Phosphateestersarewidelydistributedinanyorganism.Nucleicacids,metabolicintermediateslikeglucose-6-phosphate,energy-richsubstrates(AMP,creatinephosphate)aresomeobviousexamples.Whilemanymetabolicintermediatesareactivatedthroughthetransferofphosphategroups(e.g.,bykinases)itisequallyimportantthatphosphateesterscanalsoberapidlybrokendown.Thehydrolyticremovalofphosphategroupsfromphosphoestersiscatalyzedbyphosphatases.Manyphosphatasesarehighlysubstrate-specific,likethoseenzymesinvolvedinsignaltransduction.Anumberofphosphatases,however,cleavevirtuallyanyphosphateester.Suchunspecificenzymesfunctionmainlyinthecatabolicbreakdownofmetabolitesornutrients.DependingonthepHatwhichsuchphosphataseshaveoptimalactivity,wedistinguishbetweenacidicphosphatases(alsocalledacidphosphatases)andalkalinephosphatases.Thelatterenzymesrequiredivalentmetalionsascofactorsandarecommoninanimaltissuesandbacteria.Acidicphosphatasesarewidelydistributedinmanyorganisms,includingplants.Theyworkoptimallyat~pH5withoutadditionalcofactors.TheenzymesareclassifiedasE.C.3.1.3.2.Inthisexperiment,wewillextractanacidicphosphatasefromseedlingsofmustard(Sinapisalba)andpartiallypurifiytheenzymebyammoniumsulfateprecipiation.Mostimportantprerequisiteforanyenzymeisolationisanactivitytest.Forthisphosphatase,wetakeadvantageofthebroadsubstratespecificityanduseanartificialsubstratethatchangesitscolorafterhydrolyticremovalofthephosphategroup: SincethephosphataseisactiveonlyatacidicpHvalues,butp-nitrophenoliscoloredatbasicpHvalues,wemustchangethepHfollowingtheenzymereaction.Wewillincubatefor30minatpH4.8,andthenstopthereactionbyaddingNaOH. Youareprovidedwithaflatof5-8daysoldmustardseedlingsgrowninthedarkinabedofvermiculite. 1.Removethe5-10cmlongseedlingsfromthevermiculite,washandpatdryonpapertowel.Weighandrecordtheweight.(use25-50g). 2.Grindinamortarwith75mlofice-coldwater. 3.Makethevolumeupto150mlandtransfertheslurryintothePolytronhomogenizer.Homogenize1minatspeed5(3x20sec). 4.Filterthehomogenatethrough8layersofcheeseclothintoacoldbeakeronice.Remove1mlaliquottoanEppendorfmicrofugetube,andlabelasHOMandleaveonice. 5.Transfertheextractintoone250mlcentrifugebottle.Balanceagainstwateror,ifready,againsttheextractoftheothergroup.Alwayskeeptheextractonice. 6.CentrifugeintheSorvallsuperspeedcentrifuge(B7202)inGSArotor(r=12.5cm)at9500rpm(1,000xg)for30min. 7.Decantthesupernatant(SN1)(containsmanyorganellesandthecytosol)intoagraduatedcylinderandnotethevolume.Remove1mlaliquottoanEppendorfmicrofugetube,andlabelasSN1andleaveonice.Thiswillbeusedforsubsequentproteinandactivitydetermination.Laterfreezeat-20°C.ResUSPendthepelletwhichcontainsthecelldebrisandnucleiin10mlwater,andtakea1mlsamplelabeledP1.Discardtheremainingpellet. 8.Weightheamountofsolid(NH4)2SO4required(0.25g/mlofsolutionSN1).Recordthisamount.Leavethesupernatantina250mlbeakeronicebathandplaceonamagneticstirrer. 9.Insertacleanstirringbarandusingaspatulaaddsmallamountsof(NH4)2SO4whilestirring.Thisyieldsa40%saturatedsolution,whichprecipitatessomeproteins,butnotthephosphatase. 10.Precipitatetheseproteinsafter1hbycentrifugationin50mlcentrifugetube(approx.35mlpertube)at31,000xg(estimatetherpmvalueusingtherotorrADIus)for30minintheHermlecentrifugeat4°C(roomB8220). 11.Decantthesupernatant(SN2)intoagrad.cylinderandnotethevolume.Remove1mlaliquottoanEppendorfmicrofugetube,labelasSN2andleaveonice.Resuspendthepelletin2-3mloficecoldwater.LabelasP2andfreezeat-20°C.12.Tothesupernatant,addanother0.25g(NH4)2SO4/mloftheoriginalvolumeofSN1whilestirringslowly.Thesolutionisnow70%saturated.Thisshouldbestirredatleastfor1houroryoucanleavethisinthecoldroomwhilestirringovernight. 13.Thenextmorning,transferthesuspensiontoa50mlcentrifugetubeandspinfor30minatthespeedasinstep9. 14.Decantthesupernatant(SN3)andleaveanaliquot(1ml)inamicrofugetube.Resuspendthepelletin5mlofcolddistilledwater.Mostofthephosphataseactivityshouldbecontainedinthispellet.Takeanaliquot(0.5ml)labeledP3. 15.Divideamong4microfugetubesandremoveinsolubleproteinsbyspinninginthemicrofugeatfullspeedfor2min. 16.Carefullytransferthesupernatantintofoureppendorftubes.LabelasSN4. 17.Leaveallthealiquotsinabeakerwithyourinitialsonitinthe-20°Cfreezer. Youwillusethesefractionsforfurtheranalysis(seeflowscheme): Allfractionswillbesubjectedtoaproteinandenzymeassay.
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protein enzyme 20,000xgsed.
