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. 1977 Nov;74(11):4826–4830. doi: 10.1073/pnas.74.11.4826

Characterization of a common precursor to corticotropin and beta-lipotropin: cell-free synthesis of the precursor and identification of corticotropin peptides in the molecule.

J L Roberts, E Herbert
PMCID: PMC432048  PMID: 200934

Abstract

mRNA was isolated from cultures of AtT-20/D-16v tumor cells and translated in a mRNA-dependent reticulocyte cell-free system. The corticotropin (ACTH) product was purified by a double-antibody immunoprecipitation procedure using antisera specific for the alpha(1-24) sequence of ACTH. The product is shown by sodium dodecyl sulfate/gel electrophoresis and gel filtration on guanidine-HCl columns to be homogeneous with an apparent molecular weight (Mr) of 28,500. A product with the same molecular weight is synthesized when membrane-bound polysomes from D-16v cells are allowed to complete their nascent chains in a reticulocyte cell-free system. Mr 31,000 ACTH isolated from tumor cells has been separated into three proteins of different apparent Mr:29,000, 32,000, and 34,000. The cell-free product contains the same lysine-, methionine-, and phenylalanine-labeled tryptic peptides as the Mr 29,000 ACTH synthesized in the tumor cells. Tryptic peptide analysis also reveals the presence of the alpha(1-39) sequence in the Mr 28,500 cell-free product and suggests that there is only one copy of this sequence in the Mr 28,500 molecule.

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Selected References

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

  1. Chan S. J., Keim P., Steiner D. F. Cell-free synthesis of rat preproinsulins: characterization and partial amino acid sequence determination. Proc Natl Acad Sci U S A. 1976 Jun;73(6):1964–1968. doi: 10.1073/pnas.73.6.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Eipper B. A., Mains R. E., Guenzi D. High molecular weight forms of adrenocorticotropic hormone are glycoproteins. J Biol Chem. 1976 Jul 10;251(13):4121–4126. [PubMed] [Google Scholar]
  3. 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]
  4. Gurdon J. B., Lane C. D., Woodland H. R., Marbaix G. Use of frog eggs and oocytes for the study of messenger RNA and its translation in living cells. Nature. 1971 Sep 17;233(5316):177–182. doi: 10.1038/233177a0. [DOI] [PubMed] [Google Scholar]
  5. Jones R. E., Pulkrabek P., Grunberger D. Mouse pituitary tumor mRNA directed cell-free synthesis of polypeptides that are cross-reactive with adrenocorticotropic hormone antiserum. Biochem Biophys Res Commun. 1977 Feb 21;74(4):1490–1495. doi: 10.1016/0006-291x(77)90610-6. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Li C. H., Chung D. Isolation and structure of an untriakontapeptide with opiate activity from camel pituitary glands. Proc Natl Acad Sci U S A. 1976 Apr;73(4):1145–1148. doi: 10.1073/pnas.73.4.1145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Mains R. E., Eipper B. A. Biosynthesis of adrenocorticotropic hormone in mouse pituitary tumor cells. J Biol Chem. 1976 Jul 10;251(13):4115–4120. [PubMed] [Google Scholar]
  10. Mains R. E., Eipper B. A., Ling N. Common precursor to corticotropins and endorphins. Proc Natl Acad Sci U S A. 1977 Jul;74(7):3014–3018. doi: 10.1073/pnas.74.7.3014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Martial J. A., Baxter J. D., Goodman H. M., Seeburg P. H. Regulation of growth hormone messenger RNA by thyroid and glucocorticoid hormones. Proc Natl Acad Sci U S A. 1977 May;74(5):1816–1820. doi: 10.1073/pnas.74.5.1816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Nakanishi S., Taii S., Hirata Y., Matsukura S., Imura H. A large product of cell-free translation of messenger RNA coding for corticotropin. Proc Natl Acad Sci U S A. 1976 Dec;73(12):4319–4323. doi: 10.1073/pnas.73.12.4319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Orth D. N., Nicholson W. E., Mitchell W. M., Island D. P., Shapiro M., Byyny R. L. ACTH and MSH production by a single cloned mouse pituitary tumor cell line. Endocrinology. 1973 Feb;92(2):385–393. doi: 10.1210/endo-92-2-385. [DOI] [PubMed] [Google Scholar]
  14. Palmiter R. D. Ovalbumin messenger ribonucleic acid translation. Comparable rates of polypeptide initiation and elongation on ovalbumin and globin messenger ribonucleic acid in a rabbit reticulocyte lysate. J Biol Chem. 1973 Mar 25;248(6):2095–2106. [PubMed] [Google Scholar]
  15. Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  16. Pestka S. Inhibitors of ribosome functions. Annu Rev Microbiol. 1971;25:487–562. doi: 10.1146/annurev.mi.25.100171.002415. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Schechter I., Burstein Y. Marked hydrophobicity of the NH2-terminal extra piece of immunoglobulin light-chain precursors: possible physiological functions of the extra piece. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3273–3277. doi: 10.1073/pnas.73.9.3273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Scott A. P., Lowry P. J. Adrenocorticotrophic and melanocyte-stimulating peptides in the human pituitary. Biochem J. 1974 Jun;139(3):593–602. doi: 10.1042/bj1390593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Strauss A. W., Donohue A. M., Bennett C. D., Rodkey J. A., Alberts A. W. Rat liver preproalbumin: in vitro synthesis and partial amino acid sequence. Proc Natl Acad Sci U S A. 1977 Apr;74(4):1358–1362. doi: 10.1073/pnas.74.4.1358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Yalow R. S., Berson S. A. Size heterogeneity of immunoreactive human ACTH in plasma and in extracts of pituitary glands and ACTH-producing thymoma. Biochem Biophys Res Commun. 1971 Jul 16;44(2):439–445. doi: 10.1016/0006-291x(71)90620-6. [DOI] [PubMed] [Google Scholar]

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