轍 2
2.4 一 1.4一
0.2一
1ト1.3kb
9.5̲
7.5‑
4.4一
2.4一 1.4一
0.2一
難 鱒 ぐ 一1・2kb
ず 諒 ず ず
Fig.7.6.NorthcrnblotξmalyslsofspinachlcafandrootmRNAprobcdwiththecDNAclonesto theSAP1(A)andSAP4(B).TotalRNAandmRNAwerepreparedfromleavesandroots.In eachlaneofpanelA,20N°oftotalRNAwereputon.InpanelB,2μgofpoly(A)+‑RNAwere puton.Themigrationofsizemarkersisindicatedontheleft.
Nortleerjiblotnfral ̲ysisTheexpressionofSAPImRNAaswellasthatof SAP4vasdetectedbyNorthernblotanalysisusingrandomlyprimedcDNAinsertsas
hybridizationprobes.Eachprobewashybridizedtoa1.3‑kbRNAofSAPIand1.?‑kb ofSAP4,respectively,indicatingthatthetranscriptswerefoundinbothleavesandroots (Fig.7.6).IthasbeenreportedthatthepcacytosolicAsAPtranscript,whichwasfound tobe1050b,wasobser、'edinbothlcavesandroots,andthatsteadystateAsAP
transcriptlevelsincreasedinresponsetoseveralstressesimposedbydrought ,heat,and
paraquat(MittlerandZilinskas199?).
<←‑34kDa ぐ 一28kDa
〆'
Fig.7,7.Immiiiiohlotごmalysisofcnidecxtrac重sfromZヨ.co〃 ㏄11sthathavebeentrallsformedwith SAPIalldSAP4.Molloclollalantibodyraisedag{盛nstr:'イ818〃 αAsAPwasusedasthep1imary all〔ibodya㏄ordillgtoChaP1crIL〆 、11al了owhcadin(licatesdlcPosi吐io璽10ftheproteinreco9置ureciby thealltibody.
BacterialexpressionmidAsAPactivityAfterinductionwithIPTG,the recombinantAsAPproteinsfromSAPlandSAP4wereetpressedinE.colicells.
WesternblotanalysiswasdonewiththemonoclonalantibodiesraisedagainstEuglena AsAP,whichreactedwiththecytosolicAsAPsfromplantsources,btrtdidnotcrossreact withGP(ChapterII).AsshowninFig.7.7,theantibodiesreactedwitha34kDa
recombinantSAPlproteinanda?9kDarecombinantSAP4protein.RecombinantSAPI correlatedwellwishthecalculatedmolecularmassoftheprimaryprotein.Somereports existontheproductionofantibodiesagainstplantAsAPs(Tanakaetal.1991,Mittlerand
58
Zilinskas1991a,Koshiba1993,ChenandAsada1989).Thereisnoevidenceofcross‑
reactionwithGP.TheseresultssuggestthatSAPIwashomologouswithASAP,not GP.
AsAPischaracterizedbyaspecificelectrondonorforAsA,thoughthe chloroplasticAsAPismorespecifictoAsAthanthecytosolicenzyme(Asada1992).
Recently,WilliamandThomas{1994)havereportedarecombinantpeacytosolicAsAP expressedandpurifiedfromE.coli,whichhasenzymaticandspectralpropertiesnearly identicaltothenativeASAP.RecombinantSAPlandSAP4showedahighdonor
specificityforAsAandnoorlowactivityforelectrondonorssuchasGSH,NAL)H,and guaiacol(Table7.1),inagreementwithnativecytosalicASAPfromtea(ChenandAsada 1989)andpea{MittlerandZilinskas1991a)leaves.Incontrast,GPpurifiedfrom spinachleaves(AsadaandTakahashi1971},oxidizedpyrogallolandguaiacolathigher ratesthanAsA(Table7.1).ReconlbinantSAPユdidnotreducelmMt‑
butylhydroperoxideasanelectronacceptorinthepresenceofAsA.Theseresultsindicate thattherecombinantenzymesfromSAPIandSAP4areclearlydistinctfromGP
isozymes.
Table7.1.Effectsofdifferentelectrondonorsontherelativeactivityoftherecombinant SAPI{rSAPI},SAP4(rSAP4},andendogenousguaiacolperoxidase(GP)fromspinach.
Thepreparationandassayofrecombinantenzymesweredoneasdescribedin"Materialsand Methods."
Donor rSAPl rSAP4
RelativeActivity{°lo)*
GP
AsA Iso‑AsA GSH eyc.c NADPH NADH Pyrogallol Guaiacol
100(1.2)**
90 0 0 0 0 2370 64
100{3.6) 30
0 0 0 0 1:
10
100(1.0) 109
0 0 0 234
・':!
