Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1971 Feb 1;48(2):324–339. doi: 10.1083/jcb.48.2.324

SELECTIVE EXTRACTION OF ISOLATED MITOTIC APPARATUS

Evidence That Typical Microtubule Protein Is Extracted by Organic Mercurial

Thomas Bibring 1, Jane Baxandall 1
PMCID: PMC2108184  PMID: 5543404

Abstract

Mitotic apparatus isolated from sea urchin eggs has been treated with meralluride sodium under conditions otherwise resembling those of its isolation. The treatment causes a selective morphological disappearance of microtubules while extracting a major protein fraction, probably consisting of two closely related proteins, which constitutes about 10% of mitotic apparatus protein. Extraction of other cell particulates under similar conditions yields much less of this protein. The extracted protein closely resembles outer doublet microtubule protein from sea urchin sperm tail in properties considered typical of microtubule proteins: precipitation by calcium ion and vinblastine, electrophoretic mobility in both acid and basic polyacrylamide gels, sedimentation coefficient, molecular weight, and, according to a preliminary determination, amino acid composition. An antiserum against a preparation of sperm tail outer doublet microtubules cross-reacts with the extract from mitotic apparatus. On the basis of these findings it appears that microtubule protein is selectively extracted from isolated mitotic apparatus by treatment with meralluride, and is a typical microtubule protein.

Full Text

The Full Text of this article is available as a PDF (1.5 MB).

