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Buuetin of the shimane UniVersity (Natural science)' No' 12, pp. 20-46
44 text-figs " December' 1962 ' CSERIES B)
COMPAFATIVE PHYSIOLOGICAL STUDIES ON THE RESPIRATION OF VARIOUS YEASTS
By Kazuyoshi NISHIGAMI
Biotogical Institute' Shimane UniVersity
INTRODUCTION
In recent years, many comparative-physiological studies on respiration of yeasts have
been carried out by numerous workers. Such projects are being carried on with the
view of introducing some physiological methods in the . study of phylogenetic relations
of the yeasts
There are two important characteristics used in the classification of yeasts. These are
morphological and physioJogical properties. The former includes the taxonomic charac-
ters of vegetative and reproductive phase of cells as follows : (1) the existenence of the
spore for ~Lation, (2) type of spore formation, (3) shape of spores, (4) mode of budding,
(5) form of vegetative cells, (6) and other factors.1-2) These define the main lines in
yeast taxonomy. However, for the differentiation into spec,ies physiological characters
can not be neglected. The physiological properties are fermentation and assimilation of
carbohydrates, assimilation of nitrate, occurrence of carotinoid pigments, splittir*g of
arbutin, production of acid, splitting of fat, vitamin requirements and other properties.3)
Therefore, it is quite important to study these physiological characters comparatively in
order to analyze the phylogenetic relations of yeasts. The presently described investiga-
tions dealt with this problem.
In comparative physiological studies of yeasts. Wickerham reported comprehensive
studies about comparison of ability of carbohydrate assimilation.4) Recently, Barnett and
hi~ coworkers have described the oxidation of polyhydric alcohol and the tricarboxylic
acid(TCA) cycle intermediates.5,6)
As the means for acquiring energy in organisms, the physiological signification of the
hexosemonophosphate (HMP) cycle has come recently to attract investigaters' attention
Then studies on the corelation between the Embden-Meyerhof-Parnas (EMP) pathway
and the HMP cycle became important for elucidating the respiratory pattern of various
organisms. The early works of Warburg and his associates established the fact that
glucose~;-phosphate could be enzymically oxidized by yeast preparations to 6-phospho-
gluconic acid in the presence of triphosphopyridine nucleotide as a coenzyme.7,8) Later,
Cori and Lipmann,9) Brodie and Lipmann,lo) Horecker and Smyruiotis.,n) Glaser and
RESPIRATION OF VARIOUS YEASTS 21 Brown,12) Kornberg,13) Lipmann,14) Dickens,15) Cohen and McNair Scottl6) and many
other workers have investigated the details of the HMP cycle.17-29) Recently, investi-
gation has been made to det,ermine which is the major pathway in glucose catabolism in
various organisms, that is, whether the EMP pathway or the HMP cycle.30-33)
On the other 'hand, it is well known that there are specific inhibitors of the EMP
pathway. The inhibition mechanism of monoiodoacetic acid has been studied by
Lundsgaar,34,35) Boysen Jensen.36) Runstr6m and Alm,37) Adler. Euler and Gunther,38)
Ankel and Szulmajester.39) Stoppani, Actis, Deferrari and Gonzalez40) and numerous other
workers. On the inhibitiion mechanism of sodium fluoride, Chaix and Fromageot,41)
Sakaguchi and Baba,42) Pickett and Clifton,43) Massart and Noortgate,44) Reiner45) and
other ~vorkers have reported in various organisms. By the recent report of Hoskin23) it
was made clear that arsenite inhibits the EMP pathway more effectively than the HMP
cycle in guinea-pig brain
The object of the studies reported here, in which nine strains of yeast with various
respiratory quotient (RQ) values of glucose respiration were used, was to show from the
point of view of comparative physiology some interrelations between the values of RQ
and the following metabolic characteristics of yeasts : (1) oxidation of glucose, gluconate,
pyruvate, ethanol and the TCA cycle intermediates ; (2) inhibition by monoiodoacetic
acid, sodium fluoride and arsenite on glucose oxidation ; and (3) cytochrome components
EXPEI~Il~:ENTAL M:ETFIODS
Materials
Nine strains of yeasts were employed through the studies. They are Candida utilis,
Mycoderma cerevisiae, Schizosaccharomyces pombe. Sacchoromyces cerevisiae, , Saccharomyces
carlsbergensis. Saccharomyces sake, Rhodotorula glutinis. Torula cardida and Pichia memb-
ranae f aciens.
