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. 1973 Dec 1;59(3):601–614. doi: 10.1083/jcb.59.3.601

MOLECULES AT THE EXTERNAL NUCLEAR SURFACE

Sialic Acid of Nuclear Membranes and Electrophoretic Mobility of Isolated Nuclei and Nucleoli

H Bruce Bosmann 1
PMCID: PMC2109112  PMID: 4761332

Abstract

The molecules occurring as terminal residues on the external surfaces of nuclei prepared from rat liver by either sucrose-CaCl2 or citric acid methods and nucleoli derived from the sucrose-CaCl2 nuclei were studied chemically and electrokinetically. In 0.0145 M NaCl, 4.5% sorbitol, and 0.6 mM NaHCO3 with pH 7.2 ± 0.1 at 25°C, the sucrose-CaCl2 nuclei had an electrophoretic mobility of -1.92 µm/s/V/cm, the citric acid nuclei, -1.63 µm/s/V/cm, and the nucleoli, -2.53 µm/s/V/cm. The citric acid nuclei and the nucleoli contained no measurable sialic acid. The sucrose-CaCl2 nuclei contained 0.7 nmol of sialic acid/mg nuclear protein; this was essentially located in the nuclear envelope. Treatment of these nuclei with 50 µg neuraminidase/mg protein resulted in release of 0.63 nmol of sialic acid/mg nuclear protein; treatment with 1 % trypsin caused release of 0.39 nmol of the sialic acid/mg nuclear protein. The pH-mobility curves for the particles indicated the sucrose-CaCl2 nuclei surface had an acid-dissociable group of pK. ∼2.7 while the pK for the nucleoli was considerably lower. Nucleoli treated with 50 µg neuraminidase/mg particle protein had a mobility of -2.53 µm/s/V/cm while sucrose-CaCl2 nuclei similarly treated had a mobility of -1.41 µm/s/V/cm. Hyaluronidase at 50 µg/mg protein had no effect on nucleoli mobility but decreased the sucrose-CaCl2 nuclei mobility to -1.79 µm/s/V/cm. Trypsin at 1 % elevated the electrophoretic mobility of the sucrose-CaCl2 nuclei slightly but decreased the mobility of the nucleoli to -2.09 µm/s/V/cm. DNase at 50 µg/mg protein had no effect on the mobility of the isolated sucrose-CaCl2 nuclei but decreased the electrophoretic mobility of the nucleoli to -1.21 µm/s/V/cm. RNase at 50 µg/mg protein also had no effect on the electrophoretic mobility of the sucrose-CaCl2 nuclei but decreased the nucleoli mobility to -2.10 µm/s/V/cm. Concanavalin A at 50 µg/mg protein did not alter the nucleoli electrophoretic mobility but decreased the sucrose-CaCl2 nuclei electrophoretic mobility to -1.64 µm/s/V/cm. The results are interpreted to mean that the sucrose-CaCl2 nuclear external surface contains terminal sialic acid residues in trypsin-sensitive glycoproteins, contains small amounts of hyaluronic acid, is completely devoid of nucleic acids, and binds concanavalin A. The nucleolus surface is interpreted to contain a complex made up of protein, RNA, and primarily DNA, to be devoid of sialic acid and hyaluronic acid, and not to bind concanavalin A.

