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Title ポリアクリルアミドゲル電気泳動によるネズミマラリア原虫特異酵素の検出

Author(s) 塚本, 増久

Citation 熱帯医学 Tropical medicine 16(2). p55-69, 1974

Issue Date 1974-06-30

URL http://hdl.handle.net/10069/4160

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Tr。PicalMedicine，16（2），55－69，June，1974　　　　　　　　　　　　　　　　　　　　　55

Differential Detection of Soluble Enzymes Specific to

a Rodent Malaria Parasite, Plasmodium berghei, by

Electrophoresis on Polyacrylamide Gels

Masuhisa TSUKAMOTO

Department qfBPidemioloey．血StitutejbrTr0picalMedicine，NのgasakiUniversity

（Chief：Prof．ToshioNAEABAYASHI）

ABSTRACT: In plasma samples of mice infected with a rodent malaria parasite, NK 65strain of Plasmodium berghei, decreases in total proteins and albumin were noticed whereasincreases in enzyme activities were significant in LDH, GOT, and GPT. By means ofhorizontal electrophoresis on 5% polyacrylamide gel plates, plasma, hemolysates ofnormal or malaria-infected mice, and cell-free extracts of the parasite were compared for

various enzyme activity bands. Of several enzymes examined, the parasite extractscontain the following enzymes of which electrophoretic mobilities differ from those of

the host blood system: LDH, MDH, GDH, catalase, GOT, and esterases. With the ex-ception of the esterases, which migrate rather rapidly towards the anode, all the parasitespecific enzyme bands have relatively slow mobilities around an area between the start

line and plasma ChE position. No detectable band of parasite-specific G6PDH has beenfound except for the RBC-specific bands, suggesting that initiation of the pentosephosphate cycle in the parasite substantially depends upon the G6PDH of the host RBC.

Tosupporttheir rapidgrowth，nuClear diviSionandmultiplication・energygeneraP

tionof erythrocytic formsof malaria paraSites mustlargely depend on metabolism of

thehostblood system．Investigations onqualitative orquantitativedifferencesinenzyma－

tic featuresbetweenthehostandtheparasite areworth・While toinfer the parasite・host

Contribution No．701from theInstitute for TropicalMedicine，NagasakiUniversity

Received for publication，May24，1974

Abbreviations used are：LDH，1actate dehydrogenaSe；MDH，malate dehydrogenase；GDH，gluq

tamate dehydrogenase；SDH，SuCCinate dehydro宮enaSe；G6PDH，glucose・・6・Phosphate dehydrogen・

ase；6PGDH，6－Phosphogluconate dehydrogenase；GOT，aSPartate aminotransferase；GPT，alanine

aminotransferase；LAP，］eucineaminopeptida＄e；ChE，Cho］inesterase；RBC，erythrocyte（s）；WBC，

1eukocyte（s）；Hb，hemoglobin；Tris，tris（hydroxymethyl）aminomethane；BIS，N，Nl－methylene・

bisacrylamide；TEMED，N，N，NI，NI・tetramethylethylenediamine；NAD，nicotinamide adenine

dinucleotide；NADP，nicotinamide adenine dinucleotide phoSPhate；PMS，Phenazine methosulfate；

NBT，Nitro bluetetrazorium；EDTA，ethylenediamine tetraaceticacid（2Na salt）；BPB，bromo－

phenolblue；G・6・P，glucose・6・Phosphate；TCA，tricarboxylic acid；PBS，Phosphate・buffered

Physiologicalsaline；M．Wi，mOlecular weight

56

relationshipfroma biochemicalviewpointiInrelationtomalirialinfection，numbers of

enzymesin blood system have beeninvestigated enzymologica11y，radioisotopically，

CytOChemica王1yorelectrophoreticallybyvariousworkers（Peters，1969；1970〕．Available

informationsonthebiochemistryofmalaria，however，areSti11fragmentary，inconclusive

Orincompletetodrawadefiniteconclusion，andsometimes evenbeingcontradictory each

Otherd The reasonsforsuchaconfusion are probably due to differencesin materials

tlSed and／oT methods employed by di－fferentinvestigators．Contamination of the host

blood cells or their fragments，SuCh as platelets，WBC and RBC cytoplasm（asfood

VaCuOleswithintheparasitecytoplasm〕，intoso－Calledtlmalariaparasitefraction”mightbe

OneOfthe majorsuspicious factors，tOO．Insomecases，therefore，itisrather difficult

todefinewhetherornotreportedenzymeactiv・itiesinmalariaparasites havebeendueto

theirown enzymes．

Therecentdevelopmentofpolyacrylamidegelelectrophoresisisofgreat advantage

for distinguishing an enzyme activity band from otherisoenzymes by differencesin

molecular sizes andionic charges of proteins・Discelectrophoresishasgreatresolving

PDtentiality for theindividualsample but comparisons between separate gels are not

