Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1984 Sep 1;99(3):994–1001. doi: 10.1083/jcb.99.3.994

A 45,000-mol-wt protein-actin complex from unfertilized sea urchin egg affects assembly properties of actin

PMCID: PMC2113416  PMID: 6470047

Abstract

A one-to-one complex of a 45,000-mol-wt protein and actin was purified from unfertilized eggs of the sea urchin, Hemicentrotus pulcherrimus, by means of DNase l-Sepharose affinity and gel filtration column chromatographies. Effects of the complex on the polymerization of actin were studied by viscometry, spectrophotometry, and electron microscopy. The results are summarized as follows: (a) The initial rate of actin polymerization is inhibited at a very low molar ratio of the complex to actin. (b) Acceleration of the initial rate of polymerization occurs at a relatively high, but still substoichiometric, molar ratio of the complex to actin. (c) Annealing of F-actin fragments is inhibited by the complex. (d) The complex prevents actin filaments from depolymerizing. (e) Growth of the actin filament is inhibited at the barbed end. In all cases except b, a molar ratio of less than 1:100 of the 45,000-mol-wt protein-actin complex to actin is sufficient to produce these significant effects. These results indicate that the 45,000-mol-wt protein-actin complex from the sea urchin egg regulates the assembly of actin by binding to the barbed end (preferred end or rapidly growing end) of the actin filament. The 45,000-mol-wt protein- actin complex can thus be categorized as a capping protein.

Full Text

The Full Text of this article is available as a PDF (1,004.0 KB).

