Immunocytochemical and biochemical characterization of guanine nucleotide-binding regulatory proteins in mammalian spermatozoa☆

Michael Glassner; Jolynda Jones; Isaac Kligman; Marilyn J. Woolkalis; George L. Gerton; Gregory S. Kopf

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Immunocytochemical and biochemical characterization of guanine nucleotide-binding regulatory proteins in mammalian spermatozoa☆

机译：哺乳动物精子中鸟嘌呤核苷酸结合调节蛋白的免疫细胞化学和生化特征☆

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Polyclonalantiseradirectedagainstconservedandsubtype-specificpeptidesequencesofthe?±-subunitsofguaninenucleotide-bindingregulatoryproteins(Gproteins)wereusedtocharacterizethenatureofmammalianspermGproteinsandtodeterminewhethertheirlocalizationwasconsistentwiththeirproposedrolesinmediatingZP3-inducedacrosomalexocytosis.Mouseandguineapigspermexhibitpositiveimmunofluorescenceintheacrosomalregionusinganantiserumdirectedagainstapeptideregioncommontoall?±-subunitsofGproteins(G?±).Theimmunofluorescencedisappearsafterspermhaveundergonetheacrosomereaction,suggestingthattheimmunoreactivematerialisassociatedwiththeplasmamembrane/outeracrosomalmembraneregionoverlyingtheacrosome.ThepresenceofGproteinsinthisregionisconfirmedbythepresenceofaMr41,000substrateforpertussistoxin(PT)-catalyzed[32P]ADP-ribosylationinpurifiedplasmamembrane/outeracrosomalmembranehybridvesiclesobtainedfromacrosome-reactedguineapigsperm.ImmunoprecipitationandpolyacrylamidegelelectrophoresisofPT-catalyzed[32P]ADP-ribosylatedprotein(s)usinganti-peptideantiserageneratedagainstsequencesuniquetoGi?±1,Gi?±2,andGi?±3confirmtheexistenceofallthreeGisubtypesinmousespermextracts.IndirectimmunofluorescenceusinganantiserumdirectedagainstapeptideregionpresentinGz?±,aPT-insensitiveGprotein,demonstratespositiveimmunoreactivityinthepostacrosomal/lateralfaceregionofthemousespermhead.ThisimmunoreactivityisretainedduringacrosomalexocytosisinresponsetosolubilizedZPandthendisappearssubsequenttothisexocytoticevent.ThesedatademonstratethatGiprotein?±-subunitsarepresentintheacrosomalregionofmammaliansperm,consistentwiththeirpostulatedroleinregulatingZP3-mediatedacrosomalexocytosis,andthatPT-insensitiveGz?±isfoundinaregionofthespermheaddistinctfromthatoftheGi?±subunits.

机译：Polyclonalantiseradirectedagainstconservedandsubtype-specificpeptidesequencesofthe？±-subunitsofguaninenucleotide-bindingregulatoryproteins（Gproteins）wereusedtocharacterizethenatureofmammalianspermGproteinsandtodeterminewhethertheirlocalizationwasconsistentwiththeirproposedrolesinmediatingZP3-inducedacrosomalexocytosis.Mouseandguineapigspermexhibitpositiveimmunofluorescenceintheacrosomalregionusinganantiserumdirectedagainstapeptideregioncommontoall？±-subunitsofGproteins（G？±）.Theimmunofluorescencedisappearsafterspermhaveundergonetheacrosomereaction，suggestingthattheimmunoreactivematerialisassociatedwiththeplasmamembrane / outeracrosomalmembraneregionoverlyingtheacrosome.ThepresenceofGproteinsinthisregionisconfirmedbythepresenceofaMr41,000substrateforpertussistoxin（PT）催化[32P] ADP-ribosylationinpurifiedplasmamembrane / outeracrosomalmembranehybridvesiclesobtainedfromacrosome- PT催化的[32P] ADP-核糖基化脯氨酸的免疫沉淀和聚丙烯酰胺凝胶电泳TEIN（S）usinganti-peptideantiserageneratedagainstsequencesuniquetoGi？±1，GI？±2，andGi？±3confirmtheexistenceofallthreeGisubtypesinmousespermextracts.IndirectimmunofluorescenceusinganantiserumdirectedagainstapeptideregionpresentinGz？±，APT-insensitiveGprotein，demonstratespositiveimmunoreactivityinthepostacrosomal / lateralfaceregionofthemousespermhead.ThisimmunoreactivityisretainedduringacrosomalexocytosisinresponsetosolubilizedZPandthendisappearssubsequenttothisexocytoticevent.ThesedatademonstratethatGiprotein？±-subunitsarepresentintheacrosomalregionofmammaliansperm，consistentwiththeirpostulatedroleinregulatingZP3-mediatedacrosomalexocytosis，andthatPT-insensitiveGz？±发现在与Gi？±亚基不同的精子区域。

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《Developmental biology》 |1991年第2期|共页
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Michael Glassner; Jolynda Jones; Isaac Kligman; Marilyn J. Woolkalis; George L. Gerton; Gregory S. Kopf;
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6. Antibody specific to the alpha subunit of the guanine nucleotide-binding regulatory protein Go: developmental appearance and immunocytochemical localization in brain. [O] . K J Chang, W Pugh, S G Blanchard, 1988

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Immunocytochemical and biochemical characterization of guanine nucleotide-binding regulatory proteins in mammalian spermatozoa☆

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