Selected References

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

  1. Adelman M. R., Borisy G. G., Shelanski M. L., Weisenberg R. C., Taylor E. W. Cytoplasmic filaments and tubules. Fed Proc. 1968 Sep-Oct;27(5):1186–1193. [PubMed] [Google Scholar]
  2. Auclair W., Siegel B. W. Cilia regeneration in the sea urchin embryo: evidence for a pool of ciliary proteins. Science. 1966 Nov 18;154(3751):913–915. doi: 10.1126/science.154.3751.913. [DOI] [PubMed] [Google Scholar]
  3. BIBRING T., COUSINEAU G. H. PERCENTAGE INCORPORATION OF LEUCINE LABELLED WITH CARBON-14 INTO ISOLATED MITOTIC APPARATUS DURING EARLY DEVELOPMENT OF SEA URCHIN EGGS. Nature. 1964 Nov 21;204:805–807. doi: 10.1038/204805a0. [DOI] [PubMed] [Google Scholar]
  4. Bensch K. G., Malawista S. E. Microtubular crystals in mammalian cells. J Cell Biol. 1969 Jan;40(1):95–107. doi: 10.1083/jcb.40.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bensch K. G., Marantz R., Wisniewski H., Shelanski M. Induction in vitro of microtubular crystals by vinca alkaloids. Science. 1969 Aug 1;165(3892):495–496. doi: 10.1126/science.165.3892.495. [DOI] [PubMed] [Google Scholar]
  6. Bibring T., Baxandall J. Immunochemical studies of 22S protein from isolated mitotic apparatus. J Cell Biol. 1969 May;41(2):577–590. doi: 10.1083/jcb.41.2.577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bibring T., Baxandall J. Mitotic apparatus: the selective extraction of protein with mild acid. Science. 1968 Jul 26;161(3839):377–379. doi: 10.1126/science.161.3839.377. [DOI] [PubMed] [Google Scholar]
  8. Borisy G. G., Taylor E. W. The mechanism of action of colchicine. Binding of colchincine-3H to cellular protein. J Cell Biol. 1967 Aug;34(2):525–533. doi: 10.1083/jcb.34.2.525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Borisy G. G., Taylor E. W. The mechanism of action of colchicine. Colchicine binding to sea urchin eggs and the mitotic apparatus. J Cell Biol. 1967 Aug;34(2):535–548. doi: 10.1083/jcb.34.2.535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cohen W. D., Rebhun L. I. An estimate of the amount of microtubule protein in the isolated mitotic apparatus. J Cell Sci. 1970 Jan;6(1):159–176. doi: 10.1242/jcs.6.1.159. [DOI] [PubMed] [Google Scholar]
  11. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
  12. DIRKSEN E. R. THE ISOLATION AND CHARACTERIZATION OF ASTERS FROM ARTIFICIALLY ACTIVATED SEA URCHIN EGGS. Exp Cell Res. 1964 Nov;36:256–269. doi: 10.1016/0014-4827(64)90206-x. [DOI] [PubMed] [Google Scholar]
  13. Dunker A. K., Rueckert R. R. Observations on molecular weight determinations on polyacrylamide gel. J Biol Chem. 1969 Sep 25;244(18):5074–5080. [PubMed] [Google Scholar]
  14. Dutton G. R., Barondes S. Microtubular protein: synthesis and metabolism in developing brain. Science. 1969 Dec 26;166(3913):1637–1638. doi: 10.1126/science.166.3913.1637. [DOI] [PubMed] [Google Scholar]
  15. Forer A., Goldman R. D. Comparisons of isolated and in vivo mitotic apparatuses. Nature. 1969 May 17;222(5194):689–690. doi: 10.1038/222689a0. [DOI] [PubMed] [Google Scholar]
  16. Gibbons I. R. Chemical dissection of cilia. Arch Biol (Liege) 1965;76(2):317–352. [PubMed] [Google Scholar]
  17. Gibbons I. R., Rowe A. J. Dynein: A Protein with Adenosine Triphosphatase Activity from Cilia. Science. 1965 Jul 23;149(3682):424–426. doi: 10.1126/science.149.3682.424. [DOI] [PubMed] [Google Scholar]
  18. Goldman R. D., Rebhun L. I. The structure and some properties of the isolated mitotic apparatus. J Cell Sci. 1969 Jan;4(1):179–209. doi: 10.1242/jcs.4.1.179. [DOI] [PubMed] [Google Scholar]
  19. Hepler P. K., McIntosh J. R., Cleland S. Intermicrotubule bridges in mitotic spindle apparatus. J Cell Biol. 1970 May;45(2):438–444. doi: 10.1083/jcb.45.2.438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kane R. E. The mitotic apparatus. Identification of the major soluble component of the glycol-isolated mitotic apparatus. J Cell Biol. 1967 Feb;32(2):243–253. doi: 10.1083/jcb.32.2.243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Lindberg U. Molecular weight and amino acid composition of deoxyribonuclease I. Biochemistry. 1967 Jan;6(1):335–342. doi: 10.1021/bi00853a050. [DOI] [PubMed] [Google Scholar]
  24. Marantz R., Shelanski M. L. Structure of microtubular crystals induced by vinblastine in vitro. J Cell Biol. 1970 Jan;44(1):234–238. doi: 10.1083/jcb.44.1.234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Marantz R., Ventilla M., Shelanski M. Vinblastine-induced precipitation of microtubule protein. Science. 1969 Aug 1;165(3892):498–499. doi: 10.1126/science.165.3892.498. [DOI] [PubMed] [Google Scholar]
