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.

Week1-Day1

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.

Day2

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):

code	fraction	protein	enzyme
HOM	homogenate	+	+
SN1	20,000xgsup.	+	+
P1	20,000xgsed.	+	+
SN2	40%ASsup.	+	+
P2	40%ASsed.	+	+
SN3	70%ASsup.	+	+
P3	70%ASsed.	+	+
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