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. 1970 Dec 1;47(3):604–618. doi: 10.1083/jcb.47.3.604

ISOLATION OF RAT LIVER PLASMA MEMBRANES

Use of Nucleotide Pyrophosphatase and Phosphodiesterase I as Marker Enzymes

Oscar Touster 1, N N Aronson Jr 1, John T Dulaney 1, Herman Hendrickson 1
PMCID: PMC2108168  PMID: 5497542

Abstract

Nucleotide pyrophosphatase and phosphodiesterase I of rat liver have been found to be localized primarily in cell particulates highly enriched with respect to the most commonly accepted plasma membrane marker, 5'-nucleotidase, and therefore should themselves be assigned a plasma membrane localization. The observation that plasma membranes sediment in isotonic sucrose with both nuclear and microsomal fractions was exploited to obtain plasma membrane preparations from each fraction. Both preparations are similar in chemical and enzymic composition. Moreover, the preparative method developed in this study appears to give the best combination of yield, purity, and reproducibility available. The question of the possible identity of nucleotide pyrophosphatase and phosphodiesterase I is considered, and evidence is presented suggesting that these activities may be manifestations of the same enzyme.

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Selected References

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  1. APPELMANS F., WATTIAUX R., DE DUVE C. Tissue fractionation studies. 5. The association of acid phosphatase with a special class of cytoplasmic granules in rat liver. Biochem J. 1955 Mar;59(3):438–445. doi: 10.1042/bj0590438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ashworth L. A., Green C. Plasma membranes: phospholipid and sterol content. Science. 1966 Jan 14;151(3707):210–211. doi: 10.1126/science.151.3707.210. [DOI] [PubMed] [Google Scholar]
  3. BLIGH E. G., DYER W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
  4. Bell E. I-DNA: Its packaging into I-somes and its relation to protein synthesis during differentiation. Nature. 1969 Oct 25;224(5217):326–328. doi: 10.1038/224326a0. [DOI] [PubMed] [Google Scholar]
  5. Berman H. M., Gram W., Spirtes M. A. An improved, reproducible method opreparing rat liver plasma cell membranes in buffered isotonic sucrose. Biochim Biophys Acta. 1969 Jun 3;183(1):10–18. doi: 10.1016/0005-2736(69)90124-2. [DOI] [PubMed] [Google Scholar]
  6. CHAUVEAU J., MOULE Y., ROUILLER C. Isolation of pure and unaltered liver nuclei morphology and biochemical composition. Exp Cell Res. 1956 Aug;11(2):317–321. doi: 10.1016/0014-4827(56)90107-0. [DOI] [PubMed] [Google Scholar]
  7. Coleman R., Michell R. H., Finean J. B., Hawthorne J. N. A purified plasma membrane fraction isolated from rat liver under isotonic conditions. Biochim Biophys Acta. 1967 Sep 9;135(4):573–579. doi: 10.1016/0005-2736(67)90089-2. [DOI] [PubMed] [Google Scholar]
  8. Coleman R. Some features of the lipid composition of rat liver surface and cytoplasmic membranes. Chem Phys Lipids. 1968 Feb;2(1):144–146. doi: 10.1016/0009-3084(68)90039-x. [DOI] [PubMed] [Google Scholar]
  9. DE DUVE C., PRESSMAN B. C., GIANETTO R., WATTIAUX R., APPELMANS F. Tissue fractionation studies. 6. Intracellular distribution patterns of enzymes in rat-liver tissue. Biochem J. 1955 Aug;60(4):604–617. doi: 10.1042/bj0600604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dod B. J., Gray G. M. The lipid composition of rat-liver plasma membranes. Biochim Biophys Acta. 1968 Apr 29;150(3):397–404. doi: 10.1016/0005-2736(68)90138-7. [DOI] [PubMed] [Google Scholar]
  11. EMMELOT P., BOS C. J., BENEDETTI E. L., RUEMKE P. STUDIES ON PLASMA MEMBRANES. I. CHEMICAL COMPOSITION AND ENZYME CONTENT OF PLASMA MEMBRANES ISOLATED FROM RAT LIVER. Biochim Biophys Acta. 1964 Jul 15;90:126–145. doi: 10.1016/0304-4165(64)90125-4. [DOI] [PubMed] [Google Scholar]
  12. Erecińska M., Sierakowska H., Shugar D. Intracellular localization of phosphodiesterases I and II in rat liver. Eur J Biochem. 1969 Dec;11(3):465–471. doi: 10.1111/j.1432-1033.1969.tb00796.x. [DOI] [PubMed] [Google Scholar]
