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(1)博士学位論文 MolecularMechanismsofResistanceto PhotooxidativeDamageinAlgae. 近畿大学大学院農学研究科農芸化学専攻田茂井. 政宏.

(2) DoctoralDissertation. MolecularMechanismsofResistanceto PhotooxidativeDamageinAlgae. MASAHIROTAMOI. GraduateSchool,KindUniversity DivisionofAgriculturalScience (Major:AppliedBioscience).

(3) (英文題目) MolecularMechanismsofResistanceto PhotooxidativeDa皿ageinAlgae. MASAHIROTool. March,1999. GraduateSchoal,KinkiUniversity DivisionofAgriculturalScience Major;AppliedBioscience (Advisor:Prof.ShigeruShigeoka). (和文題目). 藻類における光・酸素毒耐性の分子機構の解明. 近畿大学大学院農学研究科農芸化学専攻田茂井政宏 (指導:重岡成教授). submittedtotheGraduateSchool,KinkiUniversity,tofulf皿therequirement fortheDoctorateDegree..

(4) Acknowledgements. IwishtoexpressmysciencegraduatetoDr.5higeruShigeoka,Professorof FacultyofAgriculture,KinkiUniversity,forhiskindguidance,valuableadvice, stimulatingdiscussionandcriticalreviewthroughouttheworkincludingthemanuscript ot`thisthesis. 1圏wishtothankDr。ToshioMitsunagaandDr.RyutaroUsumi,Professorsof FacultyofAgriculture,KinkiUniversity,forreadingtheentiretextinitsoriginalform. IamthankfultoDr.YoshihisaNakano,Dr.AkiraWadano,ProfessorsofApplied BiologicalChemistry,UniversityofOsakaprefecture,andDr.AkihoYokota,Professor ofGraduateSchoolofBiologicalSciences,NaraInstituteofScienceandTechnology,for theirkindsuggestionsantivaivabiediscussionsthroughoutthework. IamgreatlyinclebtecitoDr.Miyasaka,TechnicalResearchCenter,TheKansai ElectricPowerCo.,.forprovidingmewiththecDNAclone. IwishtothankDr.ToruTakeda,Facultyof.Agriculture,KinkiUniversity,andDr. Takahirolshikawa,FacultyofLifeandEnvironmentalScience,ShimaneUniversity,and, fortheirkindsuggestionsandvaluablediscussionsthroughouCthework. Finally,specialthanksareduetoMiyukiKimoto,AkikoMしirakatni,fortheirmany helpfulcollaborations.Thanksarealsoduetoallthepastandpresentmembersof-our laboratoryintheFacultyofAgriculture,KinkiUniversity,1'ortheirkindcooperations. ThisresearchwassupportedinpartbyagrantfromResearchFellowshipsofthe JaμmSocietyforthePromotionofScienceforYoungScientists..

(5) AOS APX AsA BSA by Cat Chl Cys D"r"1' E.coli FBPase Frui,6-P2 Fru2,6-P2 G6PDH GPX GSH IPTG K ,! κま LB NADPH,NADH,NADP+ NADP+-GAPDH. ORF PRK SDS-PAGE. SBPase Sed1,7-P2 TFA DTT. ABBREVIATIONS activeoxygenspecies ascorbateperoxidase ascorbate bovineserumalbumine basepair catalase chlorophyll cystelne dithiothreifol Escherichiacoti fructose-1,6-bisphosphatase fructose1,6-bisphosphate fructose2,6-bisphosphate glucose-6-phosphatedehydrogenase glutathioneperoxidase glutahione isopropyl(3-D-thiogalactopyranoside michaelisconstant inhibitionconstant Luria-Bertanibroth nicotinamide-adeninedinuclotideanditsoxidizedform NADP-dependentglyceraldehyde-3-phosphate dehydrogenase openreadingframe phosphoribulokinase sodiumdodecylsulfate-polyacrylamidegel electrophoresis sedoheptulose-1,7-bisphosphatase sedoheptuiose1,7-bisphosphate trifluoroaceticacid dithiothreitol.

(6) CONTENTS. Introduction. CHAPTERII. The. 1. CHAPTERI. LackofLightlDarkRegulationofEnzymesInvolvedinthe. 6. PhotosyntheticCarbonReductionCycleinAlgae. CHAPTERIII. MolecularCharacterizationantiResistancetoHydrogenPeroxideof TwoFructose-1,6-bisphosphatasesfroln5ン. η εchococc配3PCC. 7942........................................................................12. CHAPTERIV. AcquisitionofaNewTypeofFructose-1,6-bisphosphatasewith ResistancetoHydrogenPeroxideinCyanobacteria:Molecular CharacterizationoftheEnzymefromSynechocystis PCC6803. CHAPTERV. FunctionalAnalysisoftheFructose-1,6-bisphosphataseIsozymes (fbp-Iantifbp-IIGeneProducts)inCyanobacteria. CHAPTERVI. 44. EnzymaticandMolecularCharacterizationofFructose-1,6bisphosphataseinEugle」lagracilisz. CHAPTERVII. 29. 53. EnzymaticandMolecularCharacterizationofNADP-dependent Glyceraldehyde-3-phosphateDehydrogenasefromSynechococcus PCC7942.................................................................66. CHAPTERVIII. MolecularMechanismsofResistancetoHydogenPeroxideof NADP-dependentGlyceraldehyde-3-phosphateDehydrogenasefrom Chlarnydomonassp.W8078. REFERENCES. PUBLICATIONS. 88. goo.

(7) CHAPTPRI. Introduction. Livinginanoxygenatomosphereallowstheuseofoxygenintheoxidaこionor foodstoproduceenergy.However,ouroxygena〔omosphereistwo-edgedsword. ,. sincellleslow,11amelessoxidationofourtissuebyoxygen(aprocesscalled autoxidation)causesoxidativedamage.Inaddition,oxygencanbeconvertedtospecies suchassingletoxygen(102),superoxide(02'),hydmgenperoxide(H202)andh》. ノclroxyl. radicals(HO・)thatareevenmoredamaging【hanoxygen亘self;thesespeciesareof吐en collectivelycalledactiveoxygenspecies(AOS).Becauseevenantleroptimalconclilions manymetabolicprocessesproduceAOS,ourcellsareusuallyexposedtooxidative damagederivedfromAOS.Therelと)re,吐heAOSscavengingsys【emconsiderlngof variousrcdoxsubs吐anccssuchasascorbate(AsA). ,ghllatllione(GSH),tocopherohmdso. on,andenzymessuchassuperoxidedismし1tase,ca巴alase(Cat). ,AsAperoxidase(APX),. GSHperoxidase(GPX),111C)dodehydroascorba吐ereductase,dehydroascorbateredしldase. ,. 91u巳athk)neredし1c吐ase,elc.,isindispensabletOI'ownlif￠(Fig.1-1).. Eug/ena. HigherPlants. lM'α. ゜b°dytoz-・一・・1【叫鳳. 。. 纒豊曇. Chlamydomonas くしむトリ. 騰鍛1〈＼. ...cyl・. ・d〆!. Fig.1-1LocalizationofAOS-scavengingEnzymesinHigherPlants,Euglena, ChlamydomonasandCyanobacteria.Abbreviationsusedfollowing:APX,ascorbate peroxidase;SOD,superoxidedismutase;Cat,catalase;Cat-POD,catalase-peroxidase; GPX,glutathioneperoxidase;Trx-POD,thioredoxin-peroxidase.. 1.

(8) Inphotosyntheticorganisms,themostimportantofproductionofAOSaredliven byorassociatedwithlight-dependentevents.Photosyntheticcellsarepronetooxidative stressbecausetheycontainanarrayofphotosensitisingpigmentsantitheybothproduce andconsumeoxygen.Thephotosyntheticelectrontransportsystemisthemajorsource ofAOSinplanttissue(Asada1994},havingthepotentialtogenerate`02,02-,H202and HO・.Theselight・dependentgenerationofAOSistermedphotoolidativestr-ess.The eltenCtowhichthesereactionsoccurundernon-stressedconditionsisdebatablebutitis generallybelievedthattheproductionofAOSisanしmavoidableconsequenceofthe t)perationofthephotosyntheticelectrontransportchaininanoxygenatmvsphare.. み46肋01ご3〃Zρ. ズ〃y`1ro8θ. ηP61伽`1einAlgae. Longappreciatedassimpleorganismsinwhichtostudyoxygenicphotosynthesis, cyanobacteriaandeukaryoticalgaehaveinrecentyearsbecomerecognizedaso1' theoreticalalldpracticalimportanceinthephotosynthesis.Ina1galcellsaswellasill higherpunts,theproductiollofH202viaO2"canbedetectedundernormal(ズ)11ditions (PattersonandMyers1973,Miyakeetal.1991,Cohenetal.1995,Takecla1997). Eμ81θ1τ α8ハ αc'1`31acksCatandlocalizestheenzymesinvolvedintheAsA-GSH cycleincludingAPXsolelyinthecytosol(Shigeokaetal.1980x,1980b,1987x, IshikawaetaL1996).IthasbeenreportedthatasiteinphotosystemIIofEttglen`, chloroplastsistheo!'iglnorAOSfolmation(TschiersdhandOhmannl993).H2Q2 genelgatedinchloroPlastsandmitochondr重awasfoulld吐odiffusefronltheorganeilesint(〕 thecytosoltobedecomposedbyAPX(Ichikawaetal.1993}. C〃6〃. ηyご10,ηoη. αg・ θ∫納`〃珈jC9containedCat,APXandGPX.Althoughthe. ActivityofAPXwasverylowascomparedwiththatinhigherplants,theH21b'OZtest indicatedthataphotoreductanレperoxidasesystemsincludingAPXandGPXalsooperate withCat加. 小ノo(Miyakeetal.1991,Siiltemeyeretal.1993,Takedal997). CyanobacceriacanbedividedintotwogroupsrorthescavengingofH202.The. firstgroupwhichhasnoAPXevolvesOlllyl802upontheadditionofH2皿802ineilherthe Iightordark.ThisindicatesthatHZOzisscavengedbycatalasealine[eventually,this tumedouttobecatalase-peroxidase(MutsudaetaL1996)】.Secondgroupwhichhas APXevolves1602underthebathlightanddark,indicatingthereductionofH202toH20 viaaperoxiciasereactionusingphol:oreductantastheelectrondonor{Miyakeetat.1991). Ithasrenewedlybecomeclearthatthephotoreciuctant-peroxidase,thiorecloxin-peroxidase, existinbothgroups(MiyakeetaL1991,YamamotoetaLl998).. ResistanceofPhotosynthesistoHydrogenPeroxideinAlgae Judg{nggrointhesubcellulardistribtltionofthcH202-scavengingsystemandthe log-vAPXactivityiiialgae,itseemsverylikelythattheremightbeaconsiderable. 2.

(9) di∬erenceinthesusceptibilityofphotosynεhesistoH202betweenalgaeandhigherplants. Takedaetal.(1995)reportedthatthephotosynthesisofeukaryoticalgae,Eccglenccand Chlcrrriydornonas,anticyanobacteria,SynechococctcsPCC7942andSynechocystisPCC 6803,isnot. .sttscepiibletoinhibitionbyHZOZuptolmM,incomparisonwiththatof. chloroplastsofhigherplants,whichresultsfromtheresistanceofthiol-modulated enzymes,FBPase,NADP+-GAPDH,andPRKtoH202inalgalchloroplastsand cyanobacteria(Fig.1-2). Kaiser(1979)reported巳hatH2qatlowconcen〔ration(10to100μM)inhibitedlhe tlllol-1110dUlatedenzymesofthephotosyntheticcarbonreduction(PCR)cycle(Fig.13), sinceHZOZreadilyoxidizesthereducedthiolgroups.Fruclose--1,6-bisphosphatase (FBPase),NADP}-dependentglyceraldehyde-3-phosphatedehydr-ogenase(NADP+GAPDH),andphosphoribulokinase(PRK)are,illdeed,m勾ortargetsofattackbyH202 (Buchanalll980,1991).AsshowninFig.1-4,thethiol-tnocftilatecienzymesfrom in{actspinachchloroplastsweresignificantlyinhibitedby100μMH202,a8reporEec.f previously(CharlesantiHaliiwell1980}.Bycontrast,thethiolenzymesfromalgal chloroplastsandcyanobacterialcellswereonlyslightlyinhibitedbylmMH202.Thus,it seemslikelythattheinsusceptibilityofthealgalthiol-modulatedenzymestoH202isthe causeoftheresinlanceofthephotosynthesisinalgalce1】stoH202.. Fig.ト2EttectofH2020ntheRateofCO2FixationbySpinachChbroP量asts(▲),and CellsofEuglena(●)、0ん1amydomoηas(○),S.7942(E])andS.6803(■). 3.