Candida utilis, Mycoderma cerevisiae and Schizosaccharomyces pombe were received from
the Institute for Fermentation. Osaka. Saccharomyces cerevisiae was isolated from pressed
baker's yeast produced by Nippon Beet Sugar Co. Saccharomyces carlsbergensis was kindly
presented by Prof. T. Sasaki of the Faculty of Agriculture of Hokkaido University
Rhodotorula glutinis and Torula candida were given by Dr. M. Yoneyama of Hiroshime_
University. Saccharomyces sake was obtained by isolation from "moromi" of sake. Pichia
membranaefaciens was kindly furnished by Prof. I. Yokozuka of Yamanashi University
These organisms have been maintained in the writer's laboratory on agar sl/Ants contaip_-
ing "koji" juice
Culture Metho'Js
Modified He nneberg solution was used for culture of yeasts tested. This modified
solution contains 100 g cane sugar, 5 g peptone, 5 g KH2P04, 2 g MgS04, O . 5 per cent
yeast extract and 1000 ml. water. Inoculum was incubated in 300 ml. Erlenmeyer flasks
containing 50 ml. of the solution at 30'C for 16 hrs. For the culture of yeasts a recipro-
cating shaker was used
~ ~~ TTTl
22 Kazuyoshi NISI{IGAMI Preparation of Cell ,Jquspension
The organisms grown in the media above described were harvested by centrifugation,
washed with deioniz;ed water three times, and suspended in deionized water
Preparation- ~of Cell-Free . E~t~act
AL~er the disruption of washed cells by grinding with ~ sea sand, homogenate was cen-
trifuged at 3, OOO r. p. m. for _ 5 minutes to remove sea sand powder and intact cells
Then an opalescent supernatant was obtaine d
Manometric Determination
The rate of uptaken oxygen in air was determined by the conventional techniques of a
Warburg respirometer46) . The materials and their final molar concentrations in each re-
action chamber were as follows : cell suspension. O . 5 ml. ; substrate, from 10-3 M
tb 5 x 10-2 M ; phosphate buffer and acid potassium phtalate-sodium hydroxide buffer,
pH 5.9 and 4.8 respectively, 2.5xl0-2 M ; inhibitor, from 5 x l0-4 M to 5 x lO-8 M. The
voluine of solution in the reaction chamber was adjusted with deionized water to make 2 ml
in total. Respiratory inhibitor in side arm was potired' into reaction chamber at the following
times after the beginnin._a of measurement : monoiodoacetic acid, after twenty minutes : sodium
fluoride, ~thirty minutes ; arsenite, forty minutes. The center well contained O . 5 ml. of 20
per cent KOH and a fluted filter paper strip. The gas phase was air. The temperature
was 300C.
Substrates and Respiratory Inhibitors
As substrates, glucose, arabinose, gluconate,, pyruv:ate, acetate, acetaldehyde, ethanol and
the TCA cycle intermediates were employed. Oxidation of the TCA cycl.e intermediates
was tested at pH 5 . 9. The other seven substrates were tested at pH 4 . 8. Phospate buffer
and acid potassium phtalate-sodium hydroxide buffer were used at pH 5 . 9 and 4 . 8 respe-
ctively. As respiratory inhibitor, use was made of monoiodoacetic acid, sodium fluoride
and arsenite
Spectrophotometric Determinations
For estimation of cytochrome system of cell-suspensions, absorption spectra in wave
lengths ranging from 450 to 620 m/1 were observed under reduced condition of cytochromes
with SHIMAZU'S Spectrophotometer Type QB-50. Sodium dithionite was used as a
reductant
Micro-Spectroscopic Determinations
For estimation of cytochrome system of cell-suspensions, a micro-spectroscope was also
employed. Sodium dithionite was used as a reductant. These determinations were made
under anaerobical condition
Determination of Protein
Protein in cell-suspensions used in the investigations by spectrophotometer was estimated
by the method of Lubochinsky and Zalta47)
RESPIRATION OF VARIOUS YEASTS 23
EXPERIMENTA1, RESULTS
I. OXIDA TIVE ACTIVITIES OF VARIO US YEASTS ON VARIO US SUB:STl~ TES
(1) Oxidation of Glucose, Arabinose, Ethanol, Pyruvate and Gluconate
Figures I - 8 and Table I show the oxidation of substrates by eight strains of yeasts
Out of five kinds of substrates both ethanol and glucose were comparatively activ ely oxi-
dized by all the yeasts tested. Especially, glucose among the substrates examined was most
intensively oxidized by Rhodotorula glutinis, Candida utilis, Schiz:osaccharomyces pombe
and Mycodenna cerevisiae ; these organisms have comparatively low RQ values of glucose
oxidation. However, Torula candida and three other kinds of yeasts which have com-
paratively high RQ values of glucose oxidation, Sacchoromyces sake, Saccharomyces cerevisiae
and Saccharolnyces carlsbergensis, utilized e_thanol most actively. Three species of yeasts,
RhodotoruZa glutinis, Candida utilis and Mycoderma cerevisiae which utilize glucose prefer-
ably in comparison with other yeasts, oxidized gluconate rather active-ly. On the contra-
ry, four other species of yeasts scarcely utilized gluconate. Pyruvate was fairly oxidized
by eight strains of yeasts. Arabinose was slightly oxidized only by Rhodotorula gZutinis
and Torula cardida.