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Selected References

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  1. Agutter P. S. The isolation of the envelopes of rat liver nuclei. Biochim Biophys Acta. 1972 Jan 17;255(1):397–401. doi: 10.1016/0005-2736(72)90039-9. [DOI] [PubMed] [Google Scholar]
  2. BURTON K. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J. 1956 Feb;62(2):315–323. doi: 10.1042/bj0620315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berezney R., Funk L. K., Crane F. L. The isolation of nuclear membrane from a large-scale preparation of bovine liver nuclei. Biochim Biophys Acta. 1970 Jun 2;203(3):531–546. doi: 10.1016/0005-2736(70)90190-2. [DOI] [PubMed] [Google Scholar]
  4. Bernacki R. J., Bosmann H. B. Activity of a rat-liver glycoprotein: sialyltransferase utilizing desialyzed prothrombin as an acceptor in normal and hypothrombinemic animals. Eur J Biochem. 1973 Feb 15;33(1):49–58. doi: 10.1111/j.1432-1033.1973.tb02653.x. [DOI] [PubMed] [Google Scholar]
  5. Bosmann H. B., Carlson W. Identification of sialic acid at the nerve ending periphery and electrophoretic mobility of isolated synaptosomes. Exp Cell Res. 1972 Jun;72(2):436–440. doi: 10.1016/0014-4827(72)90012-2. [DOI] [PubMed] [Google Scholar]
  6. Bosmann H. B., Hagopian A., Eylar E. H. Cellular membranes: the isolation and characterization of the plasma and smooth membranes of HeLa cells. Arch Biochem Biophys. 1968 Oct;128(1):51–69. doi: 10.1016/0003-9861(68)90008-8. [DOI] [PubMed] [Google Scholar]
  7. Bosmann H. B., Jackson J. J. Glycoprotein structure: the carbohydrate of bovine corneal collagen. Biochim Biophys Acta. 1968 Nov 12;170(1):6–14. doi: 10.1016/0304-4165(68)90156-6. [DOI] [PubMed] [Google Scholar]
  8. Bosmann H. B., Kessel D. Inhibition of glycoprotein synthesis in L5178Y mouse leukaemic cells by L-asparaginase in vitro. Nature. 1970 May 30;226(5248):850–851. doi: 10.1038/226850a0. [DOI] [PubMed] [Google Scholar]
  9. Bosmann H. B., Myers M. W., Dehond D., Ball R., Case K. R. Mitochondrial autonomy. Sialic acid residues on the surface of isolated rat cerebral cortex and liver mitochondria. J Cell Biol. 1972 Oct;55(1):147–160. doi: 10.1083/jcb.55.1.147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bosmann H. B., Pike G. Z. Membrane marker enzymes: isolation, purification, and properties of 5'-nucleotidase from rat cerebellum. Biochim Biophys Acta. 1971 Feb 10;227(2):402–412. doi: 10.1016/0005-2744(71)90071-4. [DOI] [PubMed] [Google Scholar]
  11. Bosmann H. B. Platelet adhesiveness and aggregation. II. Surface sialic acid, glycoprotein: N-acetylneuraminic acid transferase, and neuraminidase of human blood platelets. Biochim Biophys Acta. 1972 Oct 25;279(3):456–474. [PubMed] [Google Scholar]
  12. Burger M. M., Goldberg A. R. Identification of a tumor-specific determinant on neoplastic cell surfaces. Proc Natl Acad Sci U S A. 1967 Feb;57(2):359–366. doi: 10.1073/pnas.57.2.359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Burger M. M., Noonan K. D. Restoration of normal growth by covering of agglutinin sites on tumour cell surface. Nature. 1970 Nov 7;228(5271):512–515. doi: 10.1038/228512a0. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Chaudhuri S., Lieberman I. The surface of the liver cell after partial hepatectomy. Biochem Biophys Res Commun. 1965 Jul 26;20(3):303–309. doi: 10.1016/0006-291x(65)90364-5. [DOI] [PubMed] [Google Scholar]
  16. Cline M. J., Livingston D. C. Binding of 3 H-concanavalin A by normal and transformed cells. Nat New Biol. 1971 Aug 4;232(31):155–156. doi: 10.1038/newbio232155a0. [DOI] [PubMed] [Google Scholar]
  17. Franke W. W., Deumling B., Baerbelermen, Jarasch E. D., Kleinig H. Nuclear membranes from mammalian liver. I. Isolation procedure and general characterization. J Cell Biol. 1970 Aug;46(2):379–395. doi: 10.1083/jcb.46.2.379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. HEARD D. H., SEAMAN G. V. The influence of pH and ionic strength on the electrokinetic stability of the human erythrocyte membrane. J Gen Physiol. 1960 Jan;43:635–654. doi: 10.1085/jgp.43.3.635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Inbar M., Sachs L. Interaction of the carbohydrate-binding protein concanavalin A with normal and transformed cells. Proc Natl Acad Sci U S A. 1969 Aug;63(4):1418–1425. doi: 10.1073/pnas.63.4.1418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Johnston I. R., Mathias A. P., Pennington F., Ridge D. The fractionation of nuclei from mammalian cells by zonal centrifugation. Biochem J. 1968 Aug;109(1):127–135. doi: 10.1042/bj1090127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. KISHIMOTO S., LIEBERMAN I. A NUCLEAR MEMBRANE CHANGE AFTER PARTIAL HEPATECTOMY. J Cell Biol. 1964 Dec;23:511–518. doi: 10.1083/jcb.23.3.511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kaneko I., Sato H., Ukita T. Binding of radioactively labeled concanavalin A and Ricinus communis agglutinin to rat liver- and rat ascites hepatoma-nuclei. Biochem Biophys Res Commun. 1972 Sep 26;48(6):1504–1510. doi: 10.1016/0006-291x(72)90884-4. [DOI] [PubMed] [Google Scholar]