(10) ラソ. 欄概㌦. Glyceraldehyde3-phosphd Dehydrogenase(GAPDH). 、. ozATPNADPH RIb・1・setへ>3-・. …pho>t3-B・. 、ph。,pho;. bisphosphateglycerateglycerate. ㌧1織Fructose1,6-:_bisphosphate. 噂. 鷹. ゜・lbul°sekl欄. ATP. 噸. …. e. 韓. 騰. 一Pi Fructose鱗>e1,61,,;sphatase) phosphate 3-Phosphoglyceraldehyde Erythrose4phosphate. r. Sedoheptulose7-Sedoheptulose1. Dihydroxyacetone phosphate. ,7-. ph°sphate 3-Phospho-. pl査bisph°sphate. glyceraldehyde. 灘. 臼g.卜3. 灘. 畿. 瑚. 《ひ・・舶灘. ReactionsofthePCRCycle. ( ろ﹂芒 8 ↑0 3 )あξ 僧ちく 010石10-5. 10°3010・610-5. 10-410く}010-610-510-4 ConcentrationofH202(M). Fig.1-4EffectofH2020ntheActivitiesofThiol-modulatedEnzymesinChloropfastof Spinach(▲),Euglena(●)andChlamydomonas(○),andinCellsofS.7942(口)andS. 6803(圏). 4. 1Q-4yQ-3.

(11) 1'2乃. ∫5・5μ ψ Thethiol-modulatedenLynleSinthePCRcycleareactivatedbyreducing. equivalentsthatcanbegeneratedphotochemicalIyviaaferredoxin-thioredoxinsystem {Buchanan1980,1991).Thus,thiol-modulatedenzymes.areinvolvedinthemetabolic regula正ionofεhePCRcydeinchloroPlas童sofhigherplants.Inpai-Cicuiar,FBPase apPeal'stobeakeyregulatoryenzylneinthenxationofCO2since玉thasthelowest activityamongthethiol-modulatedenzymesandistheprincipaltargetofattackbyH202 {CharlesandHalliwell1980). In. _thisarticles,Istudiedthefollowingtoprovideimportantinformationson. molecularpropertiesofthiol-modulatedenzymesinvolvedinPCRcycleinAlgae.Then IdiscussedthemechanismsofH202-resistancesystemand重heregu!ationof photosynthesisinalgalcells.. DErfectofligh電. ノdarkontheactivitiesoflhiol-1nodulatedenzymesinvolvedhlthe. PCRcycleincyanobacfienaandeukaryoticalgae(chapterIi,VIIandVIII) 2)Molecularpropertiesof'thiol-modulatedenzylnesincyanobacteria(chapterIII, IVandVII) 3)Physiologicalfunctiく)nsofFBPaseisozymesincyallobacteria(chapletV) 4)Molecularpropertiesofthiol-modulatedenzymesineukaryoticalgae(chapterVI andVIII). 5.

(12) CHAPTERII. TheLackofLight/DarkRegulationofEnzymesInvolvedin the .PhotosyntheticCarbonReductionCycleinCyanobacteria. Lightplaysanimportantroteintheregulationofthiot-modulatedenzymes,thatis. ,. FBPase,SBPase,NADP+-GAPDH,andPRKinthePCRcycleofthechloroplastsin higherplants(Buchanan1980).Theinvivoregulationoftheseenzymesinvolvesa syste1τ1whichconsistsoffeπedoxin,thioredoxin/orrrt,andferredoxin!thioredoxin T'edLICtclSe・Furthermore,theeffectsoflightonactivationofthethiol-modulatedenzymes canbemimickedbyDTTinvitro(Zimmermannetal.1976,BalerandL.titzko1975). L・wc・ncentrafi・ns・fH. 、0、str・nglyinhibittheCO、fixati・n・fintactchl・r・plasis. !SU且atedfromhigherplants(50%inhibitionatlOμMH 202)andthe霊hiol-modulated enzymesinthePCRcyclearemajortargetsofauackbyH 202(Kalserl976,Asadaand Takahashi1987).Aspreviouslydescribedin,chapterI,photosynthesisofEccglenrc, Chlurraytlofnoncrs,andcyanobacteria,thatis,S.7942andS.6803isnotsusceptibleto H202uptolmM,whichresultsfromtheresistanceofFBPase,NADP+-GAPDH,and PRKiilthePCRcycletoH202(Takeciaetal.1995).Thesefactsraisethequestionasto howthePCRcycleisactuallyregulatedinvivoincyanobacteria. ToexploretheregulationsystemofthePCRcycleofcyanobacteria,Istudiedthe effectsoflightandDTTtreatmentontheactivitiesofFBPase,NADP+-GAPDH,and PRKinS.794?andS.6803cells.IhavefoundthattheenzymesofthePCRcycleare notsensitivetotreatmentwithD'1'TandIrenotlightregulated.. MaterialsandMethods. 01二9`〃 τど∫'raandCGfltL〃e-一. 一SynechococcccsPCC7942ahdS)ノ. 6803wereculturedinAllen'smediumat27℃f'or5daysunderilluminadon(240μEs. ユ. を. ロユ. In)withbubblingofsterileairat81111正n(Takec.laetal.1993b).. Enz)erneCISSの!S-一. 一FBPasewasassayedbymonitoring.thereductlonof. NADP+at340nm(Zimmermannetal,1976).Inthefinalvolumeoflml,thereaction mixturecontained10σmMTris-HCl,pH8.0,10mMMgC12,05mMEDTA,0.4mM. NADP+,0.lmMFnl1,6P2,05unitsofglucose-6-phosphatedehydr-ogenase,15units ofphosphogluOoseisomerase,andtheenzyme.Thereactionwasstartedbyadditionof Frul,6-P2afterpi-eincubationfor2min.NADP+-GAPDHwasassayedinareacti()n. 6. ηθ`・ho`ツ3'∫ ∫PCC. -t.

(13) milture(lml)containing100mMTris-HCIbuffer(pH8・0)・10mMMgCI2・5mMATP, U.?mMNADPH,10mMGSH,2unitsofphosphoglyceratekinase,3mM3phosphoglyceraCeandtheenzymesolution.Themixturewaspreincしtbaledfor4minat 27°Candthereactionstartedbytheadditionof3mM3-phosphoglycerate(Latzko andGibbs1969).PRKvasessayedat27°Cbythemethodcoupledwithpyruvate kin乙聡eandlac愉tedehydrogenase.Thereactionmixturecontained50mMTris-HCl bul`Cer(pHS.0),10mMMgC12,5mMGSH,1mMATP,0.2mMNADH,SOmlvlKCI, 0.5mMphosphoenolpyruvate,2unitsofpyruvatekinase,5unitsoflactate dehydrogenase,2unifisofriboseS-phosphateisomerase,2mMribose5-phosphateand theenzymesolution(Latzko,GarnierandGibbs1970}. Chlorophyll(Lichtenthaler1987)andprotein{Bradford1976)concentrationswere measuredbythemethodsdescribedinthecuedreferences.. Preparationρ. プ6κ 亦㏄`∫4μ1伽81ight/darkcon`litivns-一. 一Tostudytheeffectsor. light!darkconditionsontheactivitiesoflhiol-1nodulatedenzymesinspiIlachleavesand cyanobacterialcells,extractswerepreparedasdescribedbelow.Spinachleavesandthe cellsof∫.7942andS.6803wereleftfor3hinthedarkbeforeilluminationatl600μEslm-2for30111111. .Auheindicatedtimes,thecrudeex￠ractswereprepal-edasdescribed. below.SpinachIeaves(1.Ogwetwt.)werehomogenizedinsmloC1001nMTris-HCI, pH8.0,containing16mMMgC12usingamortarandpestleforsmin.Thehomogenate Wc1Scentrifugedat12,000x8forl5min.Algalcells(1。Ogwetwt.)werehaハ. ・estedby. centrifugation,resuspendedinthesamebuffεr,sonicated(10KHz)forlmin,and centrifugedat12,000xgfor15min.Theobtainedsupernatantswereusedasthecrude extract.Allmanipulationsofthedark-adaptedsamplesbeforeilluminaCionwerecarried outunderagreensafeIight.. Pr6ρ. ω τ廓oη. ρプ α 〃τκ・ ∫∫`'η4DTTtreatfrzent-一. 一Chloroplastswereisolatedfrom. spitlachleavesbythemethodspreviouslydescribed{JensenandBasshamlX66).The integrityofchloroplasls,asdeterminedbytheferricyanidemethod(ShigeokaetaLl980c), wとおapPIて)x.90%.Spinachchior〔. 〕Plasts(6・0111chlorophyll)andcyanobacterialcells. (1.Ogwetwt.)weresuspendedinsandoflOUmMTris-HCI,・. ・0,00n￠ainingl6mM. MgCl2inthepresenceof20mMDTT,sonicated(10kHz)forlmin,andcentrii'tigedal 12,000xgfor15min.Thesupernatantswereusedasthecrudeextract.To completelyremoveDTT,thecrudeextractswereputonaoolumn(10cmx15cmi.d。)of SephadexG-25(Phamlacia)equilibratedwithlOOmMTris-HCI,pH8.0,containingl6 mMMgCI2saturatedwithN2gas・TheproteinfractionsataconcentrationoCapprox・12 mgPI℃tein/nllwereputintovialssatui◆atedwithN2gas・Aftertheadditionof20mM DTT,theactivitiesofthiol-modulatedenzymeswereassayedat10,20and30mins・. 7.

(14) ResultsandDiscussion. Previousstudieshaveindicatedthatthiol-modulatedenzymesinthechloroplastsof higherplantswereactivatedbylight(Kellyetal.1976,WolosiukandBuchanan1978, Andersonetal.1979).TheeffectsoflightontheactivitiesofFBPase,NADP+GAPDH,andPRKwasstudiedincyanobacterialcellsandinspinachleaves(Fig.II-1). TheactivitiesofFBPase,NADP+-GAPDH,andPRKinthecz-udeextractfromdarkadaptedspinachleaveswere8.89±0.2,125.4±4.0,and75.3±1.2.μInolh-1(mg chlorophyll)"`,respectively,whichwereabout6.6,8.0,and4.5°lo,respectively,ofthe activitiesinthecrudeextractfromlight-adaptedleavesbeforethedark.Whenthedarkadaptedleaveswereilluminated,eachactivityorthethiol-modulatedenzymesincreasedCo tllesameleveiasobservedinthecontinuouslyiUuminatedleaves.Theactlvitiesof FBPase,NADP+-GAPDH,andPRKinthecrudeextractpreparedfromthe5「.7942cells inthedarkfor3hwere79.8±5.8,679.8±12.8,and1025±19.6μmolh°1(mg chlorophyll}-1,respectively.Asimilarsituation≪asalsofoundintheenzymesinS. 6803cells.TransferofS.794?andS.6803cellsfromthedarktoilluminationat1,600 SCEs-`m-'(àiledtoincreaseeachenzymes(Fig.II-1).. Fig.II-1Effec#sofLight/DarkCondi#ionsontheActMtiesofThiol-Modulated EnzymesinSpinachLeavesandCyanobacterialCells.Spinachleavesandthecellsof S.7942andS.6803wereleftfor3hinthedarkbeforeilluminationat1600NEs"'m"2for 30min.Attheindicatedtimes,thecrudeextractswerepreparedasdescribedbelow. Thedataarethemean±SDofthreereplicates.・,FBPase; . ,NADP+-GAPDH; . PRK.. 8. ,.

(15) Thelightactivationofthiol-modulatedenzymesinthechloroplastsofhigherplants canbemimickedbytheDTTtreatmentinvitro(Zimmermannetal.1976,Baierand Latzko1975)。Whenthecrudeextractwaspreparedfromspinachintactchloroplastsin thepresence.of?OmMDTT,theactivitiesofFBPase,NADP+-GAPDH,andPRKwere 75.8±4.1,967.0±28.8,and984.2±26.3nmolmin-'(mgprotein)"1,respectively. TheremovalofDTT.causedthe.activitiesof.thethiol-modulatedenzymestodecrease dramaticallyantithesubsequentadditionof20mMDTTcausedthesetoincreaserapidly toreachthelevelsthatcorrespondedtotheinitialactivities(Fig.II-2).Theseresults agreedwiththepreviousdatareportedbyZiininerrnann(1976),Theactivitiesor FBPase,NADP+-GAPDH,andPRKpreparedfromS.7942cellswithChebuf#erinthe presenceofDTrshowed66.7±5.0,637.3±23.2,and1140.7±245nmolmln'1(mg protein)-i,respectively.However,theremovalandtreatmentofDTTha⊂11ittleeffecton theactivitiesoftheenzymesfromS.7942(Fig.II-?).Thesameresultswerealso obtainedwith山eenzymes111S.6803cells.Theseresulissuggestedthattheenzymesin thePCRcycleofS.7942,S.b803donotneedareluctanttoproducetheiractivities.. Fig.II-2EffectsofTrea#mentwithDTTontheActivitiesofThiol-ModulatedEnzymes inCrudeExtractsfrom-SpinachChloroplastsand-CyanobacterialCells.Afterthe additionof20mMDTT,theactivitiesofthiol-modulatedenzymeswere.assayeda#10,20 and30rains.Eachclosedmarkshowsthevaluesofenzymeactivitiesinthecrude extractspriortotheremovalofDTTbyacolumnchromatographyofSephadexG-25. Thedataarethemean±SDofthreereplicates.O,FBPase;n,NADP+-GAPDH;CJ, PRK.. 9.