Table I Rates of respiration on glucose, arabilrose, ethanol, pyr~vate
aud glucoceate as substrates~~
Yeast Q02 : Uptake of Oxygen (pl./mg dry wt. of yeast/hr.) RQ endog. glucose arabinose ethanol pyruvate gluconate glucose
Rhodotorula glutinis 75 27
Caudida utilis
14 92 Mvcoderma cerevisiae 10 146 Sch'izosaccharomyces pombe 42 2.4
Saccharomyces sake 48 10
Saccharomyces cerevisiae 65 11
Saccharomyces carlsbergensis 2 13 Torula candida
14 8
24 o O o o o o 7
55
88
151
21
51
79
13
37
22
75
22
11
43
13
lO
11
7 9 8 2.5 O o 0.6
4
1.2
1.1
1.l
3.5
2.0
3.1
6.5
l.3
~~ Manometer flasks contained washed yeast cells in 0.5 ml. of 10-1 M acid potassium phthalate-sodium hydroxide butfer pH 4 . 8, in the main compartments ; 2 . 5 x 10-2 M of substrate in 0.5 ml. of water in the side arms ; and 0.5 ml. of 20 per cent KOH with filter
paper in each center well. Incubated in air at 30 'C. The figures for Q02 have been corrected for endogenous respiration. 16 hrs . cultures were used. Abbreviations : endog
= no substrate added
(2) Oxidation of the TCA Cycle Intermediates
Oxidation of the TCA cycle intermediates was tested on eight strains of yeasts. Table
II shows the oxidative activities of yeasts on the TCA cycle intermediate. Succinate was
utilized by all tested organisms, though the intensities of the oxidation were not similar
Citrate was oxidized by only three kinds of yeasts. In general strains which have
higher RQ values showed relatively weak oxidative activities on the TCA cycle intermedi-
24 Kazuyoshi NISHIGAMI
200
O) E
¥ ~~
~C O QL
C~,
I OO
50 time in min,
Fig. I . Oxidation of glucose, arabi-
nose, gluconate, pyruvate and ethanol by washed resting cells of Rhodotorula glutimls. The reaction nuxtures contained
0.5ml. of cell suspension in a final volume of 2 O ml. The reactions were started at time indicated by arrow by tipping in 2 . 5 x'l0-2 M of substrate from
the side ann-.
Indication of added substrates
150
a) E ¥ oo :~
X ~ QL 50 (~
O 50 100 time in min.
Fig. 2 . Oxidation of glucose, arabi-
nose, gluconate, pyruvate and ethanol by washed resting cells of Candida utilis.
The reaction conditions were the same as those for Fig. I .
80
glucose ・-.-, ,-. -,-.
pyru v at e _ arabinose
ethanol
subsfrate
ol E ¥ 60 -~
g 40 ~' ~ c~~
~/a l'
O 50 100 t50 time in min.