  23. Keshgegian A. A., Glick M. C. Glycoproteins associated with nuclei of cells before and after transformation by a ribonucleic acid virus. Biochemistry. 1973 Mar 13;12(6):1221–1226. doi: 10.1021/bi00730a032. [DOI] [PubMed] [Google Scholar]
  24. Kornfeld S., Rogers J., Gregory W. The nature of the cell surface receptor site for Lens culinaris phytohemagglutinin. J Biol Chem. 1971 Nov;246(21):6581–6586. [PubMed] [Google Scholar]
  25. LIEBERMAN I., SHORT J. HEPATIC BLOOD SUPPLY AND CONTROL OF DEOXYRIBONUCLEIC ACID SYNTHESIS IN LIVER. Am J Physiol. 1965 May;208:896–902. doi: 10.1152/ajplegacy.1965.208.5.896. [DOI] [PubMed] [Google Scholar]
  26. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  27. Larsén C., Dallner G., Ernster L. Association of sialic acid with microsomal membrane structures in rat liver. Biochem Biophys Res Commun. 1972 Dec 4;49(5):1300–1306. doi: 10.1016/0006-291x(72)90608-0. [DOI] [PubMed] [Google Scholar]
  28. MIZEN N. A., PETERMANN M. L. Nuclei from normal and leukemic mouse spleen. III. The desoxypentosenucleic acid content per nucleus calculated from total cell counts. Cancer Res. 1952 Oct;12(10):727–730. [PubMed] [Google Scholar]
  29. Magliozzi J., Puro D., Lin C., Ortman R., Dounce A. L. Ratios of nuclear proteins to DNA for rat and mouse tumors and possible effects of cytoplasmic fibrils in isolating nuclei. Exp Cell Res. 1971 Jul;67(1):111–123. doi: 10.1016/0014-4827(71)90626-4. [DOI] [PubMed] [Google Scholar]
  30. Martin S. S., Bosmann H. B. Glycoprotein nature of mitochondrial structural protein and neutral sugar content of mitochondrial proteins and structural proteins. Exp Cell Res. 1971 May;66(1):59–64. doi: 10.1016/s0014-4827(71)80010-1. [DOI] [PubMed] [Google Scholar]
  31. McBride O. W., Peterson E. A. Separation of nuclei representing different phases of the growth cycle from unsynchronized mammalian cell cultures. J Cell Biol. 1970 Oct;47(1):132–139. doi: 10.1083/jcb.47.1.132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Molnar J. Glycoproteins of Ehrlich ascites carcinoma cells. Incorporation of [14C]glucosamine and [14C]sialic acid into membrane proteins. Biochemistry. 1967 Oct;6(10):3064–3076. doi: 10.1021/bi00862a013. [DOI] [PubMed] [Google Scholar]
  33. Monneron A., Blobel G., Palade G. E. Fractionation of the nucleus by divalent cations. Isolation of nuclear membranes. J Cell Biol. 1972 Oct;55(1):104–125. doi: 10.1083/jcb.55.1.104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nicolson G., Lacorbière M., Delmonte P. Outer membrane terminal saccharides of bovine liver nuclei and mitochondria. Exp Cell Res. 1972;71(2):468–473. doi: 10.1016/0014-4827(72)90318-7. [DOI] [PubMed] [Google Scholar]
  35. Nordling S., Mayhew E. On the intracellular uptake of neuraminidase. Exp Cell Res. 1966 Nov-Dec;44(2):552–562. doi: 10.1016/0014-4827(66)90459-9. [DOI] [PubMed] [Google Scholar]
  36. PETERMANN M. L., SCHNEIDER R. M. Nuclei from normal and leukemic mouse spleen. II. The nucleic acid content of normal and leukemic nuclei. Cancer Res. 1951 Jul;11(7):485–489. [PubMed] [Google Scholar]
  37. Robbins E., Fant J., Norton W. Intracellular iron-binding macromolecules in HeLa cells. Proc Natl Acad Sci U S A. 1972 Dec;69(12):3708–3712. doi: 10.1073/pnas.69.12.3708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. SCHNEIDER R. M., PETERMANN M. L. Nuclei from normal and leukemic mouse spleen. I. The isolation of nuclei in neutral medium. Cancer Res. 1950 Dec;10(12):751–754. [PubMed] [Google Scholar]
  39. SEAMAN G. V., HEARD D. H. The surface of the washed human erythrocyte as a polyanion. J Gen Physiol. 1960 Nov;44:251–268. doi: 10.1085/jgp.44.2.251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. SEAMAN G. V., UHLENBRUCK G. The surface structure of erythrocytes from some animal sources. Arch Biochem Biophys. 1963 Mar;100:493–502. doi: 10.1016/0003-9861(63)90117-6. [DOI] [PubMed] [Google Scholar]
  41. TSUKADA K., LIEBERMAN I. LIVER NUCLEAR RIBONUCLEIC ACID POLYMERASE FORMED AFTER PARTIAL HEPATECTOMY. J Biol Chem. 1965 Apr;240:1731–1736. [PubMed] [Google Scholar]
  42. Vassar P. S., Seaman G. V., Dunn W. L., Kanke L. Electrokinetic properties of nuclear surfaces. A comparison of nuclei from normal and regenerating rat liver. Biochim Biophys Acta. 1967 May 2;135(2):218–224. doi: 10.1016/0005-2736(67)90116-2. [DOI] [PubMed] [Google Scholar]
  43. WARREN L. The thiobarbituric acid assay of sialic acids. J Biol Chem. 1959 Aug;234(8):1971–1975. [PubMed] [Google Scholar]
  44. Zbarsky I. B., Perevoshchikova K. A., Delektorskaya L. N., Delektorsky V. V. Isolation and biochemical characteristics of the nuclear envelope. Nature. 1969 Jan 18;221(5177):257–259. doi: 10.1038/221257a0. [DOI] [PubMed] [Google Scholar]
  45. Zentgraf H., Deumling B., Franke W. W. Isolation and characterization of nuclei from bird erythrocytes. Exp Cell Res. 1969 Aug;56(2):333–337. doi: 10.1016/0014-4827(69)90022-6. [DOI] [PubMed] [Google Scholar]

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