In vivo protein phosphorylation and labeling of ATP in sea urchin eggs loaded with 32PO4 via electroporation☆

摘要

ProteinphosphorylationwasexaminedinseaurchineggsinwhichtheATPwaslabeledwith32Poverabriefperiodoftimeusingreversibleelectricalporationtogainaccesstothecytoplasm.Unfertilizedeggsfromtwospecies,LytechinuspictusandStrongylocentrotuspurpuratus,wereelectricallypermeabilizedandincubatedinthepresenceof[32P]H3PO4,underconditionsallowinglabeluptake.Aftera5-minloadingperiodtheeggswereresealedandthefateofthelabelwasmonitored.ThelabelhadequilibratedwiththecellularATPpoolwithinthe13-minperiodrequiredforloadingandresealingtheeggs.Furthermore,thisequilibriumwasmaintainedforatleast2hrbeyondtheloadingperiodineitherunfertilizedorfertilizedeggs(i.e.,thespecificactivityofATPwasthesameforfertilizedandunfertilizedeggs).WealsoexaminedthepositionofthelabelwithintheATPandfoundthat40a€“45%ofthelabelisolatedwiththeATPwasinthe?3phosphateofATPandhencewasimmediatelyavailableforproteinphosphorylation.ThelabelwasmaintainedinthispositionintheATPforatleast2hrfollowingtheloadingperiodandwasnotaffectedbyfertilization(determinedforL.pictusonly).Thephosphoproteinbandingpatternwasdeterminedbygelelectrophoresisandautoradiographyatvarioustimepointsfollowingtheloadingperiod.Therewasacontinuousincreaseoflabelincorporatedintoproteinovertime;however,thebandingpatterndidnotchange.Thispatternwasnotaffectedbyfertilization.Furthermore,inhibitionofproteinsynthesis(withemetine)hadnoeffectonthisphosphoproteinbandingpattern.Althoughtheloadingperiodwasbrieftherewassufficientincorporationoflabelintoproteinduringthistimetoobscurepotentialregulatoryphosphorylationevents.