Fig. 3 . Oxidation of scflucose, arabi-
nose, gluconate, pyruvate and ethanol by washed resting cells of Schizosac-charomyces pombe. The reaction con-ditions were the same as those for Fig. 1
500
gluconate
endogenous
CD E ¥ 200 ~ C Q)
X ~ ~ 100 C~1
~ substrate oddition
/
~ eoce~: ,~ '~ .,p c~o ,p ~et~~'b~i'~~~~~ e~P '~p
~~'ee~bS~d~'~~ ~~ e~'~~b O 50 IOO 150
time in min. Fig. 4. Oxidation of glucose, arabi-
nose, gluconate, pyruvate and~*~, ethanol
by washed resting cells of Mycoderma cerevisiae. The reaction conditions were the~~~f'ysame~as those for Fig. I .
RESPIRATION OF VARIOUS YEASTS 25
120
O) E ¥ gO ~~
C G)
x 60 O ~ (~ S C~1 SO
50 time in min.
Fig. 5. Oxidation of glucose, arabi-
nose, gluconate, pyruvate and ethanol by washed resting cells of Saccharomyces
sake. The reaction conditions were_ the
same as those for Fig. 1
120
a) E ¥- gO
~ ~Oo 60
~-
Q C~1 30
O 50 100 time in min.
Fig. 6. Oxidation of glucose, arabi-
nose, gluconate, pyruvate and ethanol bv. washed resting cells of Saccharomyces
cerevisiae. The reaction conditions were
the same as those for Fig. 1.
le
O) E ¥ 12 -~
~: ~ ~6 C~1
o
Fig. 7. xidation of glucose, arabi-
nose, gluconate, pyruvate and ethanol by washed resting cells of Saccharomyces
carlsbergensis. The reaction conditions
were the same as those for Fig. 1
substrate additlon f i
/
/
/
~ e / l
'
50 100 time in min Oxidation of glucose,
and
300
o) E
¥ ~~ c gOO
Q)
x Q.
lOO
'~s / O'
~d; ~p'
O 200 400 time in min. Fig. 8. Oxidation of glucose, arabi-
nose, gluconate, pyruvate and ethanol by washed resting cells of Toru.la candida.
The reaction conditions were as those for Fig. l.
the same
26 Kazuyosh1NISHIGAMlI
Tab1e n Rα把50グ〃8が〃κo拠o惚肋召TCλη61召初加7脇θ∂ξακ8αs8肋5〃o加5*
Yeast
Q02 Uptake of oxygen(μ1./mg dry wt.of yeast/hr.) RQ
endog. citrate ma1ate α一KG su㏄mate g1Ucose
0o%∂ξ∂o秘カ〃3 ユ4
妙60伽榊α6θ肋クs肋 101)たゐξα〃26〃θろグα解αθプ;α6クθ〃5 5.6
1己んo∂oチoグ〃2α82〃κ%ゐ 27
8σゐク203α66み0グ0〃2ツ6θ5ク0〃彫6θ 2.4
8α㏄肋70榊仰88α加 108κc加グo卿6θ86θグ助ゐクα3 11
8α60ゐα70〃zツ6θ500〃86θ7gθ犯sゐ2
44 0
0
0
0.9
0
0
3
35
26
27 4
1.9
0
1
3
17
15
20 3
2.3
0
0
9
7 ユ.1
12 1,1
3 0.94
9 1.2
1.4 3,5
1 2,0
1 3,1
3 6.5
*Compos1t1ons of contents of mano血eter f1asks were as1n Tab1e I.
Abbre▽1at1ons.endog,=no substra-te added.
α一KG=α一Keto-g1utar1c ac1d
ates.On the test by ce11イree extract of8α66肋ブo刎ツcθ50κθ切∫加 oxygen uptake in the
presence of c1trate was皿ore than twofo1d.of endogenous,whereas1ntact ce11s of the
organ1sm cou1d-not ox1d1ze c1trate (Tab1e III)
Tab]e III Rates of ox1dat1on of the TCA cycleユnter1med工ates by
ce11・free extracts of Saccharomyces cerev1s1ae栄
Yeast
Q02:Uptake of oxygen(μ1./mg dry wt.of yeast/hr.)
