withwhitelight(150μEm響2s'1)suppliedbyfluorescentlalnpfor24h.E.solistraill DH5awasculturedasprevioしtslydescribedinchapterIII.
Eηzy,η θ̀8∫ ￠y3‑一 一TheactivitiesofFBPase,NADP+‑GAPDHandPRKwere assayedaspreviouslydescribedinchapterII.
1)μr醒 α 癖oη ρプFB1)α ∫81SOZy〃ie‑一 一Allprocedureswereca面edoutat4℃.
ELCg・lencacells(20gwetwt.)wereharvestedbycentrifugation,resuspendedinlOOmlof 50mMpotassiumphosphatebuffer,pH8.0,contalning25mMDTT,1mMGSHand
10%asucrose(solutionA),andsonicated(10KHz)f̀oratotalofIUminwithnine intervalsofImineach.Tjhislysatewascentrifugedat120008for15mintoproduce
tllecrudeenzyme.Itwasthenloadedo煎 〕aDEAESephacelcolumn(2.5cmx40cm) equllibratedwithsolutionAanddevelopedwitha340ml‑lineargradientofNaCl(0‑0.5
ル
M)ataflowrateof▼2mhnin,TheactivitiesoftwoFBPaseisozymeswereresolved intothevoidvolumeandtheO.16M‑NaCIfraction.WhentheIysateQfchloroplasts
isolatedfromEìglentiwassu切 ㏄tedintoaDEAESephacelcolumn,theactivityof FBPasewasresolvedonlyonefractionatO.16MofNaCI(Fig.VI‑1).Accordingly,
theactivitycorrespondingtothecytosolicenzymes(cyFBPase)wasnotretainedonthe column.VoidfractionswereusedasacyFBPase.Theactivefractionsof
chloroplasticenzymes(cpFBPase)werecombinedandadjustedto30%saturationwith
(NH4)2Sq.ThesupernatantwaschrolnatographedonaHiLoad16110Phenyl Sepharosecolumnequilibratedwith30%(NH4)2SqinsolutionA.Thecolumnwas elutedwith100mlofalinergradientof30‑0%(NH4)2SO4atanelutionrateoflmlmin
1
.Theactivefractionswerecombinedandfractionatedwith(NH4)2SC㌧,andthepellet precipitatingbetween30and70%saturationwasdissolved1112mlofsolutionA.The enzymesolutionwaschromatographedontoaHiLoad16!60Superdex200column equilibratedwithsolutionAcontainingO.15MNaCLThepurifiedcpFBPasewas storedat‑20°Cwithouttheextensiveinactivationforseveralweeks.1)04yocり216〃 η∫46gelelectrophoresis‑一 一SDS‑PAGEwasperformedonlO%
(w/v)polyacrylalnideslabgelsaspreviouslydescribed(Takedaetal.1993x).Proteins inthegelwerestainedwithCoomassiebrilliantblueRAsoanddestainedin7%acetic acid.
Theresistunceρ ブFBA)∬6isozymes∫oH,o,一 一 一Tocompletelyremovethe DTTfromthecpFBPaseandcyFBPasepreparationsfromEccglenacellsasdescribed
above,eachex虻actwaschromalographedonaCOIL111111(1.5cmxlOcln)ofSephadexG一
25equilibratedwith100mlvtTris‑HCIbuffer,pH8.0,containing16mMMgC12.The
proteinfractionswereplacedintovialssaturatedwithN2gastogiveaconcentrationof
ロ ユ
apProximatelyl2μ9Proteinml・TheenzymespreparationswereincubatedwithH202 f‑or10mininthedarkasdescribedpreviously(Takedaetal.1995).
1∫oZ{πゴonofcDNAencodingEugienaFBPases‑一 一TotalRNAwasisolated
fromEtlglenacellsbyamodificationoftheprocedureofShirzadeganetal,(1991).
Poly(A)+RNAwaspurifiedusingthepolyATtractmRNAIsolationSystems(Promega, USA).‑TheRT‑PCRanalysiswasperformedwitholigonucleotideprimersprepared rl℃mtheI110SChighlyconservedregions{nucleotideresiduesl29‑138[5'‑
GACCCTGTGGATGG{ACT)TC{GCT)TCCAACAT(CT}GA‑3']and280‑287[S'‑
CTCATA(AGC)AG(AC)A(AGC)(CT}C(GT)(CT)AG(CT)TTTCC‑3']}of̀FBPases
fromvariousorganisms(Hureial.1992,Marcus,andHarrschl990,Rainesetal.1988, MaI℃usetaLl986,HalniltonetaLl988,Entianetal.1988).ThePCRproductswere clonedintopT7BlueT‑vector{Novagen)andsequencedbythemethodasdescribed
below.Toobtainthefull‑lengthcDNA,rapidamplificationof3'endandS'endof cDNA(S'‑RACE}wascarriedoutwith5'RACESystemforRapidAmplificationof cDNAEnds,Version2.0(GibcoBRL}inaccordance≪iththemanufacturer's instructions.ThegenespecificprimersGSP‑1{5'‑TGTTCCCCTCGTTGCAGCT‑3'), GSP‑2(5'‑GAATGGGTGTGAATGAACTC‑3'}andGSP‑3[5'‑TC(AG}ATGTTGGA (A,GC}GA(AGT)CCATCCAGAGGGTC‑3'JweredesignedfrompEuFP‑1.
NiccleotideSegecencing‐ ‐ ‐cDNAsequencingwasperformedusinga373 PRISMDNAsequencer(AppliedBiosystelns)withABIPRISMDyePrimerCycle
SequencingReadyReactionIくit(Perkin‑Elmer,FosterCity,CA).TodeCerminethe completesequence()fpEuFP,plasmidwithdeletionwaspreparedwithDeletionKit (Takara,Japan),PrimarysequenceanalysiswasperformedwithGENETYXv8.0.
HomologysearcheswereperformedIlltheGenBankdatabase,usingtheBLへST program.
Other°rraethods‑一 ‐ ‐TheoptimumpHwasdeterminedat27°Cin100mMTris‑
HClbuffer(pH7.0‑85)andglycine‑NaOHbuffer(pH85‑95)underthesameenzyme assayasdescribed.above.Toexamine‑theeffectofAMPorFru2,6‑P20nenzyme activity,eachcompoundwaspreincubatedwiththereactionmixtureatseveral concentrations{0.1,0.2,0.4,0.8,1.0,and2mM}.Theproteinwasdeterminedbythe methodofBradford(1976)withBSAasastandard.
Results
1)μrび ∫α ぬoὴ〃 π!ch̀zπ κ 彪 パz̀llionρ プF81)α ∫θISOZ)2〃zes.〃o〃 診Eugleǹ̀一 一 一During chromatographyofthecrudeextractofEuglenacellsonaDEAF‑Sephacel,theFBPase
activitieswereseparatedintoiwofractionsinvoidvolumeandatO.16MofNaCI, respectively(Fig.VI‑lA).Whenthesamemannerwascarriedoutinthecrudeextract preparedfromEuglenachloroplasts,theFBPaseactivitywaselutedassinglepeakat O。16MNaCl(Fig.VI‑1B),suggestingthattheFBPaseactivltyelutedatO」6MNaCI correspondstothechloroplasticFBPase(cpFBPase)ofEtcgleǹY.Theactivityratioor cpFBPaSetocyFBPasewasabout1:1withaCotalrecoveryof84%.Thisprocedure
≪asrepeatedthreetimescwithasimilarelutionpattern.Thepurificationprocedure
yieldedacpFBPasepreparat孟onpurifiedapProx.35.8‑foldovertheactivefractionsof cpFBPasebyDEAEsephacelwithayieldof33.2%,ThePAGEandSDS‑PAGEof
thepurifiedcpFBPaseshowedonlyonedetectableproteinbandwithamolecularmassof
‑1
43kDa(Fig.VI‑2).Thespecificactivi取thepurifiedcpFBPasewasll.7μmolmin
の ヱ
(mgprotein).GelfiltrationonaSuperdex200HiLoadl6!60columnofGAPDHwith severalstandardproteinsindicatedamolecularmassof170kDa.Thesedataindicated thattheenzymeisatetramerwithasubunitmolecularmassof43kDa.
086420 (二藁oニー三E石E仁)﹀茎鳶Φ器αmL
1.0
o.s
o,6￡
U a.aZ
0.2
・0
i1020304050607080 FractionNo.(2mltube‑1)
Fig.VI‑1ElutionPatternofcpFBPaseandcyFBPaseIsoenzymesbyHiLoadQ SepharoseColumnChromatography.ThecrudeextractswerepreparedfromEuglena ce麗s(●)andchloroplast(○)inthestationaryphaseandbadedontoaHiLoadQ Sepharose26/10columnequilibratedwithsolutionAasdescribedintheMaterialsand Methodssection.
一一一一
,NaCfconcentration
TableVI‑1ComparisonofsomepropertiesofFBPaseisozymeswishchoseofS.7942andspinach
Euglena S.7942 Spinach
ChloroplasiCyiosol FBPase‑1 FBPase‑UChloroplasiCyiosol Molecularmass(kDa)
Gelfiltration sDS‑PAGE OptimumpH Isoeiectricpoint Knvalue
Frui,6‑Pz(NM) Sed1,7‑Pz{μM) Mgt+effect
SQsvalue{mM) FBPase SBPase AMPinhibition F川2,6‑P2inhibition
n.d.
43 8.5 n.d.
6811.6 n.d.
sigmoidal
8,5t7.2 n.d.
n.d.
,,.a.
7.5 n.d.
70土1.7 n.d.
hyperbolic
0.85 n.d.
十
160 40 8.0 4.7
52士4.5 118t5.5 sigmoidal
t4±o.1 1.9土0.1
,・
37 9.Q 5.4
25t1.5
sigmoidal
8.6士0.4
isa QO 8.0 4.7
33
i50 37 8.0 5.3
2.5
sigmoidalhyperbolic
0.13 o.ot
十十
aDa重aaremeanvalUeS士SDfromthreeassays
(kDa}
94 67 43 30
饗
蓼 一4,
嚢メ
Fig.V!‑2SDS‑PAGEofcpFBPase・PurifiedfromEuglena.Theproceduresfor electrophoresisofthepurifiedcpFBPasewascarriedoutasdescribedintheMaterials andMethodssection.EachlanewasstainedwithCoomassiebrilliantblueR‑250.
TableIshowsthecolnparisonofsomepropertiesoftheELCgtenaFBPaseisozymes wiChChoseofc1110roplasCicanticytosolicFBPase#romspinach(Zimmermannetal1976, StittandHeidt1985,Stittetal.1985,Lazaroetal.1974,Zimmermannetal.1978)and
FBPasefromcyanobacteria(chapterIII,IV).Thesubstrate‑、 ・elocitycurveswith
cpFBPaseandcyFBPaseshowedMchaelis‑MententypekineticswithFrul,6‑P2at concentrationsuptolmM:theapParent1(1nvaluesofthecpFBPaseandcyFBPasefbr Fru1,6‑P2were72±4・5μMand55±1.5ACM,respectively・Tostudythesubstrate specificity,avarietyofphosphateesters{FBP,SBP,glycerate3‑phosphate,glycerate 2,3‑bisphospllate,dihydre)xyacetonephosphate,glucose6‑phospha童e,glucosel,6‑
bispllospllate,1、'uctosel‑phosphate,丘uctose6‑phosph飢e,AMP,ADP,andATP)at finalconcenCral[onof1.5mMeachwereused.Asaresit1C,bothisozymescou旧 hydrolyzeonlyFBP,butnototherphosphateesters.
2+
concentrationontheenzymeactivity,thecpFBPasehadForthee#'fecCofMg sigmoidsaturationcurves(So5valuewas85),whilethecyFBPasehadhyperbolic
saturationcurves(Sρ .5valuewasO、8)(Fig.VI‑3).ThecyFBPasewasinhibitedby AMPwithaKivalueofO.?6±0.0?mM,likethecytosolicNormofhigherplants,but
notbyFru2,6‑P2,unlikethecytosolicDorm(StittandHeldt1985,Lazaroetal.1.974).
AMPandFru2,6P2hadnoeffectonthecpFBPaseactivity'TheoptimumpHof cpFBPaseandcyFBPasewere8.5and7.5,respectively(Fig.VI‑4).
言FF°三昼口︒E=)盆≧冒"o詔島﹂
Fig.V!‑3 EffectofIvlgClConcentrationoncpFBPaseandcyFBPaseActivities
(二凱9〒三量書ε捻三ぢ器器出﹄
pH
Rg,V卜4 pHOptimumofcpFBPaseandcyFBPase
Theresistanceoffrccctose‑1,6‑bisphosphctttxseisozymestoH202‐ ‐ ‐As showninFib.VI‑5,thespinachchloroplastFBPasewascompletelyinhibitedatO.ImM
H202whichwasinagreementwiththeresultreportedpreviously(Kaiser1979,Takeda etal.1995).Incontrast,thecpFBPaseandcyFBPasewereonlyslightlyinhibitedbyl mMH202.TheseresultsindicatedtheabsenceofsusceptibilityofcpFBPaseand cyFBPaseisozymestoH202uptolmM.
Fig.VI‑5EffectsofH2020nActivitiesofcpFBPaseandcyFBPase.Spinachintact chloroplastswerepreparedaspreviouslydescribedinchapter11.ThecontrolFBPase activitiesofcyFBPase‑1(●),cpFBPase(○)andspinachchloroplasts(▲)areshownas 100%,respectively.Dataarerepresentedastheresultsofthreeassays±SD.
.Effectρ/lightontheactivitiesρ ゾ ・F13Pαr6,ノVAD1)‑GAPDH,̀〃 π!1'RKノ 〉り 〃a
Eμ818〃̀zc郁oπ)p1α ∫∫3̲一 一Theeffectoflightontheactivi巳iesofFBPase,NADP+‑
GAPDH,andPRIGwas ̲studiedinEtcglenuantispinachchloroplastsaspreviously
ciesc:ribeciinchapterII.TheactivitiesofFBPase,NADP+‑GAPDH,anciPRKindark‑
adaplecfchlo!℃Plastspreparedfrom∠ ㌃f816ὴ'cellswere60,1,495,6,alld302.5、 μmolh"1
(mgchlorophyll)'塞,respectively.TransferofEμ816ὴtch】oroplastsfromthedarkto illulninationat1,6()0μEs'肛m91failedtoincreaseeachenzymes(Fig.VI‑6).
EffectofDTTtreatmentontheactivitiesofFBPase,NADP}‑GAPDH,unclPRK
,finynEceglenachloroplasts‐ ‐ ‐TheactivitiesofFBPase,NADP+‑GAPDH,andPRK Preparedfrom」Eμ8・lendchloroplastswiththebuf#̲erinthepresenceofDTTshowed79・6
±5.0,799.8±23.2,and314.8±14.5nmolmina(mgprotein)一.̀,respectively.
However,therelnovalandtreatmentofDTThadlittleeff㏄tontheactivitiesof'the enzymeshて)mEμ81812α(Fig.VI‑7).
(}‑(=あ且20驕=・oε‑幽︒ε&鐙≧5駆o認ユ田
一180 0 10 20 30
(マ(=﹀=αO﹂〇三〇mε}マ=一〇εF一)
鵠噸=﹀精UOy匡低罰=馴堕工O氏くO,十氏OくZ
Incubationtime(min)
Rg.Vl‑6EffectsofUght/DarkConditionsontheActivitiesofThio{‑Modula#ed EnzymesinSpinachLeavesandEuglenaGeils.SpinachleavesandtheEuglenacells wereleftfor3hinthedarkbeforeilluminationat1600μEs'1m‑2for30min.Atthe indicatedtimes,thecrudeextractswerepreparedasdescribedbelow.Thedataare themean±SDofthreereplicates,●,FBPase;▲,NADP+・GAPDH;覧PRK.
Fig.VI‑7Ef#ectsofTreatmentwithDTTontheActivitiesofThiol‑ModulatedEnzymes inCrudeExtractsfromChioroplastsofSpinachandEuglena.Aftertheadditionof20 mMDT丁,theactMtiesofthio卜modulatedenzymeswereassayedat10,20and30miss.
Eachclosedmarkshowsthevaluesofenzymeactivitiesinthecrudeextractspriortothe removalofDTTbyacolumnchromatographyofSephadexG‑25.Thedataarethe
mean±SDofthreereplicates.Q,FBPase;o,NADP+‑GAPDH; ,PRK.
P,i冒 ●L曽7凸,̀巳,52̲̀監 一11‑1ユ 響息I f▲GCτC轟αC轟 τ1AG㏄AぐCCCへ66釘 τ1ACあ(冒̀「 ∂曜GC1τCぐ66C璽 ￠ctatcrrcrcrccム 為τ冨01『弓焔̀ccxrx轟C轟AIYYCAC▲CA
11070!010SO{/TO/0
6軌 晶c鳳 ㏄ 「轟τGÀc轟 τGA11鳶c6cご 勘Gα α 鉢 τ轟c6Ae:ζ ムC7Ai▲ 儒 儲 軌 に ττ6cA竃6ごcτocA66uc砿c智̀τ▲GAGGIC7
臓r欄 量τPs3■t:翻 蟹艮■5ム 幽1義c融9雪L己 ◎駈
fOloo11011011aｵOl51UoUo
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L"8暫 置G曹1」L5τ51̀666τ 「o■L「86艮Lセ レ,a
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AAGCACTGCCCCS4CCCRCCCiCACCACCAGtGACAAAGCCGGCACSACCCCACCAGC[GCGGtGGCACCMICG7GETCtCACAGGC
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5「Pz讐 顎L暢P鳥5,ΩT「7智98LB眞 為Ao5り 轟塞3
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GCTCGAC6tAτ τ6τCζムACWTAT《TTCA:ChÀ轟C写 【営OAC̀鳥 鳥CICコ6G6ACtt4iGC4GTGCTGGTCitCG鳥 ㏄ ム轟真馬CG轟轟GA㏄{:
LO"色3,0竃 「【圃1L曾 墨5Grc轟 暫乙u3富 璽81巳,
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塵「1網 τ腱5口vrf,̀066嘱 嘱竃Y5c鯛 星6圏5s凹 騨
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Fig.V1‑8 Acid
Nucleotide Sequence, nucleotideand
SequenceoftheEuFP‑16and Including
aminoacid
Upstream andDownstream
EuFP‑4and
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DeducedAmino 丁henumbersof standforthosefromtheputativeinitiationcodononeach
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Fig.V且 一9 TheDifferencesintheFBPaseProteinSequencescanbeIdentifiedwith
theSubstitutionofAnotherAminoAcidbelowtheEuFP‑16Sequence.
includedtomaximizethealignment,andthe sequence. Theasterisksshowtheconsensus
dotsindicatehomology aminoacids.
Dashesare withtheEuFP‑16
Cloningrρ プEuglenacpFBPuse一 伽4ρyFBPω6‑Eηcò枷8cD/VAs‑一 一mRNA fromEì818η αcellswasusedtopreparecDNAfromwhlcha500bppCR‑amplified
TypicalFBPasesequenceswasrecovered,Thegel‑purifiedfragmentwassubcloned intopT7blueT‑vectorandsequenced.Nucleotidesequenceofthisclone(pEuFP‑1) wasshowntobe45%homologoustothespinachchloroplasしFBPasecDNA.To
determinethefulllengthofE↓z81ε ηαFBPasecDNA,amplified3'‑and5'‑RACEcDNAs weresynthesizedusingGSP‑1{f'or3'‑RACE)andGSP‑2andGSP‑3(t̀orS'‑RACE)
showninFig.VI‑8.Intheresultofthe3'‑RACEtenindependentcloneswere
sequenced,allwerethesamesizeandwereidentical.4ntheotherhand,inresultofthe 5'‑RACEsixindependentcloneswereobtainedandsequenced,thereweretwotype length(1.OkbpandO.7kbp)andthesecloneswereidenticalelceptfor5'‑region.
Accordingly,twocDNAcloneswereobtainedandshowntocontaininsertsof1560bp {pEuFP‑1.6}and1250by{pEuFP‑4).
pEuFP‑16containeda1344by‑longORFsequencefollowedbya160‑bp3' untranslatedsequenceterminatedbypoly(A}sequence.Theencodedproteinwouldbe 449Aminoacidinlengthandhaveamolecularmassof47338Da.
Onlheotherhand,pEuFP‑4containedalO20bp‑longORFsequencefollowedby al60‑bp3'untranslと1tedsequenceterminatedbyastringofAresidues.Theencoded proteinwouldbe340aminoacidinlengthandhavea34468Da.Thededucedamino acidsegtEencesofEuFP‑4had140%homologywithEuFP‑16exceptforN‑terminal126 residues.ThepredictedaminoacidsequenceofEuFP‑16andEuFP‑4exhibiteda highlysignificanthomologywiththoseofFBPasefromwheatchloroplast(45.1%) (Rainesetal.1988),Arcrbiclopsischloroplast{50.1°lo){HorsnellandRaines199]), spinachcytosol{50.7°lo){Huretai.1992),sugarbeetcytosoi{51.4%)(HarnandDaie
1992),pigkidney(47.3%)(WilliamsandKantrowitzユ992),3̀κc伽 η〃tyressereりISIQL'
(41.2%)(Entianetal.1988)andE.colt(40.8°ro)(Hamilionetx1.1988}.
Discussion
Higherplantsusuallycontain"voisofonnsofFBPaseinchloroplastandcytoso1, respect垂vely.Ec̀818η αgraciliszalsocontainstwoisozymesofFBPaseinchloroplast (cpFBPase}andcytosol(cyFBPase).InEccglencicellstheenzymaticpropertiesoftwo
isozymeswereverydistinct.TableIIsummarizesthecomparisonofsomepropertiesof theEtcglenacpFBPaseandcyFBPasewiththoseofchloroplasticandcytosolicFBPases frolnspinach.ThepropertiesofE占̀81813αcpFBPaseandcyFBPaseweresimilarto spinachchloroplasticandcytosolicforms,respectively.
ThecloningandsequenceanalysisofcDNAsencodingcpFBPaseandcyFBPase fromE占̀glenawascan°iedout,TwocDNAclonesobtainedcontainedinsertsof1560bp
(pEuFP‑16}.and1?50by(pEuFP‑4).Asaresult,itwasconceibablethatthepEuFP‑16 encodesthecpFBPaseandpEuFP‑4encodesthecyFBPase.Interestingly,thededuced aminoacidsequenceofEuFP‑4waslOO%identicalwithEしIFP‑16exceptforN‑temlinal 126residues・Theseresuhsprovidedtwoconceivablemechanlsmsofgenerationoftwo inRNA.Firstly,theset≪omRNAsmaybetranscribedfromdifferenttranscriptionstart siteonthesamegene;thismechanismhasbeenreportedinArubiclopsisSBPase (Willinghametal.1994).Secondly,thesemRNAsmaybealternativelysplicedfroma commonpre‑mRNA.Inhigherplants,theheterogeneousC‑terminalproductionby alternativesplicinghasbeenshowninspinachchloroplasticascorbateperoxidase (Ichikawaetal.1997),ribulose‑1,5‑bisphosphatecarboxylase/oxigenase(Rubisco) activase(Wernekeetal.1995),andpumpkinhydroxypyruvatereductaseisozymes
(Hayashietal.1996),CharacterisationofgenomicDNAencodingFBPaseisozymes fromEccglenaisnowunderwayinourlaboratory.
AcomparisonofthededucedaminoacidsequencesoftheEceglenaFBPase isozymeswiththatfromspinach,wheat,andcyanobacteriademonstratesahighdegreeof homologyoverlargepartsofthemiddleandcarboxy‑terminalregionwhichisnot unexpectedconsideringtheubiquitousandcntciairoleofthecpFBPaseandcyFBl'ase.
TheonlyregiondevoidofanyhomologyisthatcontainingthemostN‑terminal126 aminoacids.Asthisregionismissingintheprotein‑derivedsequenceofthespinach enzyme,itmostprobablyrepresentsthetransitpeptideneededfordirectingthenuclear‑
encodedFBPaseproteinintothechloroplast.Homologiestothecleavagesiteofthe transitpeptideoftheRubiscosmallsubunitcanbeseenatthecorrespondingposition
where亡hesequenceofthematureELCglenacpFBPasestarts,supportingこheassumμ めn
thatthisistheactualcleavagesite.Thiswouldgiverisetoamatureproteinconsisting of410aminoacidresidueswithanmolecularmassof43000Da,whichisingood agreementwiththecalculateddataf̀orthematurecpFBPase(43kDa)purifiedfrom Eμ818η αItsmostpro董ninentfeatureistheabsenceofal4‑amino‑acidlongregion.
ThisregioncontainedthreeCysresidues(C}」s‑155,Cys‑174andCys‑179)andimplied thattheseresiduesai璽eoneoftheregulatorysi箆sof吐helightdependentactivation(Marcus etal.1988}.Theseresiduesarepresentinallplastidicformsoftheenzyme,but
cytosolicFBPaselackedtheextraaminoacidsequenceincludingthreeCysresidues.
Thelightactivationinchloroplastsofhigherplantsinvolvesareductivecleavageof
ad孟sulfidebondbyredudngequivalentsthatcanbephotochemtca韮 韮ygenera重edviaa
ferredoxln‑thior6doxinsystem(BuchananetaLl971,Buchananl990,Scheibel990).