COnVenient・Thus∫inthepresentwork，attemPtS have beenmadetocompareelectropho－

reticmobilityofseveralsoluble enzymesofthehostbloodsystem together withthoseof

malariaparasitesonaslnglepolyacrylamide gelplateinthesamerunsothat anenzyme●

bandofparasiticorigincanbedifferentiated directly from that of host originbytheir

migrationbehaviors underanelectricfield・Atleast6enzymesof parasitic origin have

SuCCeSsfullybeendistinguishedfromthose ofhostorigin．

MATERIALS AND METnODS

MのlarialStrai粥：The malaria parasite usedis NK65strain ofPlaSmodium berghei

VINCKEandLIPSobtainedfromDr．M・Suzuki，InstituteofMedicalScience，University

OfTokyo・Inearlierstagesofthepresentwork，a Strain（thenamewasnotavai1able〕

ObtainedfromDr－Q・M．Geiman，Stanford University Schoolof Medicine，WasuSed．

Theparasitesweremaintainedbyserialbloodpassageinlaboratoryalbinomice．

SamPle　凸eeparation：The mice，uSually malesweighing18鵬23g，Wereinoculated

intraperitoneally withmalaria－infected mouse blood，and when paraSitemiahadreached

40－50％（usually　5dayslater〕blood was collected from ether・aneSthetized mice by

CardiacpunctureintoanEDTA－treatedtesttube．In some cases where enzymes to be

testedwere sensitivetothemetalchelatingagentoronlysmallamountsoffreshsamples＝

Wererequlred，tailbloodwascollectedintoaheparinizedmicro－hematocritcapillarytube■

Piasmasampleswere Separated by centrifugationat2，000x g forlOminina coldroom

Or atlO，000×g for5minby a hematocrit centrifugatoratroom temperatureiThe

packedbloodcellsweresuspendedinaboutlOvolumesofanice－COldO．85％NaCIsolution

COntaining4％dextran（M・W－200，000鵬230，000）then slowly centrifugedatlO x g for

57

10minorthetubeswerejuststoodinanicebathforlhouriThesupernatant，mainly

consisted of platelets and WBC，WaS remOVed by a pipet，and the precipitate was勒

resuspendedintoaboutlOvolumesofanice－COldO・85％NaCIsolutionbufferedwithO・02M

sodiumphosphate，PH7・4（PBS）・TheRBCsuspensionwasthenpassedthroughacolumn

packed with dry cellulose powder（Whatman Column Chromedia CF12）in order to

furtherremoveanycontaminatedWBCfromthesample（FultonandGrant，1956；RiChard

andWilliams，1973）。TheresultanteluatecontainedpracticallynoWBCwhereasremoval

oflymphocyteswasstillincomplete・TheRBCfractionwassedimentedatl，500x gfor

lOmin，andthepackedRBCfractionwaslysedbysuspendingintolO volumesof＄aline

containingO．5％saponinat30。Cthenfollowedbycentrifugationatl，500x gforlOmin

atroomtemperature．subsequently，theparasitesfreedfromthehostRBCwerewashed

4timeswiththeice－COldPBSbycentrifugationsatl，500x gforeachlOmin・Atthis

stageoftheprocedurestheparasitepreparationstillpossessedtheinfectiousability・The

finalpacked parasite fraction was then storedin a deepfreezerat－700Cuntilused・

Cell・free extracts of the parasites were obtained by atleast2cyclesoffreezing・and・

thawingfo1lowedbyhomogenization with a Teflon・glass homogenizerinanicebathand

centrifugedat20，000×gfor30minatOOC・Finallytheextracts were COnCentrated to

anoriginalvolume of packed parasites by a disposable membrane filter kit，Minicon

（AmiconCorp．〕．HemolysatesfromnormalorinfectedRBCwereobtainedbysuspending

thepackedRBCinto5－10volumesofglassdistilledwaterfollowedbycentrifugation・

QuantitaiionqFPlaSma丹oteinSandEnFymeS：Plasma samples freshlypreparedfrom

normalormalaria－infectedmice（5⊥6daysafterinoculation）werecomparedforamounts

ofproteinsand activities of some enzymes by a commercialclinicalexamination kit，

UNIKITRapidBloodAnalyzer System（ChugaiPharmaceuticalCoi・Ltdi）・Theitems

testedwereasfollows：tOtalprotein，albumin，Cholesterol，LDH，GOT，GPT・andLAP・

ElectrqphoreSiS：5％polyacrylamide gelplates，0・9mm thickness，Were prepared by

mixingthefollowingstock＄01utionsin2：1：1volumeratiointhi＄Order（modificationof

the method of Ogita，1965）：

AiAcrylamide，mOnOmer　　19・O gBIS l．O g

Distilled water to200　ml

B．Tris　　　　　　　　　　　　9．12　g

lN HCl　　　　　　　　　12．O ml

TEMED l．O ml

DiStilled water tolOO ml

C．Ammonium persulfate　　120　mg

Distilled water lOO ml

ThestocksolutionsAandB canbe stored withoutthelossof polymerizing capabilityin

darkbottle＄inaIefrigeratorforatleastonemonth・ForthesolutionC，however・itis

desirabletoprepare fresh every week・Todetect catalase一perOXidase bands，meaSured

58

amountsofstarchwereaddedintothegelmixture before polymerization．An electrode

bufferusedforeithercathodeoranode was a O．3M boric acid州0．05N NaOH solution，

PH8・3・HorizontalelectrophoresiSwaSCarriedoutinarefrigeratorataconstantgradient

Ofvoltage：10V／cm for oxidoreductases and15V／cm for proteins orother enzym世

Adistance between filter・PaPer Wicks at both electrodes was routinely7．Ocm，anda

distance between sample ditches and an edge of the cathodalwick wasl．Ocm．A

diagramoftheSetuPforthe gelplate electrophoresisiS givenin Figurel．A sma11

dropletofaO・05％BPBsolutionwasutilized as a visible marker for the discontinu。uS

bufferfront－　Whenthefrontmarkerhadmoveda5cmdistancefromtheoriginalStart

王ine towards the anode，the electrophoretic run was terminated・Disc electrophoresis

described by Davis（1964）and modified by Dietz eial．（1970）for separating LDH

isoenzymeSWerealsoperformed．

Glass pla上e・・・．．

†tparailmI†日b（∋e

Polya肝ylamide

gelplate

C由l叩血弧e．ノ

sheet

・Ditch for sample

application

巨＝：ニニ

Figil・　Diagram of set up u＄edforhorizontalelectrophoresis．

DeteciionqfProteinsorEnEymeAciivityBandS：Protein bands were stained by O．5％

AmidoBlacklOBinamiⅩtureOfm已thanoldWaterdaCeticacid（5：4：1），and destained

With7％acetic acid at50q600C・Incubating mixture solutions for staining enzyme

activitybandswerepreparedin dark just before theterminationoftherun，andunless

Otherwise stated gels wereincubated at370C・Thedarknessduringthepreparationof

Staimingsolutionsandtheincubationperiodisquiteimportant especially for thedetection

Ofdehydrogenases and esterases・Freshly preparedincubation mixtures should bepre－

Warmed just priorto theincubationinawater bath at370CiThis procedureisquite

effectiveespeciallyfordehydrogenases to galn moreintensified activity bands because■

usual1ythebufferstock solutionsarestoredinarefrigerator．Inthe case of eSterases

OrCatalaseNPerOXidase，a COldbuffersolutionisratherpreferabletouseasanincubation

mediumwithoutwarmlng・Afterdetectingtheactivity bands or protein bands，thegel

WasSandwichedbetweentwocellophanesheet＄Whichwereimmersedinadvancein O．5柵

1％glycerol，attaChed to a glass plate，thenallowedtoformadriedfilminair。Care

mustbe takento avoidleavlnganyair bubblesbetweenthe shee世■

Recipes usedforvisuali2ing enLyme aCtivitytands arelistedtelow‥

59

Lactate dehydrogenase O－1MPhosphate buffer，PH7．4　　　　50ml

（LDH，ECl．1．1．27〕　　　　　　　60％SodiumI）L－1actate　　　　　　　　　3mlNAD　　　　　　　　　　　　　　　　　　　20mgNBT　　　　　　　　　　　　　　　　　　15mg

PMS lmg

KCN　　　　　　　　　　　　　　　　　　15mg

Malatedehydrogenase Same as LDIl．butsubstitute3仰mg of

（MDH，ECl．1．1．37）　　　　　　　sodium DL・malate forlactate．

Glucose・6－phosphatedehydrogenase O．1MTrisqHClbuffer，pH8．5　　　　　50ml

（G6PDH，EClil．1．49）　　　　　SodiumG・6・P・3H20　　　　　　　　　　50mgNADP　　　　　　　　　　　　　　　　　　20mg

NBT　　　　　　　　　　　　　　　　　　15mgPMS lmg

EDTA　　　　　　　　　　　　　　　　　　　20mg

MgC12・6H20　　　　　　　　　　　　　50mg

Fresh blood samples fortifiedwith NAI）P and2・merCaptOethanol（0．1mg and

lpIperml，re＄PeCtively）wereused．

Glutamatedehydrogenase O．1MTris，Citratebuffer，pH8，5　　　　50ml

（GDH，ECli4．1．4）　　　　　　　Sodium LLglutamate lOOmgNADP　　　　　　　　　　　　　　　　　　　30mg

NBT　　　　　　　　　　　　　　　　　　15mg

PMS lmg

CatalaSe（ECl．11．1．6〕＆　　　　　　A：2％KIin2％acetic acid

Peroxidase（ECl．11．1．7）　　　　　B：0．03％H202

Weighanappropriate amountofsolublestarch（finalconcentration，0．5％〕；

dissolveinto smallamounts of waterin abeaker；heatit shortly untilthe

solution becomes transparentandthe volume of wateris reduced as smal1

asnegligible；thenprepare agelmixtureinthisbeakerasusual．Immediately

following electrophoresis，immerse gelsintd anice・COldsolutionoftheO・3M

boratebuffery pH6．5，for40min．Then，keepgelsinthesolutionA forl

min；Washwith coldwater3times；andimmersegelsintothesolutionBuntil

white（catalase〕anddarkblue（peroxidase）band＄apPearagainst ablue・Violet

background．Since the colorintensity changes rapidly，Photo pictures for

record should be taken as soon as possible when the colorintensity has

reached an optimum．

Aspartate aminotransferase A：0．1Mphosphatebuffer，PH7．4　　50ml（GOT，EC2．6．1．1）　　　　　　　　　　L－Aspartate　　　　　　　　　　　200mg

2・0ⅩOglutarate　　　　　　　　　　　20mg

PyridoxalphosPhate　　　　　　　　20mg

B：0．1M PhoSphate buffer，pH7．4　10mlFast blue B（or Fastviolet B）　　50mg

60

‡ncubategelsinthemixtureAat37OCforlOmin，then add the SOlution B

andfurtherincubate for30N60min at37OCin dark．When dark blueor

Vioietbandsappear，Wash七hegelwithwaterseveraltimestoremove excess

brownish backgroundstain．

Esterases（EC3・1・1・－〕　　　　A：ahNaphthylacetate　　　　　　50mgAcetone　　　　　　　　　　　　　　3ml

B：0・1MPhosphate buffer，PH6．8　　40ml

C：0．1MPhosphatebuffer，pH6－8　20ml

FastblueB（OrFastvioletB）　30mg

Place the substrate（A〕in an acetone－PrOOf reaction vesselin dark and

dissolveitwithacetonefollowedbythecoldphosphatebuffer（B〕；immersea

gelplateimmediatelyintothisemulsionatroomtemperature；2－3minlater，

addthesolutionCandkeepdark．Occasionalgentle aggitations of thecon・

tainerwereeffectivetopreventtheadhesionトOf the gelonto the bottomof

l

thecontainer・Usuallywithin5qlOmin，darkbrown－Violetbands appear at

room temperature－　Whenthe colorintensity has reached the mostdesirable

One，Stillcontinue theincubation for3N5min more；then wash the gel

rapidly with water2d3times and fixitin7％aceticacid．Someexcess

dark blCkground may be reduced during the fixation・Exposure tolight

Shouldbeminimiてed duringalltheprocedures，Otherwiseit may cause the

daTker backgroundi

RESULTS

Plasma Protein and Fnzyme Levels

Resultsofquantitativeanalysisonplasmaproteinsand some enzyme activities are

SummarizedinTablel・ExceptformeasurementofLDHlevels，undiluted plasma sam・

pleswere used・Itisofinteresttonote thatthe plasma LDHlevelseveninnormalmice

areextremely higher than a normalrange of LDHlevelsinhuman plasma．Thusin

CaSeSOf mouse plasma LDH，Plasmasamples were diluted　5・OrlO－fold with the PBS．

Decreasesinto七alproteinand albuminlevelsinmalaria－infected mouseplasma（parasiteq

miaat7thday，60M75％〕wereobservedinaccordance with resultsreportedbyprevious

investigators（Sadun eial．，1965．1966；TellaandMaegraith，1965；Sizaretet al．，1971〕，

althoughthe formerwaSnOtSignificantatthe5％1evel．Ontheotherhand，Statistically

highly signifficantincreasesin enzyme activities of LD世　GOT and GPT were shownin

plasma of malari計infected mice．

Electrophoretic Patterns

Inearlier＄tageS Of七hisinvestigation，disc electrophoresis was mainly employed

and resultedin resolution of sharp protein bands within each gel．However，inlater

61

Tablel．　Quantitative comparison of plasmabiochemicalvalues

between normaland malaria・infected mice

It。m Mice N窓ごf　95％豊呂よn孟m．ts unit

T。ta】pr。t。in Normal　　　18　　　　　5．21土0．33　　　　　g／100■ml

lnfected　　　　　16　　　　　　　4．73土0．27　　　　　　　　　〝

Albumin Normal　　　　18　　　　　　　3．31土0・13　　　　　　　　〝

Infected　　　　　16　　　　　　　2．97土0．13＊　　　　　　　　〝

ch。1ester。1　　　N。rmal ll lOO．4土12．3　　　　　mg／100ml

Infected　　　　　12　　　　　　108．7土15．3　　　　　　　　　〝

LDH Norma】　　　　　21　　　　　　2935土　282　　　　　Wrob］ewski

Tnfected　　　　　16　　　　　　13050土1624＊＊　　　　　　　〝

GOT NorlTlal　　　　14　　　　　　　93．1土10．4　　　　　　Karmen

Infected　　　　　14　　　　　　257．4土35．0＊＊　　　　　　　　〝

GPT Normal　　　　　15　　　　　　　38．2土　7・1　　　　　　　　〝

Infected　　　　　15　　　　　　120．5土37．2＊＊　　　　　　　　〝

LAP Normal　　　　12　　　　　　103．8土13．4　　　　　Goldberg－

Tnf，．e．十，1if l只　　　　　　　　127n　＋13．畠　　　　　　　　　Ruenburg

＊〕Significantly different from the normalcontrolat the5％1eve】・

＊＊〕Significantly different from the norma］controlat thel％1evel・

horizontalelectrophoresis on gelplateswas mostlycarried outto compare exactmobilities

