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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
Right
NAOSITE: Nagasaki University's Academic Output SITE
http://naosite.lb.nagasaki-u.ac.jp
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