IhavepreviouslydescribedthatSynechococciesPCC794?FBPase‑II(FBPase‑II)have noCysresiduesandisnotregulatedbyDTTinvitaりantilightilluminationinvivo.SoI studiedtheeffectsoflightandDITtreatmentontheactivities6fcpFBPase,NADPH‑
GAPDH,SBPase,andPRKinEuglenach】oroplasis.Theseenzymesfromsplnach
c;hloroplasts≪sereactivatedbylightilluminationinvivaorDTTtreatmentinvitro{Fig.
VI‑5,VI‑6).However,treatmentoflightorDTThadlittleeffectontheactivitieso#' enzymesinEecglenachloroplasts.Moreover,cpFBPasefromELCglenctwasresistanttol InMH2021iketheS.フ942FBPase‑II.TheseresultsstlggestedthaUheenzymesinthe
PCRcycleofEμ818η αarenotregulatedbyaferredoxin/tllioredoxillsystem.
AsprokaryoticcellsincludingcyanobacteriaarenotcornpartinentalizedthePCR cycleisnotenclosedinaseparateorganelle.BecausetheFBPaseandGAPDHmust
functionbothinthephotosynthesisandgluconeogenesis,thelightregulationmechanisms mightnotneedfortheseenzymesinprokaryoticcells.However,eukaryoticalgaehave chloroPIastsinwhichperforlnphotosynthesissimilartohigherplants.Accordingly another‑reguEationsystembylightforthiol‑modulatedenzymesandphotosynthesismight benecessaryforeukar)10ticalgae,WedeleCal.(1997)havereportedthatPRKand NADPH‑GAPDHoligomerizedontothenonenzymaticpeptideCP12inhigherplant chloroplasts.Avilanetal.(1997)havereportedthatCP12existinChlarnydvmoncrsand formcomplexwithGAPDHandPRKundernon‑reducing{dark}conditions.Together withformationofcomplex,theactivityofPRKdramaticallydecreases.Iftheformation anddissociationoftheseoomplexoccurinchloi‑vplasts,andtheactivityofPRKis
regulated,carbonnowlnthePCRcyclecanregulateb》 ・redoxmodulation.Wedeletal .
havesuggestedthatlightregulationofPCRcycleactivityviaNADPH‑mediated reversiblePRK/CP12/GAPDHcomplexdissociationisconservedinallphotosynthetic organism.Theelucidationofthisregulatorymechanismsmayoffernewideasinstead offerredoxin!thioredoxinsystem.Iamtryingtoexaminewhetherthissystemarepresent andregulatecarbonflowinchloroplastsofEuglena.
Summary
InEccglenngraciliszcells,chloroplastYCandcytosolicfructose‑1,6‑bisphosphatase isozymes,designatedcpFBPaseandcyFBPase,havebeenresolvedbychromatography
onaDEAFSepharosecolumn.ThecpFBPaseandcyFBPasehavebeenpurifiedto
electrophoretichomogeneityfromthecells,respectively.ThecpFBPaseandcyFBPase hadrespectivemolecularmassesof172kDaand154kDa.andwerecomposedoffour identicalsubunits.TheapparentKrnvaluesofcpFBPaseandcyFBPaseforfructose
l,6‑bisphosphatewere72±45and55±1.5μM,respectively.cyFBPasewas inhibitedbyAMPwithaKivalueofQ.26mM,butcpFBPasewasnotaffectedbyAMP.
ThecpFBPaseandcyFBPasefromEuglenacellswereresistanttolmMhydrogen peroxideunlikethelightactivatedhigherplantchloroplastenzymes.ThecDNAs encodingcpFBPaseandcyFBPasewereclonedfromthe」ELCglenct.A1107‑bpopen readingframe,encodingprematurecpFBPaseof369aminoacidresidues(approx.
molecularmassof42kDa)wasobserved.ThenucleotidesequenceofthecyFBPase geneshowedanopenreadingframeof1017‑bpthatencodesaproteinof̀339aminoacid residues(approx.molecularmassof37.2kDa}.Interestingly,thededucedaminoacid sequenceofEicglenacpFBPasewas100°loidenticalwithcyFBPaseexceptforN‑terminal 30residues.