9082
GSH:reducedglutatluone,Cytc:reducedcytochrotnec
*皿ieperoxidaseactivityforAsAwasshownas100%ofactivity .
**Specificactivity(μ1nolmin‑lmgprotein1)giveninparentheses
Onthebasisofthedatareportedhere,IconcludedthatSAPIcanbeclassifiedasa newtypeofASAPgene.Detailedstudiesonthedetectionandsubcellularlocalizationof endogeneousproteinfromthenewSAPlremaintobedone.
Summary
TwocDNAsclones(SAPIandSAP4)encodingascorbateperoxidasewere
isolatedfromacDNAlibraryusingthemonoclonalantibodiesraisedagainstascorbate peroxidasepurifiedfromEuglenagracilisZ.SAPlcontainedanopenreadingframe encodingaproteinof309aminoacidsandacalculatedmolecularmassof34471Da.The deducedaminoacidsequenceofSAPlshovedratherhigherhomologywiththecytosolic ascorbateperoxidasefromplantsourcesandSAP4thanwithbacterialperoxidasesand classicalplantperoxidases.SAP4containedanopenreadingframeencodingaproteinof 250aminoacidsandacalculatedmolecularmassof27625Da,inagreementwithspinach cytosolicascorbateperoxidase{WebbandAllen1995}.Thecompletecodingsequence oftheSAPlandSAP4wereexpressedinE.coli.Theperoxidsaeactivityofrecombinant SAPIproteinwasi.6‑foldhigherwithascorbatethanwithguaiacol,whichwassimilar tothoseofSAP4.NorthernblotanalysisshowedthatascorbateperoxidasemRNAsfrom SAPIandSAP4wereexpressedinleavesandroots.HereIconcludethatSAPIisanew typeofcytosolicascorbateperoxidasefromspinach.
60
CHAPTERVIII
CloningandSequenceAnalysisofacDNAEncodingChloroplastic AscorbatePeroxidasefromSpinach
ASAPisknowntohavetwotypesofisozymes,thatis,chloroplasticandcytosolic forms(Asada1992).Inangiospermchloroplasts,ASAPoccursinthestromaasa
solubleform(sAsAP}andalsointhethylakoidsasamembrane‑boundform{tAsAP).
TheenzymaticandmolecularpropertiesofASAPisozymeshavebeencharacterizedand areclearlydifferentfromthoseofguaiacolperoxidase(GP)fromhorseradish{Asada 1992}.
Recently,manipulationoftheexpressionofanti‑oxidativeenzymesincluding superoxidedismutaseandglutathionereductasebygenetransfertechnologyhasprovided newinsightsintotheroleoftheseenzymesinchloroplastsbyallowingthedirect investigationoftheirfunctionsandinteractions(Foyeretal.1994b,Allen1995}.cDNAs forcytosolicASAP(cAsAP},encodedbynucleargenesareisolatedandcharacterized frommanyplantsources,includingpea(MittlerandZilinskas1991b,1992)and Araゐidopsis(KuboetaL1992,1993).However,nocDNAsthatencodechloroplastic AsAPshaveyetbeenidentified.Forthisreason,ithasnotbecomeclearhowthe regulationsystemofchloroplasticASAPgenesrespondtoenvironmentalstress.
InChapterVII,IreportedtwocDNAclonesencodinganewtypeofcAsAP (SAPl)andhavealreadydeterminedcAsAPfrommaturespinachleavesusing
monoclonalantibodiesraisedagainstEκ8̀ε ηαAsAPasaprobe.Here,Idescribethefirst completecloningofachloroplasticASAPcDNAfromspinachgreeningcotyledonsusing
theEuglejiamAb.
MaterialsandMethods
1VlaterialsSpinachseeds(∫ 御 α磁016roc6のweregerminatedonmoistgauze at15°Cinthedark.Thecotyledonsfromseedlingsgrownfor4‑5daysinthedarkwere
transferredtoillumination(140μEm2sec曜1)for24hrtoobtainthegreeningcotyledons.
Allchemicalswerereagentgradeandusedwithoutfurtherpurification.
ConstructionandscreeningofcDNAlibraryTotalRNAwasisolatedfrom greeningcotyledonsofspinachseedlings(5.Og,wet!wt)asdescribedinChapterVII.A cDNAwassynthesizedusingacDNAsynthesiskit{Amersham,UK)andacDNA
librarywasconstructedinλgtllasdescribedl)ythesupplier(Amersham,UK).The spinachcDNAlibraryinλgtllwasscreenedbymonoclonalantibodiesraisedagainst EuglenaASAP(ChapterII).ThecDNAinsertfromanimmunopositiveclonewas
subclonedintotheplasmidvectorpBluescriptSK(+)andusedasahybridizationprobeto obtainlongercDNAclonesfromthesamelibrarybyplaquehybridization.The
nucleotideofthelongestcDNAclonewassubclonedintopBluescriptSK(+)and sequencedbythemethodasdescribedinchapterVII.AcDNAclonecontainingthe5' endofthechloroplasticASAPmRNAwasobtainedby5'RACEPCR(Edwardsetal.
1991)usingnestedantisenseprimerssynthesizedtosequencesdownstreamoftheN‑
terminalprimerposition.
PurificationofstrornalascorbateperoxidaseIntactchloroplastswereisolated fromthefreshspinachleavesbypercolldensitycentrifugation(lshikoetal.1992).The purificationofsAsAPfromintactchloroplastswasthendoneaccordingtoNakanoand Asada(1987}.TodeterminetheN‑terminalaminoacidsequence,partiallypurified sAsAPwasseparatedbySDS‑PAGEandtheresolvedproteinswereelectroblottedontoa PVDFmembrane(Millipore,USA).TheblotwasstainedinO.1%Coomassiebrilliant blueR‑250andthemainbandcorrespondingtothesAsAPprotein(34kDa}wascutout witharazorblade.TheN‑terminalsequencesoftheexcisedsAsAPbandwas
performedbyautomatedEdomandegradationusingamodel477Asequencer(Applied Biosystems,USA).
ResultsandDiscussion
InordertoisolateacDNAcloneforputativechloroplasticASAP,theEuglenaanti AsAPmAbs(EAPI,EAP2),whichcross‑reactedwithchloroplasticASAP(ChapterII), wasusedasaprobe.
A
B
0 336
H
0 1287
tAsAP
1
一一一 一一 一 一 一一一一 一 一一一 一 一伽 レ
一 一 一
1407
一 一 一 一
Fig.8.1.PartialrestrictionmapandcloiungstrategyofcDNAencodingthylakoid‑boundASAP (tAsAP)fromspinach.tAsAPcDNAwasconstructedfromthesequenceoftwooverlapping cDNAclonesAandBobtainedbyRACE‑PCRusingnestedantisenseprimerssynthesisedto sequencesatpositions317‑336byand372‑391bydownstreamoftheS‑terminalposition(see Fig.8.2).Restrictionenzymesareshownasfollows:N,Ndel;Sa,Sacl;Sc,Scal.Arrows indicatethedirectionandextentofsequencing.
62
GAAAAACCACCCAATCTCACTCACTTTCTCTCTCTATATTTTCAAACCACCACCCCGCAA CCAATGACTGATCGTCTAGCAATGGCATCCTTCACTACCACCACCGCCGCCGCTGCATCT
MASFTTTTAAAAS
CGTCTGCTTCCTTCTTCTTCCTCCTCCATCTCTCGACTTTCTCTCTCCTCTTCTTCCTCC
RI」LPSSSSS工SRLS工 」SSSSS
TCCTCCTCCTCACTCAAATGTCTCCGATCATCTCCACTCGTCTCTCACCTCTTCCTTCGA SSSSLKCLRSSPLVSHLFLR
CAGAGAGGAGGTTCAGCTTATGTGACGAAGACGAGGTTTAGCACGAAATGCTACGCTTCT
QRGGSAYVTKTRFSTKCYAS
▲
GATCCTGCGCAGCTGAAGAATGCTAGGGAAGATATTAAAGAGCTTCTTCAATCTAAGTTC DPAQLKNAREDIKELLQSKF
TGTCATCCTATTATGGTTCGCTTAGGTTGGCACGATGCCGGTACTTATAATAAGGACATT CHPIMVRLGWHDAGTYNKDI
AAAGAATGGCCACAAAGAGGTGGAGCCAATGGCAGTCTGAGCTTTGATGTTGAGCTCAGG KEWPQRGGANGSLSFDVELR
CATGGAGCTAATGCAGGTCTTGTTAATGCCCTGAAACTTCTACAGCCCATAAAAGACAAG HGANAGLVNALKLLQPIKDK
TACTCTGGAGTTACATATGCAGATCTATTCCAGCTGGCTAGTGCTACTGCAATAGAGGAG YSGVTYADLFQLASATAIEE
GCTGGTGGTCCAACAATACCCATGAAGTATGGAAGAGTGGATGCCACAGGGCCGGAGCAG AGGPTIPMKYGRVDATGPEQ
TGCCCAGAAGAAGGAAGGCTTCCTGATGCTGGACCTCCTTCACCTGCTCAACATCTACGT CPEEGRLPDAGPPSPAQHLR
GATGTTTTCTACAGAATGGGTCTTGATGATAAGGATATAGTAGCATTATCTGGAGCACAT DVFYRMGLDDKDIVALSGAH
ACGTTGGGAAGGTCTAGACCTGAACGCAGTGGTTGGGGCAAGCCAGAGACTAAATACACG TLGRSRPERSGWGKPETKYT
AAAGATGGACCTGGAGCTCCAGGAGGGCAGTCATGGACTGCGGAGTGGTTGAAGTTTGAT KDGPGAPGGQSWTAEWLKFD
