12. Latham H., Darnell J. E. Distribution of mRNA in the cytoplasmic polyribosomes of the HeLa cell. J Mol Biol. 1965 Nov;14(1):1–12. doi: 10.1016/s0022-2836(65)80224-8. [DOI] [PubMed] [Google Scholar]
  13. Lin B. J., Haist R. E. Insulin biosynthesis: effects of carbohydrates and related compounds. Can J Physiol Pharmacol. 1969 Sep;47(9):791–801. doi: 10.1139/y69-133. [DOI] [PubMed] [Google Scholar]
  14. Lisowska-Bernstein B., Lamm M. E., Vassalli P. Synthesis of immunoglobulin heavy and light chains by the free ribosomes of a mouse plasma cell tumor. Proc Natl Acad Sci U S A. 1970 Jun;66(2):425–432. doi: 10.1073/pnas.66.2.425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Peters T., Jr, Fleischer B., Fleischer S. The biosynthesis of rat serum albumin. IV. Apparent passage of albumin through the Golgi apparatus during secretion. J Biol Chem. 1971 Jan 10;246(1):240–244. [PubMed] [Google Scholar]
  16. Rabinovitz M., Freedman M. L., Fisher J. M., Maxwell C. R. Translational control in hemoglobin syntheskis. Cold Spring Harb Symp Quant Biol. 1969;34:567–578. doi: 10.1101/sqb.1969.034.01.064. [DOI] [PubMed] [Google Scholar]
  17. Redman C. M. Biosynthesis of serum proteins and ferritin by free and attached ribosomes of rat liver. J Biol Chem. 1969 Aug 25;244(16):4308–4315. [PubMed] [Google Scholar]
  18. SIEKEVITZ P., PALADE G. E. A cytochemical study on the pancreas of the guinea pig. 5. In vivo incorporation of leucine-1-C14 into the chymotrypsinogen of various cell fractions. J Biophys Biochem Cytol. 1960 Jul;7:619–630. doi: 10.1083/jcb.7.4.619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. STAEHELIN T., WETTSTEIN F. O., OURA H., NOLL H. DETERMINATION OF THE CODING RATIO BASED ON MOLECULAR WEIGHT OF MESSENGER RIBONUCLEIC ACID ASSOCIATED WITH ERGOSOMES OF DIFFERENT AGGREGATE SIZE. Nature. 1964 Jan 18;201:264–270. doi: 10.1038/201264a0. [DOI] [PubMed] [Google Scholar]
  20. Sabatini D. D., Blobel G. Controlled proteolysis of nascent polypeptides in rat liver cell fractions. II. Location of the polypeptides in rough microsomes. J Cell Biol. 1970 Apr;45(1):146–157. doi: 10.1083/jcb.45.1.146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sherr C. J., Uhr J. W. Immunoglobulin synthesis and secretion. V. Incorporation of leucine and glucosamine into immunoglobulin on free and bound polyribosomes. Proc Natl Acad Sci U S A. 1970 Aug;66(4):1183–1189. doi: 10.1073/pnas.66.4.1183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Tanese T., Lazarus N. R., Devrim S., Recant L. Synthesis and release of proinsulin and insulin by isolated rat islets of Langerhans. J Clin Invest. 1970 Jul;49(7):1394–1404. doi: 10.1172/JCI106357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Van Ventrooij W. J., Henshaw E. C., Hirsch C. A. Nutritional effects on the polyribosome distribution and rate of protein synthesis in Ehrlich ascites tumor cells in culture. J Biol Chem. 1970 Nov 25;245(22):5947–5953. [PubMed] [Google Scholar]
  24. Vuust J., Piez K. A. Biosynthesis of the alpha chains of collagen studied by pulse-labeling in culture. J Biol Chem. 1970 Nov 25;245(22):6201–6207. [PubMed] [Google Scholar]
  25. Young D. A. Glucocorticoid action on rat thymus cells. Interrelationships between carbohydrate, protein, and adenine nucleotide metabolism and cortisol effects on these functions in vitro. J Biol Chem. 1969 Apr 25;244(8):2210–2217. [PubMed] [Google Scholar]

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