(16) TilelightactivationofNADP+-GAPDHinpees(GapA)involvesthereductive cleavageofaclisullldebondbetweentheCys-18andCys-285(Lieta1.1994).In addition,thechloroplasticFBPaseofhigherplantscontainedtwocysteineresidues separatedbyonlyfouraminoacidresidues(Cys-Val-Val-Asn-Val-Cys}inwhichthe aminoacidsequencewasoneoftheregulatorysitesofthelight-dependentactivation. (Marcusetal.1988).JudgingfromtheaminoacidsequenceoftheNADP+-GAPDH froln∫.7942,∫,6803andC〃. 伽. ン40脚. ηα∫sp.W80acysteilleresiduecorrespondingto. Cys-285inpeaswasabsent(PacoldetaL1995,chapterVII,VIII)・Asdescribedinthe ne刈chapter,S.7942cellscontainedtwoFBPaseisozymes(FBPase-IandFBPase-II), andshattheFBPase-Icouldhydrolyzebothfructose1.,6-bisphosphateandsedoheptulose 1,7-bisphosphatewithalmostequalspecificactivities(chapterIII).TheFBPase-IIgene lackedthesepotentialredox-sensitivecysteines.Inaddition,thenucleotidesequenceof theFBPase-Ifrom5'.7942andS.6803showednosimilaritieswiththoseofany previouslyknowntypicalFBPases,indicating;thatthepotentialcysteineresiduesinvolved intheferredoxin!thioredoxinsystemdidnotexistintheFBPase-Iprotein(chapteI'III). InthecaseofPRKof∫.7942and∫.6803,theN-terminussequencesincludingtwo cysteineresiduesresponsibleforthelightactivationshowedthatthoughthetwocysteines, thatis,Cys-16andCys-55wereconserved,thel70rl8aminoacidresidueswere missingamongthe40residues.foundinthePRKofhigherplants(Wadanoetal.1995). ThesedataindicatedthattheIeisconsiderabIestructuraldifferences111巳hevicinityofthe chiolgroupsinvolvedinthelightactivationbetweenthechloroplaslicthiol-modulated enzylnesofhigherplantsandthoseof5.7942andS.6803.BasedOllthedatareported hereandthemolecularcharacterizationofFBPase,NADP+-GAPDH,andPRK,itis conceivablethatthePCRcycleofsomecyanobacteriaisnotregulatedbylight. Tosustainthephotos》1ntheticCO. CO. 2fixationratecorrespondingtoaboutl50μ1nol. Z11-`(rngchloc'ophyll)一ìnhigherplants,theactivitiesofFBPase,NADP+-GAPDH,. andPRKinthePCRcyclerequired33,200,andlOO%oftherateofCO2rixation, respeclive(y(RobinsonandWafer1981).Accordingly,theacliviiiesofFBPase, NADP+-GAPDH,andPRKindark-adaptedspinachleaveswerenot:enoughtosustain thephotosynthe重icCO. 2fixation(Fig.II-1).TheratesofphotosyntheEicCO2assimilation. inS.7942andS・6803cellswere1345±10・5,andl26±?・8μm・tCO、h-1(lng chlorophyll)"`,respectively{Takedaetal.1995).Accordingly,thetheoreticalactivities. ofFBPase,NADP+-GAPDH,andPRKof∫.7942are44,4,269,and135μmo1/h!mg chlorophyU,respectively,Theactivitiesoftheenzylnesboth'inthelightanddarkadaptedS.7942cells≪erenotaltered{Fig.II-1),suggestingthatthePCRcycleinthis orgallismissumcientlyoperatinginthephotosyntheticCO2fixation.Asimilarsituation. 10.

(17) alsooccurredinthePCRcycleofS.6803cells.Thesefactsraisetheqt皇estionastohow thePCRcycleincyanobacteriaisregulatedinthedark,ifnecessary.IChasbeen repol嘘edthattheferredoxin!thioredoxinsysCemispresentinsolnecyanobacteriaspecies, suchas5'ynechococctcs(Schmidt1980)and/Vostoc(AustinetaL1992),andthatthe FBPaseofSynechococcccs6301andNostocmttscorltrnisactivatedbythioredoxin (Schmidt1980,Austinetal.1992,Yeeetal.1981).However,thedataavailable showedthatthePCRcycleofsomecyanobacteriaisnotregulatedbylight,suggesting thatitmayhaveanalternativeregulationsystemdifferentfrolnthelight!darkregulationof thiol-modulatedenzymesinthe. _chloroplastsofhigherplants.Somemetabolitesmight. mainlyregulatetheenzymesinvolvedinthePCRcycleofcyanobacteiia.Itisworth notingthattheFBPaseisozymeFBPase-IfromS.7942andS.6803 FBPaseandSBPaseacti、. _containsboth. ノities,issensitivetochangeinthelevelofMgCI. hlhibitedbyalowlevelofAMP(chapterIII,IV). Summary. 一D uringthePCRcycleofSynechococcusPCC7942andSynechocystisPCC6803,. FBPase,NADP+-GAPDH,andPRKwerenotsensitivetotreatmentwithDTT,a reducingagent,rnvitroandwerenotregulatedbylightinvivo,unlikethechloroplastic enzymesofhigherplants.TheseresultsindicatethatthePCRcycleinthecyanobacterial cellslnaynotbeactuallyregulatedbylightinVIVOeveniftheferredoxin/thioredoxin systemjspresent.. 11. 2,andis.

(18) CHAPTERIIl. MolecularCharacterizationandResistancetoHydrogenPeroxideof TwoFructose-1,6-bisphosphatasesfc・omSynechococcicsPCC7942. Fructose-1,6-bisphosphat<ise(FBPase;EC3.1.3.11),whichcatalyzesthe formationoffructose6-phosphate(Fru6-P)byhydrolysisoffnictose1,6-bisphosphate (Fru1,6-P2)islocatedinthechloroPlastandcytosolofhigherplantsandparticipatesin thePCRcycle,gluconeogenesis,andoxidativepentosephosphatepathwaysof carbohydratemetabolism(Zimmermannetal.1976,Kellyelal.1982,Preis1982, Marcusetal.1987).ThepropertiesofthechloroplasticFBPaseareclearlydistinctfrom thoseol`cytosolicFBPasewithrespecttolight-dependentactivationbywayo1à ferredoxin/thioredolinsystemandinsensitivitytoAMPinhibition(Marcusetal.1987, Buchanan1980)・Inphotoautotrophicprokaryoticcellsincludingcyanobacteria,aspecial regulatorymechanismoftheFBPaseactivitymayberequiredbecauseinthesecells,due toalowerdegreeofcvmparcmentation,thePCRcycleisnotclosedinaseparate organelle(Gerblingetal.1984).PreviousstudieswithpartiallypurifiedFBPasesfrom cyanobacteriasuchasSynechococcicsleopoliensishaveleadtoconflictingresultsinthe kineticpropertiesoftheenzyme(Bishopl979,Gerblingeta1.1985).Aa;ordingly,the lackofinformatlononmolecularcharacterizationofFBPasehaslimitedthe understandingot`theregulatorypropertyinthecyanobacterialPCRcycle. Asdescl-ibedinchapterI,photosynthesisofeukaryoticalgae,Each・lendand Chlcrrrayrlorrzoncrs,andcyanobacteria,SynechococcccsPCC7942andSynechocystisPCC 6803,isnotsusceptibletoinhibitionbyH202uptolmM,incomparisonwiththatof chloroplastsof-higher-plants,whichresultsfromtheresistanceofthiol-modulated ellLymes,FBPase,NADP+-GAPDH,andPRKtoH20zinalgalchloroplastsand cyanobacteria(Ta.keda.etal.1995). Inthischapter,IdescribethecharacterizationoftheFBPaseisoenzymes(FBPase-I andFBPase-II)purifiedfl℃mε,7942andthecloningandnucleotidesequenceofthe genesencodingbothisoenzymes.Theenzymaticandimmunologicalpropertiesandthe nucleotideseque11㏄forFBPase-IclearlyshowthatthisformwithSBPaseactivity belongsEOanewtypeofFBPase.Ialsoinvestigatedwhetherthepurifiedisoenzymes andtherecombinantforlnsexpressedinE.coli、serestESCeptibletoinhibitionbylmM x202.. 12.

(19) MaterialsandMethods. ルlcrteric'is-一. 一Frul,6-P2,D-fructose2,6-bisphosphate(Fru2,6-P2),D-. sedoheptulose1,7-bisphosphaCe(Sed1,7-P2),AMP,pyruvatekinase,lactLite dehydrogenase,fructose-6-phosphatekinase,glucose-6-phospha柾)dehydrogenase, H202,phosphoglucoseisomerasewerefromSIGMACHEMICAL,CO.,USA, GENECLEANIIKitwasfromBIO101,INC,,USA,andBALB/cmicewasfroln CLEA,Osaka,Japan.Themolecularbiologyreagentsandenzymeswereanalytical gradeandwerepurchasedfro111commercialsources.. 0際9αzな〃a〃24c〃. π`形一一一Synechococcc`S'PCC7942wasculturedasdescribed. inchapterII.E.coli,strainsJMIUgandBL21(DF3)plysS,wasculturedat37°Cin Luria-BeItanibroth(LB)(L/IiUerl972).. Eπzy〃aeu∬uys-一. 一FBPaseandSBPasewasassayedaspreviouslydescribed. inchapterII.Oneunitofenzyrneactivitywasdefinedasthea1皿ountofenzymethat hydrolyzeslmmolofs∫ubstrate.ProteindeterminationwasbytheIneぬodofBradford (Bradford1976)usingBSAasastandard.. P111"!f1Ctll1QIZρ1°FBA)`rse-IISOenZytrte-一. 一AIIprocedureswerecarriedoutat. 4°C,S,フ942cells(32gwetwt)fromthestationaryphase(5days)wereharvestedby centrifugation,resuspendedin100mlof50mNipotassiumphosphatebuffer,pH8.0, containing2.Sil11VIDTT,1nllviGSHand10%sucrose(SUIULIOnA)andsonicated(10 KHz)foratotalofIOminwithfourintervalsof2mineach.Thislysatewascentrif'uged atl800008for20minproducingthecrudeenzymewhichwasloadedontoaHiLoad. ロ . 26/10QSepharosecolumn(FPLCsystem,Pharmacia)equilibratedwithsolutionAand developedwitha340ml-lineargradientofNaCl(0-0.6M)SCaIOWtateof4mlmin. TheactivitiesoftwoFBPaseisoenzymeswereelutedastwoseparatedpeaksatO.24N・. 五. (FBPase-1)andO.34M(FBPase-II)ofNaCLTheacfiivefl'actionsofFBPase-Iwere combilledandadjustedto30%saturationwith(NH4)2SC㌧.Thesupernatantwas chromatographedonaHiLoad16!10PhenylSepharosecolumnequilibratedwith30% (NH4)2SqinsolutionA・Thecolumnwaselutedwith100mlofalineargradientof30一. ロ . 0%(NH4)2SC㌧atanelutionrateoflmlmin・Theactivei士acεionswerecombinedand fractionatedwith(NHS)2Sq,andthepelletprecipitatingbetween30and70%saturation wasdissolvedin2mlofsolutionA.Theenzylnesolutionwaschromatographedontoa HiLoadl6!60Superdex200columnequilibratedwithsolutionAcontainingO・15M NaCI.TheactivefractionswereconcentratedtoatinatvolumeofO.Smlbyultrafiltration (AmicollPM-30)andloadedUtllOa2㌧5「-ADPSepharosecolumn(0.8cmx4cm). 13.

(20) egLtilibratedwithsolutionA.About30minlater,thecolumnwaselutedwithsolutionA -i. and1.2mlfractionscollectedataflowrateof?OinlhTheptirifieclFBPase-Iwas staredat-20°rCwithoutextensiveinactivationforseveralweeks.. Polycrcrylcrrraielegelelectrophoresis‐. ‐. ‐Discgelelectrophoresiswith7.5°lv. ロユ. (WIV)polyacrylamideatpH9.4wasperformedaspreviouslydescribed(Shigeokaetal.. 1987b).Electrophoreslswascarriedoutataconstantcurrent(2mAgel)with bromophenolblueasthemigrationmarker.SDS-PAGEwasperformedonl2.5%(w!v). polyacrylamideslabgelsaspreviouslydescribed(Takedaetal.1993x).Proteinsinthe gelwerestainedwithCoomassiebrilliantblueR-250anddestainedin7%aceticacid. Esti〃 π4'oηof〃aolec〃!ω. ・vveight-一. 一ThemolecularweightofFBPase-Iand. FBPase-IIwasdetermilledusingaHiLoadl6!60Superdex200prepgradecolumn equilibratedwithsolutionA. ,containingO.15MNaClandcalibratedwithmolecular. markers(MW-GF-1000}fromSigma.7ndeterminingthemolecularweightofasubunit bySDSIPAGE(Takedaetal.1993a),anLMWkitEfrolnPhannaciawasし. 聖sedasthe. standard.. Digestionρ!」F君1)≪e-1α. η4peptideseparation-一. 一ThereducedandS-. carboxylnethylated(MarcusetaL1981)FBPase-1(10nmol)wasdissolvedin125μ10f 100mMTris-HCI,pH9.Oanddigestedbyincしlbationwith50pmolofAchromobcu・ter iysylendopeptidase(WakoChemicals,Japan)for12hrat37°C.Theresultingpeptide. リユ. mixtureswereseparatedbyreverse-phaseHPLCcolumnofμBondasphere5μC18300A (3。9xl50mm,Millipore).GradientelutionwasdoneatO5mlminwithO.1% cri「luoroaceticacid(TFA)inwaderandO.1%TFAin60%acetonitrile.. Aη`'1)23∫30ゾ`〃}1∫ηo`κ ・ ∫`!38(1μθηcθ 一一一Theamilloacidseqしleveeanalysisofsix isolatedpeptides(P-1toP-6)ofFBPase-IandtheN-terminalregionofFBPase-Iwas performedbyautomatedEdmandegradationonanAppliedBiosystems492Agas-phase proteinsequencerusingthestandardprogramandreagentsprovidedbythemanufacturer.. ム・01伽 genes‐. η 伽`Zη. μdε0∫ 〃8∫89``θ ηC8伽4ン. ∫∫34F8Pω. ε一lan`1朋Pω. ε一11∫50ε ηzyηZε. ‐ ‐ThechromosomalDNAwasisolatedfromtheS.7942cells(wetwt.1.5. g)bythemethodofWillialns(Williams1988).Theoligonucleotideprimers correspondingtotheaminoacidsequenceoftheP-1peptideandtheN-terminusof FBPase-Iweredesigneda.ndsynthesized(JBioS,Co.,Japan>asfollows;5聖 (CT)TC(AG}CC(AG}AT(GC)A(AG}(AG)CC(GC)GT-3'and5'-AT(CT)GA(AG)GT. 14. 一.