endog. c1trate nユa1ate α・KG succinate
∫肌肋ク0仰㈱鮒ωゐξ〃 0,35 0,85 0.1 O.75 0.3
*Manometer f1asks contamed ce11-free extract m05m1of10-M phosphate buffer pH 59,m the mam compartments Other compos1t1ons were as1n Tab1e II
(3) Ox1dat1on of tlhe Tr1carboxyr1c Ac1d Cyc1e Intermed.1ates m Var1ous Concentrat1ons
Cα〃滅α肋〃5was used1n the exper1ments,{or1t ox1d1zed血ost actwe1y the TCA cyc1e
mtermed。ユates among the e1ght strams emp1oyed- As substrate,c1trate,皿aユate,α一keto-
g1uta1ate and succmate were em-p1oyed As a contro1,ox1dat1on of g1ucose1n▽ar1ous
concentrat1ons was exam1ned- In generaユ,to血amta1n act1Ye resp1rat1on th1s organ1sm-
requ1red co皿parat1∀e1y h1gh concentrat1o:ns of those substrates The oxldlat1on of g1ucose
was皿ax1蛆aユat67×10-31M[of concentrat1on C1trate was ox1d1zed 皿ost act1ve1y at
13xlOi21M1o{concentratエon The M1chae11s constants of ox1d.at1ons of both g1ucose and.
c1trate were2×10-31M[ On the other hand,m case of ox1dat1on of皿a1ate andα一keto-
g1utarate,gradua11ncrease o{the ox1dat1ve rate was seen1n the tested一蝸nge of concemta-
t1on froIn1O■3to5×10}2M The ox1dat1on of succ1nate mcreased s11ght1y m the range
of concentration from2×10・3to5×10‘21Ml(Figures9-10).
RESPIRATION OF VARIOUS YEASTS 27
lOO
80.⊆
E○
ぐ①60E三べ
.⊆
Φ 40よoO-
0 20
gluCOSe
Ci甘rO寸e
mOlO予e
50
40ピ
EO⑩\③30E三ユ
.1≡
Φ20oo-
O 1O
α・ke寸O・91u寸OrOfe
SuCCinOfe
10・引!5刈0・31旧×1◎・2 125xI0・2a3XlO・2 5XlO-2
2X10,3 10・2 2×!O・2
COnCen寸r0†lOn Of Su⑪S廿Q含e ln ~8
F1g g Ox1dat1on of g1ucose,c1trate
and.ma1ate1n var1ous concentrat1ons by
washed resting ce11s of Cα〃〃α 秘〃ゐ.
The react1on二m1xtures conta1ned05m1of ce11suspens1on1n a f1na1 vo1ume of
2.0m1.
lO’3//5刈O・3113刈O引2酬O一・3洲O一・ 5×lO一・
2×lO・3 10・2 2X1O-2
concen寸ro千1on of subs予r〇十e in M
F]g lO Ox1dat1on ofαketo g1utarate
and succ1nate1n Yar1ous concentrat1ons
by washed restmg ce11s of0例6肋α肋伽
The reacr1on血1xtures contamed05m1of suspens1on1n a f1na1yo1u工ne of20m1
旧㌃、、、、干、。青、。、、、看、。、
θOO
べ
ζ・一600Φヱ呈
ま400δ S・帥k・
200
↓ R・9I・刑・i・
0 50 100 150 200 250
青im魯inmin.
F1g ll Companson of ox1dat1onprocesses of ethano1 by 8α66ゐoグo〃η6硲
8α加and R〃odo乏07〃α8〃κ伽s. The re-
act1on m1xtures conta1ned05m1of ce11
suspe:ns1on m a fma1vo1ume of20m1
The react1ons were started at t1me
md1cated by arrow by t1ppmg1n25×10’2M1of ethano1from the s1de arm
800
べ 600.⊆
Φヱ
◎400αコ
0200
↓s・b・1・o帖od州◎・
P・ei…b・f・di・㏄・1・1;\、\
proincuboオed in ◎{honol
050100!5020025C オime in min.
F1g 12 Compar1son of pre1ncubat1on
efεects on ethano1ox1d1z1ng act1v一ユty of
washed rest1ng ce11s of R肋∂oチo榊1α
g1〃棚3. The organisrns were preincu・
bated 1n25×10-2M of ethano1or ace・
tate for4hrs The react1on cond1tユons
were the same as tbose for F1g11
28 Kazuyosh1NISHIGAMI
斗 40◎.ε
⑩主Oaコ200
0
0 50 100
fime in min.
Fig.ユ3. Effect of ch1oramphenico1
on premcubat1on1n ethano1 Washedrestmg ce11s of Rゐ0∂0サ07”α82”2励3we「e
pre1ncubated1n the mユxed-so1ut1on of
2.5×10’2M ethano1and200γ/m1.ofch1oramphen1co1for4hrs The react1oncondユt1ons were the sa皿e as those for
Fig.11.
300
ユ.⊆
Φ200ヱoα3ω lOO
O
↓s・bsf・ole
Oddi刊◎n
↓
p1-eincubOi6d
に in OC6七0+e
non・十reo{6d
『
pr6incu1コlo壬6d
in e寸honcI
050100150200250 サime in min.
Fig.14. Comparison of preiηcubation
effects on acetate ox1d1z1ng act1▽1ty by
washed resting ce11s of 亙肋∂o玄07〃α
g肋吻z8. The organ1sms were pre1ncu-
bated1n25×10-2Mofethano1orace-tate for4hrs The react1on cond1t1ons
were the same as tbose for F1g 11,
except that2.5×10-2]M1of acetate was
emp1oyed-as substrate
lOO
べ仁
呈£ 50
sδ
0 50
fime in min.
lOO
F1g15 Compar1son of pre1ncubat1oneffects on aceta1dehyde ox1d1z1ng act1Y1ty
by washed rest1ng ce11s of R脆o∂oまo〃2α
8肋榊3 The organ1sms were pre1ncu-
bated1n25×10-2M of acetate,ethano1
and aceta1dehyde for4hrs.The reaction
cond1t1o二ns were the same as those for
F1g 11, except that 25×10■2Ml of
aceta1dehyde was emp1oyed as substrate。
RESPIRATION OF VARIOUS YEASTS 29
n. EFFECTS OF PRElNCUBATION IN ETHANOL AND ACETATE
(1) Ethanol and Acetate Oxidizing Activity
An adaptation phenomenon was seen in the case of ethanol oxidation by Rhodotorula
glutinis which has relatively low RQ values, whereas Saccharomyces sake exhibited active
oxidation as soon as ethanol was added (Figure 11) . The maximum rate of ethanol
oxidation by Rhodotorula glutinis arose about 130 minutes after the addition of substrate
Figure 12 shows the effects of preincubation in ethanol or acetate. By means of preincu-
bation for four hours in 2 . 5 x 10 -2 M of ethanol, Rhodotorula *"lutinis acquired an ability
to oxidize ethanol without any lag phase. That is to say, the oxidation of ethanol pro-
ceeded analogously like that by Saccharomyces sake. Similarly, this organism by preincu-
bation with acetate obtained the capacity to oxidize ethanol instantaneously (Figure 12)
The acquisition of this ability was not disturbed by 200 T/ml. of chloramphenicol (Figure
13) . Untreated strain of this organism has only weak ability to oxidize acetate. After
about three hours of contact with acetate, gradual increase of activity of acetate oxidation
was observed. Four hours of preincubation in 2 . 5 x' 10-2 M of acetate gave vigorous
ability of acetate oxidation. Such acquired ability to oxidize acetate was seen also on
the strain which had been preincubated in 2 . 5 x l0-2 M of ethanol (Figure 14)
(2) Acetaldehyde Oxidizing Activity
Figure 15 exhibits the effect of preincubation on the acetaldehyde oxidizing activity of
Rhodotorula glutinis. This organism, Rhodotorula gZutinis, has only weak ability of oxi-
dation of acetaldehyde. . Howev. er, the procedure of preincubation either on ethanol,
acetaldehyde or acetate gave active ability to oxidize acetaldehyde (Figure 15)
III. EFFECTS OF INHIBITORS ON RESPIRATION OF VARIOUS YEASTS
(1) Inhibition by Monoiodoacetic Acid
Rhodotorula glutinis.
Monoiodoacetic acid in a concentration of 5 x l0-4 M exerted only a slight inhibition upon
oxidation of glucose. Even by 5 x 10-3 M of monoiodoacetic acid this organism exhibited
fair respirattory activity. This organism was most insensitive to monoiodoacetic acid
among eight kinds of yeasts tested (Figure 16) . On C02 outpu.t also, monoiodoacetic
acid showed extremely weak inhibition effect (Figure 17)
Candida utilis.
