Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1975 Aug 1;66(2):305–315. doi: 10.1083/jcb.66.2.305

Preparation and purification of polymerized actin from sea urchin egg extracts

PMCID: PMC2109559  PMID: 1095598

Abstract

Isotonic extracts of the soluble cytoplasmic proteins of sea urchin eggs, containing sufficient EGTA to reduce the calcium concentration to low levels, form a dense gel on warming to 35-40 degrees C. Although this procedure is similar to that used to polymerize tubulin from mammalian brain, sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows this gel to have actin as a major component and to contain no tubulin. If such extracts are dialyzed against dilute salt solution, they no longer respond to warming, but gelation will occur if they are supplemented with 1 mM ATP and 0.020 M KCl before heating. Gelation is not temperature reversible, but the gelled material can be dissolved in 0.6-1 M KCl and these solutions contain F- actin filaments. These filaments slowly aggregate to microscopic, birefringent fibrils when 1 mM ATP is added to the solution, and this procedure provides a simple method for preparing purified actin. the supernate remaining after actin removal contains the other two components of the gel, proteins of approximately 58,000 and 220,000 mol wt. These two proteins plus actin recombine to form the original gel material when the ionic strength is reduced. This reaction is reversible at 0 degrees C, and no heating is required.

Full Text

The Full Text of this article is available as a PDF (2.6 MB).

