potassiumphosphatebuffer,pH7.5,containing?.5mMDTT,1mMGSHanti10°la sucrose,andsonicated(10kHz)foratotalof10minwithfourintervalsof2mineach.
Thislysatewascentrifugedat12000gfor15inin.Theobtainedcrudeenzymewas loadedontoaDEAF‑cellulosecolumn(2.8cmx40cm)equilibratedwiththepotassium phosphatebufferanddevelopedwitha300mi‑lineargradientofKC1(0‑0.5M}.The activefractionswerecombinedandadjustedto30%saturationwith(NH
4)2SO4・The supernatantwaschrolnatographedonaPheny1‑SepharoseHR515column(FPLCsystem,
Pharmacia)equilibratedwith30%(NH
4)2SO4inthe501nMpotassiulnphosphatebuffer, pH75,conta{ning2.51nMDTT,lmMGSHandユ0%sucrose。Thecolumnwas
elutedwithlOOmlofalineargradientof30‑0%(NH
4)2SO4atanelutionrateof11111 miri'・Theactivef・acli・nswerec・mbinedandf・acti・natedwilh(NH
、)、SO、,andthe pelletprecipitatingbe"veen30and70%saturationwasdissolvedin2mlofthe54mM
potassiumphosphatebuffer,pH7.5,containing2.5mMDTT,1mMGSHand10°10 sucrose.TheenzymesolutionwaschromalographedontoaSuperdex200HiLoad 16160columnequilibratedwiththe50mMpotassiumphosphatebuffer,pH7.5, con重aining25mMDTr,11nMGSH,10%sucroseandO.15MNaCI.Theactive
fractionswereconcentratedtoafinalvolumeofO.5mlbyultrafiltration(AmiconPM30) andloadedontoa2',5'‑ADPSepharosecolumn(0.Scmx4cm)equilibratedwiththe50 mMpotassiumphosphatebuffer,pH7.5,containingZ.5mMDTT,1mMGSHand
lO%sucrose.TllecolulnnwaselttCeclWftllImlofthepotassiumphosphatebunセr containingln1MNADP+antithenwith10m1ofthepotassiumphosphatebuffer.The enzymefractionswerecollectedandchromatographedontoaSephadexG‑25column (?cmx15cm)equilibraCedwiththe104mMTris‑HCIbuffer,pH8.0,toremovethe NADP+.Thepurifiedenzymewasstoredat‑20gCwithoutextensiveinactivationfor severalweeks.
Pal)2̀κ κy!̀〃η'̀18gelelectroplzoresis‑一 一Discgelelectrophoresiswith75%
(WN)polyacrylamideatpH9.4wasperformedaspreviouslydescribed(Shigeokaetal.
198フb),Electrophoresiswascarriedoutataconstantcurrent(21nAgel'1)with bromophenolblueasthe‑migrationmarker.SDS‑PAGEwasperformed.on12.S%
(W!V)polyacrylamideslabgelaspreviouslydescribed(Takedaetal.1993a).Proteins inthegelwerestainedwithCoomassiebrilliantblueR‑250anddestainedin7%vacetic acid.
Est〃 η6ぜゴonρ プmolecularweight‑一 一ThemolecularwelghtofGAPDHwas estimatedbyusingaSuperdex200HiLoadl6160column(2.4cmx90cm)equilibrated
with50mMpotassiumphosphatebuffer,pH7.5,containing2.5mMDTT,1mMGSH,
10%sucroseandO.15MNaCI,andcalibratedwithbluedextran12000kDa},
thyroglobulinfrombovine(66.9kDa),apoferritinfromhorsespleen(44.3kDa),B‑
amylasefromsweetpotato(?OkDa),alcoholdehycirogenasefromyeast{15kDa), albuminfrombovineserum{67kDa)andcarbonicanhydrasefrombovine‑erythrocytes (30kDa).ForthemolecularweightofasubunitbySDS‑PAGE,phosphorylasebfrom x‑abbitmuscle{94kDa),albuminfrombovineserum(67kDa),ovalbuminfromeggwhite (43kDa),carbonicanhydrasefrolnbovineertythrocytes(30kDa),trypsininhibitorfroln soybean(20.1kDa)anda‑lactalbuminfrombovinemilk(14.4kDa)wereusedas
stalldards.
Analysisρ ノcaninoacidsegacence‑一 一ThearninoacidsequenceoftheN‑
terlninalregionoftheenzymewasdeterminedbyanautomatedEdmandegradationusing aModel477Agasphaseproteinsequencer(ShigeokaandNakano1991).
鵡 麟躍
(kbp)
"
輔 騨 雛
3㊨26
鵜
'硝離 2.5
1.0
Fig.VII‑1GenomicSouthernAnalysisofDNAfromS.7942.GenomicDNAwas
digestedwithdifferentristrictionenzymesasindicatedabovethePaneandsubjectedto Southernanalysis.ThePCRproductwasdigoxigeninlabeled‑andusedas
hybridizationprobes.DetailsoftheprocedurearedescribedintheMaterialsand Methodssection.ThesizesofDNAmolecularmassstandardsareindicated.
Isolccttioǹrnclncccleotide389L̀8ηoεcinnlysis{ゾGAS)DH8・ene‑一 一一 一The
chromosomalDNAwasisolatedfromtheS.794?cells(wetweight1.5g)asdescribed previously(Williams1988)。Theoligonucleotideprimerscon'espondingtothemost highlyconservedregions(residues13‑32[51‑GT(AG)GC(AC)ATCAATGG(AT)T丁TG G‑3']and452‑474[5'‑GTTGGTGGTGCAAGAAGCATTGC‑3'])ofNADP‑and
NAD‑dependentGAPDHfromvariousorganisms‑{Brinkmannetal:1989,Shinetal.
1991,BranlantandBranlant1985}weredesignedandsynthesizedbyJBioS,Co.,Japan.
ThePCRproductsusingtheseprimerswereusedasaprobetoscreenthefull‑length
DNAfragmentincludingthegeneofGAPDHfromtheS.7942chromosomalDNA digestedwithEcoRI,BarnHI,Hindlll,XbaI,BstPIorStyI(TAKARASHUZO CO.,LTD,JaPan)at5units(μgDNA)‑1.Thesouthernblothybridizationwas
completed ̲accordingtotheinstructionsofAmersham.Asaresult,therewasonlyone fragmenthybridizingwiththeprobeineachLane{Fig.VII‑1}.A6.6kb‑BcrmHI
fragmentincludingtheGAPDHgenewasisolatedfromtheagarosegelusingthe GENECLEAN⑪IIKit(BIO1011NC.,USA),subd・nedinめapBlue・criptIISK(+) vector,andsequencedusingthedideoxychainprimermethodmodifiedfordoable strandedplasmidDNA.TheoverlappinginsertDNAfragmentswereobtainedby st bcloningafterrestrictiondigestion.
ExpressionofGAPDHgeneinE.colitrnclptcrificationofrecoynbinantenzyme‑一 一一一‐FortheconstructionoftheplaslnidtoexpresstheclonedGAPDHgene
,thefull‑
lengthproteinencodingDNAfragmentswereamplifiedbyPCR.Twooligonucleotide primerscontained/VdeIrestrictionsitewiththeATGtranslationinitiationcodonand B̀〃 ηHIrestrictionsite(boldsequence),5'‑AGGGTATAGGCATATGACGATTCG‑3冒 (nucleotideresidues‑6‑17)and5'‑AGCATTTGGATCCCTGAACC‑3'(nucleotide residuesll72‑1191).ThePCRproductsweredigested≪iththe/VoleIandB̀肥HI restricUonenzynles,andligatedintoa/VdeI!BcanHI‑digestedpEI'3aexpressionvector followedbyitstransformationintotheE.colistrain,BL?1(DE3)pLysS.TheE.soli ceりsweregrownwithshakingat37℃untilanA6000fO.6‑0.7inLBmediumcontaining 50ysgml"̀ampicillinand34ysgml"̀chloramphenicol.Theexpressionwasthen
inducedwithaddingisopropylβ 一D。thiogalactoside(IPTG)toO.4mMandshakingfbr6 hat37℃.Thecells(2.29wetweight)wereharvestedby㏄ntrifugationat5008・for10
min,suspendedin50mMpotassiumphosphatebufFer,pH7.5,containing2.5mMDTT, 1mMGSHalldlO%sucrose,anddisruptedbysonication(10kHz)for3min.This lysatewascenirif̀ugedal18000g.for20min.Theobtainedcrudeenzymewasused foranalysisoftotalsolubleproteinofE.colibySDS‑PAGEandthenadjustedto30%
saturationwith(NH4)2SO4.ThesupernatantwaschromatographedonaPhenyl‑
SepharoseHR515cohlmnequilibratedwith30%(NH4)2SO4inthe50mMpotassium
phosphatebuffer,pH7.5,containing2.5mMDTT,1mMGSHand10%sucrose.