AATTCCTATTTCAAGGACATCAAAGAP,AAGAGAGATGCAGATTTGCTTGTTTTGCCAACT NSYFKDIKEKRDADLLVLPT
GATGCTGCTCTTTTCGAAGATCCGTCTTTCAAGGTATATGCAGAGAAATATGCAGCTGAC DAALFEDPSFKVYAEKYAAD
CAAGAAGCATTTTTCAAGGATTACGCTGAAGCCCATGCCAAACTCAGCAACCAAGGAGCC QEAFFKDYAEAHAKLSNQGA
AAATTTGACCCTGCTGAGGGTATCACTCTTAATGGAACCCCTGCCGGAGCAGCTCCAGAG KFDPAEGITLNGTPAGAAPE
AAGTTTGTAGCAGCCAAGTACTCATCTAACAAGAGATCAGAGCTTTCGGATTCTATGAAG KFVAAKYSSNKRSELSDSMK
GAAAAGATTCGCGCTGAATATGAAGGTTTTGGAGGTAGCCCTAATAAGCCTCTACCAACA EKIRAEYEGFGGSPNKPLPT
AACTACTTCCTAAACATTATGATTGTGATTGGAGTTTTGGCAGTTCTATCATATCTTGCG
NYFLNIMIV工GVLAVLSY】 ⊃A
GGAAATTGATTTGTGGTTTGATGAGTTTTTTTCCATTTATAAATATAACGGCAGTTGATT GN
ATATGAAAAAAAAAAAAAAAAAAAAムA
Fib.8.2.
60 120
13 180
33 240
53 300
73
360 93 420 113 480 133 540 153 .i1 173 ..1 193 720 213 780 233 1 253 900 273
・.1 293 1020
313 1SI 333 1140
353 zoo 373 1260
393 1320
413 i3so
415 1407
Nucleotidesequenceofspinachthylakoid‑boundASAPanddeducedan血oacidsequences.The aminoacidsequencededucedfromanopenreadingframeisshownbelow血enucleotidesequences.The
arrow五ndicatestheputativecleavagesiteofthetransitpeptide.TlleputativeIV.glycosilationsite,Asn‑X.
Ser!Thr,areunderlined.ThenucleotidesequencedatareportedinthischapterwillappearintheDDBJ,
EIViBLandGenBazil:nucleotidesequence(iatabaseswi血 血efollowingaccessionnumberD77997.
A950bpcDNAfragmentwasisolatedfromaλgtllcDNAlibraryconstructedwith
librarywasrescreenedbyplaquehybridizationusingthe950bycDNAfragmentasa probe.Oneclonewitha1287bp‑lengthinsertwasselectedforsequenceanalysis,which lackedthe5'endcontainingtheputativestartcodon.Weemployed5'RACEtoamplify theunknownsequenceatthe5'endofthecDNAclone.Theresulting336byfragment wasfoundtocontain216byofoverlappingsequencewiththeformerclone.Thewhole nucleotidesequenceconsistedof1407by(Fig.8.1).Itisnotpossibletounambiguously deducethetranslationalstartforthespinachchloroplasticASAP‑precursorprotein.The openreadingframeprecedingthematurepolypeptideincludestwoin‑phasemethionine residuesatnucleotidepositions64‑66and82‑84.Thesequencescontiguouswiththese, thefirstmethioninecodons,matchtheconsensussequencesforplanttranslationinitiation sites(A/GA/CXATGG)withpurineat‑3andGatposition+4beingthemostcritical
{Lutckeetal.1987}.So,IassumedthattheATGcodonatposition82‑84actsasthe initiatorcodon.Thecompletesequencerevealedtheopenreadingframeof1248by encoding415aminoacids{Fig.8.2).Thecalculatedmolecularmassoftheencoded proteinwas45015Da.
4.0 02 00 ︒0逡
壼 罵 α o も ﹀ エ
一4.0
0 goo 200 300 400
Residuenumber
Fig.8.3.Hydropathyprofileofthededucedaminoacidsequenceofspinachthylakoid‑bound ASAP.HydrophobicitywasanalyzedbydieGENETYXsoftwareprogram,asdescribedKyteand Doolittle{1982),forawindowsizeofnineaminoacidresidues.Thehydrophobicdomainsare abovethezeroline.
ThecorrespondingsequencewiththepurifiedtAsAPfromspinachleaveswas foundinthefirst20aminoacidsequenceofthededucedaminoacids71to90(Miyakeet al.1993}.So,thepositionofdeducedaminoacidsbetween70and71wasdefinedas thecleavagesite.Thecalculatedmolecularmassofthepredictedmatureproteinwas 37710Da.ThetAsAPsolubiiizedandpurifiedfromspinachleaveswasestimatedtobea molecularmassof40000±2000Dabygel‑filtration(Miyakeetal.1993}.Furthermore, hydropathyanalysisshowedthatthepredictedmatureproteinhasonemajorhydrophobic
64
region{residues380‑415)attheC‑terminousdomain,whichmaybethedomainforthe bindingtothethylakoidmembranes(Fig.8.3).Theseresultsclearlyindicatethatthe isolatedcDNAcloneencodestAsAPofthespinachchloroplasfis.
S.tAsAP S.cAsAP s.sApx
S.tAsAP T.sAsAP S.cAsAP S.SAPi
S.tAsAP T.sAsAP S.CAsAP S.SAPI
S.tAsAP T.sAsAP S.cAsAP S.SAP1
S.tAsAP T.sAsAP S.cAsAP S.SAP1
S.tAsAP T.sAsAP S.cAsAP S.SAP1
ロ
1:MASFTTTTAAAASRLLPSSSSSiSRLSLSSSSSSSSSLKCLRSSPLVSSLFLRQRCSA!S
! :1̲.
1:灘
★・
1;;雛蕪 難 難1繋;1;;叢
*****
難 難 離 醸;;;;織;欝
★ ★ ☆ ★ ★ ★ ★ ★ ☆ ★ ★ ★ 嚢 費 ★ ★ ★ ★灘
鳶 ★ 嚢・
;;;灘 鍵難1灘;1;1雛;雛 灘鐵 鍵
★ ★ ★ ★ ★ ★ ★ 嚢 ★ ★ ★ ★ ★ ★ ★ ★ ★ ☆
;;;;難 攣;;;;難;;;:;;;;;懸;;講1轄
★ 嚢 ★ ★ ☆ ★ ★ ★ ★ ★ ★ ★ ★ 禽 ★
;;;;騰購 欝 鰭;:::1::1::灘
*****************
S.tAsAP360:YSSNKRSELSDSMKEKIRAEYEGFGGSPNKPLPTNYFLNIM工VIGVLAVLSYLAGN S.SAP1266:KTEAVQCNTDMLDPMQ工,EMVAAQAATDTYNMPIYTAVNCNSLRD
Fig.8.4.Alignmentofthededucedanvnoacidsequencesofspinachthylal:oid‑boundAsAPwith thepartialaminoacidsequenceofteastroinalASAPandthededucedaminoacidsequencesof spinachcytosolicAsAPs.S.tAsAP,spinachthylakoid‑boundascorbateperoxidase(thisstudy);
T.sAsAP,[eastromalascorbateperoxidase(Chenetal.1992);S.cAsAP,spinachcytosolic ascorbateperoxidase(ChapterVII);S.SAP1,spinachcytosolicascorbateperoxidaseisozyme (ChapterVII).Aminoacids,whichareidenticaltothespinachthylakoid‑boundascorbate peroxidase,areshaded.Dashesareincludedtomaximizeali;nment.Theasterisksshowthe consensusaminoacids.ThedistalandproximalHisresiduesareshownbyheavydots.
ThesequenceupstreamoftheN‑terminusofthematureAsAPproteinencoded74 aminoacidresidues,withapredictedmolecularmassof7322Da,whichhadseveral
featurescommontomostchloroplastictransitpeptides.Likethetransitpeptidesofthe majorityofchloroplasticprecursors,thetransitpeptideofthetAsAPprecursorcontained fewacidicresidues,wasrichinserineandthreonine,andhadanetpositivecharge.It
alsohadapotentialtoformanamphiphilic(3‑strandclosetotheputativeprocessingsite
{deBoerandWeisbeek1991).
Fig.8.4comparesthepredictedaminoacidsequenceencodingspinachtAsAPwith thoseofotherAsAPs.ThetAsAPfromspinachdoesexhibitahighlysignificant
homology(82.4%)overa108residualregiontopartialaminoacidsequencesofsAsAP purifiedfromtealeaves(Chenetal.1992}.ThededucedspinachtAsAPhada46.5%
and40.2%homologywiththatofcAsAPandSAPlfromspinach,respectively.
Moreover,thededucedspinachtAsAPproteinshowed46.5°loaminoacididentitywith theArabidopsiscAsAP{Kuboetal.1992)and43.8%identitywithpeacAsAP{Mittler andZilinskas1992}.AsAPandyeastcytochromecperoxidase(CCP)havebeen classifiedasamemberoftheclassIplantperoxidasefromitsaminoacidsequenceand hasbeenpartofthelineageofprokaryoticperoxidases{Welinder199?).Thededuced spinachtAsAPwas34.6%identicalover240aminoacidswithyeastCCPandhadless homologywiththeclassicalplantperoxidasesuchasGP.Thisisalsothecaseforthe otherpreviouslydescribedAsAPs(Kuboetal.199?,Chenetal.1992,ChapterVII).