  13. Evans W. H. Fractionation of liver plasma membranes prepared by zonal centrifugation. Biochem J. 1970 Mar;116(5):833–842. doi: 10.1042/bj1160833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Futai M., Mizuno D. A new phosphodiesterase forming nucleoside 5'-monophosphate from rat liver. Its partial purification and substrate specificity for nicotinamide adenine dinucleotide and oligonucleotides. J Biol Chem. 1967 Nov 25;242(22):5301–5307. [PubMed] [Google Scholar]
  15. Galliard T., Michell R. H., Hawthorne J. N. Incorporation of phosphate into diphosphoinositide by subcellular fractions from liver. Biochim Biophys Acta. 1965 Dec 2;106(3):551–563. doi: 10.1016/0005-2760(65)90071-8. [DOI] [PubMed] [Google Scholar]
  16. Glaser L., Melo A., Paul R. Uridine diphosphate sugar hydrolase. Purification of enzyme and protein inhibitor. J Biol Chem. 1967 Apr 25;242(8):1944–1954. [PubMed] [Google Scholar]
  17. Graham J. M., Higgins J. A., Green C. The isolation of rat liver plasma membrane fragments. Biochim Biophys Acta. 1968 Mar 1;150(2):303–305. doi: 10.1016/0005-2736(68)90173-9. [DOI] [PubMed] [Google Scholar]
  18. HOLLMANN S., TOUSTER O. Alterations in tissue levels of uridine diphosphate glucose dehydrogenase, uridine diphosphate glucuronic acid pyrophosphatase and glucuronyl transferase induced by substances influencing the production of ascorbic acid. Biochim Biophys Acta. 1962 Aug 13;62:338–352. doi: 10.1016/0006-3002(62)90048-3. [DOI] [PubMed] [Google Scholar]
  19. JACOBSON K. B., KAPLAN N. O. Distribution of enzymes cleaving pyridine nucleotides in animal tissues. J Biophys Biochem Cytol. 1957 Jan 25;3(1):31–43. doi: 10.1083/jcb.3.1.31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kashnig D. M., Kasper C. B. Isolation, morphology, and composition of the nuclear membrane from rat liver. J Biol Chem. 1969 Jul 25;244(14):3786–3792. [PubMed] [Google Scholar]
  21. LUFT J. H. Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol. 1961 Feb;9:409–414. doi: 10.1083/jcb.9.2.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lansing A. I., Belkhode M. L., Lynch W. E., Lieberman I. Enzymes of plasma membranes of liver. J Biol Chem. 1967 Apr 25;242(8):1772–1775. [PubMed] [Google Scholar]
  23. Lieberman I., Lansing A. I., Lynch W. E. Nucleoside triphosphate pyrophosphohydrolase of the plasma membrane of the liver cell. J Biol Chem. 1967 Feb 25;242(4):736–739. [PubMed] [Google Scholar]
  24. MAGGIO R., SIEKEVITZ P., PALADE G. E. STUDIES ON ISOLATED NUCLEI. I. ISOLATION AND CHEMICAL CHARACTERIZATION OF A NUCLEAR FRACTION FROM GUINEA PIG LIVER. J Cell Biol. 1963 Aug;18:267–291. doi: 10.1083/jcb.18.2.267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Michell R. H., Harwood J. L., Coleman R., Hawthorne J. N. Characteristics of rat liver phosphatidylinositol kinase and its presence in the plasma membrane. Biochim Biophys Acta. 1967 Dec 5;144(3):649–658. doi: 10.1016/0005-2760(67)90053-7. [DOI] [PubMed] [Google Scholar]
  26. NEVILLE D. M., Jr The isolation of a cell membrane fraction from rat liver. J Biophys Biochem Cytol. 1960 Oct;8:413–422. doi: 10.1083/jcb.8.2.413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Neu H. C. The 5'-nucleotidase of Escherichia coli. I. Purification and properties. J Biol Chem. 1967 Sep 10;242(17):3896–3904. [PubMed] [Google Scholar]
  28. PATTERSON M. K., Jr, TOUSTER O. Intracellular distribution of sialic acid and its relationship to membranes. Biochim Biophys Acta. 1962 Jan 29;56:626–628. doi: 10.1016/0006-3002(62)90623-6. [DOI] [PubMed] [Google Scholar]
  29. PHILLIPS A. H., LANGDON R. G. Hepatic triphosphopyridine nucleotide-cytochrome c reductase: isolation, characterization, and kinetic studies. J Biol Chem. 1962 Aug;237:2652–2660. [PubMed] [Google Scholar]
  30. Pfleger R. C., Anderson N. G., Snyder F. Lipid class and fatty acid composition of rat liver plasma membranes isolated by zonal centrifugation. Biochemistry. 1968 Aug;7(8):2826–2833. doi: 10.1021/bi00848a019. [DOI] [PubMed] [Google Scholar]
  31. Razzell W. E. Polynucleotidase activity of animal and plant tissue phosphodiesterases. Can J Biochem. 1968 Jan;46(1):1–7. doi: 10.1139/o68-001. [DOI] [PubMed] [Google Scholar]