ThecolumnwaselutedwithlOOmlofalineargradientof30‑0%(NH4)zSO4atan elutionrateoflmlmin‑1.Theactivefractionswerecombinedandfractionatedwith (NHS}2SO,andthepelletprecipitatingbetween30and70°losaturationwasdissolvedin 2mlofthe50mMpotassiumphosphatebuffer,pH7.5,containing2.5mMDTT,1mM
GSHandlO%sucrose.TheenzymesolutionwaschrolnatographedanloaSuperdex 200HiLぐ}ad16!60columnequilibratedwiththe50mMpotassiumphosphatebしiffer,pH
7.5,containing2.5mMDTT,1mMGSH,10°losucroseandO.15MNaCI.Theactive fractionswerecombined,adjustedtoO.05MNaCIandloadedontoMonoQHR1.0/10 columnequilibratedwiththe50mMpotassiumphosphatebuffer,pH7.5,containing2.5 mMDTT,1mMGSH,10%sucroseandO.05MNaCl.Thecolumnwaselutedwith
100mlofalineargradientofO.05‑1MNaClatanelutionrateoflmlmin'.Theactive fractionswerecombinedandconcentratedtoafinalvolumeoflmlbyultrafiltration (AmiconPM‑30).
TocompletelyremovetheDTTfromtheenzymepurifiedfromtherecombinant cellsaswellastheenzymepurifiedfromtheS.794?cells,theextractswere chromatographedonacolumn(15cmx10cm)ofSephadexG‑25equilibratedwith100
mMTris‑HCIbuffer,pH5.0,containing16mMMgC12.Theproteinfractionswere PlacedintovialssaturatedwithN2gastogiveaconcentrationofapproximatelyl2ン9 proteinmr1.Thepul漁edrecombinantandnativeenzymeswereincubatedwithH202 ror10mininthedarkaspreviouslydesc【7bedinchapterIIL
ResultsandDiscussion
CharacterizcrtivnofNADP‑dependentGAPDH‑一 一Th6purificationschemeof S.7942GAPDHusingthefive‑stepprocedureissummarizedinTable1.The
purrl・icationprocedureyieldedaGAPDHpreparationpurifiedapproximately173‑fold overthecrudeenzymewishayie1dofl4.9%Thispurificationwasrepeated山ree timeswithsimilarresults.DuringpurificationofGAPDHfromS.7942,theenzyme activitywaselutedfromeachcoiuinnasasharpandsinglepeak,indicatingthatS.794?
containsonlyonetypeofGAPDH.ThePAGEandSDS‑PAGEofthepurifiedenzyme
showedonlyonedetectableproteinband(Fig.VII‑2).ThespecificactiniEycfpurified
enzymewi吐hNADPHwasl50.5μmolmin‑1(lngprotein)一 霊,whichwasthesameorde整 ・
c)rlnagnitudeasthatofGAPDHfromhigherplants(Cerff1978).Theenzymealso showedspecificactivitywithNADH,whichwere26.9μmolmin‑1(lngprotein)'1.The enzymereactionwithNADPHandNADHfollowedMichaelis‑Mententypekinetics.
Fromthedoublereciprocalplots,theapparentKrnvalueoftheenzymelorNADPHand NADHwere62±45and420±10.5μM,respectively.Theseresultsindicatethat NADPHisthephysiologicalelectrondonorfor‑S.794?GAPDH.
ル10伽̀1̀〃 〃ì榔 伽 め ・sisc〃idsequenceofthelt1‑tern痂̀泌̀剛 η0傭̀'region‑一 一
Gelfiltration‑onaSuperdex200HiLoad16/60columnofGAPDHwithseveralstandard proteinsindicatedamolecularmassof160kDa.Thesubunitmolecularmassofthe enzymewasestimatedtobe41kDafromSDS‑PAGE,indicatingtheenzymeisatetramer withasubunitmolecularmassof41kDa(Fi8.VII‑2)。TheaminoacidsequencesofN一
terminustothe24thresidueofthepurifiedenzymewasdeterminedbyanautomated Edmandegradation,andthesequencewasasfollows:T‑1‑R‑V‑A‑1‑N‑G‑F‑G‑R‑1‑G‑R‑
N‑F一 しR‑c‑w‑F‑G‑R‑Q‑。
TableVII‑1PurificationofGAPDHfromS.7942
Step
TotalproteinTotalactivity (mg)(Nmolmin‑1)
SpecificactivityPurificationYield (Nmolmin‑1(fold)(%)
(mgprotein)一)
Crude1156.2 DEAEcellulose100.2 30%(NH4)2SO466.8 PhenylSepharose11.3 Superdex2004.21 2",5"‑ADPSepharose1.00
1008.5 757.2 643。8 499.1 322.9 150.5
1 7.56
・.1
44.2 76.9 150.5
1.0 8.7 11.0 50.8 88.4 173.0
100.0 75.1 63.8 49.5 32.0 14.9
(A) MW
(kDa) 97.4 66.3
(B)
42.4 鋤 蜘E41
.0
30.0 十
Fig.VII‑2TheProceduresfor日ectrophoresisofthePurifiedEnzymewereCarried OutasDescribedintheMaterialsandMethodsSection.PAGE(A)andSDS/PAGE(B)of purifiedenzyme(3fig)stainedwithCoomassiebrilliantblueR‑250.
IsolationcmdchnrcrcterizationofDNAencodingGAPDH‐ ‐ ‐Therestriction mapandsequencingstrategyforthe6.6kbp‑BranHIfragmentcontainingtheGAPDH
8eneareshowninFig.VII‑3.ThenucleotidesequenceoftheGAPDHgeneconsisted of1140byORF,encodingforapeptideof38Uaminoacidresidues(Fig.VII‑4).The initiationcolonisGTGinsteadofATG,similartothesituationobservedfor PhytophthorainfestnnsgprlAgene(Moonetal.199?).Thecalculatedmolecularmass ortlleclonedsequenceinthegeneofGAPDHwas41,304Da,whichwasclosedto41
kDaoftheGAPDHsubunitdeterminedbySDS‑PAGE.‑Thededucedaminoacid sequencecoincidedwithN‑terminusoftheNADP‑GAPDHpurifiedfromS,794?.
Sixteenbasepairsupstreamfromtheinitiationcolonisaputativeribosomalbinding site,theShine‑Dalgamosequence,ACGAGGTheG+CcontentoftheGAPDHgene
was54.1°70,≪ 」hichwassimilartothepercentageofG+Cbasepairsfoundinthegenomic DNAfromS.794?.
王6qΦ
≡℃贈ミ
こい吐
一屯qく
一=◎瑠曇
一旬ど
≡め鳴ミ
一&ど至ε醒
}
一
、＼ガ ....L一゜'°°°'きゆ
い一ロ ↑一
Fig.VII‑3TheThickBarRepresentstheLocationoftheGAPDHGene ,with3'and5'
Endsindicated.丁hesubfragmentco耐ainingGAPDHgenewassubjectedto sequenceanalysiswiththestrategyindica#edinthelowerportion.