(21) (GC)GT(GC)GA(AG)CA(AG)GC-3'.Theprimerscorrespondingtothemosthighly conservedregions{nucleotideresidues129-138[5'-GACCCTGTGGATGG(ACT)TC (GCT)TCCAACAT(CT)GA-3']and?80-?87[5'-CTCATA(AGC)AG(AC)A(AGC)(CT) C(GT)(CT)AG(CT)TTTCC-3']}ofFBPasesfromvariousorganisms(Huretal.199?, Marcus,andHac-r-sch1990,Rainesetal.1988,Marcusetal.1986,Hamiltonetal.1988. ,. EntianetaL1988)weremade.ThePCRproductsobtainedwilhtheseprilllers、vereused asprobestoscreentllel'ull-1engthDNAfragmentillcludingtheFBPase-IandFBPase-II genesfromtheS.7942chromosomalDNAduesledwi吐hEcoRI,B`〃nHI,〃. 加dIII,. Xbcal,BstPI・rStyl(TAIくARASHUZO,Japan)aCsunitsyCgDNA一. 且.TheS・ulhem. blochybridizatio!1wascompletedaccordingtotheinstructionsofAmersham(Fig. .III。1);. therewasonlyonefragmenthybridizingwiththerespediveprobeilleach量ane.A3.4 kbp-PstIlhlgmelltincludillgtheFBPase-Igeneanda5.5kbp-Bω1zHIrragment includingthcFBPase・. ・IIgenewereisolatedf1'omtheagarosegeiusing吐heGENECLEAN. IIKit,subclonedintoapBluesciiptIISK(+)vector(STRATAGENE)antisequenced usingthedicieoxychalnprimermethodmodiriedfordoしlble-s吐randedplasmidDNA.The overlappillginsertDNAfmgmentswereob吐ainedbystlbclollingallerres[rictdigestion antipartialdeletionbyexonucleaseIII.. 圃 . ＼斜. ダ d. §. "岬. 調◇. 論韓織ら禽 (kbp). 20 3.5. 20. 1.0. 5.5 3.0. Fig.III-1GenomicSouthernanalysisofDNAfromS.7942.GenomicDNAwas digestedwithdifferentristrictionenzymesasindicatedabovethelaneandsubjectedto Southernanalysis.ThePCRproductsofFBPase-1(A)andFBPase-II(B)were digoxigeninlabeledandusedashybridizationprobes.Detailsoftheprocedureare describedintheMaterialsandMethodssection.ThesizesofDNAmolecularmass standardsareindicated. E,rpre.∼ ・lionρ. プFBPccse-16∠. η`1FBA)ω8-118・8〃8∫!/2E.coli-一. cons吐ructionoftheplasmidtoexpresstlleclonedFBPase-Igene,thefull-len8{hprotein encodingDNAfragmentswasamplifiedbyPCR.Twootigonucteotideprimers containedtheNileIrestrictionsitewiththeATGtranslationinitiationcolonandtheBcara HIrestrictionsite(boldsequence),5'-CCATATGGAGAAGACGATCGGTCT-3'. 15. 一Forthe.

(22) (nucleotideresidues-4-2U)and5'-AACTGCGGGATCCATGAGGCCG-3'(nucleotide residues1164-1185).ThePCRproductsweredigestedwiththeNdeIandBonaHI restrictionenzymesandligatedintoaNdeI!BnrraHI-digestedpET3aexpressionvector (Novagen)rollowedbyits1ransformationintotheEcolistrain,BL21(DE3)pLysS {Stticlierelal.1990).TherecombinantE.solicellsweregrownwithshakingat37°Cio. ロ -1. anA6000fO・6-0・7inLBmediumcontaining50μgmlalnpiciUinand34μgml chloramphenicol.FortheconstructionoftheplasnmidfortheFBPase-IIgene,two oligonucleotitieprimers,5'一. 一CTTCATGGCTCAATCCA-3'(nucleotideresidues-6-17). and51-AGGACGAGAAGAGCAAAG-3冒(nucleotideresiduesIl72-1玉91)wereused forPCR.TheproductswerebltlntedwiththeMangBeanNucleare,andligatedintoa EcoRI-digestedantithenMungBeanNuclease-bluntedpKP150Uelpressionvector. . く. (MkietaLl987),followedbyidstransformationintotlleE.colistrain,JMlO9.The. recombinantE.solicellsweregrowninLBmediumcontaining50ysgmlampicillin. Eachexpressionwasinducedbyaddingisopropylb-D-thiogalactopyranoside(IPTG)to O.4mMandshakingfog-6hat37°C.. Pc〃ゆ卯 α 厩oη. ρプreco〃ibincurtFBノ)α. ∫8-1`〃14FβP`68-111S・oen,ン. 〃38∫. 一一一. 一The. rec;ornbinant(FBPase-ll)E'.colicells(1.6gwetwt.)wereharvestedbycentrifugationat 5008rorlOmin,resuspendedin8mlofsolutionAandsonicated(10KBz)foratotai oflminwithfourintervalsof15seceach.Thislysatewascentrifugedat-12000gfor l5minandthecrしldeenzymeobtainedwasusedforanalysisoftotalsolubleproteinof E.cullbySDS-PAGEandthensubmittedtothesamepurificationprocedurebyaHiL.oad 26!loQSepharosecolumn,fractionationwith(NH,,)2so4,HiLoad16110Phenyl SepharosecolumnandHiLoad16160Superdex200colulnnasthatlbrthenative FBPase-1.ThepurificationprocedureyieldedarecombinantFBPase-IIpreparation purifiedapprol.3.8-foldoverlllecrudeenzyme,givingafinal?9.6%vrecoveryofthe activity,TheSDS-PAGEofthepuliriedFBPase-IIshowedonlyonedetectableprotein band(datanotshow11).Thespecificactivityof'thepurifiedrecombinantFBPase-IIwas 75μm・lmin°. 丘(mgPr・tein)一. ・.There・. ・mbinantFBPase-lwaspa・. 吐iallypuriliedby. fractionationwith(NH4)2SO4andaHiLoad16!10PhenylSepharosecolumn.. 1)reps〃. τ漉oη(ifantibodies`'8α`nstFBPase-1α. η4」FBP6四. nativeFBPase-IandrecombinantFBPase-IIproteinswereusedtoprepareeachantibody. Tlleenzymeprotein(500μgeach)suspendedinsolutionAwasdialyzedwithO.9% (w!v)salinesolution(21)for24hrandemulsifiedwiththesamevolumeofFreund響s completeadjuvant.FiveBALB/cmalemice(6weeksold)wereimmunizedby. -1. subcutaneousinjections(50μginjection)twiceattwo-weekintel'vals.Oneweekafter. 16. θ一Z1-,一. 一Thepurified.

(23) thesecondinjection,bloodwascollectedfromthetallveinofeachmouseandserumwas separatedfromtheblood.Theseseraweretestedbyenzyme-linkedimmunosorbent assay,andeachhightitermousevasgiventhelestinjection.Fourdaysa/tertheIasi injection,bloodwascollectedfromtheorbitalveinofeach-mouseandserumwas separatedfromtheblood・. 1'mrriunoblotting-‐. ‐ProteinswereseparatedusingSDS-PAGE(12.5%gels). with?-tnercaptoethanolandblottedontoanImmobilon-Ptransfermembrane{PVDF, poresizeO.45Vim,No.IPVH304FO,Millipore,Bedford,MA}usingasemidry electrobloiiingsystemaccordingtothemanufacturer`sinstructions(Bio-Rad). lmmunoblottingwasperformedaccordingtothemeEhodofShigeokaetal.(1991). AntibodyagainstmouseIgs,conjugatedwithperoxidase(BoehringerMannheimGmbH, Gerlnany),wasusedasthesecondantibody.. τ加. 肥8競. 伽c8ρ. プFBP`rseisoenzンrraestoH202-一. 一Tocompletelyrelnovethe. DTTfromtheFBPase-IandFBPase-IIpreparationsfromtheS.794?cellsandthe recombinantE.colicells,eachextractwaschromatographedonacofumn(1.5cmlIU cm)ofSephadelG-25equilibratedwith100mlviTris-HCIbuffer,pH8.U,containing16. ロ . mMMgCl2.Theproteinl了ac廿on$werepIacedintovialssariiratedwi吐hN2gastogivea. concentrationofapproximately12μgproteinIn「Thepurifiedrecombinantandnative enzymeswereincubatedwithH202forlOminlnthedarkasdescribedprevious1)ノ (Takeclaetal.1995).. Results. 5「θP`〃 τπめ η`〃zdch`〃. τκ彪擁zoπoηofFB・Pα. ∫8-1rrnrl1遅3P`∬6-Z1.〃o'ηS・7942-一. 一. TheFBPaseactivitieswereseparatedintot、vofractionsbyQsepharosecolumn chl・omatographyastwopeaksatO.24M(FBPase-1)andO.34M(FBPase-II)orNaCl (Fig.III-2).Theactivityratiool'FBPase-ItoFBPase-IIwas12:lwithatotalI'ecovery of74%.Thisprocedurehasbeenrepeatedthreetimeswithasimilarelutionpattern・The purilicationschemeof∫.7942FBPase。Iusirlgthefive-stepprocedureissulnmarizedin TableI.ThepurificationprocedureyieldedaFBPase-Ipreparationpurifiedapprox. 35.S-#òldovertheactive#'racoonsofFBPase-lbyQSepharosewithayieldof33.2%.. -1. ThePAGEandSDS=PAGEofthepurifiedenzymeshowedonlyonedetectableprotein band(Fig.III-3).ThespecificactivityofthepurifiedFBPase-Iwasll.7μmolmin (mgprotein}-1.. 17.

(24) (Σ )6 ①Z. 204060. 1. 80. FractionNo.(4.25ml/tube). Fig.III-2ElutionPatternofFBPase-IandFBPase-IIIsoenzymesbyHiLoadQ SepharoseColumnChromatography.ThecrudeextractswerepreparedfromS. .7942. cellsinthestationaryphaseandloadedontoaHiLoadQSepharose26/10column equilibratedwithsolutionAasdescribedintheMaterialsandMethodssection ・,FBPase;O,SBPase;一. .. 一一一,NaCIconcentration. TableIII-1PurilicationofFBPase-IwithactivitiesolIructose-1,6-bisphosphataseandsedoheptulose-1,7-bisphosphatasefrom Sン ηθoわocoocロ57942. Step. TotalproteinTotalactivitySpecilicactivityPunhcationYield (mg)({imolmin-)({imolmin・mgprotein-)(fold)(%) FBPaseSBPaseFBPaseSBPaseFBPaseSBPaseFBPaseSBPase. Crudeextract. 2919. 221.7. 1797. Oorsooss. 461,5. 150.7. 157.0. 0.327. 210.0. 12.9. 307.7. 144.4. 150.4. 0.469. 0,;.. 18.1. 134.6. 140.1. 7.44. 7.74. 6.7. 87,0. 905. 130. 13.5. 4.3. 50.1. 52.1. 117. 12.1. QSepharose peak-1(FI) peak-2(FII) 30%(NHa)2SO4(FI) PhenylSepharose(FI) Superdex200(FI) 2",5'-ADPSepharose(FI. 0.340. 1.00. 1.00. 100. 1.43. 95.8. 958. 22.8. 89.3. 89.2. 398. 39.7. 57.7. 57.6. 35.8. 35.6. 33.2. 331. zza. 1.43. B. A. 麓. (kDa) 94 67 43. 撫. 30. 憎40. 鞠嚇・. 20 十. 100. 0061. 14. Fig.III-3PAGEandSDS-PAGEofFBPase-1.Theproceduresforelectrophoresisof thepurifiedFBPase-IwerecarriedoutasdescribedintheMaterialsandMethodssection. PAGE(A)andSDS-PAGE(B)ofthepurifiedenzyme(3Ng)werestainedwith CoomassiebrilliantblueR-250.. 18.