of similar bands to each other．Electrophoretic patterns of proteinsin the host and

parasitesamples on5％polyacrylamide gelplates are schematicallyillustrated together

Withthose of enzyme activity bandsin Figure2，Where position ofthe Hbis shown as a

reference protein so that relative mobility of each band can be easily compared．

Proteins

Mouse plasma：Separation of plasma proteins by disc electrophoresis was effective

and atleast16bands were detected．　Whereas there were considerableindividualvaria－

tionsinprotei・nPatternS（since noinbredmouseline was used〕，nOCOnSistent qualitative

difference was observed between normaland malaria－infected mice．Resolution of plasma

proteins by horizontalelectrophoresis on gelplate wasless sensitive but stil110－12bands

COuld be recognized．Allthe mouse plasma proteins were notimmunologicallyidentified，

but onthe basis of analogy to human plasma proteins，SeVeralcharacteristic proteinsin

the mouse plasma could be recognized to be prealbumin，albumin，Hb，tranSferrin，alpha－

2macroglobulin，and so on．

Pibergheiextracts＝　By disc electrophoresis，atleastllproteinibands were

detectedin accordance with results reported by severalworkers（Chavin，1966；Corradetti

eial．，1971；Desowitz et al．，1966；Spira and Zuckerman，1966）．Horizontalelectro－

phoresis also resultedin the separation of8－9bands．The major one migrated withthe

buffer front，and other minor bands were distributed from the post－albumin areato the

OriginallineiSince noimmunologicaltest has been performed，itis very difficultto

62

reveaiwhetherotherm克orhandsfoundintheparasitee封ractfractionare realy parasi・

ticoriginorjuStduetohost RBCcomponents．

LDH

Normaimouseplasmacontainsalltheknown5isoenzymes of LDH，amOng Which

LDHq5isthe ma10rOne・Extractsof malaria parasites possessedanLDH band di＄tinct

from the host LDHiSOenZymeS，i．e．，the parasite LDH band hada slower electror

phoreticmobilitythananyknown LDH bands of the hostincludingthatofspermatozoa

（A11en，1961；Goldberg，1965）．The mobility of LI）H band foundin mouse RBCis

Simiiartobutslightlyslowerthanthatofthe plasma LDHp5．Inaddition to usualLDH

bands・Plasmasamples obtaimed from heavilyinfected miceoftencontainedtheparasite

LDHandRBC・LDHwhichappeared to beasif anintense tailing of theplasma LDH・5

ban乱　The significantincrease of LDH activityin plasma of malaria・infectedmiceis

accountable for the releaseof the enzymeinto blood stream bothfromtheinfected host

RBCandfromtheparasites．The parasite extracts also often possessed not only the

PaTaSite－SPeCificLDHbutalsothehostLDH・5band even after thorough washingofthe

parasites freedfrom thehost cells・Thusthis maynotbe atedmicalcontaminantofthe

host RBC component but may be released from ttfood vacuolesU within　the parasite

CytOPiasm・Mobilityofthemalaria parasite－SPeCificLDHbandcorresponded to an area

aroundtheplasma ChE band and to one of the protein bands detectedintheparaSite

extracts・Discelectrophoresisof amiⅩture Of host and parasite samples by the modified

technique ofDietz et al．（1970〕thus effectivelydemonstrated allthe　6separated LDH

bandsim a single gel．Phisphumvidhiand Langer（1969〕，uSing NYU・2strain ofP．

berghei，repOrted the presence of a distinct LDH bandinthe paraSite extractsiThe

illu＄仕ated curve for LDH of the mouse RBC bya scanning densitometer，however，is

COmPletely different from resultsofthe presentwork．

MDH

王nnormalmouseplaSma，usual1yonlyone majorMDHbandS Whichmigratedfaster

than the transferrin band butslowerthan the Hb，and a minor bandnear the buffer front