  32. SEARCY R. L., BERGQUIST L. M., JUNG R. C. Rapid ultramicro estimation of serum total cholesterol. J Lipid Res. 1960 Jul;1:349–351. [PubMed] [Google Scholar]
  33. Schiller S., Hille F. Nucleotide diphosphate sugar pyrophosphatase activity in the skin of alloxan-diabetic rats. Biochim Biophys Acta. 1969 Apr 1;177(2):377–379. doi: 10.1016/0304-4165(69)90156-1. [DOI] [PubMed] [Google Scholar]
  34. Schnaitman C., Erwin V. G., Greenawalt J. W. The submitochondrial localization of monoamine oxidase. An enzymatic marker for the outer membrane of rat liver mitochondria. J Cell Biol. 1967 Mar;32(3):719–735. doi: 10.1083/jcb.32.3.719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schneider W. C., Kuff E. L. The association of deoxyribonucleic acid with liver microsomes. J Biol Chem. 1969 Sep 25;244(18):4843–4851. [PubMed] [Google Scholar]
  36. Skipski V. P., Barclay M., Archibald F. M., Terebus-Kekish O., Reichman E. S., Good J. J. Lipid composition of rat liver cell membranes. Life Sci. 1965 Sep;4(17):1673–1680. doi: 10.1016/0024-3205(65)90213-4. [DOI] [PubMed] [Google Scholar]
  37. Song C. S., Rubin W., Rifkind A. B., Kappas A. Plasma membranes of the rat liver. Isolation and enzymatic characterization of a fraction rich in bile canaliculi. J Cell Biol. 1969 Apr;41(1):124–132. doi: 10.1083/jcb.41.1.124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sottocasa G. L., Kuylenstierna B., Ernster L., Bergstrand A. An electron-transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study. J Cell Biol. 1967 Feb;32(2):415–438. doi: 10.1083/jcb.32.2.415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Stein Y., Widnell C., Stein O. Acylation of lysophosphatides by plasma membrane fractions of rat liver. J Cell Biol. 1968 Oct;39(1):185–192. doi: 10.1083/jcb.39.1.185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Takeuchi M., Terayama H. Preparation and chemical composition of rat liver cell membranes. Exp Cell Res. 1965 Oct;40(1):32–44. doi: 10.1016/0014-4827(65)90287-9. [DOI] [PubMed] [Google Scholar]
  41. Thines-Sempoux D., Amar-Costesec A., Beaufay H., Berthet J. The association of cholesterol, 5'-nucleotidase, and alkaline phosphodiesterase I with a distinct group of microsomal particles. J Cell Biol. 1969 Oct;43(1):189–192. doi: 10.1083/jcb.43.1.189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. VENABLE J. H., COGGESHALL R. A SIMPLIFIED LEAD CITRATE STAIN FOR USE IN ELECTRON MICROSCOPY. J Cell Biol. 1965 May;25:407–408. doi: 10.1083/jcb.25.2.407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Vignais P. M., Nachbaur J. A critical evaluation of the contamination, by lysosomes, of preparations of outer membrane of mitochondria. Biochem Biophys Res Commun. 1968 Oct 24;33(2):307–314. doi: 10.1016/0006-291x(68)90785-7. [DOI] [PubMed] [Google Scholar]
  44. WARREN L. The thiobarbituric acid assay of sialic acids. J Biol Chem. 1959 Aug;234(8):1971–1975. [PubMed] [Google Scholar]
  45. Wallach D. F., Kamat V. B. The contribution of sialic acid to the surface change of fragments of plasma membrane and endoplasmic reticulum. J Cell Biol. 1966 Sep;30(3):660–663. doi: 10.1083/jcb.30.3.660. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wattiaux-De Coninck S., Wattiaux R. Nucleosidediphosphatase activity in plasma membrane of rat liver. Biochim Biophys Acta. 1969 Jun 3;183(1):118–128. doi: 10.1016/0005-2736(69)90135-7. [DOI] [PubMed] [Google Scholar]
  47. Weaver R. A., Boyle W. Purification of plasma membranes of rat liver. Application of zonal centrifugation to isolation of cell membranes. Biochim Biophys Acta. 1969 Apr;173(3):377–388. doi: 10.1016/0005-2736(69)90003-0. [DOI] [PubMed] [Google Scholar]
  48. Weiss L., Mayhew E. Ribonuclease-susceptible charged groups at the surface of Ehrlich ascites tumour cells. Int J Cancer. 1969 Sep 15;4(5):626–635. doi: 10.1002/ijc.2910040507. [DOI] [PubMed] [Google Scholar]
  49. Widnell C. C., Unkeless J. C. Partial purification of a lipoprotein with 5'-nucleotidase activity from membranes of rat liver cells. Proc Natl Acad Sci U S A. 1968 Nov;61(3):1050–1057. doi: 10.1073/pnas.61.3.1050. [DOI] [PMC free article] [PubMed] [Google Scholar]

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