(25) Thec;ompar-isonof-somepropertieso#'zheS.794?FBPase-IantiFBPase-II(native antirecombinant}withthoseofchloroplasticandcytosolicFBPasefromspinach (Zimmermannetall976,StittandHeldtI985,StittetaL1985,L誕zaroetal.1974, Zimmermannetal.1978}areshowninTableII.Thesubstrate-velocitycurveswith FBPase-IandFBPase-IIshowedMchaells-MenCentypekineticswithFru1,6-P2at concentrationsuptolmM:theapparentKmvaluesoftheFBPase-IandFBPase-IIfor Fru1,6-P2were52±4・5μMand25±15μM,respectively,Theisoelectricpointsof FBPase-IandFBPase-IIwere4.8and5.4,respectively,whichareinagreement、vith thosecalculatedfromdeducedaminoacidsequences.TheMgCl2-velocitycurveswith FBPase-IandFBPase-IIweresigmoidal;theapparentSo.svalueswere1.4±0.1mM alld8.6±0.4mM,respectively,suggestingthatFBPase-IandFBPase-IIactivitieswere particularlysensitivetochangeintheMgC12concentration.TheFBPase-Iwasinhibited byAMPwithaKivalueofO.26±0、02mM,Iikethecytosohclbr星110fhigherpIa醐,but notbyFru2,6-P2,unlikethecytosolicform(StittandHeldtl985,LazaroetaL1974). AMPandFru2,6-P2hadnoeffectontheFBPase-IIactivity.TheoptimumpHof FBPase-IIwas9.0,whiletheactivitiesatpH8.OandpH9.5wereapprox.67and85%v, respectivelyofthatatpH9.0.. TableEII-2ComparisonofsomepropertiesofSynechococcus7942FBPase-IandFBPase-11 withthoseofspinachFBPases Spinach. S.7942. ChloroplastCytosol. FBPase-!FBPase-11 NativeRecombinantsNativesRecombinant. Molecularmass(kDa) 160. Aso. 160. ao. 37. 40. OptimumpH. :f. 8.0. Isoeiectricpoint. 4.7. 5.3. 33. 2.5. Gel#filtration SDS-PAGE. 150 40. 37. ・ .;. 37 9.0. 5.Ob. 5.4. 5.6b. EGr,value Frui,6-P2(μM>. Mgt{effect. 25土t5c. 52土4.5c 118土5.5c. Sed1.7-P2(μM). SigmoidalHyperbolic. Sigmaidal. Sigmoidal. AMPinhibition Kvalue(mM). nil. 2.4. 0.26tO.22. nik. a.s. nil. nil. nil. nil. ni!. Fru-2,6-P2inhibition Kvalue(uM) aPartiallypurifiedenzyme bDataarecalculatedfromdeducedaminoacidsequences cDataaremeanvalues±SDfromthreeassays. TheactivitiesofFBPase-IextractedfromS.7942cellswithbothFni1,6-P2and sedl,7-P2wereelutedwithO.24M-NaclbyQsepharosecolし1mllchromatography(Fig・ III-?}.ThespecificactivityoftheactivefractionsofFBPase-IwithSed1,6-P2was. 19. nil.

(26) simi[arlolha吐wi吐hFru1,6-P2(TableI).NoSBPaseactivitywasroしmdinanyether huction・Duringfur吐herPurificと1tions巳epsorFBPase-1,!heSBPaseacこivitywaselu吐ed fromeachCOIU11111asthesamesharpandsinglepeakastheFBPasc-Iac吐ivitywithFru 1,6-P2;thepuriricationandyieldofSBPaseactiviしyover吐heactivefractionsofFBPase-I byQsepharosewasallnostthesameasthoseoftheFBPaseactivi巳y(TableI).These resultsindicatedthatasingleproteindesignatedFBPase-Iisresponsibleforthetwo. ロ . activ而es.ThepurifiedFBPase-IsllowedaspeciricSBPaseactivityof12. .114molmim-'. (mgprotein)TheSedI,7-P2-velocitycurvewithFBPase-Ialsowasahyperbolicwith aκ. "、of118±5.SAM. Asshe)wninFig.III-4A,theantibodyraisedとlgainstthenativeFBPase-Icrc)ss.. reactedwiththepurifiednativeFBPase-Ibutnotwiththepartiallypurifiednative FBP乙. 遣e-II.Simnarly,theantibodyraisedagainstthe1℃c;ombinanlFBPase-IIdldnol. cross-reac;lwi【11thepuririednativeFBP乙use-1(Fig.III-4B). ,whi且eitreac:led、. partiallypuriricdnativeFBPase-II(lane3)andthepuririedrecombinantFBP乙. 聡e-II(da巳a. llUI.shown).TheantibodytoFBPase-Ialsorecognizedasingle42kDaproteininthe crudeextractofSynccho`ツSIISPCC6803,whichcorrespondedtoaFBPasepurified fromS.6803cells(chapterIV).However,bothantibodiesFailedtocross-reactwiththe partiallypuririedFBPasesfromspinach(1ane5).. (kDa). A. B. 42d鱒..',・43 40麺. 鱒峰. ・・、. 37. 、・ 30. 無'・、 ≧、20. 12345. Fig.III-41mmunoblotAnalysisofFBPasesUsingAntibodiesRaisedAgainstthe. ロ. PurifiedNativeFBPase-1(A)andRecombinantFBPase-II(B).Eachenzyme(3Ng)was subjectedto. .SDS-PAGEinslabgel.Detailsoftheprocedurearedescribedinthe. MaterialsandMethodssection.Lanes:1,purifiednativeFBPase-1;2,partiallypurified nativeFBPase-II;3,crudecellextractofS.6803;4,partiallypurifiedspinachchloroplast enzyme;5,partiallypurifiedspinachcytosolenzyme.. ム401ε α 〃αρ ηω3`〃2α4y5∫5-一. 一GelfiltrationonaHiLo乙1dl6160Superdex200. columnofFBPase-IandFBPase-IIwithseveralstanciarciproteinsindicatedamolecular massof160kDaand150kDa,respectively.Thesubunitmolecularmassof吐heFBPaseIwaseslimalecitobe40kDafromSDS-PAGE(Fig.III-3). ,while巴hatof巳heFBPase。II. waseslinuileci[Ube37kDafromimmしmoblotanalysisusing巳hepartiallypurificdnaUve FBPase-II(Fig.III-4B),whichwasinagreementwithapredictedmolecularmassofthe. 20. ∼・i吐hthe.

(27) clonedsequenceintheFBPase-IIgenedescribedafterward.Thesevaluesaresimilarto thoseofthechloroplastic(40kDa)andcytosolic(37kDa)FBPases#nomspinachwhich aretetrameric(Lazardetal.1974,Zimmermannetal.1978).Theaminoacidsequences oftheN-terminusandsixpeptidesofthepurifiedFBPase-Ienzymewereasfollows:Nterminus,MEKTIGLEIIEVVEQAAIASAGLM;P-1,TGLIGESRESNIARLQP-2, LMGK;P3,GGLFAAPDF;P-4,LAAPPAAK;P-5,DTVHMFDDVK;P-6,TVSLPL.. 15・olulioncrnclc乃. ω τκ!8万zα. ゴoη. ρfD2VAencoclingFIBPω8∫308ηzyη26∫. 一. restrictionsitesandsequencingstrategiesforthe3・4kbp-PstIfraglnentincludillgthe FBPase-Igeneandthe5.5kbp-B`〃. ηHIfragmentincludingtheFBPase-IIgeneare. showninFig.III-5.ThenucleotidesequenceoftheFBPase-Igeneconsistedofa1068 byopenreadingframe(ORF),encodingforapeptideof356aminoacidresidues(Fig. III-6).ThecalculatedmolecularmassoftheclolledsequenceinthegeneorFBPase-I was38251Da,whichwasclosetothemolecularmassoftheFBPase-Isubunit determinedbySDS-PAGE.ThededucedaminoacidsequencewincidedwiththeNterminusandsixpeptic.fesoftheFBPase-IpurifiedfromS.794?shownbythe underlines.TheaminoacidsequenceoftheS.794?FBPase-Igenehasnohomology withthoseofthetypicalFBPasesfromvarioussources(Hureta1.1992,Marcus,and Harrschl990,Rainesetal.1988,Marcusetal.1986,HamiItonetal,1988,Entianetal. 1988).Marcusetal.(1989)havereportedtheaminoacidsequenceoCsiloftheprepared peptidesfromS.leopoliencesFBPaseformB.Interestingly,theaminoacidsequences ofthepeptidesshowanidentitywiththoseofthe5'.7942FBPase-Ililtheregionsshown bytheshadowinFig.III-6・. ↓. レ. =避. ー . 500bρ. 王 E爵. 覧 __司 ---.<-一.一ーレ一レーレう. 3. 一. 一煽ε の. =♂ ト. 5 王 ε qo q. A. 一一一一一一一一1レ州. レーq巳一一一一 . 一 :≧一レ一. ぐ一一一一. レ. i_i. B. 工E避. 3. コ. ↓. 輌需 ηく〒﹁. チ. 至 ε弼. → 500by u. Fig.III-5SequencingStrategiesforFBPase-IandFBPase-IIGenes.Thethickbars representthelocationoftheFBPase-1andFBPase-ligenes,with3'and5'ends indicated.ThesubfragmentscontainingFBPase-IandFBPase-IIgeneswere subjected#osequenceanalysiswiththestrategyindica#edinthelowerportion.. 21. 一. 一The.

(28) 一171-161-151-141-i31-121-111-141-91 CGTCGCGCGCfiCCATGCCCGCAGCTGCGCCTTTGATGCCGCGGAAGATATTGCCGCCAACTAACGATANNAGTCACTGCGATCGCAACTA. 一81-71輌61-51-41-31鴨21口11-l AAGCCAGAGATGTGAGGAGGGGATCCGGCCTTTGGATGACTCAACTGTTGGAATCCCCAGAAGCAATCATCCGTAAGGAGTCAGGACGGC -35-10SD lQ2030405060708090 GTGGAGAAGACGATCGGTCTCGAGATTATTGAAGTTGTCGAGCAGGCAGCGATCGCCTCGGCCCGCCTGATGGGCAAAGGCGAAAAGAAT MEKTIGLEIIEVVEOAAEASARLMGKGEKN. 100110120130140150160170180 GAAGCCGATCGCGTCGCAGTAGAAGCGATGCGGGTGCGGATGAACCAAGTGGAAATGCTGGGCCGCATCGTCATCGGTGAAGGCGAGCGC EADRVAVEAMRVRMNΩVEMLGR灘. 垂鱗、翻・ ≡ 購購鰯;轍. 舞. 議. 190200210220230240250260270 GACGAAGCACCGATGCTCTATATCGGTGAAGAAGTGGGCATCTACCGCGATGCAGACAAGCGGGCTGGCGTACCGGCTGGC肌GC田GGTG. 縣1綴撚魏. ・鱗. 鱗. 鱗・翻_養. 罷. 霧. 猶莚. 馬. 諜. 藷DADKRAGVPAGKLV. 280290300310320330390350360 GAAATCGACATCGCCGTTGACCCCTGCGAAGGCACCAACCTCTGCGCCTACGGTCAGCGCGGCTCGATGGCAGTTTTGGCCATCTCCGAG E工DIAVDPCEGTNLCAYGΩPGSMA・VLAISE. 370380390400410420430440450 AAAGGCGGCCTGTTTGCAGCTCCCGACTTCTACATGAAGAAACTGGCTGCACCCCCAGCTGCCAAAGGCAAAGAGACATCAATAAAGTCC K購'轍. 一. く'轍:邑;:・. 灘"離:1:鱗KLAAPPAAKGKETSエKS. 460470980490500510520530540 GCGACCGAAAACCTGAAAATTCTCTCGGAATGTCTCGATCGCGCCATCGATGAATTGGTGGTCGTGGTCATGGATCGTCCCCGCCACAAA ATENLKILSECLDRATDSLVVVVMDRPRHK. 550560570580590600610b20630 GAGCTAATCCAAGAGATCCGCCAAGCGGGTGCCCGCGTCCGTCTGATCAGCGATGGTGACGTTTCGGCCGCGATCTCCTGCGGTTTTGCT ELzΩ. 呂. エRΩAGARVR鱒. 辮騰.鱗. 羅,翻. 、顯. 類. 麩A棄. 薄. C鑛. 葛Jj. 640650660670680690700710720 GGCACCAACACCCACGCCCTGATGGGCATCGGTGCAGCTCCCGAGGGTGTGATTTCGGCAGCAGCAATGCGTTGCCTCGGCGGGCACTTC. 鱗灘灘. 鵜!、鍛1嚢. 魏. 籍. ・GAAPEGVエSAAAMRC羅. 辮,、,;1彊舞㈱. 730740750760770780790800810 CAAGGCCAGCTGATCTACGACCCAGAAGTGGTCAAAACCGGCCTGATCGGTGAAAGCCGTGAGAGCAACATCGCTCGCCTGCAAGAAATG. 薫;轟繹雛購1;雛議難≡ll難鐘難鍵巌ミ黛議灘鱗ミ ≡ 灘'"雛. 灘犠1鍵. 820830840850860870880890900 GGCATCACCGATCCCGATCGTGTCTACGACGCGAACGAACTGGCTTCGGGTCAAGAAGTGCTGTTTGCGGCTTGCGGTATCACCCCGGGC. 藤1'徽'購. 鱗 ≡ll;鯉lll識;鱗lll鯉ll簸llllll灘鰭;瓢1;麟灘犠麟SGΩEVLFAACGエTPG 9109209309409509bO970980990. TTGCTGATGGAAGGCGTGCGCTTCTTCAAAGGCGGCGCTCGCACCCAGAGCTTGGTGATCTCCAGCCAGTCACGGACGGCTCGCTTCGTT. LLMEGVRFFKG・sR?9SLVエSSQSRTAR雛1韓ll. 10001010ユ020103010401050106010701080 GACACCGTTCACATGTTCGACGATGTCAAAACGGTTAGCCTGCCGTTAATTCCTGATCCCAAATGGCGGCCGGRGCGGTAGAACGGGTAT 藤;霧. き:1;:1:・.;::1:;:}.:1::1::ll:;;三':'1':'・:::・:・::° ・"凸_:1;;.里__玉. 乙__旦__工L_ΣL-.LI・PDPKwRPER禽. 109011001110112011301140115011601170 AGCTCGATCGCTTCGGTCGTTGTTTTTCAGCGAATCCATTTGCGATCGCTTTTCAAACCCTTTTTTCGTCAACCTTCTTTAAACGGCCTC. Fig.111-6NucleotideSequenceoftheFBPase-iGeneandDeducedAminoAcid Sequence,includingUpstreamandDownstreamElementsTheaminoacid sequencesoftheN-terminalregionandthesixpeptides,whichareidenticaltothe sequencesdetemlinedusingtheautomatedEdmandegradation,areshownbythe underlines.Thenumbersstand#orthosefromtheN-terminalfirstresiduesoneach line.Potential-35.-14andShine-Dalaarnosequencesareindicated.Theshadows Potentia卜35,Shine-Dalgarnosequencesareindicated representthe aminoacidregionswhichareidenticaltothesequencesofsixofithe preparedpeptidesfromS.ノ. θoρo〃encesFBPaseformB(Marcusetal.1989).. 22.