Inordertoanalyzetherelationshipsamongplanttypeperoxidases,aphylogenic treewasconstructedaccordingtoHein'salignmentalgorithm(1‑rein1990).Asillustrated inFig.8.5,tAsAPwasmorecloselyrelatedtothecAsAPsandyeastCCPthantoother perolidases.ThisresultclearlysupportsthefactthattAsAPbelongstobetheclassI peroxidaseaswellascAsAPs.SuchahighdegreeofhomologyamongAsAPssuggests thatAsAPgenesmighthaveevolvedfromthesameancestralgeneandhavebeenfar fromclassicalplantperoxidasesuchasGPintermsofmolecularevolution.
S.tAsAP
S.cAsAP
S.SAP1
Y.CCP
P.Mn‑P
HRP
Fig.8.5.Phylogetvctreeforspinachthylakoid‑boundandcytosolicAsAPsandotherplanttype peroxidases.Thedendrogranlwasgeneratedbycoitiparisoiioftheknownacniiioacidsequences a㏄ordiヨ1gtoHeill(1990).Thele皿gthofthebrallchcsisproportiollaltotlleevdutiollary divergence.S.tAsAP,spinachthylal:oid‑boundascorbateperoxidase(thisstudy);S.cAsAP, spinachcytosolicascorbateperoxidase(ChapterVII);S.SAP1,spinachcytosolicascorbate peroxidaseisozyme(ChapterVII);Y.CCP,yeastcytochromecperoxidase(Kaputetal.1982);
PMn‑PPharierochaetecんysosporii〃 ηmanganese‑dependentperoxidase(Peaseetal」989);HRP, horseradishperoxidase(Welinder1976}.
66
Distal{His‑104)andproximal(His‑233)histidineresiduesattheactivesiteare indicatedinFig.8.4bydots.TheclassIperoxidasessharethecommonfeaturesofthe distalhistidinesite(R‑L‑A‑W‑H).ThetAsAPalsohadhighlyconservedresiduesinthe sequence,exceptforoneresidueatthepositionofglycine‑102.Theproximalhistidine siteofthetAsAPagreedverycloselywiththatofteasAsAP(Fig.8.4}.ThetAsAP
containedtwoputativeN‑glycosilationsiteswhichfollowthegeneralruleofAsn‑X‑
Thr/Ser(KornfeldandKorenfeld1985}.TeasAsAPshowsthatitssugarcontentis lowerthanO.25%andisnotaglycoprotein{ChenandAsada1989).TheputativeN‑
glycosylationsitesofspinachtAsAPmaynotbeglycolated.
InordertomakecomparisonswiththeprimarystructuresofthechloroplasticASAP isozymes,sAsAPwaspurifiedfromisolatedintactspinachchloroplasts.ThesAsAP showedamolecularmassof34000Daasamonomerjudgedbygel‑filtrationandSDS‑
PAGE.TheaminoacidsequenceoftheN‑terminalresiduesofthematuresAsAPfrom spinachwasdeterminedasfollows:YASDPAQLKNAREDIKELLQ.Interestingly,the first20aminoacidsofspinachsAsAPexhibitedacompleteconsensussequencewiththat ofspinachtAsAP.ChenandAsada(1989)reportedthemolecularmassofteasAsAP
wasestimatedtobe34000Daandhadapproximately309aminoacidsdeducedfromits aminoacidcomposition.Fromtheaminoacidsposition71to3790fthepredicted
tAsAP,whichcorrespondedtothatofteasAsAP,itispossibletocalculatethemolecular massof33885Da.Furthermore,theaminoacidcompositionofspinachtAsAP
determinedfromitspredictedaminoacids(residues71‑379)wassimilartothatoftea sAsAP(datanotshown).Theresidual36aminoacidsresiduesoftheC‑terminalregion (residues380‑415)hadahydrohobicdomainasdescribedabove.Recently,weisolated thecDNAencodinganewtypeofcAsAP(SAP1},whichisdifferentfromthatofthe
alreadyknowncAsAP(ChapterVII).SixASAPisozymeswerefoundinbellpeppers andacomparisonoftheseisozymesshoweddifferencesinitsgrowthconditions {Schantzetal.1995).AnovelAsAPisozymewasfoundtobelocalizedonthe
membranesofmicrobodiesinpumpkin(Yamaguchietal.1995).Takingintoaccountthe datareportedsofarandthepresentfindings,itseemslikelythattheASAPofspinachisa multigenefamilyandthereareatleastmorethanthreeASAPgenes,twoofwhichmaybe
encodedbynearlyidenticalchloroplasticgenes,differingonlyinthepresenceorabsence ofthe3'codingregions,whichconstructahydrophobicregionattheG‑terminusdomain intheASAPprotein.
