Vol.8,No.2(1957) 221
STUDIES ON/PYRUVATE AND CARBOHYDRATE
METABOuSMS BY LACTIC ACID BACTERIA
ⅠⅤ.Analysis of Dismutation by L.fermentum
KeiYAMANAXA(LaboIatOryOfTechnicalMicrobiology)
(ReceivedJanuaIy8,1957)
TheapplicabilitytobacteriaoftheEMBDEN−MEYERHOF(EMl)schemeofcarbohydratedi早Simi1ation has beeninvestigated by many woIkeIS.,Since WARBURG(1)in1931demonstIated the occurrence of so−
Ca11ed“ゐi5‘henf‘r7rCnt,”namely glucose・6・Phosphate(G−6−P)dehydrogenasein yeast,itsimportancein
aerdbic dissimjlation of carbohydrate has beenincIeaSed greatly‖ Asit was elucidatedthat a number・Of bacteriahadtheabilitytooxidizeG−6−P・SOthenewschemeRforhexoseoxidationwere 且MscIleme
For example,glucose fermentation bylactic acid bacteria occurs by atleast two di茸erent routes Homolactic species,the streptococciand homolactobaci11i,COnVert hexose tolactate by the EM.pathwaY Incontrast,fdrmentationofhexosebyheterolacticoIganisms(LeuconoSiocandLaciobacilli)ischaracteTizeすby
theequimolaIfoImationoflactate,ethanolandcaIbondioxideqUNSALUSandG‡甲S(1952)(ヱ,予〉obtainedlthe
result ofisotopic analysis with C141abe11ed gh!COSe by Leuc,n2eSenterOides,by Z.eucde.Airanicum,Lfern;enii,
andエbentoateti(uSく小… Fe‡mentation ofglucose・・lHClよand glucose・3,4−C14reVealed thatcarbonユ.ofglucose
appears as carbon dioxide,and caIbon2as methylgIOup Of ethanol.Fuzthermore,it was free from al・
dolase(盲)butcontairledG−6−Pdehydrogenase(O・7)and6−phosphogluconate(6−PG)dehydrogenase(8)in Leuc
me・SenterOiders,From these results,itis proposed that glucose may be decomposed via so:calledanae’zdbic hexosc monophosphate pathway,Although the demonstration of enzymes and unknowninteImediates be・
longio such oxidative scheme by het畠主olactic acid bacteIia aIe aS yetincomplete
A new dismutation of gluconate(glucosc)and pyruvate by Lh fernlentum has been presentedin a
previous paper(9).In suchexpeIimcnts,metabo王ic gas changewas measuredwithaWARBURGmanOmeter,
While analyses of aliquoteⅥere Carriedoutinothermacroexperiments thusitwasdifEculttocomparethese
data fo工 di#erent manner
In this paper,gaSeOuS metabolism measu;ed with a WARBURG manOmeter andanalyzed metabolites inthis vesselof WARBURGin orde工tOde丘nefurthe:eXaCtly,and seveIalconditionsof colorimetricdeterm・
inations for pyruvate,1actate,diacetyl(DAC)and acetylmethylcarbinol(AMC)were descIibed.
二眼a乞erial$and恥乞et王10ds
伽血re L”feYmenium,ahetero・・iermente:,hasbeenusedthroughouttheexperimentslTheorgan子$P,
grownin3L of malt extract(4∼6%as sugar content)with4%of CaCO3.for40 hours at370C,WaS harvested by centrifuge,After washing the bacteria cells by distilled water for twice,thewetcellpe11ets
WeIe dIied over CaC12undeIreduced pressure
REanome七ric procedtlreS Manometric experiments were carried out by・COrlVentional− WARBURG
technique,aS describedin a previo11S paper(10)・0Ⅹygen uPtake and CO2eVOlution weremeasuredat3フOC
acco工dirlg tO the procedures descIibedbyUMBREIT,BuRRISandSTAUFFER(11).The reactionmiⅩtuIe WaS
incubated for600r9C)min,at Which period reaction was stopped by tipping ofO2mlof20 % trichlor・ acetic acid(TCA)fIOm2zld side arm,and then the amount ofliberatedCO2WaSreadh NoCO2retention WaS Observedin endogeneous andin the case of sugar added onlyl ReactionmiⅩtureS WereremOVedand
TecIlり Bl軋Fac.AgI・1.XagawalJniv.