COuld be detectedi Hemolysates of the host RBC showed one major band of which

mobilitycoTreSpOndedtothe transferrin，In malaria－infected mouse plasma，the major

MDH band shifted to the transferrin area．Under the routine alkaline buffer condition

employed．the parasite extracts possessed anadditionalmajor MDH band ofwhich mobilu

ity was correspondingtothat of the malarialLDH band．By using acidic buffer condi－

tionsB however，theparasite LDH and MDH bands could successfullybeSeparated each

Other：for example，0．03MTris－maleate gelbuffer，PH5．4，Or O．05M：Na2HPO4－Citrate

gelbuffer，pH3．8，andO．1MTris－maleate electrode buffer，pH6．5；Wherethe parasite

LDH migrated towards the anodalside andthe MDH bands towards the cathodalside．In

theiatter cases9　the major MDH band　畠eParatedinto atleast　2distinct bands．More

ex七ensive Studies should be performed on the malarialMDHin future．　Under the

63

PROTEIN

1∴D H

M D H

G6p D H

G D H

CATALASE＆

pEROXIDASE

G O T

T

ESTERASE

plasma（No：rm叫

p．b肝油eie疲七ract

plasma（N肝m叫

Plasma（Infected）

P．b朗唱h扇既tra¢t

Hemolys慮e（Nom叫

Plasmai脚Ormal）

PlaSma（Infected）

P．be王■如ei既traCt

HemolyS如e脚Orm叫

plasm乱（Norm叫

P。bergheiextract

Hemolysate（Normal）

pl乱Sm乱（N肝mal）

P。bergheiextract

plasm乱（Normal）

Pibergheiextract

Hemolysate（Normal）

plaSm乱（Normal）

plasma（Iaeeted）

P。bergheiextract

H畠molysa七e脚om叫

Plasma（Norm叫

P．bergheiextract

Figi2．　Comparison of electrophoretic patterns for protein and enzyme bandsin maSTTudium

be唱hei，nOrmaland malaria－infected」mOuSe Plasma（0：origin，F：buffer front〕．

64

experimentalcondition＄fortifiedwithNADP and MnC12，nO ttmalic enzyme”band was

detected bothin mouse plasmaandinthe parasite extracts．

G6PDH

Usually3G6PDHisoenzyme bands could be detectedin plasma samplesobtained

from uninfected mice：One at the transferrin position，Other just at the Hb band，and

anothor r）ear the buffer front－　Malaria－infected mice usually showed plasma G6PDH

Pattern Where the　2nd band waS mOreintensein color after staining for the enzyme

activity；thismightbe duetohemolysis bythe heavyinfection becausethisintense band

COrreSpOnded to that foundin RBC hemolysates．Cell・free extracts of the parasites

possessedlower G6PDH activitythanthatof plasma，andonlyone activity bandhad been

detected ofwhichpositioncorrespondedtothe host RBC G6PDH bandat just below the

Hb band．

GDH

At王east3GDHisoen叩meSCOuld bedetectedinmouse plasma，Whereasnoactivity

band could be foundin hemolysates of normalmouse RBCi The soluble extracts of

malariaparasitespossessed atleastone GDH band，Whichmigratedveryslowly（slightly

lower than plasma ChE）and no corresponding band was showninthe host blood materials，

indicatingthatthis GDHbandwasspecifictothe malaria parasite．SeveralGDH，S Which

requiredifferentcoenzymes are knownin animals and plants・Inthis malaria species，

NÅDP wasaneffective coenzymewhereas NAD wasnot；Onlyveryweak band couldbe

detected with NAD．Thus the parasite・SPeCific GDH seemstobeanNADP・dependent

GDH：ECl．4．1．4．

Catalase＆Peroxidase

Therewere noremarkable qualitative differencesinisoenzyme patternsofplasma

Or RBCbetweennormalmice and malaria－infected ones，though malariaHparaSitized Rt8C

Showed ratherstrong catalase activity．Cell－free extractS Of the malaria parasite hada

Singlecatalasebandofwhichmobilitywasslightly faster thanthatofRBChemolysates，

andno corresponding catalaseband was detectedinthehostplasmasamples．No specific

PerOXidase bandotherthanthe Hb was foundininfected mouse bloodor malaria parasite

extracts。

GOT

NormalmouSe plaSma showedone major enzyme activity band at the transferrin

areaandaloweractivitybandclose tothe origin．NormalRBCposse去sed asingle major

GOT band which migrated rather　日lowly thanthe major band foundin plasma．Plasma

Samples obtainedfrom malaria－infected mice showed a rather singleintense band which

COVered GOT band areas of bothplasma and RBC．Sometimes the slowest band near the

Originshowed heavycolorintensityiExtractsof thehostcell－free parasitesdemonstrated

anobviouslyco］ored bandclosetoa transparentband whichlocatediust adjacent to the

65

anodalsideoftheorigin．Anadditionalbandwhichslightlymigratedtowards thecathode

was also observed．Thus plasma samples taken from mice with heavy parasitemia

Showedahigher GOT activityof both parasitic andhost origins．

Esterases

There aremanyvariations of esteraseisoenzymesinactivityorinnumberof bands

among mice，gelconcentration，and substrate used（Hunter andStrachan，1961〕・When

a－naphthylacetateisused as a substrate，7－9bands of so－Called non－SPeCificesterases

