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. 1980 Jan 15;186(1):35–45. doi: 10.1042/bj1860035

Polyribosomes in isolated liver cells. Preparative procedures, effects of incubation and correlation with protein synthesis.

A J Dickson, C I Pogson
PMCID: PMC1161501  PMID: 6154459

Abstract

Methods have been derived which permit the isolation of undergraded polyribosomes from isolated rat liver cells. Under the conditions used the polyribosome profile of hepatocytes immediately after isolation was essentially identical with that from intact liver. However, during incubation of cells in complex physiological media there was a progressive dissociation of polyribosomes. The addition of a variety of factors that produce reaggregation of polyribosomes in rat liver in vivo did not prevent dissociation during cell incubations. Although large polyribosomes were lost most rapidly, the albumin-synthesizing capacity of isolated cells was not selectively lost when compared with total protein synthesis. The significance of these results for the use of isolated hepatocytes in the study of liver protein synthesis is discussed.

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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. Distribution of radioactivity between the acid-soluble pool and the pools of RNA in the nuclear, nonsedimethable and ribosome fractions of rat liver after a single injection of lebaled orotic acid. Biochim Biophys Acta. 1968 Aug 23;166(1):48–57. doi: 10.1016/0005-2787(68)90489-9. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Bloemendal H., Benedetti E. L., Bont W. S. Preparation and characterization of free and membrane-bound polysomes. Methods Enzymol. 1974;30:313–327. doi: 10.1016/0076-6879(74)30034-1. [DOI] [PubMed] [Google Scholar]
  4. Clemens M. J., Pain V. M. Hormonal requirements for acute stimulation of rat liver polysome formation by amino acid feeding. Biochim Biophys Acta. 1974 Sep 13;361(3):345–357. doi: 10.1016/0005-2787(74)90377-3. [DOI] [PubMed] [Google Scholar]
  5. Crane L. J., Miller D. L. Plasma protein synthesis by isolated rat hepatocytes. J Cell Biol. 1977 Jan;72(1):11–25. doi: 10.1083/jcb.72.1.11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. DEBRO J. R., TARVER H., KORNER A. The determination of serum albumin and globulin by a new method. J Lab Clin Med. 1957 Nov;50(5):728–732. [PubMed] [Google Scholar]
  7. Daskal I., Ramirez S. A., Ballal R. N., Spohn W. H., Wu B., Busch H. Detergent lysis for isolation of intact polysomes of Nivikoff hepatoma ascites cells. Cancer Res. 1976 Mar;36(3):1026–1034. [PubMed] [Google Scholar]
  8. Dickson A. J., Pogson C. I. The metabolic integrity of hepatocytes in sustained incubations. FEBS Lett. 1977 Nov 1;83(1):27–32. doi: 10.1016/0014-5793(77)80634-0. [DOI] [PubMed] [Google Scholar]
  9. East A. G., Louis L. N., Hoffenberg R. Albumin synthesis by isolated rat liver cells. Exp Cell Res. 1973 Jan;76(1):41–46. doi: 10.1016/0014-4827(73)90416-3. [DOI] [PubMed] [Google Scholar]
  10. Ernest M. J., Chen C. L., Feigelson P. Induction of tyrosine aminotransferase synthesis in isolated liver cell suspensions. Absolute dependence of induction on glucocorticoids and glucagon or cyclic AMP. J Biol Chem. 1977 Oct 10;252(19):6783–6791. [PubMed] [Google Scholar]