222
anddeproteinized bycetrifugation”Aliquots wereassayed as describedbelowトRecoveIies ofmetabolites fromvesselofWARBURG,Were90and96.9∼98.3%withoutandwith washing three times by5%TCA,
respectively
Determinations For the deteImination of pyIuVate,the method of FRIEDEMANN and HAUGEN(12) wasused.Standard ofpymvatewasestimatedbythemethodofCLIFT−CooK(13)… HydIaZOneOfpyruvate formedwasextracted by8mlofxyleneinstead ofbytoluene,COloured with NaOH,5miofl5Nre・ placed by2mlof4N DAC and AMCwereidentified both by VoG‡S・PROSKAUER reaCtion(14)and by
iormationofnickel・dimethylglyoxime,deteIminedbythemethodasdescribedby WHITE and KRAMP‡IZ(Z5), and that of EGGLETON et alh(1(;)。The foImeImethod wasless sensitive than thelatter。Forstandard of
these methods,DAC from WakoCo.WaS disti11ed and estimated by the method of VAN NIEL(17)Distil・
1ate from one or twomiof aliquot was collectedin 負ve to ten ml,then one or two mlof this distillate WaSuSedfor determination小 AMCwasdetermined as DAC,distiユ】ed by oxidationwith additionof20mlof 20%FeCl3SOlution,amOuntS OfAMCvascalculated fromtotalamounts of DACandamounts of freeDAC
Res111tsfor recoveIy OfDACfor distillationwas sho・ Wnin Tablel.
Tablel Recovery of diacetylby distillation
lfDACfound慧㌫ど
dnt (鵬) (min) (ml) (%) 4 10 15−ユ6 500 12I)AC was used245.2FLgin each cases・
DAC waseasily recoveredby distillation for10min completely.For thesetwomethods,pyruVatedid notinterferewithin2000pg,andglucose also did nota#ectwithin2500pg,eVen When added to each DACsolutionafterdisti11ation.ForVoGES・PROSXAUER(V.P”)reaction,aliquotswereusedwithoutTCA
Or With10%metaphosphoric acid,While this reaction wasinterfeIedby TCA to form a blue colour.For the methodofEGGLETONetal.,eだectof time forcoIouring,WaSteStedunder20∼25OC,The same colo11r densities we王e Obtainedbetween 10∼20minafter alkalineα・naphthoIsolution was added.Lactate was determinedbythe method modiRed by BARKER and SuMMER・ SON(ユ8〉.In this method,Visualization was performed by Oい1mi of p・hydroxydiphenyl and0.,05miof4%CuSO4・The opticaldensi− ties obtainedまⅠOm125JLg OflactateandOnO5 mlof4%CuSO4,With vaIied amo11ntS Ofp・ hydIOXydiphenyl:0.3フ5byO.05mlof♪HDP, 0.41by O.lmland OA(15by O.15 ml,and COlouring time at300C was25 min.This time was not eぽectedat20∼40min whereby Opticaldensities obtained O0フ3 at O min. 0り434at15min,0。439at2(〕min,043フat30 mi1】,and O.{436at40min.However,irlthe presence of pyruvate,reprOducible res111ts COuld not beachievedwiththisoriginaltech・ nlque,eSpeCiallyin thecaseofsmallamount Oflactic acid withlarge amount of pyruvic acid・・Thenseveralmodi負cationsofthispro・ Table 2 DAC determination as a function of time
Time(min) 5 10 20 30 40 60
Optical density “ 0 2 ︻h︶l C︺3 00 02 フl OC3 0〇 52 72 ︵03 00 1 82 0C3 〇 鵬20 3 0 ABBy the method of EGGLETON eial
A,S530withIto electrophqtometer,DAC25.8JLg B,at530mp,withShimadzuspectrophotometer,mOdel
QB50,DAC14.O pg
Table3 The eぽect of successive coppeI・lime treat・ ments on the colour obtained with pyruVic acid samples
0√DけS70m〃・
acid (1)After (ⅠⅠ)After (IlI)After
(pg/鮎st Without one copper・tWO COpper−threecopper・
Smi) treatmentlime ljme lime
treatment tIeatment treatment
20 0.09フ 0.036
5U O.215 0..036 0023 0.018 1∞ 0..440 0.061 0.022 0.018 200 0.890 0い064 0.035 0、027 400 0い○フ4 0.038 0.032
(Ⅰ)4“フmlof standard solution(COntaining20up to4(刀 pg of pyIuVic acid)+0”3miof20%CuSO4+Ca(OH)2‖(ⅠⅠ) 3mi of supernatant sollユtion from(Ⅰ)+0・3 mi of 20% CuSO4+Ca(OH)2,Which volume make to5‖Oml,(lII)3mi Of supeL.natant SOlution fr’Om(ZI)another are same as王L
Cl.5mlof supernatant solution are taken for analysisin each case,and opiticaldensities areIead at570mp ac− COIding to the method ofBARKER and SuMMERSON.