Co〃3ρ ω τμ∫v{v̀〃 πψソSIS6ゾ̀〃Winòuìlsegacencesq1'OAPD1ノ ノYO〃zv̀JJ"1'ULIS5・o泥1℃ ・L'S
‐ ‐ ‐FigureVI‑4comparesthededucedaminoacidsequencesoftheS
.7942GAPDH gene.withαherGAPDHgenesfrolnvariousorganisms.Thededucedaminoacid
sequenceoftheS.794?GAPDHgenewas66.9%,67.0°loand.45.9%identicaltotype
GapA,GapB,andGapCofAπ 痂4ρ ρ3∫∫,respectively(Shinetal,1991).Slmilar
resultswerealsoobtainedwithGapA(65.7%),GapB(66.1%),andGapC(44.6%)of Tobacco(Shinetal.1986).TherewasarelativelylowsimilaritybetweentheS.794?
GAPDHandF.coliNAD‑dependentform(48.St%1identity)(Doolittleetal.1990).
Theseresultsshowedthattheaminoacidsequenceol̀theS.7942GAPDHgenehada higherhomologywiththeNADP‑dependentandchloroplasticfoF‑mthanwiththeNAD‑
dependentandcytosolicNorm.Thisviewisalsosupportedbythedonorspecificityfor NADPH;thespecificactivityandtheKmvaluesforNADPHasdescribedabove.
Thesubunitsofん てめ ∫̀10ρ5'∫GapAandGapBhadapproxima吐ely14and35alnino acidN‑terminalextensions,respectively,whichindicatefeaturescommontothetransit
peptidesofcy重osolicprecursorsofproteinsimportedacrossthechloroplasticenvelope (Brinkmannetal.1989,Shinetal.1991).Unlike.GapAandGapB,theS.7942
GAPDHgenehadnotransitpeptides.Futhermore,itisinterestingtonotethattlleC‑
terminaloftheS.7942GAPDHgeneaswellastheGapBgenewaslongerthanthoseof thechloroplasticalldcytosolicforlns.The42‑residueC‑terlninalregionofthegene
fromS.7942GAPDHhadaverylowhomologywitha28‑residueC‑terminalregionof GapBandothersequencesintheSwiss‑Protdatabase.
BothGapAandGapBarecharacterizedbythefiveCysresiduesattheconsex・ved positionsasfollows:33,167,171,291,and302inArcrbidopsisGapA;however,GapC lackedthethreeCysresiduescorrespondingto33,291,and302(Sh三netaL199ユ).In theS.7942GAPDHgene,thetwoCysresiduescorrespondingto291and302were absent,butfourCysresiduesat24,78,155,and159werepresent.
一49ムAGCTTAATAGGTATTTACACCTGAムCG(:ACGTACGAGGGTムTムGGGCA・
SP GTGACGATTCGAGTTGCGATCAATGGCTTTGGCCGTRTTGGCCGGAATTTTCTCCGTTGC
麗
TGGTTTGGACGGCAGAACACCGATCTAGAGGTTGTGGCCATTAACAACACCTCGGATGCA
碗FGRQ口?DLsVVム:【 蕊NTSDム
CGGACGGCTGCTCACCTGCTGGAGTACGACTCTGTTCTCGGCCGGTTCAACGCCGACATC
R?A為 腿LLEYDSVLGRFNAP工
AGCTACGACGAGAATTCGATCACCGTCAACGGCAAGACGATGAAAATCGTCTGCGATCGC SYDBNSITVNGKTMRIVCpR
AACCCCCTCAACCTGCCTTGGAAAGAGTGGGATATCGATCTCGTCATTGAATCTACAGGT NPLHLP珊KE秘D工DLVI鱈STG
GTGTTCGTCACCGCTGAAGGCGCATCCAAGCACATCCAAGCCGGGGCCAAGAAAGTTCTG VFVTAEGASKHiQAGAKRVL
ATCACGGCTCCTGGTAAAGCCGARGGTGTCGGCACCTACGTCATCGGTGTCAACGATTCG xTムPGKハEGVGTYV工GVNDS
GAATACCGCCACGAAGACTTCGCAGTCATCAGCAATGCAAGCTGCACCACCAACTGCTTA EYRH遅DFムVIsN△SCT㌘NCム
GCACCGGTCGCCAAAGTTCTGCATGACAACTTTGGCATCATCAAAGGCACGATGACCACC APVAKVL8RNFGIIKGTMTT
ACCCACAGCTACACGCTGGACCAGCGCATCTTGGACGCCAGCCACCGTGATCTACGTCGG THSYTLDQRILDASHRDLRR
GCTCGGGCTGCAGCCGTTAACATCGTTCCCACCACGACCGGCGCTGCTAAAGCCGTTGCT
ARハ ムAVNIVPTTTGAムKムVA
TTGGTGATCCCCGAGCTGAAAGGCAAACTAAACGGGATTGCGCTGCGCGTTCCTACGCCA LVIPELKGKLNGIALRVPTP
AACGTGTCTGTCGTTGACTTGGTGGTTCAAGTCGAGAAACCGACGATCACTGAGCAGGTC NVSVVDLVVQVEKPTITEQV
AATGAAGTCCTGCAAAAAGCTTCTCAAACGACGATGAAGGGCATCATCAAGTACTCGGAT
NE .VLO区 蕊SOT磐MKGIIxYSD
CTGCCCTTGGTCTCTTCCGACTTCCGGGGTACTGACGAGTCTTCGRTCGTTGACTCCAGC LPLVSsDPRGTDESsTVDSS
CTGムCCTTGGTAATG〔 撒GGCGATC管CGTC肌AGTAA冊GC冊GG肝 ハCG為C樋CG^GTGG
I」 歴LV麗DGDLVKV工A田YDN日 り
GGCTACAGCCAACGAGTTGTCGACTTGGCTGAACTGGCCGCTCGCAAATCGGGCCGCTTA GYSQRVVDLAELAARKSGRL
号 GGACTTGTTCAAACCCTCTTAATTTCTAGTCAAGATAGCCTCCCATTAACGGGAGGCTTT GLVQTLLISSQDSLPLTGGF
TTTGTGGCCAAGAGAGAACACCCCAGGAGCCAAGATTCGATCAATCCATCAGGCCCCGAT FVAKRENPRSQQSINPSGPD
TAGGGTGAAATGACGGATTTTCAATCCCCTAGGTTCAGGAATCCAAATGCTATTAGGGCT 倉
GAATTTCAGTTGGCTGAAAGCAGAGCCAATCAGCTCGCCACCTATAATTCAGAGAAGTGR CGATTAGGAGCGATCGCCGCATGGCTTTGCTGGAGACCCGCACCGAGCCAATGGTGCTCA ムCム!『GGGCCC1330
一1
60 20
izo 40
180 so 240
ea 300 100
360 124
420 iao 480 160 540 180 600 zaa 660 220 Aso 240
T80 260
eaa ssa 900 300 960 320 2020 340 1080 360 1190 380 izaa
1260 1320
Fig.Vll‑4TheAminoAcidSequenceofN‑terminalRegion,WhichareIdenticaltothe SequencesDeterminedUsingtheAutomatedEdmanDegradationofthePuri#ied Enzyme,areUnderlined.ThenumbersstandforthosefromtheN‑terminalfirst residuesoneachline.Theputativeribosomebindingsite,theshine‑Dalgarno sequence,isdoublyunderlined.
8
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240・,LD,̲̲TFKTA.A・SYK・ 一吻 ・MK‑QAS・GS・A…GYT・ ・EV・ ・T・ ・QGDTH.SIF・AGAGI肌
236:...M..F....E.R.IT‑.,..Q.L‑K.AS.GP...YSELQ...S.YQGT.A.SIV.A...L..
236・.肌.A・LT・ 考 ・F￡VNRPTTV・.1・ ・Lr,xaA・EQAP・Q…GYEER・ …1・YKDDPR・SI・ ・ALS・ ・.v
283:..1..TAI..S,‑TV..PVK.N...LLLQKAA‑QGAFH..V.YTEL...NHDPH.AIV.GTQ.R.S 238:。...曹...,.E.P.IT‑.Q..EVLQKA‑SQTTM...工KYS.L....S.。.GT。E.SIV...L。.
☆ ★★ ★☆ 壷 ☆ ☆
318:GDDMVKVIAWYDNEWGYSQRVVDLADIVANNWK
342・,̲̲V・ ・̲.̲.・ …̲肌.・SK・PG配AVGSGDPLED彰KTNPADε 喚VYD
308:S,KF..LVS..。..,...S,....工VRMSKA 308:NSNFF..V...N...,,MLS 304:.N.L...M...L...EL..EK.V 305:DETQ..IL...VN.M.E..RK..LSL.
3063.AHLI.TLV.霧...̲FAN.肌.TT囲.TVAFR
306:DG.L...。..,...ELA.RKSGRLGLVQTLLISSQDSLPLTGGFFVAKREHPRSΩDSI四PsGPD
★ ★ 貨曹★ 索★ ★
Fig.VII‑5TheDififierencesintheGAPDHProteinSequencescanbeIdentifiedwith theSubstitutionofAnotherAmino‑AcidbelowtheS.7942GAPDHSequence.
Dashesareincludedtomaximizethealignment,andthedotsindicatehomologywiththe
S.7942GAPDHsequence.Theas#erisksshowtheconsensusaminoacids.
TheresistanceofrecombinantandnativeenzymesofS.7942toH202‐ ‐ ‐ AfterinductionwithIPTG,therecombinantproteinsfrolntheGAPDHgenewere
expressedinE.colicells.AsshowninFig.VII‑6,therecombinantproteincorrelated wellwiththecalculatedmolecularmassofthenativeenzymepurifiedfromS.794?cells.
TheE.co〃enzymeshowedaNADPH‑dependentGAPDHactivityof13.4μmolmin'1
(mgprotein)"'.In GAPDHactivityof GAPDHactivity.
preparationpurified
57.0%recoveryoftheactivity onedetectableproteinband wishNADPHwas149.8ycmolmin theput‑ifiednativeenzyme.
contrast,theE.coliasacontrolshowedaNADH‑dependent 2.9μlnolmin‑1(mgprotein)'1,butnotheNADPH‑dependent
ThepurificationprocedureyieldedaRecombinantGAPDH approximately11.2‑foldoverthecrudeenzyme,givingafinal
° °
.TheSDS‑PAGEofthepurifiedenzymeshowedonly Thespecificactivityofthepuc‑ifiecirecombinantenzyme
in‑'(mgprotein)"̀,whichwasinagreementwiththatof
1 2 3 45
(kDa) 94 67
43 鐵
癖
㌔︑↑
30
、総灘
Fig.VII‑6ProductionoftheGAPDHinE.colibytheGAPDHGene.Protein
,thelysateofE.standards(lane1),purifiedenzyme(lane2),thelysateofE.coli(lane3) colitransformedwiththeGAPDHgeneandincubatedintheabsence(lane4)and
presence(lane5)oflPTGweresubjectedtoSDS‑PAGE.EachlanewasstainedwithTh
earrowindicatesCoomassieBrilliantBlueR‑250anddestainedwith7%aceticacid theGAPDH.
Ihavepreviouslydemonstratedthatthethiol‑modulatedenzymes,fructose‑1,6‑
bisphosphatase,NADP‑GAPDH,andribulose‑5‑phosphatekinaseinchloroplastsof
Eccglena乙mdC〃 伽 リノ40〃 εoηα∫andincrudeextractsofcyanobacteria,S.7942and SynechocystisPCC6803areresistanttoH202(Takedaetal.1995).HereIstudiedthe
effectsofH2020ntheactivitiesofthenativeGAPDHpurifiedfromS.7942cellsandthe recombinantGAPDHpurifiedfromE.colicells.AsshowninFig.VII‑7,boththe nativeandrecombinantGAPDHswereonlyslightlyinhibitedbythelmMH202,
indicatingtheabsenceofsusceptibilityoftheGAPDHs亡oH202uptolInM ,In
contrast,thespinachchloroplastGAPDHwascompletelyinhibitedatO .1mMHzO2,
whichwasinagreementwiththeresultreportedpreviously{Takedaetal .1995).