(29) Thenuclcotidescquenceof吐heFBPase-IIgeneshowedal10RFc)flOl7bp巳ha巳 encodcsapπ. て)teinof339aminoacjds,whichwasinagreemen巴. 、、・i巳h吐hedatareported. Previously(NewlnannetaLl995)・Thecalculaèdmolecularmassor吐hecloned sequenceonhisgenewas37190Da,whichwasidenticaltothoseofthena吐iveand rec;onibinanlFBPase-IIenzymes.Thededucedaminoacidsequencescoincidedwi吐hthe N4erminusofulenative乙mdrecombhlalltellzymes(NH3-M-A-Q-s-T-T-s-E-T-H-T-R). Thcseresultsindicatethauherecombinantenzymeisidenticalto【heFBPと. 聡c-IIprotein.. ThcdeducedaminoacidsequenceoftheS.7942FBPase-IIgeneexhibiしedhomologous sequenceswi吐hthoseoftypicalFBPasesrromwheatchloroplast(38」%)(Rainesdal. 1988),Arcrbiclopsischloroplast(42.1%)(HorsnellandRainesl991),spinachCylUsUI (43.7%)(HuretaLl992),sugarbeetcytoso1(43.4%)(HarnandDaiel992),pigkidney (39.3%)(W川iamsandKanlrowitzl992),∫`κ. ・chω40〃iyc・8∫c8148v'5惚(37.2%)(Entianel. aLI988)alldE,CU〃(44.8%)(H乙mlihonetal.1988),. EΨr8∬'o〃. ρ1ア. β1)`乙y81SUeYIZ,y))iC'genes"zEcoli-一. 一Ablerinductionwith. IPTG,tllercc⊂)mbillantproteins丘om吐heFBPase-IandFBPase-IIgeneswereexpressed illE.α)〃cells.AsshowninFig.III-7,therecombinantp1'αeillsorFBPase-Iand FBPasc-IIcorrelatedwellwiththecalculatedmolecularmass(、f40anしi371:Da, rcspectivcly.TheE.c・olirecombin乙inlFBPasc-IandFBPase-IIshowcdFBPascactivi吐ies 。f2.72・ndl.98μm()lmin°1(mgPmteln)・1,respectively.Inc・ntrusいhe8cθ. 〃,. BL21(DE3)pLysSandJM109asacontrolsh()wedenzymeactivitiesofl.2a11d6.8nmol 111111-1(mgprotein)-1,respectively.TherecombinantFBPase-IalsocontailledanSBPase adivityor2.83μmolmilrl(117protein)'1.Theellzymologicalpropertiesorthe recomblllan吐FBPase-IandFBPasc-IIwerethesameasthoseorthenaliveisoenzymes (TableI). AB (kDa)(kDa) 94 67_. 、,・. ぐ一40. 4. 7σ 9 6. 3 4﹁ー 30、. 囎. 43 醐齢. 20. くト37. 30 1134. 567. Fig.III-7ProductionoftheFBPaseisoenzymesinE.colibytheFBPase-1(A)and FBPasell(B)genes.Proteinstandards(lanes1,5),purifiedFBPase-1(lane2),the lysateofE.colistrain,BL21(DE3)pLysS(lane3),thelysateofrecombinant(FBPase-1)E_ coli(lane4),thelysateofE.colistrain,JM109(lane6),thelysateofrecombinant (FBPase-II)E.coli(lane7)weresubjectedtoSDS-PAGE.Eachlanewasstainedwith CoomassieBrilliantBlueR-250anddestainedwith7%aceticacid.Arrowsindicatethe FBPase-IandFBPase-II. 23.

(30) Theresistance`ゾr8co〃. め加`〃ttαz6!η. α万vθ. ∫∫08ηzy〃38∫. ρプS.7942toH,Oz-一. 一. AsshowninFig.III-8A,thespinachchloroplastFBPasewascompletelyinhibitedatO.1 mMH202,whichwasinagreementwiththeresultreportedpreviously{Takedaetal. 1995).Incontrast,thenativeandrecombinantFBPase-IandFBPase-IIwereonly slightlyinhibitedbylmMH202.-TheSBPaseactivityofFBPase-Iwasalsonot inhibitedbylmMH242,whiletheenzymeactivityofspinachchloroplaslswas(Fig.IIISB).TheseresultsindicatedtheabsenceofsusceptibilityofFBPase-IandFBPase-II isoenzytnestoH202ttptolmM.. Fig.III-8EffectsofH20zonActivitiesofRecombinantandNativeFBPase-1and FBPase-IIinS.7942.RecombinantFBPase-IIwaspurifiedfromE.coli,while recombinantFBPase-!waspartiallypuri#ied.ThenativeFBPase-Iwaspurified#romS. 7942cells,whilethenativeFBPase-IIwaspartiallypurl#ied.Spinachintactchioropiasts arepreparedaspreviouslydescribedinchapteril.ActivitiesofFBPases(A)for FBPase-1,FBPase-IIandspinachchloroplastsandSPBases(B)forFBPase-1and spinachchloroplastsweredeterminedasdescribedintheMaterialsandMethods section.ThecontrolFBPaseactivitiesofnativeFBPase-i(may,recombinantFBPase-1 ([]),nativeFBPase-ll(●),recomblnantFBPase・ll(○)andspinachchlorop[asts(▲)are shownas100%,which.are11.7±0.6,6.7±0.2,7.2±0.2,7.5±0.1andO.11±0.01 μmolmin曽1(mgprotein)-1,respectively.TheSBPaseac重ivitiesofnativeFBPase-1(■), recombinantFBpase・1(□)andspinachchEoroplasts(▲)are10.5±. α5,6.5±. O.068±0.003μmoImin-'(mgprotein>'1,respectively.Dataarerepresentedasthe resultsofthreeassays±SD.. Discussion. Autotrophictissuesofhigherplantsusuallycontaintwoisoformsoftheenzyme, oneinthecytosoiandoneintheplastids.InSynechococcusPCC7942,anobligate autotroph,thecellularFBPase.activitywillinvolveboththePCRcycleand gluconeogenesis.TheFBPasesfromS.794?etistedasisoenzymes,FBPase-Iand FBPase-II(Fig.III-2).FBPase-Irepresentedthem勾orpartofthetotalextractable activity;thiswasalsothecaseintheformBFBPaseisoenzymesinS.leopoliensis (GerblingetaLl985).TableIIS1117117ユarizestheoomparisonofsomepropertiesoftheS 7942FBPase-IandFBPase-IIwiththoseofcl}loroplasticandcyCosoiicFBPasesfrom. 24. α4and.

(31) spinach.Withrespecttothesigmoida1effectofMg'一+,theIackofFru2,6-P2inhibition andtheKrravalues1òrFru1,6-P2,bvthFBPase-IandFBPase-IIinS.794?weremore similartothechloroplasticformthantothecytosohcformofhigherp豆an重s.In photoautvtrophiceukaryoticcells,thecytosolicenzymeisallostericallyregulatedby AMP,whereasthechloroplasticenzymeisregulatedviaacovalentredo-modificationof proteintlliols/disulfidesmediatedbyferredoxin-thioredoxinreductaseandthioredoxins (Buchananetal.1971,Buchanan1990}.TheFBPase-IactivitywasinhibitedbyO.1 mMAMP,whereas. .CheFBPase-IIactivity/wasnot;thusinthisrespectFBPase--Iwas. similartothecytosolicenzymeratherthanthechloroplasticFBPase. TheFBPase-IpurifiedfromS.794.2wasfoundtohydrolyzeFru1,6-P2andSed l,7-P2:thekineticconstants,specificactivitiesandK〃avaluesweresimilarforboth substrates(TableII).Amongprokaryotes,activitywithbothsugarbisphosphateshas beenrepol覚edfor3ン R加408ρ. η8chococα. ∠5'leopolie'zsis(Gerblingetal.1986),. θ占κ10η20η64∫(SpronggateandStachowl982),ノVUC'CI」"L!1ì(AmaclliandBoqien. 1979}andHydrogenomoncrs{AbdelalandSchlegel1974).. .TheSouthernblot. hybridizationoftheSynechocystisPCC6803chromosomalDNAdigestedwithmany restrictionenzymesusingaprobeoftheFBPase-IIgeneindicatedthattherewasonlyone fragmenthybridizingwiththeprobeineachlane(Fig.III-1).TheantibodytoFBPase-I isolatedfromS.794?cross-reactedwitha46kDabandcorrespondingtoaFBPase presentincrudeextractsfromS.6803(Fig.III-4A).Thesefindingssuggestshat FBPaseoftheFBPase-ItypecancatalyzebothbisphosphatasereactionswithinthePCR cycleandmaybewidelydistributedincyanobacteria. ThededucedaminoacidsequenceoftheS.7942FBPase-IIgenehada considerablesequencesimilaritytothecytosolicandchloroplasticformsofvarious sour㏄s,indicatingthatFBPase-IIisatypicalFBPasetype,unlikeFBPase-1.Thefailure ofthecrossreacdvityofanlibociiesagainstFBPase-IandFBPase-IIwithCheoPPosite lSOtoi'n1SimpliedChatbothisoenzy111eshavenocommonepitope,thatis,dhighly antigenicregionrecognizedbyeachantibody(Fig.III-4).RecenIEy,ithasbeenreported thattheFBPase-IIgenewaslocatedupstreamofazwfgeneencodingglucose-6phosphatedehydrogenase(Newmannetal・1995)・ThecloseproximityoftheFBPase-II genetothez毘. プgenesuggestedthatFBPase-IIaswellasglucose-6-phosphate. dehydrogenasemaybeinvolvedintheoxidativepentosephosphatepathwaytooxidize glucose6-phosphatetoCO2inS.7942(Newmannnetal.1995,HorsnellandReines 1991).However,itisworihnotingthatsomeoftheenzymaticregulatorypropertiesof FBPase-IItestedweresimilartothoseofthechloroplasticformofhigherplants(Table II)andthatthetotalextractableen2ymeactivityofFBPase-IIwasverylowcompac-edto thatorFBPase-1(TableI),. 25.