Summary
AcDNAcloneencodingthylakoid‑boundascorbateperoxidasewasisolatedfroma spinachcDNAlibraryconstructedbygreeningcotyledonsfromseedlingsusingthe monoclonalantibodyraisedagainstEuglenaascorbateperoxidaseasaprobe.ThecDNA containedanopenreadingframeencodingamatureproteinof345aminoacidswitha calculatedmolecularmassof37,710Daprecededbyatransitpeptideof70aminoacid residues.Thededucedaminoacidsequencehad40‑46010and34.6%homologytothe otherknownascorbateperoxidasesfromplantsourcesandcytochromecperoxidasefrom yeast,respectively.
68
CHAPTERIX
Conclusion
InEuglefiagracilis,whichlackscatalase,theantioxidantenzymesinvolvedinthe AsA‑GSHcycleoccuronlyinthecytosolbutnotinthechloroplasts.Theuseofthis
organismasamodelspeciesforstudyingtheeffectofalteredlevelsofASAPinvolvedin protectionagainstoxidativestresshasmeanedthatabetterknowledgeisrequiredof ASAPandofitsregulation.
EuglenaAsAPwaspurifiedtohomogeneity.AlthoughtheEuglenaAsAPshowed closelysimilarenzymlogicalpropertiestothoseofcytosolicASAPisozymefromhigher plants,theenzymepossesseditsownproperties;thehighmolecularweightandthe diferenceofaminoacidsequenceoftheN‑terminus.Especially,itisworthnotingthat theErcglenaASAPalsoreducedt‑butylhydroperohideandcumenehydroperoxideasan electronacceptorinthepresenceofAsA.Tomyknowledge,EugleriaAsAPisthefirst ASAPwhosesubstrateisanorganichydroperoxideaswellasH242inphotosynthetic eukaryotes.InChapterIII,theASAPactivitywasnotobservedinFe‑deficientEuglena
cells,andthatlipidperoxides(thiobarbituricacid‑reactivesubstances)inFe‑deficientcells wereapproximately2.6‑foldgreaterthanthoseinFe‑sufficientcells.Theseresults
suggestthattheErr.glenaAsAP,likeglutathioneperoxidaseinanimalsand Clilarnydotnotrcrs,mayservetoprotectthecellmembranebyreducingtheperoxide compoundsgeneratedendogenouslyfromunsaturatedfattyacids.N‑‑terminalaminoacid sequenceofEuglenaASAPshowednosignificantsimilaritytoanyotherAsAPsfrom
higherplants.FromtheresultofpartialaminoacidsequencesofE配81ε ηαAsAP,
however,theenzymeexhibitedahighdegreeofhomologytosequencesofcytosolicand chloroplasticAsAPsinhigherplants,suggestingthatEuglenaASAPgeneisalso comprisedinthesameancestralgene.
ChapterIIIandChapterIVshowthatbothFeandlightbecomeimportantregulatry factorsforAsAPexpression.Fe‑deficientEatglenacellsaregoodmodeltoinvestigatethe molecularmechanismofASAPexpression.TheAsAPactivityvasnotfoundinFe‑
deficientcellsasstatedabove.TheadditionofFetotheFe‑deficientcellscausesthe AsAPactivitytoincreasebytwophase,theformerwastheactivationofASAPandthe laterwasdenovosynthesisofASAPprotein.Illuminationofdark‑grownEuglenacells causedanincreaseintheAsAPactivity.TheactivitiesofenzymesrelatedtoAsA‑GSH cyclesuchasSAD,MDAsAreductase,DAsAreductase,andGSHreductasewerealso foundtoriseparalleltotheincreaseinASAPactivity.Theincreasedleveloftheenzyme activitiesinAsA‑CrSHcyclewereattributedtosynthesisdenovooftheirproteins.These
factsclearlyindicatethatelevatedlevelsofantioxidativecomponentscanbeconsideredan earlyphysiologicalresponseofEugleraacellstoremoveH202generatedinvivo.
ItisaninterestingproblemwhytheEuglenacellslocalizeAsAPonlyinthe cytosol,butnotinthechloroplasts,toscavengeH202generatedircvivo.ChapterV demonstratedthatH20?formedinEugletcachloroplastsandmitochondriadiffusesfrom theseorganellesintothecytosol.Onthebasisoftheseobservations,Fig.9.1showed
theworkingmodelsofprotectivesystemagainstH202inEuglerraandhigherplantcells.
