The Purification of a 50 KDa Protein-actin Complex from Unfertilized Sea-urchin (Strongylocentrotus Purpuratus) Eggs

Overview

Journal Biochem J

Specialty Biochemistry

Date 1989 Feb 1

PMID 2930488

Citations 1

Authors

R M Golsteyn

D M Waisman

Affiliations

Soon will be listed here.

Abstract

The 100,000 g supernatant from the unfertilized egg of the sea urchin Strongylocentrotus purpuratus has been fractionated on DEAE-cellulose and analysed for Ca2+-binding activity by the Chelex-100 competitive Ca2+-binding activity assay. The major peak of Ca2+-binding activity was subjected to further purification and the Ca2+-binding protein responsible for this Ca2+-binding-activity peak has been isolated and characterized. Non-denaturing polyacrylamide-gel electrophoresis (PAGE) followed by 45Ca2+ autoradiography suggested a molecular mass of 80 kDa for the Ca2+-binding protein. SDS/PAGE revealed that the 80 kDa protein consisted of a 1:1 molar complex of proteins of 50 and 42 kDa. The 42 kDa protein was identified as actin. The complex was not dissociated by extensive dialysis against an EGTA-containing buffer. The EGTA-stable complex was named '50K-A'.

Citing Articles

The 50 kDa protein-actin complex from unfertilized sea-urchin (Strongylocentrotus purpuratus) eggs. Interaction with actin.

Golsteyn R, Waisman D Biochem J. 1989; 257(3):817-22.

PMID: 2930489 PMC: 1135661. DOI: 10.1042/bj2570817.

References

Laemmli U . Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259):680-5. DOI: 10.1038/227680a0. View

Chou Y, REBHUN L . Purification and characterization of a sea urchin egg Ca2+-calmodulin-dependent kinase with myosin light chain phosphorylating activity. J Biol Chem. 1986; 261(12):5389-95. View

Steinhardt R, Epel D . Activation of sea-urchin eggs by a calcium ionophore. Proc Natl Acad Sci U S A. 1974; 71(5):1915-9. PMC: 388353. DOI: 10.1073/pnas.71.5.1915. View

CHAMBERS E, PRESSMAN B, Rose B . The activation of sea urchin eggs by the divalent ionophores A23187 and X-537A. Biochem Biophys Res Commun. 1974; 60(1):126-32. DOI: 10.1016/0006-291x(74)90181-8. View

Vacquier V . The isolation of intact cortical granules from sea urchin eggs: calcium lons trigger granule discharge. Dev Biol. 1975; 43(1):62-74. DOI: 10.1016/0012-1606(75)90131-1. View

Simpson R, Neuberger M, Liu T . Complete amino acid analysis of proteins from a single hydrolysate. J Biol Chem. 1976; 251(7):1936-40. View

Bradford M . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72:248-54. DOI: 10.1016/0003-2697(76)90527-3. View

Head J, Mader S, KAMINER B . Calcium-binding modulator protein from the unfertilized egg of the sea urchin Arbacia punctulata. J Cell Biol. 1979; 80(1):211-8. PMC: 2110296. DOI: 10.1083/jcb.80.1.211. View

Coluccio L, Sedlar P, Bryan J . The effects of a 45 000 molecular weight protein from unfertilized sea urchin eggs and its 1:1 actin complex on actin filaments. J Muscle Res Cell Motil. 1986; 7(2):133-41. DOI: 10.1007/BF01753414. View

10.

Ohnuma M, Mabuchi I . The 45K molecular weight actin-modulating protein from sea urchin eggs forms a complex with actin in the presence of calcium ions. J Biochem. 1986; 100(3):817-20. DOI: 10.1093/oxfordjournals.jbchem.a121776. View

11.

Tokuda M, Khanna N, Waisman D . Purification of novel calcium-binding proteins from bovine brain. Methods Enzymol. 1987; 139:68-79. DOI: 10.1016/0076-6879(87)39075-5. View

12.

Golsteyn R, Waisman D . The 50 kDa protein-actin complex from unfertilized sea-urchin (Strongylocentrotus purpuratus) eggs. Interaction with actin. Biochem J. 1989; 257(3):817-22. PMC: 1135661. DOI: 10.1042/bj2570817. View

13.

Towbin H, Staehelin T, Gordon J . Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979; 76(9):4350-4. PMC: 411572. DOI: 10.1073/pnas.76.9.4350. View

14.

Lambin P, Fine J . Molecular weight estimation of proteins by electrophoresis in linear polyacrylamide gradient gels in the absence of denaturing agents. Anal Biochem. 1979; 98(1):160-8. DOI: 10.1016/0003-2697(79)90721-8. View

15.

Epel D, Patton C, Wallace R, Cheung W . Calmodulin activates NAD kinase of sea urchin eggs: an early event of fertilization. Cell. 1981; 23(2):543-9. DOI: 10.1016/0092-8674(81)90150-1. View

16.

Hamaguchi Y, Hiramoto Y . Activation of sea urchin eggs by microinjection of calcium buffers. Exp Cell Res. 1981; 134(1):171-9. DOI: 10.1016/0014-4827(81)90474-2. View

17.

Vacquier V . Dynamic changes of the egg cortex. Dev Biol. 1981; 84(1):1-26. DOI: 10.1016/0012-1606(81)90366-3. View

18.

Steinhardt R, Alderton J . Calmodulin confers calcium sensitivity on secretory exocytosis. Nature. 1982; 295(5845):154-5. DOI: 10.1038/295154a0. View

19.

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; 92(6):1853-62. DOI: 10.1093/oxfordjournals.jbchem.a134115. View

20.

Waisman D, Rasmussen H . A reexamination of the chelex competitive calcium binding assay. Cell Calcium. 1983; 4(2):89-105. DOI: 10.1016/0143-4160(83)90038-6. View