(32) Kaiser(1979)hasdescribedthatH202atlowlevels(10μM)inhibitsthethiolmodulatedenzymesofthePCRcycleinthechloroplasisofhigherplants,b㏄auseHzO2 readilyoxidizesthereducedthiolgroupsoftheenzymes.Ihavedemonstratedthatthe thiol-inocitilatedenzymes,FBPase,NADP-GAPDHandribulose-5-phosphatekinase,in thechloroplastsofÈicglenccandC/zlcrrraydornoncrsandinthecrudeextractsof cyanobacteria,S.7942andS.6803,areresistanttoHzO2{Takedaetal.1995).Inthe precedingpaper,IhaveconfirmedthatthenativeGAPDHpurifiedfromS.794?ceits andtherecombinantGAPDHpurifiedfromE,colicellsareresistanttoH202(chapter VII).AsshowninFig.III-8A,thespinachchloroplastFBPasewascompletelyinhibited atO.lmMH202,whichwasinagreementwiththeresultsreportedpreviously(Kalser l979,CharlesandHalliwe111980).Incontrast,thenativeandrecombinantFBPase-I andtherecombinantFBPase-IIwereonlyslightlyinhibitedbylmMH202.Asdescribed above,theactivitiesofFBPase-IwithFru1,b-P2andSed1,7-PZsuggested.theclosely coupledregulationof`bothactivitiesinthePCRcycle.H202alsohadnoeffectonthe SBPaseactivityofFBPase-1{Fib;.III-8B}.Theseresu]tsshowedthattheSBPaseas wellasFBPaseisoenzymesofthePCRcycleinS.794?areresistanttoH202uptol mM.. ユハ. ユヨヨ. ユフ . spCh WCh spcy Sucy 箋SC EC F一工工. 離羅羅讐翻難難1 ComparisonofAminoAcidSequenceAroundPositionsEquivalenttotheFig.III-9. CysResiduesResponsibleforLight/DarkRegulationofFBPasesincludingS.7942 wheatchloroplast(WCh)FBPase-II. .Spinachchloroplast(SpCh}(Marcusetal.1990) (R釦,esetal.・::),・pinach・yt・s・1(SpCy)(Hu・etd・1992),sugarbeet・yt・s・1(S・Cy) (Harnetal.1992),Saccharomycescerevisiae(Sc)(En#ianetal.1988}andE.c°lt(Ec)th. atareacidsAmino(Hamiltonetai. .i988)aretakenfromthereferencescited. identicalwithS.7942FBPase-llareshaded.Dashesindicatedale#ion.Thenumber abovethesequenceofspinachchloroplastFBPaseindicatelocationsoftheCys residues(Marcusetal.1988,1990).. Thelightactivationofthiolenzymes,FBPase,SBPase,GAPDHandPR.K,inthe PCRcycleinhigherplantchloroplastsinvolvesareductivecleavageofadisulfidebond byreducingequivalentsthatcanbephotochemicallygeneratedviaathioredoYinfαTedoxinsystem(Buchananetal.197ユ,Buchanani990,ScheibeI990).Whyarethe. 26.

(33) thiolenzymesincludingtheFBPase-IandFBPase-IIresistanttoH202inS.7942?The nucleotidesequenceoftheFBPase-IgeneshowedthatFBPase-Iisdistinctfromany previouslyknownFBPasesandbelongstoanewtypeofFBPase・Consequently,the resistanceorFBPase-ItoH202isnotexplainedbythepositionsofpotentialredoxsensltiveCyspairspredictedbytertiarystructureofchloroplasticFBPases(Lieta1. 1994}.Marcusetal.(1988)havereportedthatthespinachchioroplastFBPasecontains twoCysresiduesseparatedbyonly4aminoacidresidues(Cys-Val-Val-Asn-Val-Cys) andimpliedthatthisaminoacidsequenceisoneoftheregulatorysuesofthelight dependentactivation.ThespinachcytosolicFBPaselackedtheextraaminoacid sequencepresentinchloroplasticFBPase,includingtwoCysresidues.Recently,the crystalstructureofspinachchloroplastFBPaseandthepredictedtertiarystructureof wheatchloroplastenzymehaveindicatedthatadisulfidebridgebetweentwoCys residuesofCys-155,Cys-i74andCys-179inthespinachenzymeisresponsibleforthe ligllt-darlLregulation(Lietal.1994,Villeretetal.1995}.AsshowninFig.III-9,These potentiairedox-serlsitlveCys,arenotconservedinFBPase-IIor5'.7942.Accordingly, theresistanceofFBPase-IItoH202isduetothelackofthethiolgroupswhicharefoしmd inthechloroplastFBPasesfromhigherplants.IChasbeenreportedthatthelight activationofGAPDHinpeas(GapA)involvesareductivecleavageofadisし. 山ldebond. beteeentheCys-18andCys-285(Lieεal.1994).IntheGAPDHgeneofS.7942,a Cys. .residuecorrespondingtoCys-285inpeaswasabsent;however,Cys-?Owaspresent. (ChapterVII).Recently,theGAPDHsfromAn`め. 磁 ηìvariabilisandSynechoeystisPCC. 6803havebeenfoundtolackoneoftheCysresidues(Pacoldetal.1995}.ThetwoCys residuesareconservedinS.794?PRK,con-espondingtotheresiduesCys-16antiCys55inthespinachenzymerequiredforactivationbylight.However,170r18aminoacid residuesweremissingamongthe40residuesfoundinhigherplantPRK(Wadanoetal. 1995}.Basedonthedatareportedhereandotherdatasofar,itseemslikelythatthere maybeconsiderablestructuraldifferencesinthevicinityofthethiolgroupslnvolvedin theligh亡ac亡ivationofthechloroplasticenzylnesofhigherplantsandthoseofS・7942. Furthermore,aquestionraisesastow'hetherthethiolenzynlesofthePCRcycleln cyanobacteriaincluding5'.7942aresensitivetotreatmentwithDTrinvitrc)andare regulatedbylightactivationinviりo.Thethioredoxin-ferredoxinsystemhasbeenpresellt inSynechococcttsandNostoc(Wangeretal.1978,Yeeetal1981),whereastheFBPase ofA〃. α 脚'`3nit!〃013(5ン. η66加co`c〃. のwasnoεaαivatedbyDTT-treaimentandllght-. treatment(DugganandAnderson1975).CrawfordeCal.(1.984)havereportedthatthe PCRcycleisregulatedbylightinacyanobacteria(Nostocrficcscor°LCrrt)buCnotin anoxygenicphotosyntheticpurplebacteria.Whilefurtherrovesligationisrequiredto film至yanswerthequestion,thedatapresentlyavailablesuggestthatthePCRcyclein samecyanobacteriaincludingS.7942isnotregulatedbylight.. 27.

(34) Summary. In5yη. εo加cocα. ∫5PCC7942ceUs,twocataly江icFBPaseisoenzymes,designated. FBPase-IandFBPase-II,havebeenresolvedbychromatographyonaHiLoad26110Q SepharosecolumnatO.24MandO.34MofNaCl,respectively;theformerrepresented themajorpartofthetotalextractableenzymeactivity.FBPase-Ihasbeenpurifiedto electrophoretichomogeneityfromthecells.FBPase-IandFBPase-IIhadrespective molecularmassesof160kDaand150kDaandeachenzymewascomposedoffour identicalsubunits.FBPase-IhydrolyzedbothFruI,6-P2andSed1,7-P2,whereas FBPase-IIhydrolyzedonlyFrul,6-P2.TheapParentKyravaluesofFBPase-Iand FBPase-IIforfructose1,6-bisphospllatewere52±4.5told25±1.5ACM,respectively. FBPase-IwasinhibitedbyAMPwithaKivalueofO.26mM,butFBPase-IIwasnot affeαedbyAMP.TheFBPase-Ifailedtocross-readbywesternblottingwiththe antibodyraisedagainstFBPase-II;similarly,theFBPase-IIdidnotreactwiththe FBPase-Iantibody.ThegenesencodingFBPase-IandFBPase-IIwereclonedfromthe chromosomalDNAofSynechococcusPCC7942.AIO68-bpORF,encodingFBPase-I or356arninoacidresidues(approx.molecuIarmassof38.3kDa). ,wasobserved.The. nucleotidesequenceoftheFBPase-IIgeneshowedanopeDreadingframeof1017-bp thatencodesaproteinof339aminoacidresldues(apProx.1nolecularlnassof37.2kDa). TherecombinantenzymesexpressedinEscherichicccoliaswellasthenativeenzymesof FBPase-IandFBPase-IIfromSynechococcLisPCC7942cellswereresistant1:01mM hydrogenperoxideunlikethelightactivatedhigherplantchloroplastenzymes.. 28.

(35) CHAPTERIV. AcquisitionofaNewTypeofFructose・1,6-bisphosphatase withResistancetoHydrogenPemxideinCyanobacteria: MolecularMechanismsoftheEnzymefromSytxechocystrsPCC6803. Inprokaryoticcellsincludingcyanobacteria,aspecialneedforaselectivecontrol oftheFBPaseactivitymayexistbecauseinthecells,duetoalowerdegreeofcompartmentation,thePCRcycle_isnotenclosedinaseparateorganelle.Ihavefound thatSynechococcccsPCC794?,anobligateautotroph,containstwoisoforinsofFBPaseIandFBPase-IIandtheprimarystructureol`FBPase-Iisdit-1'erenCt`romthoseol`typical chloroplastandcytosolicFBPasesfromeukaryoticcells(chapterIII).TheFBPase-I showednotonlytheFBPaseactivitybutalsotheS$Paseactivity.Thededucedamino acidsequenceoftheFBPase-IIgeneof&7942hadaconsiderablesim翻a1'itytothe cytosolicandchloroplasticfonnsorvarioussources,indicatingthattheFBPase-IIisa typicalFBPasetype,Inphischapter,thequestioncanberaisedastowheCheranovel FBPaselikeFBPase-Iisgenerallydistributedinphotosyntheticorganisms. Inthischapter,IextendthesurveyofthenewtypeofFBPase,thatis,FBPase-Iin cyanobacteriaintennsofthegenomicSouthernbioitingwithaprobefrolntheFBPase-I geneandtheWesternblottingwithanantibodyagainsttheFBPase-IproεeinfrolnS. 7942.Interestingly,IfotmdthattheFBPase-IexistsinSynechocystis6803,Anab`gnu 7120,andPlectonernaわo':y`肥. 〃ra.Second,Istudytheeffectofgrowthoonditionson. theactivitiesoftheFBPase-IisozymeandtheenzymesinvolvedinthePCRcycle, emphasizingtheimportantphysiologicalroleot`theFBPase-IenzymeinthePCRcycle andgluconeogenesisofS.6803cells.Finally,Ifocusonthemolecularcharacterization oftheFBPase-Igene.. MaterialsandMethods. ルf6π8廠Z3___Aη. の αθη07120,」PZθ. αoη 翻 α 加町y`臨`mwereobtainedfromDr.. Miyake(NaraResearchInstit<tteofScienceandTechnology).Thematerialsusedwere obtainedfromthesources.Themolecularbiologyreagentsandenzymeswereanalytical gradeandwerepurchasedfromcommercialsources.. 0へ96〃 ②'ぶ'ηandct〃tore-一 7120,and1)lecto'2e〃zαborソ. ∼Cyanobacteria,S)2η6chα:y51〃3PCC6803,Aη6め`昭. ηα. 伽L槻wereculturedphotoautotrophicallyinAIIenlsmediuln 2. s-1)withthebubblingofsterileairat8at27°CrorSdaysunderillumination(240SCEm l!min(Takedaetal.1993b).HeterotrophiccultureswereobtainedbyculturingS.6803. 29.

(36) inAllen'smediumcontaining10tnMglucoseunderincompletedarknessreceivinga dailybriefpulseoflight(NavarroandFlorencio1996).E.coli,strainsDHSawas culturedaspreviouslydescribedinchapterIII.. prε ρoπ 厩oηcゾcrt`rleextract-一. 一Thealgalcellswereharvestedby. centrifugation,washedonce,suspendedin50mMpotassiumphosphatebuffer(pH8.0) containing?.5mlviDTT,1mMglutathioneand10%sucrose(solutionA)andsonicated at10KHzforatotaloflminwithfiveintervalsof10seceach.Theselysateswere centrifugedat12000gfor15min.Eachcrudeenzymeobtainedwasusedforthe enzymeassayandforanalysisbyWestemblotwithantibodyagaillsttheFBPase-Iand FBPase-IIprotein.ForthepreparationofcrudeextractforG6PDHandpyrophosphatedependentphosphofructo-kinase(PPi-PFK),50mMpotassiulnphosphatebuffer(pH S.0)wasused.. En,)ノ. 〃z8`鯉3のIS-一. 一一FBPaseandSBPasewereassayedasdescribedinchapter. III.Tostudythesubstratespecificityoftheenzy1:ne,bothbisphosphataseactivities weremeasuredbythediscontinuousassayinwhichPireleasevasmeasuredatfixed intervals.Thereactionmixtures(finalvolume2.1nil)contained100mMTris-HCI(pH 8.0),1.5mMphosphateesters{asspecified),10mMMgC12,andpurifiedenzymes accordingtoGerbling{Gerblingetal.1986).NADP+-GAPDHandPRKwere assayedaspreviousdescribedinchapterII.G6PDHactivitywasmeasuredina reactionmixture(1ml)containing100mMTris-HCIbuffer(pH8.0),10mMMgC1 2, 4.4mMNADP+,1mMglucose6-phosphate,andtheenzymesolution(Domagkanti Chilly1975}.PPi-PFKwasassayedbycouplingformationofFru1,6-P 2tvthe olictationC)fNADHthroughaldolase,triosephosphateisolllerase,arldα. 一. glycelgophosphatede1、ydrogenase.Thereactionmixturecontained50mMTris-HC1(pH 8,0),lmMFru6-P,1μMFru2,6-P. 2,5mMMgCl2,0.2mMNADH,1mMEDTA,. 0.4unitsofaldolase,6unitsoftliosel)hosphateisomerase,1.2unitsofα. 一. glycerophosphatedehydrogenase,2.SmMNaPPi,andtheenzymesolution{Yuanetal. 1988}.. P配rのcα. 加. η. ρプ. 、EBPα. ∫θ一1ブ π 珈S.6803-一. 一AllproceduresofFBPase. purifica塾ionwerecarriedoutat4℃.Synechocystis6803cells(10gwetwt,)grown photoautotrophicallywereharvestedbycentrifugation,resuspendedinlOOmlofsolution Aandsonicated(10KHz)foratotalof10minwithfourintervalsof2min.Thislysate wascentrifugedatl20008・forlOmin,Thecrudeextractwasultracen口. 層ifugedat. 30.