  26. Mazia D., Dan K. The Isolation and Biochemical Characterization of the Mitotic Apparatus of Dividing Cells. Proc Natl Acad Sci U S A. 1952 Sep;38(9):826–838. doi: 10.1073/pnas.38.9.826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mohri H. Amino-acid composition of "Tubulin" constituting microtubules of sperm flagella. Nature. 1968 Mar 16;217(5133):1053–1054. doi: 10.1038/2171053a0. [DOI] [PubMed] [Google Scholar]
  28. Moore S. Amino acid analysis: aqueous dimethyl sulfoxide as solvent for the ninhydrin reaction. J Biol Chem. 1968 Dec 10;243(23):6281–6283. [PubMed] [Google Scholar]
  29. ORNSTEIN L. DISC ELECTROPHORESIS. I. BACKGROUND AND THEORY. Ann N Y Acad Sci. 1964 Dec 28;121:321–349. doi: 10.1111/j.1749-6632.1964.tb14207.x. [DOI] [PubMed] [Google Scholar]
  30. Olmsted J. B., Carlson K., Klebe R., Ruddle F., Rosenbaum J. Isolation of microtubule protein from cultured mouse neuroblastoma cells. Proc Natl Acad Sci U S A. 1970 Jan;65(1):129–136. doi: 10.1073/pnas.65.1.129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Puszkin S., Berl S. Actin-like properties of colchicine binding protein isolated from brain. Nature. 1970 Feb 7;225(5232):558–559. doi: 10.1038/225558a0. [DOI] [PubMed] [Google Scholar]
  32. Sakai H. Studies on sulfhydryl groups during cell division of sea-urchin eggs. 8. Some properties of mitotic apparatus proteins. Biochim Biophys Acta. 1966 Jan 4;112(1):132–145. doi: 10.1016/s0926-6585(96)90015-1. [DOI] [PubMed] [Google Scholar]
  33. Shapiro A. L., Viñuela E., Maizel J. V., Jr Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem Biophys Res Commun. 1967 Sep 7;28(5):815–820. doi: 10.1016/0006-291x(67)90391-9. [DOI] [PubMed] [Google Scholar]
  34. Shelanski M. L., Taylor E. W. Isolation of a protein subunit from microtubules. J Cell Biol. 1967 Aug;34(2):549–554. doi: 10.1083/jcb.34.2.549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Shelanski M. L., Taylor E. W. Properties of the protein subunit of central-pair and outer-doublet microtubules of sea urchin flagella. J Cell Biol. 1968 Aug;38(2):304–315. doi: 10.1083/jcb.38.2.304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Stephens R. E., Linck R. W. Comparison of muscle actin and ciliary microtubule protein in the mollusk Pecten irradians. J Mol Biol. 1969 Mar 28;40(3):497–501. doi: 10.1016/0022-2836(69)90168-5. [DOI] [PubMed] [Google Scholar]
  37. Stephens R. E. On the structural protein of flagellar outer fibers. J Mol Biol. 1968 Mar 14;32(2):277–283. doi: 10.1016/0022-2836(68)90009-0. [DOI] [PubMed] [Google Scholar]
  38. Stephens R. E. Reassociation of microtubule protein. J Mol Biol. 1968 Apr 28;33(2):517–519. doi: 10.1016/0022-2836(68)90210-6. [DOI] [PubMed] [Google Scholar]
  39. Stephens R. E., Renaud F. L., Gibbons I. R., Stevens R. E. Guanine nucleotide associated with the protein of the outer fibers of flagella and cilia. Science. 1967 Jun 23;156(3782):1606–1608. doi: 10.1126/science.156.3782.1606. [DOI] [PubMed] [Google Scholar]
  40. Stephens R. E. The mitotic apparatus. Physical chemical characterization of the 22S protein component and its subunits. J Cell Biol. 1967 Feb;32(2):255–275. doi: 10.1083/jcb.32.2.255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Stephens R. E. Thermal fractionation of outer fiber doublet microtubules into A- and B-subfiber components. A- and B-tubulin. J Mol Biol. 1970 Feb 14;47(3):353–363. doi: 10.1016/0022-2836(70)90307-4. [DOI] [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. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
  44. Weisenberg R. C., Borisy G. G., Taylor E. W. The colchicine-binding protein of mammalian brain and its relation to microtubules. Biochemistry. 1968 Dec;7(12):4466–4479. doi: 10.1021/bi00852a043. [DOI] [PubMed] [Google Scholar]
  45. Wilson H. J. Arms and bridges on microtubules in the mitotic apparatus. J Cell Biol. 1969 Mar;40(3):854–859. doi: 10.1083/jcb.40.3.854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. ZIMMERMAN A. M. Physico-chemical analysis of the isolated mitotic apparatus. Exp Cell Res. 1960 Sep;20:529–547. doi: 10.1016/0014-4827(60)90122-1. [DOI] [PubMed] [Google Scholar]
  47. Zahler W. L., Saito A., Fleischer S. Removal of structural protein from mitochondria. Biochem Biophys Res Commun. 1968 Aug 13;32(3):512–518. doi: 10.1016/0006-291x(68)90692-x. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES