A protein from abalone sperm dissolves the egg vitelline layer by a nonenzymatic mechanism☆

[摘要] Unfertilizedabaloneeggs(Haliotisrufescens)possessanelevatedfibrousglycoproteinaceousvitellinelayer(VL)about0.6μminthickness.SpermbindtotheVLbythetipofalargeunreactedacrosomegranule.Afterbinding,thetipofthegranuleopensandthesolublecontentsarereleasedontotheVL.Aholeabout3μmindiameterthenformsintheVLintheareaofthedischargingacrosome.UltrastructuralobservationsshowtheholetobefilledwithattenuatedVLfibers.Thespermthenswimsthroughtheholeandinteractswiththeeggplasmamembrane.Thesolublecontentsofabaloneacrosomescanbeobtainedbyinductionoftheacrosomereactioninhigh-calciumseawater.Twomajorproteinsofsubunitmolecularweights13,000(13K)and15,000(15K)arefoundinthesupernatantafterremovalofthereactedspermbycentrifugation.Gelanalysisofwholespermshowsthesetwoproteinsarethemajorcomponentsofthecell.The13Kproteincanbepurifiedonthebasisofitssolubilityatlowerionicstrength.Thisproteinisapotentsolubilizer(lysin)ofeggvitellinelayers.Characterizationofthe13Klysinyieldsanisoelectricpointofabout9,basicaminoacidsaccountingfor19.6%ofitsweight,anegativePASreaction,anondenatured-molecular-weightestimateof17,000,thepresenceofexposedhydrophobicregions,andalackofenzymeactivity.Thelyticactionofthe13Kproteinisrapidlyinactivatedbyboiling,showingthatthenativeconformationisnecessaryforactivity.ThelysindoesnotdegradethemacromolecularcomponentsoftheVL.Itdoesnotproducereducingsugars,peptides,lysophosphatides,orSHgroups.AturbidometricassayforlysinactivitywasdevelopedusingisolatedVLsand13Klysin.WhenlysinisaddedtoVLsinseawaterthedissolutionactionoccursforonly15–30secbeforeabruptlystopping.MixingvariousamountsoflysinwithaconstantamountofVLsshowsthatthelysindissolvesVLsbyastoichiometric,noncatalytic(nonenzymatic)mechanism.Forexample,about11μgoflysinarerequiredforthecompletedissolutionof63μgofVLprotein(theVLis36%protein).AnidenticalconclusionwasreachedbyK.Haino-Fukushima(1974,Biochim.Biophys.Acta,352,179–191)workingwithan8.8Klysinofanotherarcheogastropod,Tegulapfeifferi.IsolatedabaloneVLsarecomposedofaboutfivemajorglycoproteinsranginginmolecularweightfrom32to44K.Highionicstrengthsuchas2MKCldoesnotsolubilizeVLs,butagentswhichdestroyhydrophobicbondsbetweenmacromolecules,suchasNaSCN,dimethylsulfoxide,andheat,areVLsolubilizers.ExposedhydrophobicportionsofthelysinmightbindtothehydrophobicregionsofVLglycoproteinsandcompetitivelydissociatetheVLfibersfromeachother,thus,destroyingtheVL'sstructuralintegrity.Stoichiometricmechanismsformakingholesinegginvestmentsmaybemorebiologicallyattractivethanenzymaticmechanisms.Astoichiometricreactionwouldbequicklyself-limitingandnondegradativetoothercellsurfacecomponents.

[学科分类] 生物科学（综合）