Vol二8,No∴2(195L7) 223
Cedure were made accoIding to the technique described by HuLLIN and NoBLE(19)り As seenin Table3, COpPer・lime treatment was repeated壷or two or three times,Whereby the value of colour development due to pyruvate became smal1and constant which may be deduced fェom thefinalopticaldensity
The absorption spect川m Of the aldehyde・b−hydroxydiphenylcomplex shows a sharp maximum at570 mjL aSillustIated by the fo1lowing opticaldensities obtained from8pg oflactic acidundertheconditions described above at wavelengths between460to620mJL:460,OhO54;480,OhOフ2;500,01フフ;520,0,353;530,
0.4eO;540,0640;550,0.754;560,0,860;565,0。S95;5フ0,0.900;580,0835;590,0.678;600,0い471;610,0.302; 620,016l.The absorptjon curve Ofalkaline2,4・dinitIOphenylhydrazone of pyIuVate Shows also a sharp maximum at4E;O m〃′by the壬ollowing opticaldensitiesobtained壬Ibm2012pgofpyuvicacid at wavelengths
between3eOto580mIL:380,0165;400,0260;420,0422:430,0482;435,0,503;440,0520;450,0・528;455, 0”520;460,Oh508;4eO,0408;500h O”36フ;540,0,335;530,0235h Andthe absorption curveof DAC by the methodofEGGLETONetal.showsfollowingopticaldensitiesobtainedfIOm224pgofDACatwavelengthsbe・ tween320to600m〃:320,02∂フ;340,02e6;360,02∋5;380,○269;400,0り215;420,0192;440,0/190:460,0い236; 4eO,0367;500,○539;520,0・642;525,0・642;530,0642;535,0638:540,0・622;560,0448;5∈氾,0小ユ58;600,0.007
To deteImine colorimetric,byS480,at450mp for pyIuVate,byS530,at530mp forDAC and AMC, and by S580,at5フOmpforlactatewereusedwithelectIicphotometerandwithShimadzu spect【Ophotometer, m−odelQB50,reSpeCtively
Pentose was detected by the BIAL、’sIeaCtionintIOduced by ME・
JBAUM,aSgivenin UMBREIT,BuR− RIS and STAUFFER(11)”Nitrogen COntent Of cells was determined by the method of micro−KJEHLDAHL
ResⅦlts and D紬eussions I.DismuねtiomwitIlIn七aet
Cells
ltis evidentinFig,l.that glu− COSeandgluconate are dissimilated by intact celis grown in mait ex- tract,and the respiratory quotient values(RQ)foIglucose and glu・ conatearelh14andl89,reSpeCtive・ ly
Since the formation o土1actic acidisIeCOgmized both from glu・ COSe and gluconateandtheyieldof COz for gluconate is gIeater than that for glucose,SO the RQ Valuelor gluconateis as g工eat a− bout two til羊eSaSthatfor glucose, it is therefore suggested that the glucoseisfermentedbythisorgan・ ism via some scheme containlng gluconateoritsderivativesasinter−
Fig‖1Gaseous metabolism ofdismutation byintact ce11s ⑳−−⑳CO2eVOIved,○−00ゴCOnSumed
l・Pyruvate50FLM+glucose40pM,2”Pyruvate50pMエ gluconate40p〃,3Pyruvate50FLM:4.Glucose40jLM1
224 Tech.Bu11.,Fac.Agr.,:Kagawa Univ. mediates oxidatively. Pyruvateis