canbe distinguishedinnormalmouseplasma．The major esterasebandsare analbumin・

associatedarylesterase and aplasmaChEiBetween these2major bands．usually3－5

esterase bands can be observed．

CellNfree extracts of parasites showed a single major esterase band，Of which

positioncorresponded to a post・albumin area where no major esterase bands couldbe

observedinnormalmouse plasma，WBC or RBC．Tlms thi＄　eSteraSe SeemS tO be a

parasiteqspecific enzyme．

GENE蛹AL CoNSIDERATION

Itis wellknown thatthehost RBC」is richin enzymes of both glycolysis andpen・

tose phosphate cycle－　The host cell－free P・bergheialso can metabolize14C－1abeled

glucose predominantlytolactate（Bowmanet al－，1961；Bryant et al．，1964〕，indicating

the possession ofa completesetof the enzymesin glycolytic pathwayin this malarial

species．Electrophoretically，uPtOthe date，atleast2enzymes of glycolysis havebeen

demonstrated to occurin Piberghei：glucose phosphateisomerase，EC5．3．1．9（Carter－

1970；Wa11iker et al．，1971〕and LDH（Sherman，19e62；Nagarajan，1968；Phisphumvidhi

and Langer，1969〕．Results of the pre日ent WOrk also confirm the presence of the

parasite－SpeCific LDHi

Bryant et ali（1964〕haveshown that radioactivity of14C－glucoseis alsoincorporated，

in1esser extent，into aspartate，glutamate and alaninein free parasites，SuggeSting the

presence of a transamination system whichis connected to the glycolysISiTheresult●

reported here evidently demonstratesthe possession of GOT of parasiticorigin．Electro・

Phoretic demonstration of GPTin the extracts of P．berghei，however，has not been

SuCCeSSfulyet．

Forthese years therehave been some confusions on whether or not malaria para－

Sites possess the Krebs，TCA cyde．butitis now recognized that erythrocytic forms of

mammalianmalariaparasites cannot metabolize pyruvateinto citratevia acetyl－CoA and

lack severalmembers of the TCA cycle whereas avian malarial　species seem to possess

the TCA cycle（Bryant and Voller，1961；Nagarajan．1968；Peters，1969，1970〕．The

PreSenCe Of only2　enzymes of the TCA cycle has been reportedin P．berghei：SDH

（Nagarajan，1968；Howells eial．，1970）and MDH（Sherman，1966，Nagarajan，1968）．

How to connectthe glycolysis to the TCA cycle or to the transaminationin this malaria

parasite metabolismis stillwrappedin mystery，although　Nagarajan（1968，ⅠⅠⅠ）have

66

discuSsed on severalpossible pathways・Atleast under the experimentalconditions

empIoyedp nodetectableNADPNmalic enzyme pathway has been foundin bothparasite

extractSandthehostbloodcomponentsinaccordance with results obtainedbyNagarajan

（1968，I）o Thusspecuia七ionsbyother plausible pathways，eSPeCially CO2fixationfrom

PhosphoenoIpyruvate to oxaloacetate or from pyruvate to oxaloacetate，aWait to be

demonstrated directly．

Another example of confusior］S On the glucose metabolismis whether ornotP．

bergheipoBSeS＄eSitSOWnG6PDH which acts asa keyenzymetothepentose phosphate

」CyC呈e・Sherman（1965）has reported that when parasitemia beyond78％the G6PDH

deficiencywasobservedin P・berghei，infected mouse蛹BC・Working on free parasite

ex七rac七S Of this species，Langer et al．（1967）have demonstrated the existance of the

P昆raSiteNSPeeific G6PDHand6PGDH which d主fferin electrophoretic mobilityfromthose

OfthehostRBC血　Onthecontrary，Theakston and Fletcher（1971〕havereported，uSing

theNstrainofP・bergheiandelectroncytochemicaltechniques，thatnoG6PDHactivity

WaSObserved within the parasite cytoplasm except for foodvacuoles・Morerecently，

accordingtoTheakston andFletcher（1973〕，their unpublished data obtained by electro－

Phoresis omCe1logelhaveshownthe presence of onlythe erythrocyticG6PDHbandinthe

extracts of the malaria parasite used．Result of the present work withthe NK65strain

OfPibergheiisalsoinaccordance with thisobservation・Oneoftheplausibleexplana・

tions to understand such a discrepancy may be the strain－SPeCific differences（i．e．，

geneticvariations）within a species or even within a subspecies．Carter（1970〕has

beautifuliydemonstratedanexampleofsuchelectrophoreticvariationsofseveralenzymes

invarious strainsof P．berghei（s・1・），andHowells et al．（1970）have shown a signifi・

Cant differencein the presence of SDH activity betweenchloroquinesensitive N strain

and resistant RC and NSlinesi Furthermore，enZyme aCtivity may or may not be

detectable duringthe parasite’slife cycle（Howells，1970）．Unfortunately，Lang・er et ali