(37) 1800008for20min.Thesupernatantwasloadedontoa日iLoadl6!10Qsepharose column(FPLCsystem,Pharmacia}equilibratedwithsolutionAanddevelopedwitha 160-m目ineargradientofNacl(o-o,6M)atanowrateoflm1!min.Theactive fractionswerecombinedanda(加stedto30%saturationwith(NH. 4)2SO4・The. supernatantwaschromatographedonaHiLoad16/10PhenylSepharosecolumn equilibratedwith30%(NH4)2SO4insolutionA.Thecolumnwaselutedwith120-ml lineargradientof30-0%(NHS}2SOatanelutionrateoflml/min.Theactivefractions werecombinedandfractionatedwith(NH. 4)so,andthepelletprecipitatingbetween30. and70%saturationvasdissolvedin2mlofsolutionA.Theenzymesolutionwas chrotnatographedontoaHiLoad16/10SuperdeY?00columnequilibratedwithsolution AcontainingO.15MNaCI.Theactivefractionswerecombined,adjustedtoO.05M NacIwithsolutionA,andloadedontoaMonoQHR5!5c61umnequilibratedwith solutionAanddevelopedwitha24-mllineargradientofNaCl(0.05-0.6M)ataflow rateofO.8ml!1nin.The2.4-mlpuri#'iedFBPase-Iwasstoredat-20°C.. Pvlyruy)〃`〃yais/68・el618c跡oノ. 班o形. ∫∫∫. 一. 一. 一Dlsc8eleleclrophoresiswith7.5%. (W!V)polyacrylamideatpH9.4wascarriedoutaspreviouslydescribed(Shigeokaetal. 1.987b).Electrophoresiswasperformedataconstantcurrent{2mA/gel)with bromophenotblueas-themigrationmarker.SDS-PAGEwasperformedon12.5% (w!v)polyacry1amldesヱabgelaspreviouslydescribed(Takedaetal.1993a).Prαeinsin tllegelwerestained≪ithasilver-stainingreagent.. E辞 ∫ 〃2{ガ∫oη ρズ〃30」θc`f1αrweight-一. 一ThemolecularweighCofFBPase-Iwas. determinedusingaSuperdex200HR10130columnequilibratedwithsolutionA containingO.15MNaCIandcalibratedwith-molecularmarkers(MW-GF-1000}from Sigma.ForthemolecularweightofasubunitbySDS-PAGE,anLMWkitEFrom Pharmaciawasusedasthestandard.. Ancめ23'30ブ`rrriinoacid3θgLònce-一. 一TheaminoacidsequenceoftheN-. termillalregionoftheenzymewasdeterminedbyautomatedEdmandegradationonan AppliedBiosystems492Agas-phaseproteinsequencerusingstandardprogramand reagentsprovidedbythemanufacturer.. lyrzriiccnoblotting‐. ‐ ‐ProteinswereseparatedusingSDS-PAGE{12.5%gel). andblottedontoanImmobilon-Ptransfermembrane(PVDF,poresizeO.45」 IPVH304FO,Millipore,Bedford,MA)us111gasemidryelectroblotCingsysEemaccording tothernanufacturer璽sinstructions(Bio-Rad)(Shigeokaeta1.1991).Immunoblotti119. 31. μm,Nα.

(38) wasperfonnedwiththeantibodiesralsedagainstS,7942nativeFBPase-Iand recombinantFBPase-IIasdescribedinchapterIII.Goatanti-mouseIgsantibodies conjugatedwithperoxidase(BoehringerMannheimGmbH,Germany)wereusedasa secondaryantibody.. Theresistance(iftheactivitiesσ. 溜P膨. 伽4∫BP`掴. θt・H、0、. 一一一T・. completelyremovetheDTTfromtheFBPase-1purifiedfromS.6803cells,theenzyme preparationwaschromatographedonacolumn(1.5x14cmi.d.}ofSephadexG-25 (PhalmaciaBiotechnology,UpPsala,Sweden)equilibratedwith100mMTris-HCIbuffer (pH8.0}containing16mMMgCl2.Theproteinfractionswereplacedintovials sa加ratedwithN2gastogiveaconcentrationofapProximatelyl2μ9Protein!ml・The PurifiedenzymewasincubatedwithH202for10mininthedarkaspreviouslydescribed (Takedaetal.1995}.. Soccthenzhybriり4∫z磁oη cyanobacterialcells(、. 一一一ThechrolnosomalDNAwasisolai￡dfrom. ヘノetweightl5g)bytllemethodaspreviouslydescribed(Williams. 1988).Digoxigenin-labeledprobeswerepreparedfromtheS.794?FBPase-Iand FBPase-IIgeneaccordingtotheprotocolsuppliedwiththeDIGDNALabelingKit (BoehringerMannheimGmbH,Germany).ThechromosomalDNAdigestedwithApcc I,BarnHI,EcoRI,HindIII,KpnIandPstI,wereseparatedbyagarose-gel electrophoresis(1°logels}andblottedontoaHybond-N+transfermembrane(Amersham Internationalplc,England)usingaModel785VacuumBlotteraccordingtothe manufactLirer'sinstructions(Bio-RadLaboratories,NewYork).Hybridizationvas performedforl6hat55℃.Detectionsofdigoxigenln-labelednucleicacidswere performedbyenzymeimmunoassayandenzyme-catalyzedcolorreactionusingDIG NucleicAcidDetectionIくlt(BoehringerMannheimGmbH,Germany).. ノVcccleoticlesegtrenceCUZCめISISρ/F、B、Pα. ∫8-1gene/cornS.6803-一. oligonudeotideprimerscorrespondingtotheaminoacidsequenceoffiheN-terminusand theP-1peptide(nucleotideresidues28-47[5'-AT(CT}GA(AG}GT(GC}GT(GC)GA (AG)CA(AG)GC-3りand774-757[5'一(CT)TC(AG)CC(AG)AT(GC)A(AG)(AG)CC (GC)GT-3']of∫.7942FBPase-IwereusedtoobtainaninitialDNAIragmentamplified fromgenomicDNAbyPCR(chapterIII).ThePCRamplificationwasperformedina lOOμlreactlonlnixtしlrecontaining1μMofeachprimer,200μMofeachdNTP,30ng of5.6803genomicDNA,10mMTris-HCI(pH8.3),50mMKCし15mMMgCl2,and 2.5unitsofrecombinantTrulDNApolymerise(TOYOBO).PCRconditionsconsisted ofaninitialdenaturationperiodofsminat94°Cfollowedby30cyclesat94°Cforlmin,. 32. 一The.

(39) 58°Cforlmin,and7?°Cforlmin.ThePCRproductsweresize-fractionatedona 1.5%(W/V)agarosegel,andthebandcorrespondingtotheexpectedsize(360bp)was cutout.TheDNAfragmentwasisolatedfromtheagarosegelusingtheGENECLEAN IIKIC(BID101,Inc.),clonedintoapT7BlueT-Vector(Novagen)andsequencedbythe dideoxychainprimermethodwithM13forwardandreversesequencingprimers.Ihave confirmedthatthenucleotidesequenceoftheDNAfragment,which-wasdesignated pSsFPI,was76.6%identicaltothatoftheS.7942FBPase-Igene.Thisresult indicatedthatthepSsFPlisapartofthefu11-lengthDNAfragmentoftheFBPase-1-gene. Recently,the-wholegenomicDNAfromSynechocystisPCC.6803ha.5been characterizedbytheKazusaDNAResearchInstitute(Kanekoetal.1996).Iobtained thedaEaonalOkbpnucleosidesequenceincludingthepSsFP1・'Inordertoanalyzethe FBPasegenefromS.6803,thefull-lengthFBPaseIIgenewasamplifiedbyPCR.with primerscorrespondingtotheaminoacidsequenceoftheN-terminusandC-terminusof theFBPase-1,subclonedintothepT7BlueT-vector(pSsFP2)andresequencedusingthe abovemethod.. 0伽. η π幽0439-一. HCIbuffer(pH7.0-一. 一TheoptimumpHwasdeterminedat27℃in100mMTris一8.S)andglycine-NaOHbuffer(pH8.5‐9.5)underthewine. enzymeassayasdescribedabove.ToexaminetheeffectofAMPorFru2,6-P20n enzymeactivity,eachcompoundwaspreincubatedwiththereactionmixtureatseveral concentrations{0.1,0.2,0.4,0.8,1.0,and2mNn.TheisoelectricpointofFBPase-I wasdeterminedbyisoelectricfocusingusingMode1111MniIEFCell(Bio-Rad) accordingtothemanufacture'sinstruction.Theproteinwasdeterminedbythemethod ofBradford(1976)withBSAasastandard.. Results. Tノ昭occttrrenceρ. ブEBPα. ∫ε一1加cyα. ηoZ)tccteria-一. oftheFBPase-IandFBPase-IIenzymesincyanobacteriaandeukaryoticalgae,the SouthernhybridizationwithgenesoftheFBPase-IandFBPase-IIastheprobefrom SynechococctcsPCC794?wascarriedout.AsshowninFig.IV-1,therewasonlyone fragmenthybridizing≪iththe_FBPase-IgeneorFBPase-IIgeneineachcyanobacteria, SynechocystisPCC6803,Anabcrena7120,andPlectonemaboryancim.InWestern b且otting,theantibodyraisedagainsttheFBPase-IofS・7942cross-reactwithaprotein band(42kDa)correspondingtoanFBPase-Iineachcrudeextractflて)mthe cyanobactelialcells(Fig.IV-2A)・However,theantibodyagainsttheFBPase-IIproteln failedtocross-reactwithaproteinband(37kDa)correspondingtoan-FBPase-IIineach crudeextractfromthecyanobacterialcells(Fig.IV-2B).Thecellextractfrom. 33. 一Toelucidatetheoccurrence.

(40) cyanobacterialcellsgro、. へ'nphotoautotrophycallywaspassedthroughaMonoQHR515. columnequilibratedwithsolutionAanddevelopedwitha24-mllineargradientofNaCI (0-0.6M)ataflowrateofO.8ml/min.InthecrudeextractfromSynechococccesPCC 7942,theFBPaseactivitieswereelutedwithO.22(FBPase-1)andO.29M(FBPase-II) NaCIwithatotalrecoveryof85°lo(Fig.IV-3-IA).Theelutionpatternsweresimilarto thoseoftheFBPase-IandFBPase-IIwhichwereresolvedbychromatographyonaQSepharosecolumnatO.?4andO.34MofNaCI,respectively(chapterlll).Inthecase ofSynechocystisPCC6803,An`め6昭. ηα7120andPlectone〃i`どboリ. ノ〃2`〃'2,theFBPase. activitywaselutedwithO.22MNaClwithatotalrecoveryofapproximately85°lo(Fig. IV-3-?A,3A,and4A).Noactivityoftheenzymewasfoundinanyotherfraction. TheactivhiesoftheenzymeswithbothFru1・6-P2andSedl・7-P2wereequallyelutedat O.22MofNaCI.Theseprocedureshavebeenrepeatedfourtimeswithasimilarelution pattern.TheantibodytotheFBPase-Iproteincross-reactedwiththe4?-kDaproteinina fractionatO.??MNaCI,whiletheantibodytotheFBPase-Ilproteintailedtocross-react withthe37-kDaproteininthefractionatO.22MandintheviciniWofO.29Mandinany otherfractionsofMonoQ(Fig.IV-3-1B,2B,3B,and4B).. A. 1-1. 1-2. 1-3. 23 9.4 6.5 4.3 2.3 123456. 123456. 123456. 123456. 123456. 123456. B 4.r O 39 6 2 3 nO. 4 ,、- (∠. Fig.IV-1GenomicSouthernAnalysisofDNAfromCyanobacteria.EachgenomicDNA fromSynechocysiisPCC6803(1-1),Anabaena7120(1-2),andPlecionemaboryanum (1-3)wasdigestedwithApa_1(lane1),BamHI(lane2),EcoRI(lane3),Hindlll(lane4), KpnI(lane5),PstI(lane6)andsubjectedtoSouthernanalysis.ThegenesofS.7942 FBPase-1(A)andFBPase-II(B)weredigoxigeninlabeledandusedashybridization probes.DetailsoftheprocedurearedescribedintheMaterialsandMethodssection. ThesizesofDNAmolecularmassstandardsareindicated. 34.