Inhigherplants,H20iseliminatedbyASAPlocatedinchloroplastsandcytosoland catalaselocatedinmicrobodies.Ontheotherhand,inEuglerracells,H202generatedin chloroplastsandmitochondriamustdiffusefromeachorganelleintothecytosolandthen decomposebytheAsA‑GSHcycleincludingAsAP.ThediffusionofH202from
chloroplastsintothecytosolaswellastheH202‑scavengingbyASAPseemstobea protectivesystemagainstoxidativestressinEacgleruccells.
Higherplants
助g'eηacellS
Fig.9.1MetabolismofHZOainhigherplantsandEuglenacells.
70
Inhigherplants,thesituationtoinducetheresponseofantioxidativeenzymesto environmentalstressesisoftenconsiderablycomplicatedbythepresenceofalarge numberofisoenzymeforms.Forexample,ASAPislocatedinchloroplasts,microbodies andcytosol。Inkomatsuna(i/∬icaRαPα)leaves,chloroplasticAsAPwasalow content(approximately20%}comparedwithcytosolicASAPandkomatsunaleaves containonlyonetypeofcytosolicisozyme.Itislikelythatkomatsunaisalsotheuseful plantmodeltoinvestigatetheresponseofcytosolicASAPtoalterationsintheoxidative stresses.
Atthepresenttime,onlyonetypeofcytosolicASAPisclonedandnocDNAsthat encodechloroplasticAsAPshaveyetbeenidentified.Forthisreason,littleisknown aboutthemolecularmechanismofAsAPisozymesunderlyingtheirresponseto environmentalstresses.oneofmonoclonalantibodies{EAPI)raisedagainstpurified EugleraaASAPcross‑reactedwithbothcytosolicandchloroplasticAsAPisozymesin higherplants,indicatingthatEuglenaASAPisimmunologicallyrelatedtoAsAPisozymes inhigherplantandthatEAPIbecomeagoodprobeto̲screenacDNAencodingAsAP
isozymesfromhigherpunts ..Actually,ChapterVIIandChapterVIIIshowedcDNA cloningofcDNAsencodingtwocytosolicASAPisozymes(SAPlandSAP4)anda
cDNAencodingthylakoid‑boundAsAPofspinachleavesusingEAPIasaprobe.SAPl wasidentifiedtobeanewtypeofAsAPisozyme,Comparisonofthededucedamino acidsequencesofthreeASAPisozymesshowedapproximately45%identityamongthese isozymes,suggestedthatAsAPisozymeinspinachisamultigenefamily.ThesecDNAs andmonoclonalantibodiespreparedinthisstudywouldprovideexcellentprobesfor studyingthemolecularmechanismofAsAPgenesresponsetoenvironmentalstresses.
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PUBLICATIONS
Originalpapers
Ishikawa,T.,Takeda,T.,Shigeoka,S.,Hirayama,0.andMitsunagaT.{1993)
HydrogenperoxidegenerationinorganellesofEuglercagracilis.Phytochemistry, 33:1297‑1299.
Ishikawa,T.,Takeda,T.,Shigeoka,S.,Hirayama,0.andMitsunagaT.(1993)
RequirementforironanditseffectonascorbateperoxidaseinEuglenagracilis.
PlantSci.,93:25‑29.
Ichikawa,T.,Sakai,K.,Takeda,T.andShigeoka,S.{1995}Cloningandexpression ofcDNAencodinganewtypeofascorbateperoxidasefromspinach.FEBSLett., 367:28‑32.
Mutsuda,M.,Ishikawa,T.,Takeda,T.andShigeoka,S.{1995)Subcellular
localizationandpropertiesofL‑galactono‑y‑lactonedehydrogenaseinspinach leaves,Biosci.Biotech.」B'oc」hen.,59:1983‑1984.
Ishikawa,T.,Takeda,T.,Kohno,H.andShigeoka,S.(1996)Molecularcharacter‑
izationofEreglenaascorbateperoxidaseusingmonoclonalantibody.Biocliim.
Biophys.Acta,inpress.
Ichikawa,T.,Sakai,K.,Takeda,T.andShigeoka,S.Cloningandsequenceanalysis ofacDNAencodingchloroplasticascorbateperoxidasefromspinach.
inpreparation.
Ishikawa,T.,Takeda,T.andShigeoka,S.Purificationandcharacterizationof cytosolicascorbateperoxidasefromkomastuna(iJ∬icarapの.inpreparation.
Proceedings
Takeda,T.,Ishikawa,T.andShigeoka,S.{1994)TheH202‑scavengingsystemand tolerancesystemtoinalgae.InFrontiersofreactiveoxygenspeciesinbiologyand medicine,edsbyAsada,K.andYoshikawa,T.,pp143‑146,Elsevir,Amsterdam, TheNetherlands.
lshikawa,T.,Takeda,T.andShigeoka,S.(1996)Molecularcharacterizationof ascorbateperoxidasefromEuglenagracilisZ.Curr.Res.Photosynth.,inpress.
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