（1967）didnotmentionthename of the parasite strain used，and Carter（1970〕didnot

report any about G6PDHisoenzymes－Therefore，itis stilluncertaln whether the G6pI）H

bandfoundinthe parasite extractsistrulyorlglnatedfrom the malaria parasiteorisJuSti　　●

derived from anir唱eSted host RBC materialwithinthe parasite ttfoodvacuoles”．

Presenceof the NADP－dependent GDHinthis malaria parasite has been reported

enzymologicaiiyby Langeret al．（1970〕whiletheydidnotmention aboutits electropho・

reticcharacterist豆cs．Results of the present work thus electrophoretica11y confirm the

evidence that the paras主tic GDHiS distinct from the host enzyme．Itis genera11y

recognized by some workers that GOT and GPTlevelsin blood Serum Or plasma

Significantlyincreasein malaria・infected rodents（Sadun et al．，1965，1966；Wellde et al．，

1966〕．HoweverS there was no experimentalproof to distinguish whether suchincreases

inGOTlevelbeingderived from disintegrated RBC or fromthe parasite．The present

WOrk reported here again eviderltly demonstrates that both the host enzyme and the

ParaSitic one can be attributed to theincreased enzymelevelin plasma－Itis now

interestingto know thatthe reaction betweenglutamate and　2－0ⅩOglutarateis catalyzed

67

byboththetransaminaseandthedehydrogenase，SuggeSting animportant role ofthese

compoundsinthemetabolismof P・berghei・

In conclusion，electrophoretic comparison seems to be one of themost effective

resorts．atpresent，tOdiagnoseparasite－SPeCificenzymesfromthoseofhostorigin・

AcE．NOWLEDGMENTS

Iwouldliketoexpress my sincere thanks to Dr・Zen－ichiOgita・Departmentof

Genetics，Osaka University，forhisvaluable suggestions and technicaladvicesonthe

electrophoresis；to Prof．Quentin M．Geiman，Departmentrof Preventive Medicine，

StanfordUniversitySchoolofMedicine，fora rodent malarialstrainwhichhasbeenused

in earlier partsofthepresentwork；andtoDr・Mamoru Suzuki・Institute of Medical

Science，Universityof Tokyo，fortheNK65strainoftheparasite・IamgratefultoProf．

Toshio Nakabayashiforhiscontinuousinterestandencouragementtothiswork；to Miss

ReikoIkedaandMiss Fumiyo Tsuchiefortheircooperative■as＄istance as undergraduate

researchers fromtheFaculty of Pharmacology，NagasakiUniversity・Thelaboratory

assistance of MissAkikoIShidain preparing experimenta］materialsi＄e＄PeCia】1y appre・

ciated．Iam alsoindebtedto，Dr．Fumiya Murakamiand Dr．Yasuo Nakajima fortheir

advice on quantitative measurements of plaSma LDHleve］s・

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ポリアクリルアミドゲル電気泳動によるネズミマラリア原虫特異酵素の検出

塚本増久(長崎大学熱帯医学研究所疫学部門〕

マラリア原虫を感染させたマウスでは正常マウスに較べ血漿の総蛋白質量,アルブミン量などが低下

する傾向が見られたが,一方乳酸脱水素酵素やアミノ転移酵素などの活性は著しく上昇した.その原

因が感染に伴なう宿主血液系における変化によるものであるのか,あるいは寄主自身の産生する酵素

によるものであるのかはこれらの測定値だけからは区別することができない.そこでマラリアの生化

学とくに寄主宿主関係についての基礎的知見を得るためにポリアクリルアミドゲルによる電気泳動を

行ない,正常マウス,マラリア感染マウスの血漿,赤血球,分離マラリア原虫などの可溶性蛋白質や

酵素の泳動像について比較検討を試みた.その結果,乳酸脱水素酵素,リソゴ酸脱水素酵素,グルタ

ミン酸脱水素酵素,カタラーゼ,グルタミン酸オキザロ酢酸アミノ転移酵素,エステラーゼなどにお

いてマラリア原虫に特有と考えられる活性泳動帯が認められた.また感染マウス血漿で著しい活性上

昇が見られた乳酸脱水素酵素やグルタミン酸オキザロ酢酸アミノ転移酵素では,赤血球の崩壊に伴な

う宿主側の酵素とマラリア原虫自身に由来する酵素の双方がその原因となっていることが証明された.

マラリア原虫の解糖系に隣接する代謝系についての報告,とくにブドウ糖6燐酸脱水素酵素の有無に

ついては,従来多くの混乱が見られたが,今回用いられた系統のマラリア原虫抽出液中には宿主赤血

球の酵素と一致する活性帯しか検出されなかった.このように同一ゲル上での電気泳動による比較は

寄主宿主間の酵素系の異同を識別する上で極めて有力な手段であることが示された.

熱帯医学　第16巻　第2号　59-69昆1974年6月