Selected References

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

  1. Arnold J. M. Cleavage furrow formation in a telolecithal egg (Loligo pealii). I. Filaments in early furrow formation. J Cell Biol. 1969 Jun;41(3):894–904. doi: 10.1083/jcb.41.3.894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Borisy G. G., Olmsted J. B. Nucleated assembly of microtubules in porcine brain extracts. Science. 1972 Sep 29;177(4055):1196–1197. doi: 10.1126/science.177.4055.1196. [DOI] [PubMed] [Google Scholar]
  3. Bullard B., Dabrowska R., Winkelman L. The contractile and regulatory proteins of insect flight muscle. Biochem J. 1973 Oct;135(2):277–286. doi: 10.1042/bj1350277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Good N. E., Winget G. D., Winter W., Connolly T. N., Izawa S., Singh R. M. Hydrogen ion buffers for biological research. Biochemistry. 1966 Feb;5(2):467–477. doi: 10.1021/bi00866a011. [DOI] [PubMed] [Google Scholar]
  5. HUXLEY H. E. ELECTRON MICROSCOPE STUDIES ON THE STRUCTURE OF NATURAL AND SYNTHETIC PROTEIN FILAMENTS FROM STRIATED MUSCLE. J Mol Biol. 1963 Sep;7:281–308. doi: 10.1016/s0022-2836(63)80008-x. [DOI] [PubMed] [Google Scholar]
  6. Hatano S., Kondo H., Miki-Noumura T. Purification of sea urchin egg actin. Exp Cell Res. 1969 May;55(2):275–277. doi: 10.1016/0014-4827(69)90492-3. [DOI] [PubMed] [Google Scholar]
  7. Hatano S., Oosawa F. Extraction of an actin-like protein from the plasmodium of a myxomycete and its interaction with myosin A from rabbit striated muscle. J Cell Physiol. 1966 Oct;68(2):197–202. doi: 10.1002/jcp.1040680214. [DOI] [PubMed] [Google Scholar]
  8. Hatano S., Oosawa F. Isolation and characterization of plasmodium actin. Biochim Biophys Acta. 1966 Oct 31;127(2):488–498. doi: 10.1016/0304-4165(66)90402-8. [DOI] [PubMed] [Google Scholar]
  9. Inoué S., Borisy G. G., Kiehart D. P. Growth and lability of Chaetopterus oocyte mitotic spindles isolated in the presence of porcine brain tubulin. J Cell Biol. 1974 Jul;62(1):175–184. doi: 10.1083/jcb.62.1.175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kane R. E. Direct isolation of the hyaline layer protein released from the cortical granules of the sea urchin egg at fertilization. J Cell Biol. 1970 Jun;45(3):615–622. doi: 10.1083/jcb.45.3.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kane R. E. Hyalin release during normal sea urchin development and its replacement after removal at fertilization. Exp Cell Res. 1973 Oct;81(2):301–311. doi: 10.1016/0014-4827(73)90519-3. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Miki-Noumura T. An actin-like protein of the sea urchin eggs. II. Direct isolation procedure. Dev Growth Differ. 1969 Dec;11(3):219–231. doi: 10.1111/j.1440-169x.1969.00219.x. [DOI] [PubMed] [Google Scholar]
  14. Miki-Noumura T., Kondo H. Polymerization of actin from sea urchin eggs. Exp Cell Res. 1970 Jul;61(1):31–41. doi: 10.1016/0014-4827(70)90254-5. [DOI] [PubMed] [Google Scholar]
  15. Miki-Noumura T., Oosawa F. An actin-like protein of the sea urchin eggs. I. Its interaction with myosin from rabbit striated muscle. Exp Cell Res. 1969 Aug;56(2):224–232. doi: 10.1016/0014-4827(69)90006-8. [DOI] [PubMed] [Google Scholar]
  16. Morgan J. Microfilaments from amoeba proteins. Exp Cell Res. 1971 Mar;65(1):7–16. doi: 10.1016/s0014-4827(71)80043-5. [DOI] [PubMed] [Google Scholar]
  17. Perry M. M., John H. A., Thomas N. S. Actin-like filaments in the cleavage furrow of newt egg. Exp Cell Res. 1971 Mar;65(1):249–253. doi: 10.1016/s0014-4827(71)80075-7. [DOI] [PubMed] [Google Scholar]
  18. Pollard T. D., Ito S. Cytoplasmic filaments of Amoeba proteus. I. The role of filaments in consistency changes and movement. J Cell Biol. 1970 Aug;46(2):267–289. doi: 10.1083/jcb.46.2.267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Pollard T. D., Korn E. D. Filaments of Amoeba proteus. II. Binding of heavy meromyosin by thin filaments in motile cytoplasmic extracts. J Cell Biol. 1971 Jan;48(1):216–219. doi: 10.1083/jcb.48.1.216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Rebhun L. I., Rosenbaum J., Lefebvre P., Smith G. Reversible restoration of the birefringence of cold-treated, isolated mitotic apparatus of surf clam eggs with chick brain tubulin. Nature. 1974 May 10;249(453):113–115. doi: 10.1038/249113a0. [DOI] [PubMed] [Google Scholar]
  21. Schroeder T. E. Actin in dividing cells: contractile ring filaments bind heavy meromyosin. Proc Natl Acad Sci U S A. 1973 Jun;70(6):1688–1692. doi: 10.1073/pnas.70.6.1688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Schroeder T. E. Cytokinesis: filaments in the cleavage furrow. Exp Cell Res. 1968 Oct;53(1):272–276. doi: 10.1016/0014-4827(68)90373-x. [DOI] [PubMed] [Google Scholar]
  23. Schroeder T. E. The contractile ring. I. Fine structure of dividing mammalian (HeLa) cells and the effects of cytochalasin B. Z Zellforsch Mikrosk Anat. 1970;109(4):431–449. [PubMed] [Google Scholar]
  24. Schroeder T. E. The contractile ring. II. Determining its brief existence, volumetric changes, and vital role in cleaving Arbacia eggs. J Cell Biol. 1972 May;53(2):419–434. doi: 10.1083/jcb.53.2.419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Shelanski M. L., Gaskin F., Cantor C. R. Microtubule assembly in the absence of added nucleotides. Proc Natl Acad Sci U S A. 1973 Mar;70(3):765–768. doi: 10.1073/pnas.70.3.765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Szollosi D. Cortical cytoplasmic filaments of cleaving eggs: a structural element corresponding to the contractile ring. J Cell Biol. 1970 Jan;44(1):192–209. doi: 10.1083/jcb.44.1.192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. THOMPSON C. M., WOLPERT L. THE ISOLATION OF MOTILE CYTOPLASM FROM AMOEBA PROTEUS. Exp Cell Res. 1963 Oct;32:156–160. doi: 10.1016/0014-4827(63)90078-8. [DOI] [PubMed] [Google Scholar]
  28. Tilney L. G. Actin filaments in the acrosomal reaction of Limulus sperm. Motion generated by alterations in the packing of the filaments. J Cell Biol. 1975 Feb;64(2):289–310. doi: 10.1083/jcb.64.2.289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tilney L. G., Marsland D. A fine structural analysis of cleavage induction and furrowing in the eggs of Arbacia punctulata. J Cell Biol. 1969 Jul;42(1):170–184. doi: 10.1083/jcb.42.1.170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Weisenberg R. C. Microtubule formation in vitro in solutions containing low calcium concentrations. Science. 1972 Sep 22;177(4054):1104–1105. doi: 10.1126/science.177.4054.1104. [DOI] [PubMed] [Google Scholar]

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

RESOURCES