Selected References

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

  1. Adelman M. R. Physarum action. Observations on its presence, stability, and assembly in plasmodial extracts and development of an improved purification procedure. Biochemistry. 1977 Nov 1;16(22):4862–4871. doi: 10.1021/bi00641a018. [DOI] [PubMed] [Google Scholar]
  2. Begg D. A., Rebhun L. I. pH regulates the polymerization of actin in the sea urchin egg cortex. J Cell Biol. 1979 Oct;83(1):241–248. doi: 10.1083/jcb.83.1.241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bensadoun A., Weinstein D. Assay of proteins in the presence of interfering materials. Anal Biochem. 1976 Jan;70(1):241–250. doi: 10.1016/s0003-2697(76)80064-4. [DOI] [PubMed] [Google Scholar]
  4. Bonder E. M., Fishkind D. J., Mooseker M. S. Direct measurement of critical concentrations and assembly rate constants at the two ends of an actin filament. Cell. 1983 Sep;34(2):491–501. doi: 10.1016/0092-8674(83)90382-3. [DOI] [PubMed] [Google Scholar]
  5. Bonder E. M., Mooseker M. S. Direct electron microscopic visualization of barbed end capping and filament cutting by intestinal microvillar 95-kdalton protein (villin): a new actin assembly assay using the Limulus acrosomal process. J Cell Biol. 1983 Apr;96(4):1097–1107. doi: 10.1083/jcb.96.4.1097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bray D., Thomas C. Unpolymerized actin in fibroblasts and brain. J Mol Biol. 1976 Aug 25;105(4):527–544. doi: 10.1016/0022-2836(76)90233-3. [DOI] [PubMed] [Google Scholar]
  7. Bretscher A., Weber K. Villin is a major protein of the microvillus cytoskeleton which binds both G and F actin in a calcium-dependent manner. Cell. 1980 Jul;20(3):839–847. doi: 10.1016/0092-8674(80)90330-x. [DOI] [PubMed] [Google Scholar]
  8. Burgess D. R., Schroeder T. E. Polarized bundles of actin filaments within microvilli of fertilized sea urchin eggs. J Cell Biol. 1977 Sep;74(3):1032–1037. doi: 10.1083/jcb.74.3.1032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Carlsson L., Nyström L. E., Sundkvist I., Markey F., Lindberg U. Actin polymerizability is influenced by profilin, a low molecular weight protein in non-muscle cells. J Mol Biol. 1977 Sep 25;115(3):465–483. doi: 10.1016/0022-2836(77)90166-8. [DOI] [PubMed] [Google Scholar]
  10. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed] [Google Scholar]
  11. Cooper J. A., Buhle E. L., Jr, Walker S. B., Tsong T. Y., Pollard T. D. Kinetic evidence for a monomer activation step in actin polymerization. Biochemistry. 1983 Apr 26;22(9):2193–2202. doi: 10.1021/bi00278a021. [DOI] [PubMed] [Google Scholar]
  12. Glenney J. R., Jr, Kaulfus P., Weber K. F actin assembly modulated by villin: Ca++-dependent nucleation and capping of the barbed end. Cell. 1981 May;24(2):471–480. doi: 10.1016/0092-8674(81)90338-x. [DOI] [PubMed] [Google Scholar]
  13. Hasegawa T., Takahashi S., Hayashi H., Hatano S. Fragmin: a calcium ion sensitive regulatory factor on the formation of actin filaments. Biochemistry. 1980 Jun 10;19(12):2677–2683. doi: 10.1021/bi00553a021. [DOI] [PubMed] [Google Scholar]
  14. Hatano S., Owaribe K. Some properties of Physarum actinin. A regulatory protein of actin polymerization. Biochim Biophys Acta. 1979 Jul 25;579(1):200–215. doi: 10.1016/0005-2795(79)90099-0. [DOI] [PubMed] [Google Scholar]
  15. Higashi S., Oosawa F. Conformational changes associated with polymerization and nucleotide binding in actin molecules. J Mol Biol. 1965 Jul;12(3):843–865. doi: 10.1016/s0022-2836(65)80332-1. [DOI] [PubMed] [Google Scholar]
  16. Hinssen H. An actin-modulating protein from Physarum polycephalum. I. Isolation and purification. Eur J Cell Biol. 1981 Feb;23(2):225–233. [PubMed] [Google Scholar]
  17. Hosoya H., Mabuchi I., Sakai H. Actin modulating proteins in the sea urchin egg. I. Analysis of G-actin-binding proteins by DNase I-affinity chromatography and purification of a 17,000 molecular weight component. J Biochem. 1982 Dec;92(6):1853–1862. doi: 10.1093/oxfordjournals.jbchem.a134115. [DOI] [PubMed] [Google Scholar]
  18. Isenberg G., Aebi U., Pollard T. D. An actin-binding protein from Acanthamoeba regulates actin filament polymerization and interactions. Nature. 1980 Dec 4;288(5790):455–459. doi: 10.1038/288455a0. [DOI] [PubMed] [Google Scholar]
  19. KASAI M., ASAKURA S., OOSAWA F. The cooperative nature of G-F transformation of actin. Biochim Biophys Acta. 1962 Feb 12;57:22–31. doi: 10.1016/0006-3002(62)91073-9. [DOI] [PubMed] [Google Scholar]
  20. Kilimann M. W., Isenberg G. Actin filament capping protein from bovine brain. EMBO J. 1982;1(7):889–894. doi: 10.1002/j.1460-2075.1982.tb01265.x. [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. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  23. Mabuchi I. Effects of muscle proteins on the interaction between actin and an actin-depolymerizing protein from starfish oocytes. J Biochem. 1982 Nov;92(5):1439–1447. doi: 10.1093/oxfordjournals.jbchem.a134068. [DOI] [PubMed] [Google Scholar]
  24. Mabuchi I. Electron microscopic determination of the actin filament end at which cytochalasin B blocks monomer addition using the acrosomal actin bundle from horseshoe crab sperm. J Biochem. 1983 Oct;94(4):1349–1352. doi: 10.1093/oxfordjournals.jbchem.a134480. [DOI] [PubMed] [Google Scholar]
  25. Mabuchi I., Spudich J. A. Purification and properties of soluble actin from sea urchin eggs. J Biochem. 1980 Mar;87(3):785–802. doi: 10.1093/oxfordjournals.jbchem.a132808. [DOI] [PubMed] [Google Scholar]
  26. Maruyama K., Kimura S., Ishi T., Kuroda M., Ohashi K. beta-actinin, a regulatory protein of muscle. Purification, characterization and function. J Biochem. 1977 Jan;81(1):215–232. doi: 10.1093/oxfordjournals.jbchem.a131438. [DOI] [PubMed] [Google Scholar]
  27. Nagata K., Sagara J., Ichikawa Y. Changes in contractile proteins during differentiation of myeloid leukemia cells. II. Purification and characterization of actin. J Cell Biol. 1982 May;93(2):470–478. doi: 10.1083/jcb.93.2.470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nishida E., Sakai H. Kinetic analysis of actin polymerization. J Biochem. 1983 Apr;93(4):1011–1020. doi: 10.1093/oxfordjournals.jbchem.a134224. [DOI] [PubMed] [Google Scholar]
  29. Pollard T. D., Mooseker M. S. Direct measurement of actin polymerization rate constants by electron microscopy of actin filaments nucleated by isolated microvillus cores. J Cell Biol. 1981 Mar;88(3):654–659. doi: 10.1083/jcb.88.3.654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schroeder T. E. Dynamics of the contractile ring. Soc Gen Physiol Ser. 1975;30:305–334. [PubMed] [Google Scholar]
  31. Siegel L. M., Monty K. J. Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductases. Biochim Biophys Acta. 1966 Feb 7;112(2):346–362. doi: 10.1016/0926-6585(66)90333-5. [DOI] [PubMed] [Google Scholar]
  32. Southwick F. S., Tatsumi N., Stossel T. P. Acumentin, an actin-modulating protein of rabbit pulmonary macrophages. Biochemistry. 1982 Nov 23;21(24):6321–6326. doi: 10.1021/bi00267a043. [DOI] [PubMed] [Google Scholar]
  33. Spudich A., Spudich J. A. Actin in triton-treated cortical preparations of unfertilized and fertilized sea urchin eggs. J Cell Biol. 1979 Jul;82(1):212–226. doi: 10.1083/jcb.82.1.212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Spudich J. A., Watt S. The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J Biol Chem. 1971 Aug 10;246(15):4866–4871. [PubMed] [Google Scholar]
  35. 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]
  36. Tilney L. G., Hatano S., Ishikawa H., Mooseker M. S. The polymerization of actin: its role in the generation of the acrosomal process of certain echinoderm sperm. J Cell Biol. 1973 Oct;59(1):109–126. doi: 10.1083/jcb.59.1.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tobacman L. S., Korn E. D. The kinetics of actin nucleation and polymerization. J Biol Chem. 1983 Mar 10;258(5):3207–3214. [PubMed] [Google Scholar]
  38. Vacquier V. D. Dynamic changes of the egg cortex. Dev Biol. 1981 May;84(1):1–26. doi: 10.1016/0012-1606(81)90366-3. [DOI] [PubMed] [Google Scholar]
  39. Wang L. L., Bryan J. Isolation of calcium-dependent platelet proteins that interact with actin. Cell. 1981 Sep;25(3):637–649. doi: 10.1016/0092-8674(81)90171-9. [DOI] [PubMed] [Google Scholar]
  40. Wegner A. Head to tail polymerization of actin. J Mol Biol. 1976 Nov;108(1):139–150. doi: 10.1016/s0022-2836(76)80100-3. [DOI] [PubMed] [Google Scholar]
  41. Wegner A., Savko P. Fragmentation of actin filaments. Biochemistry. 1982 Apr 13;21(8):1909–1913. doi: 10.1021/bi00537a032. [DOI] [PubMed] [Google Scholar]
  42. Yin H. L., Stossel T. P. Control of cytoplasmic actin gel-sol transformation by gelsolin, a calcium-dependent regulatory protein. Nature. 1979 Oct 18;281(5732):583–586. doi: 10.1038/281583a0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES