EstimationofProteinDisul＆de−isomeraseActivity BasedonproteinRe払1ding＊

Bul】．Fac．Bioresources，MieUniv．  

No．8：59−63   March26．1992  

EstimationofProteinDisul＆de−isomeraseActivity   BasedonproteinRe払1ding＊  

KatsuzumiOKUMURA，YoshiakiMIYAKE＊＊，HiroshiTAGUC川  

andYoshihideSfllMABÅYASHI   

索ThisworkwassupportedinpartbyGrant−in−AidforScient漬cResearch（No．61760077）  

ftomtheMinistryofEducation，Science，andCulture，Japan  

輌Presentaddress：CentralResearchLaboratory，PokkaCorp．，Shikatsu−Cho，  

Nishikasugai一糾n，瓜chi4軋Japan  

LaboratoryofBiolog】CalChemistry．FacultyofBioresources，  

MieUniversity，Tsu，Mie514，Japan  

Abstraet  

Proteindisul鋸e−isomerase（PDI）isassociatedwiththerefbldingofproteinsthathavemis−matehed   disuはdebondsthroughthethioトdisul鋸einterchangereaction．Whentheenzymeactivitywithreduced・  

denaturedRNaseas the substratewaspIotted女〉ranalysisorthe駄sトOrderkinetics，theaccelerated  

regencrationofRNaseactivitygave】inearplotsforboththeFPIconcentrationandreactiontime．This  

activity（90％），SOitshouldbesuitablefortheinvestigationofPD王inl1ibitorsandinhibitorsofprotein   refolding，andforresearchintothere餌dingprocessofotherproteinsubstrates，SuChasrecombinarlt  

1（ey words：prOtein disultide−isomerase，PrOtein refoldhg，first−Order kinetics，disulfide bond，  

RNaseactivity  

IlltrOdu（：tion  

ProteindisulBde−isomerase（PDI，EC5．3．4．1）isacatalystthatformsnativedist11員debondsofsecreted  

（ECl．8．4．2）2）．TheprimarystruCtureOfPDIhasbeenidenti鮎d3）andanalysishasshownthatitsstructureis   thesameasthatofβ−Subunitsofprolyi4−llydroxylase（ECl．14．11．2）4），thyroidhormonebindingprotein5），  

iodothyronine，5，rmonodeiodinase6），andglycosylationsitebindingprotein7）・Thus，theessentialphysiological  

如nctionsofPDIareof払terest8，9）  

PDIassociatedwiththefoldingofproteins，inwhichtheinactivefomoftheproteinisconvertedtothe  

activeformbyformationofstabiedisul鋸epairs．PDIcanbeusedtorefoldrecombinantproteinsexpressed   andmadeinsolubleinbacterialO）．ItcouldbeusedinbothinvivoandinvitrostudiesoftheroleofPD‡inthe  

fbldingofvariousproteins，includingrecombinantproteins．OnlyafewreportsontheassayofPDIactivitygive  

AcceptedOctober31，1991   

60   KatsuzumiOKUMURA，YoshiakiM‡YAKE，HiroshiTAGuC用andYoshihideS川MA鋸YASt！l  

linearresults，andthereisnomethodtomeasuretheisomerizationactivityacctlrately．Themostreliable   rnethod女）rtheassayofPDIactivitywouldbethedirectmor血0ringofthedisul餌eexchangereaction，buta   Suitablesubstratehasnotbeenidentihed．Twomethodsaremainlyusednowforthisassay．Oneisbasedon   thedetectionofthe splitthgofinsu血intoits twocomponentchainsll），andthe otherisbasedon the  

regenerationofenzymeactivityfrom scrambled ribonucleaseA（RNase），Whichhasrandomiypaireddisultide   bondsandisenzymatica11yinactive12）．Thefbmermethoddoesnotreaecttheextentofproteinrefolding．  

ThelatterisbasedontheregenerationofRNaseactivity，butitindirectlydetectsminorchangesinRNase  

RNaserefolding．AmethodtoassayPDIactivitybasedonproteinrefbldingisneededifPDIistobeusefdlfor  

theregenerationofrecombinantproteinnotinitsnativeconformation．Inthispaper，WerepOrtanaSSay  

methodthatgiveslinearplotsforPDIactivitybasedonl）rOteinrefoldingbytheuseofthereducedpdenaturedand  

scrabmledfbrmsofRNase．  

M乱terialsandMethods   

PDIwaspuri鮎dfromfreshbovineliverbythemethodofLambertandFreedman13）・Thereaction   mixture contained，ha totaivolume of200pl，0．1MTris一斑Clbuffer（pH7．8），0．1mMoxidizedformof   glutathione（GSSG），200FLg／mlreduced−denaturedRNase，andPDI．Thereactionwasstartedbytheaddition   ofthesubstrate（theRNase）andproceededat250c．Attimes，20−iLlportionsofthereactionmixturewere  

takenandthereactionwasstoppedbyitsdi王utionwithO．2Msodiumacetatebu拝er（pH5．0）containinglmg／mi  

bovineserumalbumin（BSA，Sigma，なactionV）．EachdilutedsamplewasthenassayedforRNaseactivitybya   slightlymodifedmethodofAn蝕senetal14）・Thereactionmixturefortheassaycontained，inatota量volumeof  

l．Oml，0．1M sodiumacetate buffer（pH5．0），2mg／miRNA（sodium salt，Kohjin），0．25mg／mlBSA，and   enzyme．Thereactionwascarriedoutat370candstoppedbytheadditionofO・25rnlofO・83Nperchloricacid，  

fbuowedbycooungofthemixtureinice．Aftercentr漁Igationatl，700×gfor5min，SuperrlatantWaSdiluted20  

times with distilled water andits absorbance at260nm was measured，Reduced−denatured RNase was  

preparedbythemethoddescribedelsewhere15）withbovinepancreaticribonucleaseA（Sigma，TypeトA）not  

血沈herpuri鮎d．TileprOteinconcentrationwasassayedat275nmwithuseofmolarextenctio11COe統cientsof  

9，300and8，100fornativeandreduced−denaturedRNase，reSpeCtively16）．  

Rcsultsiln（11）iseussion  

FigurelshowsthecourseofthePDIreactionwithreduced−denaturedRNaseasthesubstrate・The  

regenerationofRNaseactivitywasacceleratedinproportiontotheconcentrationofPDI・Thesereactions  

wereinthepresenceofO・1mMGSSG，buttheRNasewasregeneratedunderotherredoxconditions，SuChasin   thepresenceofboth4mMreducedfomofglutathione（GSH）andO．4mMGSSG（datanotshown）・The  

effectsofPDIwereneghgibleinreactionmixtureswithoutoxidoreductivereagents・   

ThecourseoftheregeneratedactivitywasreplottedinFig．2Aforanalysisofthe鮎st〜Orderkinetics・The   pattem ofthe regeneration ofreduced−denatured trypsinogengives a sim並arplot17）・The acce王erated  

regenerationofRNaseactivitygavelinearplotsateachPDIconcentration・WhentheresultsinFigr2Awere   

EstimaとionofPD王Activity  

nV     れU nU  

︵ざ︶uO；巴望￠謬∝  

Fig．1．Efkcts of PD王on the Regeneration of RNase   Actわity．The course ofthe PDIreaction with   reduced−denaturedRNaseasthesubstrateisplot・  

ted．Tlle PDIconcentrations were O（◎），10  

（○），20（A），and30〃g／ml（△），Eachpercen−  

tageofRNaseactivitywascalculatedre王ativeto  

︵∝−ロ≡︶＼≡∪−﹃  

蒜T⊥岩こ＼冨︻u一  

10   20    30   PDl（〃g′ml）  

0  

2D    40    60   Time（min）  

Fig，2．First−OrderKineticAnalysisoftheRegenerationofRNaseActivity（A）andtheDependenceon軌ePDI   Concentration（8）．  

（A）ThedatainFig．1werereplottedby釦st・Orderkinetics．SymboIsarethesameasinthelegendof   Fig．1．Risthepercen払geofregeneratedRNa＄eaCtivityateachpoinL（B）Thedatafor亡王IeCOntrOI  

SamPle（A）subtractedandtheresultsareplottedagainstthePDIconcentrationforeachreactiontime．  

Fromthebottom，theほnesareforreactiontimesoflO，20，30，40，8nd60min．  

PlottedagainstthePDIconcentration（Fig．2B），therelationshipwasalmostlinearintherangeofconcentrations   upto30pg／mlPDI．Whenthereactiontimewas20min，therangeoflinearitywasuptoatleast60FLg／rnlPDI  

（datanotshown）．Moreover，theplotscoverhighregenerationofRNaseactivity（about90％）．Thismethod   fortheassayofPD王activitycouldalsobeusedinreactionmixturescontainingscrambledRNaseasasubstrate，  

inwhichcasedithiothreitolorGSHwasaddedinsteadofGSSGandtheamountofPI）lusedwassma払er（datanot  

Shown），However，preParationofscrambledRNasetakestirne．Thereactionshouldbefor20−30minfor   PDIassaywhenreduced−denaturedorscrambIedRNaseisusedasthesubstrate．   

TheothermethodsIO・11）describedabovemaybesuitableforkineticstudiesofthisenzyme．Themethod   

62   KatsuzumiOKUMURA，YoshiakiMIYAKE，HiroshiTAGt3Cl＜ilandYoshihideSⅢMABAYAS王v11  

proposedhere，however，iss血pler，mOreuSeful，andconvenientforstudiesontheeffectsofPD王orltheprotein   refoldingprocess．ItisalsosuitableforcomparisonoftherefoldingabilityofproteinswithPD川keactivity，  

suchasthioredoxin15），ThemethodwouldbesuitablefortheinvestigationofPDIinhibitorsandinhibitorsof  

proteinreねIding，andforresearchintotherefbldingprocessofotherproteinsubstrates・  

Referen（三eS   

l）F7そEEDMAN，R．B．Nativedisulphidebondformationinproteinbiosynthesis：eVidencefortheroleofproteindisulphide    isomerase，r柁刀ゐβわc如札Sれ9（10）：438−441（198射．  

2）DuclくWOlm（，W．C．andA．E．KITABC軋 王nsu主inmetabolismanddegradation，Endocr・Reu・，2‥210p233（1981）・  

3）EDMAN，）．C．，L．ELuS，R．W．BLAC王IElモ，R．A−RoTHandW．J．RuYm汎 Sequenceofproteindisulphideisomeraseand   血piicationsofitsrelationshjptothioredoxれ肋知和，317：267−270（1985）・  

4）KolVU，J．andK．MYLLYLA，Interchaindisul鋸ebondfomaionintypesIand王Iprocollagen，］・Biol・Chem・．262（13）：  

6159−6164（1987）．  

5）YAMAUCIil，K．，T．YAMAMO′rO，H．HAYAS11l，S．KoYA，rI．TAKIKAWÅ，K．ToYOSHIMÅandR・HoR王UCⅢ・Sequenceof   membrane−aSSOCiatedthyroidhomonebilldingproteinfrombovineliver：itsidentitywithproteindisulphideisomerase，  

β玩ゐg臥βゆ妙ざ．ガgぶ．C抑‡〃‡購．∫146（3）：1485−1492（1987）−  

6）BoÅDO，R．JリD．A，CAMPBELLand工．J．CJ】IOP肌 NucIcotidesequenceofratliveriodothyronine5′−rrWnOdeiodinase   

（5′MD）：itsidentityⅥ7iththeproteindisul鋸eisomerase，β加如肌βf坤毎ぶ・虎βざ・Co，乃刑鉦払，155（3）：1297−1304（1988）・  

7）GEETllA−HAE〜lB，M．，R．NorvA，H．A．KAl）LANandW．J．LENNARZ．Glycosylationsitebindingprotein，aCOmpOnentOf    oligosaccharyitransferase，ishighlysimilartothreeother57lくdluminalproteinoftheER，Ce仏独1053−1060（1988）・  

8）PA王N，R．I王．Acaseofmistakenidentity，〟αJ緑柁，328：298−299（1987）・  

9）FREEr）MÅN，R∴臥Proteindisul魚deisomerase：multiplero‡esinthcmodi鮎ationofnascentsecretoryproteinproteins，  

CβJg，57：1069−1072（1989）．  

10）OKUMURA，KりY．M王YAKE，H．WAKAYAMA，K．MuRAYAMA，T．MIYAIくE，K．SE′rO，H・TAGUCrllandY・SⅢ湖ABAYASIIl・  

E鮎ctsofproteindisul魚deisomeraseontherefoldingofhumanpro−urOkinaseclonedandexpressedinEscherichiacoli・  

A伊た．βねJ．Cゐg沼り52（7）：1735−1739（1988）・  

11）KATZEN，H．M．and F．TrE・rZE．Studiesonthe speci鮎ityand mechanismofaction ofhepaticglutathione・insulin   transllyむogenase，ノ．都8〜．C如猟り別肌15）：356ト3570（1966）・  

12）Ⅰ以ほSTON，A，L．andR．B，FREEDM卿．Thioトprotebldisulp王扇deoxidoreductase，βわc舶用．ん159：377−3朗（1976）・  

13）LAMBEl汀，N．andR．B．FREEDMAN．Structuralprotertiesofhomogeneousproteindisulphide−isomerasefrombovine    liverpuri鮎dbyarapidhiかyieldingprocedure，Biochem・J。213：225−234（1983）・  

14）AがFエNSEN，C．B．，R．R．REDFIELD，W−L．CliOA′rE，T．PAGEandW．R．CAlモROLL．Studiesonthegrossstructure，  

cross・linkages，andteminalsequencesinribonuclease，ノ・βわJ・Cゐ紳乙，2椚：20ト210（195礼  

15）P（GIET，V．P．andB．J．Scllt）STElモ．thioredoxin・Catalizedrefoldingofdisul鋸e−COntainingproteins，Proc・Na払Acad・Sci・  

ぴ．5．Aり83：7643−7647（1986）．  

16）KoNIS軋Y．andH暮A．ScliEI汰GA．Regeneratiol10fribonucleaseÅ打omtllereducedprotein・1・Conぬmationalanalysis   ofthcintemediatesbymeasurementsofenzymaticactivity，0画Caldensity，andoptica暮rotation，Biochemis吻19：1308−  

1316（1980）．  

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EstilⅥationofPDIAc貢vity  

タンパク質のRefbldingに基づいたProtein   Disu払de−isomerase活性の評価   

奥村 先細・三宅 義明＊・田口  麗・嶋林 率英   三親犬学廃物資源学部  

率税所属 ポッカコーポレーション中央研究所  

ProteinDisu泊dc−isomerase（PDI）は，thiol・disulfide交換反応により誤ったdisul餌e結合を持つタンパク質のrefold−  

i昭を促進する。本酵魔の基磐として還元変性RNaseを用い，その活性の全体的な復元過程を駄sトOrderkineticsに   よりプロットしたところ，PD王浪度並びに反応時問の間者について麗絶間係が得られた。この評価法は，簡単で，商   いrefoldi11g備約90％）まで包括することから，遺伝子組換えタンパク粟をはじめとする様々のタンパク質のre一   払Idingに対するPD‡の効果の解析，並びに．PDIの阻客剤およびタンパク貿のrefoldi咽に対する阻害園子の検討な  

どに有効な方法と考えられる。