  11. Feldhoff R. C., Taylor J. M., Jefferson L. S. Synthesis and secretion of rat albumin in vivo, in perfused liver, and in isolated hepatocytes. Effects of hypophysectomy and growth hormone treatment. J Biol Chem. 1977 Jun 10;252(11):3611–3616. [PubMed] [Google Scholar]
  12. Fishman B., Wurtman R. J., Munro H. N. Daily rhythms in hepatic polysome profiles and tyrosine transaminase activity: role of dietary protein. Proc Natl Acad Sci U S A. 1969 Oct;64(2):677–682. doi: 10.1073/pnas.64.2.677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Grant A. G., Black E. G. Polyribosome aggregation in isolated rat-liver cells. A comparison of extraction and incubation techniques. Eur J Biochem. 1974 Sep 1;47(2):397–401. doi: 10.1111/j.1432-1033.1974.tb03705.x. [DOI] [PubMed] [Google Scholar]
  14. Gribnau A. A., Schoenmakers J. G., Bloemendal H. Purification of rat liver RNase inhibitor and its effect on polyribosome integrity. Arch Biochem Biophys. 1969 Mar;130(1):48–52. doi: 10.1016/0003-9861(69)90007-1. [DOI] [PubMed] [Google Scholar]
  15. Jeejeebhoy K. N., Ho J., Greenberg G. R., Phillips M. J., Bruce-Robertson A., Sodtke U. Albumin, fibrinogen and transferrin synthesis in isolated rat hepatocyte suspensions. A model for the study of plasma protein synthesis. Biochem J. 1975 Jan;146(1):141–155. doi: 10.1042/bj1460141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jefferson L. S., Korner A. Influence of amino acid supply on ribosomes and protein synthesis of perfused rat liver. Biochem J. 1969 Mar;111(5):703–712. doi: 10.1042/bj1110703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Khairallah E. A., Mortimore G. E. Assessment of protein turnover in perfused rat liver. Evidence for amino acid compartmentation from differential labeling of free and tRNA-gound valine. J Biol Chem. 1976 Mar 10;251(5):1375–1384. [PubMed] [Google Scholar]
  18. Korner A., Gumbley J. M. Impaired ability of ribosomes to react with polyuridylic acid after hypophysectomy of the rat: removal of the disability by a cell sap factor. Nature. 1966 Jan 29;209(5022):505–506. doi: 10.1038/209505a0. [DOI] [PubMed] [Google Scholar]
  19. Marston F. A., Pogson C. I. A simple and rapid assay for tyrosine aminotransferase. FEBS Lett. 1977 Nov 15;83(2):277–280. doi: 10.1016/0014-5793(77)81022-3. [DOI] [PubMed] [Google Scholar]
  20. Morgan E. H., Peters T., Jr The biosynthesis of rat serum albumin. V. Effect of protein depletion and refeeding on albumin and transferrin synthesis. J Biol Chem. 1971 Jun 10;246(11):3500–3507. [PubMed] [Google Scholar]
  21. Nandi Majumdar A. P., Fjendbo Jorgensen A. J. Response of well-fed adrenalectomized rats to tryptophan force-feeding on hepatic protein and RNA synthesis. Biochem Med. 1976 Dec;16(3):266–276. doi: 10.1016/0006-2944(76)90032-6. [DOI] [PubMed] [Google Scholar]
  22. Palmiter R. D. Magnesium precipitation of ribonucleoprotein complexes. Expedient techniques for the isolation of undergraded polysomes and messenger ribonucleic acid. Biochemistry. 1974 Aug 13;13(17):3606–3615. doi: 10.1021/bi00714a032. [DOI] [PubMed] [Google Scholar]
  23. Peavy D. E., Taylor J. M., Jefferson L. S. Correlation of albumin production rates and albumin mRNA levels in livers of normal, diabetic, and insulin-treated diabetic rats. Proc Natl Acad Sci U S A. 1978 Dec;75(12):5879–5883. doi: 10.1073/pnas.75.12.5879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ramsey J. C., Steele W. J. A procedure for the quantitative recovery of homogeneous populations of undegraded free and bound polysomes from rat liver. Biochemistry. 1976 Apr 20;15(8):1704–1712. doi: 10.1021/bi00653a018. [DOI] [PubMed] [Google Scholar]
  25. Reddi K. K. Nature and possible origin of human serum ribonuclease. Biochem Biophys Res Commun. 1975 Nov 3;67(1):110–118. doi: 10.1016/0006-291x(75)90290-9. [DOI] [PubMed] [Google Scholar]
  26. Seglen P. O. Preparation of isolated rat liver cells. Methods Cell Biol. 1976;13:29–83. doi: 10.1016/s0091-679x(08)61797-5. [DOI] [PubMed] [Google Scholar]
  27. Seglen P. O. Preparation of rat liver cells. II. Effects of ions and chelators on tissue dispersion. Exp Cell Res. 1973 Jan;76(1):25–30. doi: 10.1016/0014-4827(73)90414-x. [DOI] [PubMed] [Google Scholar]
  28. Seglen P. O. Protein degradation in isolated rat hepatocytes is inhibited by ammonia. Biochem Biophys Res Commun. 1975 Sep 2;66(1):44–52. doi: 10.1016/s0006-291x(75)80292-0. [DOI] [PubMed] [Google Scholar]
  29. Seglen P. O., Solheim A. E. Valine uptake and incorporation into protein in isolated rat hepatocytes. Nature of the precursor pool for protein synthesis. Eur J Biochem. 1978 Apr;85(1):15–25. doi: 10.1111/j.1432-1033.1978.tb12208.x. [DOI] [PubMed] [Google Scholar]
  30. Sidransky H., Verney E., Sarma D. S. Effect of tryptophan on polyribosomes and protein synthesis in liver. Am J Clin Nutr. 1971 Jul;24(7):779–785. doi: 10.1093/ajcn/24.7.779. [DOI] [PubMed] [Google Scholar]
  31. Stirewalt W. S., Wool I. G., Cavicchi P. The relation of RNA and protein synthesis to the sedimentation of muscle ribosomes: effect of diabetes and insulin. Proc Natl Acad Sci U S A. 1967 Jun;57(6):1885–1892. doi: 10.1073/pnas.57.6.1885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Stone A. B. A simplified method for preparing sucrose gradients. Biochem J. 1974 Jan;137(1):117–118. doi: 10.1042/bj1370117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Swan P. B., McGown E., Richardson A., Henderson L. M. Factors affecting ribosomal aggregation in the isolated perfused rat liver. Am J Clin Nutr. 1971 Jul;24(7):777–778. doi: 10.1093/ajcn/24.7.777. [DOI] [PubMed] [Google Scholar]
  34. Tanaka K., Sato M., Tomita Y., Ichihara A. Biochemical studies on liver functions in primary cultured hepatocytes of adult rats. I. Hormonal effects on cell viability and protein synthesis. J Biochem. 1978 Oct;84(4):937–946. doi: 10.1093/oxfordjournals.jbchem.a132207. [DOI] [PubMed] [Google Scholar]
  35. Thomas G., Siegmann M., Bowman P. D., Gordon J. The isolation and analysis of polysomes and ribosomal RNA from cells growing in monolayer culture. Exp Cell Res. 1977 Sep;108(2):253–258. doi: 10.1016/s0014-4827(77)80032-3. [DOI] [PubMed] [Google Scholar]
  36. Wall R., Lippman S., Toth K., Fedoroff N. A general method for the large-scale isolation of polysomes and messenger RNA applied to MOPC 21 mouse myeloma tumors. Anal Biochem. 1977 Sep;82(1):115–129. doi: 10.1016/0003-2697(77)90140-3. [DOI] [PubMed] [Google Scholar]
  37. Wunner W. H., Bell J., Munro H. N. The effect of feeding with a tryptophan-free amino acid mixture on rat-liver polysomes and ribosomal ribonucleic acid. Biochem J. 1966 Nov;101(2):417–428. doi: 10.1042/bj1010417. [DOI] [PMC free article] [PubMed] [Google Scholar]

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