also王eadily oxidizedtoacetateinae・ robically by pyruvic oxidase of this OIganism as alIeadyshownin a pre・ vious pape工(10).As seeninTable4, smallamountsofDACandAMCfrom
pyIuVate are Obtained,identi丘ed by theformatior)OfNi・dimethylglyoxime, Table4Analysis of dismutation of pyruvate and glucose by
intact cells. 1ⅠⅠ ⅠⅤ Ⅰ (witb glucose) (浣雲3別品三豊)(霊∑芝;) Substrate PyIuVate(pM) 50”85Eぼect of 5085 Glucose(pM) 40 glucose O PyI・uVate IemOVed(〝朗) 48.6 十35..0 1356 CO2eVOlved(〝凡才) 42,2 +28・2 14小0 02COnSumed(〝朗)13.2」−11.1 2.1 9.25 01 8.25 0.1 ーーーー「− 0 and by theVoGES−PROSKAUERreaC・  ̄ J二ユ5 formed(〝〟) 0‖J5+ 0 0.13
AMC formed(JLM) 0”96+0.83 tion,afterdistillationwithandwithout
20%FeCl$
In addition,Fig”lalsoindicated the remarkable increase of carbon dioxideevolutionfromthe mixture of pyIuVate and glucose or gluconate, whereas O2COnSumption was alittle decreased.A result of this reaction byintact cellsis shownin Table 4. Lactic acid which occurein this
0 0 3.3フ 0 Lactateformed(pM)2l.0 +2l.0 0 BIAL’s test + + RいQい 3..20 6い67 1小12 1.0 C・RecoveIy鴇 122 33.O
Reactionperiod,90min,andNcontentofcel王per cup wasO小/6mg 場C−Recorveryis expressed by following equation
2×(DAC・AMC+02)+1actate
C・RecoveIy(%)=
pyruvate removed
reactionwasidentiRed by papeIChromatography,the sample were developped byascendingchromatogIaph
using BuOH:aCetic:H20(4:ユ:2)as soIvent,and visuali2:ing the spots with BPB−ethanoIsolutionFrom Table4,theincreasedamountsofCO2p工Oduction from glucose and pyruvateis 28」2pM,that of pyruvate removedis35OILM.and the formation oflactateis2l10pM,While the formation of both DAC andAMC areincIeaSed sユightly.In generaいt js unable toexpect to obtain a typicalresult byintact cells,While
glucoseaddedasasole substate is degraded byintact cells,SO de・
tails of this reaction
willbe elucidated by 山ied ceユユs. 2.DismlltatioIIWith Dl・iedCeils ThesimilaIgaSeOuS metabolismis observ− ed with dried cells as glVeninFig2,andthe e仔ect of adding variq oussugaISO点CO2prO・ duction from pyruvate and sugaI:Sislistedin Table5 As notedpIeVious− 1y:9)the additions of gttlCOSe,fructose,man− nose,maltose,andglu−
Fig‖2 Gaseous metabolisms of dismutation by dried cells −O COゴeVOIved,−▲Oi2COnSumed
A.lPyruvate50pM+glucose20lLM, 2・Pyr 50〃M+fructose20pM,
3。Pyrl5〔)FLM十galactose20JJ・M
B.l.Pyruvate5C)pM+・maltose20pM,2h Pyr”50FLM+sucrose20pM,
3・Pyr.50神灯+ユactose20/J肱
C‖ ⊥い PyIuVate50FLM+gluconale2C)JLM,2.Pyr50pM十mannOSe20pM, 3Pyr‖50ILM+Ⅹylose2C)FLM,4PyruVate5C FLM
Vol.8,No.2(195フ)
Table5 E仔ectofsugarsonCO2prOductionindismu− tation system.
225
COnate tO pyruVate rise the accelerativeevo・ 1ution of CO2,although no e鱈ectiveIeSults aエe eVer Obtained with galactose,SuCrOSe, lactose,and xylose.The analyticaldetails of these Ieactions are presented in Tables
6a−6c
In experiment with pyIuVate and gl11COSe in Table6a,the amo11ntSOf pyruvateIemOVed, CO2prOduced,andlactate foImedin the pre・ SenCe Ofglucose were386pM,33。O pプ吼and 30r18 p爪先IeSpeCtively;the amountsin the COrreSPOnding contIOlrun,in which glucose plin60min increased ratio(%)
CO2 02 CO2 02 ︵∪80∪︵つ1﹂︵〇9︵○︻b︻ノ 091つん00909∩︶ ﹁l l l l ユ l 1 0237︵U180︵D3 ∩︶335∩︶24196 11⊥ll l11⊥⊥ ﹁⊥ PyIuVate 5【】0−640 260−295 +gl11COSe 840 290 +fIuCtOSe 850 350 十mannose 910 370 +galactose 640 30〇 +Ⅹylose 700 311; 十maltose 860 255 +SuCrOSe 640 275 +1actose 56〔) 245 +・gluconate 945 28〕
Table6 Dismutation of pyruVate and various sugars by dIied cells (Reaction’period,60min,Ce11used,15mg per cups) a。With glucose,fructose,and galactose
1V (witbo扇sugar)
Ⅰ(+glucose) ⅠⅠ(+fructose) lII(+galactose)
E庁ect 45.2 0f 20 glucose Effect E償ect 45.2 0f 45.2 0f 20 frutose 20 galactose Stlbstrate Pyruvate SugaI 38.6 ・+13りユ. 35,8 十10.4 23=2 −2..3 ⅠemOVed CO2eVOlved 33・0 + 7.9
334 十e3
25・・2 ・+0.1 02COmSumed 11..3 −0.3 13”フ +2‖L ll.フ +0.ユDAC foImed Ol.20
AMC formed O”47
Lactate foImed 30.18 0.12 − 005 0い33 − 0,.11 1フ“94 +1Lフ.94 十 0.03 十0い03 十3018 0.1フ 0 0り24 ・− 020 L49 +1“49 BIAL’s test 寸十 + ± RQい 2.92 2.44 2.15 C・Recoveェy(%) ユ01 b小 With maltose,SuCrOSe,andlactose ⅠⅤ (Wit!10ut S11ga!) Ⅰ(+maltose) Ⅰ王(+SuCrOSe) ⅠⅠⅠ(+1actose)
Eぽect Eぽect 45い9 0f 45.9 0f 20 maltose 2つ sucrose SubstIate Pyruvate SugaI E仔ect 459 0f 20 1actose 395 +18.2 22‖9 +1.6 185 −2..7 Ⅰ・emOVed CO2eVOIved 34.0 +lL1 25rl +・21−2 22‖0 −0.9 02COnSumed lO.1 −0・、1
108 十06
9小フ ー0.5 0.12 − 0.C2 0,32 − 010 0 0DAC formed O.24 AMC for・med へ70 Lactate formed 33..9 + ○。.10 + 0.28 + 33..9 013 −001 0.29 − 0..13 0 0 BIAL’s test 十1− R−Q.. 336 2.25 C・RecoveIy 86.4 98。3 109 110い8
TechBull.Fac.Agr”Kagaw∂Univ 226
C一With gluconate,mannOSe;and xylose
Ⅰ(+gluconate) lI(+mannOSe) ⅠⅠⅠ(+Ⅹylose) (without S11gaI)
Substrate Effect E任ect E庁ect
ate ”9 ・9 ”9 49
glate ma。Se Ⅹse
Pyruvate 3フ.フ +109 39.1ユ. +12け3 30.2 +33 26 9 IemOVed + 4り6 22.8 十 35 9.0 358 ・+13.0 2フ4 14.1 + 56 〕2。5 CO2eVOIved 37ll +14l3 02COnSumed ll0 +20 0。25 ・十 008 0.76 十 0.11 21フ0 + 2,フ○ 007 ・− 0け10 039 − 0…26 1387 +ユ.3…87 DAC foI・med O.24
AMC formed O.81
Lactate f0Ⅰmed 19い33 + 0‖07 十 0.16 +・1.9い33 BIAL’s test l十 2.45 221 RいQ、 3.38 94い8 93 8 C−Recovery 89小4
was omitted,WeIe255pM,251FLM,andOILM・SOtheIeSpeCtiveincreasedamountsof pyruvateIemOVed, CO2prOduced,andlactateformedcausedbytheadditionofglucosewe工e13・lpM・7・9lLM・and3018〃・M・
respectively・Asis showninTables6a−6c,thereactionproceededrapidly to astable end−POint,SO the
postulatedunknownintermediateinthedismutationsystemwasrapidlydegIadedbelowthestageofacetate
andlactate undeIthe condition used.In agreement with the mac工O eXPerimentsin the previous paper(9〉,
detai1s of the dismutation weIe elucidated more exclusively壬rom the fact thattheratioofincreasesofCO2 evoIved,pyruVateIemOVed,andlactatefollmedwasnearlyl:l;2
One maythen suggestlogically thefollowingover−allsequencefor this dismutationbyLfermenlum
This sequence of reaction wouldyield thatabovemen・ tioned ratioisl:l:2。SincetheobseIVed工atiosarel.0
:06:2.3with giucose,ユ.。0:1、3:l.78withgluconate,itcan be concluded that thisdata危tthissequence。Thepze・
