Sea urchin embryo fertilization envelope: Immunological evidence that soluble envelope proteins are derived from cortical granule secretions☆☆☆

[摘要] Althoughstructuralstudiessupportthehypothesisthattheseaurchinembryofertilizationenvelopeisderivedfromthepreexistingvitellineenvelopetemplateandstructuralproteinssecretedduringthecorticalreaction,biochemicalevidenceisminimal.Weusedanimmunologicalapproachtodeterminethesubcellularoriginofproteinswhichwereextractedfromthefertilizationenvelope.FertilizationenvelopeswereisolatedfromStronglyocentrotuspurpuratusembryos30minpostinseminationandextractedwith6.0Murea-0.15M2-mercaptoethanol,pH10.5,for10minat80°C.Extractedproteinswereexhaustivelydialyzedagainst0.015M2-mercaptoethanol-0.100MTris-HClatpH8.6andmixedwithFruend'scompleteadjuvantpriortoinjectionintofemaleNewZealandwhiterabbits.Theantiserumwhichwaspreparedcontainedantibodiestosixmajorandtwominorpolypeptidesinthesolublefertilizationenvelopefractionbasedontwo-dimensionalsodiumdodecylsulfateimmunoelectrophoresis.Extractsofvitellineenvelopesandextractsofunfertilizedeggsurfaceswhichareknowntocontainvitelineenvelopeproteinsdidnotformimmunoprecipitateswithantiserumagainstsolublefertilizationenvelopepolypeptides.Extractsofisolatedcorticalgranulesandthesecretedparacystallineproteinfractionformedfourandthreeimmunoprecipitates,respectively,whichshowedcompleteidentitywiththesolublefertilizationenvelopepolypeptidesbasedonrocket-lineimmunoelectrophoresis.Two-dimensionalsodiumdodecylsulfateimmunoelectrophoresisofcorticalgranuleextractandthesecretedparacrystallineproteinfractionshowedacomplexpatternofimmunoprecipitates,butamajorfindingwasthatcorticalgranulescontaina193,000-daltonpolypeptidewhichwasnotfoundintheparacrystallineproteinfraction.Theseresultssuggestthatproteolyticprocessingofacorticalgranuleprecursoroftheparacrystallineproteinfractionoccursduringfertilizationandthatnotallofthecorticalgranulepolypeptidesareincorporatedintothefertilizationenvelopebymeansofdi-andtrityrosinecrosslinkswiththevitellineenvelopeproteins.

[学科分类] 生物科学（综合）