Glucose
Y Gluconate肌−>(6・Phosphogluconate) PyIuVate
1r
CO2+(Pentose・phosphate)/、\Lactate
〆′ \
C2(Acetate) C8(Lactate) sentdatadonotpermittodecide thissequence more
de丘nitely,however,HoRECKER andSMYRNIOIIS(2〇−22)have reported the董01lowing equations using6PG
andlactic dehydorogenases from yeast
6PG+TPN+→ ribulose−5−phosphate+TPNH+H++CO2
TPNH+H十−l−pyruVate一寸1actate
Sum(l)+(2)6−Phosphogluconate+・pyruVate→工ibulose・5・phosphate+lactatc+CO2
⊥ 2
Sincethe fermentation did not go to completion,itis not unlikely that appreciable quantitiesofinter・
mediates accumulate,SO tO eStablish the nature of theintermediate productsandultimatelytoevaluatethe
signi丘cance of the pathwayin carbohydratemetabolism by L・/’ermentum,detectionofpentoseexpectedas
anendproductofthisdismutationIeaCtion was undertakenin these aliqo11tS,SOitwasequally positivein
whichdismutationwasproceededlIncontract,in other aliquotsin whichdismutationwasneverobserved,it wasscaIeCely detectedby BIAL・sreactionEvidencehasbeenpIeSented,hoⅣeVer,thata ketopentose・Phos− phateisthesubstanceactuauy splitbyL♪enioISuSintoC3andC2units(23),SOaketopentosephosphateisp王0−
bablyexpectedastheendproduct for this dismutation system from phosphorylated gluconate,also as
comparedwiththe data of yeastenzymes by HoRECI(ER andSMYRNIOrISAsalready mentioned,this unknownintermediate willbe rapidly degraded to a stable end products,namely acetateandlactate,SOthe
fomationof ketopentose phosphate whichis expected to accumulatewi11bethe subjectof the succeeding
Voll8,Noり2(195フ) 22フ On the basis of these considerations,itis suggested that ketopentose phosphateis a product of this
dismutation system by L,fermenium,and thatits formation maybeinitialstepinthedegradationsequent色 Of6PG
Stlmmary
The new dismutationofpyruVate and gluconate by L。f■eYmenlum,a hetero・type Oflactic acid bacteIia, WaS mentionedinapreviouspaper(9)小Inthatpaper,inordertoel11Cidatethisreaction,tWOdi#erentmethods Were employed,that of the manometry for gaseous metabolism,and of tbe macro oscillationforanalyzing metabolites except gases
This paper dealed a quantitative analysis foraliquots of vessels of WARBURG,and seveIalconsider・
ations for the conditions of colorimetric determinations of DAC,AMC,andlactate,eSPeCially forlactate deteImination of sample contaminated with alarge amount of pyIuVate(Seein Table3).
The similar results for dismutation of pyruvate was obtained withglucose,fructose,mannOSe,maltose, andgluconate,SO the ratio ofincreases of pyruvate removed,CO2eVOIved,andlactateformedwasl:l:2・ In these cases,DAC anc AMC werelittleincreased.In contIaSt,the cells did not catalyze similar dis− mutationwithgalactose,SuCrOSe,lactose,and xylose‖ Moreover,by using the BIAL,s reaction,PentOSe was detected onlyin the cases whendismutations wereobserved」・ThissubstancewhichIeaCtWith orcinol might be a pentose phosphate(unpublished)
The signi丘cance of this dismutation and the formation of pentose phosphatein the fermentation of hexose by L.fermenium was discussed
AckItOWledgement
The a11thor wishes to express his gratitude to Prof.・HIKATAGIRI,Ky6to Umiversityforhis contin11ed interest and encouragementい He also wouldlike to thank ProfりTu KuROXAMIfor his helpfuladvise,and
Prof.,K…KATAKURA and Prof.S”KAWAMURA for many valuable suggestions and for use of technical equipments
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