Monoiodoacetic acid of 5xl0-4 M exerted little inhibition upon oxidation of glucose
However, this organism suffered remarkable inhibition by 5 x 10-3 M monoiodoacetic acid,
namely, its respiratory activity was completely inhibited at 60 minutes after addition of
the inhibitor (Figure 18) . The C02 evolution suffered conspicuous inhibition by 5 x l0-4
M of monoiodoacetic acid. But perfect inhibition even by 5 x 10-3 M of monoiodoacetic
acid (Figure 19) was not seen. The endogenous C02 evolution was scarcely inhibited
by 3 X l0-8 M. In general, the endogenous respiration of wild yeasts such as Rhodotorula
and Candida is more stable against monoiodoacetate inhibition than the respiration of
30 Kazuyosh1NIS亘IGAM1
100
80
③ε
\一60べ⊆
Φ皇◎
牛40o。
コ
O20
↓MlA
↓
↓
oddi+ion
7〃
.グ
〃ク
。〆
〆”,.〃ケ!
帥d◎9.
12◎
lOO
↓MlA
①
E80二弐ζ
巾60コo.
ち◎ ↓δ40
0
2◎ ↓
oddi+ion 夕
.〃〆
φダ
!
〆 〆
〆
end◎9.
グ
0 20 4◎ 60 80 0 2◎ 4◎ 60 8◎
fime in min. 刊me in min.
F1g16 The effectof mon010doacet1c F1g17 The effectof mon010doacet1c
ac1d on02uptake by washed rest1ng ac1d on C020utput by washed rest1ngce11s of児肋∂o乏oグ〃αg2〃多〃3.The reaction ce11s of況肋6o〃グ〃o8伽左多伽3・The reaction
m1xtures contanmed05m1of1O-1M cond1t1ons were the same as those for
buf重er pH48 and 05m] of10-1M1 F1g16,except that05m1of de1omzedg1ucose1n a f1na1vo1u血e of20m1The water was p1aced 1nstead of KOHreact1ons were started at t1me1nd1cated so1ut1on m center we11
by arrow by t工pPmg m var1ous concen・
trat1ons of mon010doacet1cac1d並om the
s1de arms Concentrat1on of add.ed mon010doacet1c ac1d(from Flg16to F1g31)
nOn ・一・一…・…・………… 3×10-3
5×10’4 . 一・・一・・一・・一・・一・・一・・一・ 4×ユ0-3
1×10-3 一一 一一・ 5×10-3
_一_一_一_一一一_一_._一_ 2×10-3
1OO
⑲80E\
弐ε・’60
⑩
ヱ◎
αコ40
0
20
↓MlA
↓
↓
oddi廿ion
・・二
〃
〆
4ノー end◎9...一・
0 20 40 6◎ 80
fime in min
F1g18 The effectof mon010doacet1c
ac1d-on02uptake by washed restmgce11s of Cα〃〃o 〃〃伽. The reaction
cond1t1ons were the same as those for
Fig.16.
140
120
⑰ε100二弐
仁 80
コ
α
青6◎o◎
040
20
↓MlA
↓
c1ddiセion
〃;;タ「
end◎9.
0 20 40 60 80
fime in mi同.
F1g 19 The effect of加on010doacet1c
acヱd on C020utput by washed restmg
cens of C肋励伽 〃〃眺. The reaction
cond1t1ons were the sa㎜e as those for
Fig.17。
RESPIRATION OF VA~IOUS YEASTS 31 exogenously supplied substrate.
Schizosaccharomyces pombe
Upon the oxidation of glucose, 5 x lO-4 M of monoiodoacetic acid exerted only a little
inhibition. However, the respiration was inhibited completely at 5 x l0-3 M (Figure 20)
The endogenous respiratory activity was extremely weak. The activity of C02 evolution
was inhibited at 5 x l0-4 M rather severely. That activity wa~* inhibited perfectly at 4 X
10-3 M (Figure 21).
Saccharomyces cerevisiae.
Upon the oxidation of glucose, 5 X 10-4 M of monoiodoacetic acid exerted some con-
spicuous inhibition. The respiration was inhibited almost perfectly at 3 x 10-8 M. As to
C02 output, intense inhibition was seen at 3 x 10-8 M (Figures 22, 23)
Saccharomyces sake.
Upon the oxidation of glucose, 5 X l0-4 M of monoiodoacetic acid exhibited fairly severe
inhibition. The respiration was inhibited almost perfectly at 3 >'. 10-8 M (Fgigure 24)
Similar inhibition was seen of C02 evolution (Figure 25)
Saccharomyces carlsbergensis.
Upon the oxidation of glucose, 5 x 10-4 M of monoiodoacetic acid exerted severe
inhibition (Figure 26) . As to C02 evolution, monoiodoacetic acid inhibited it perfectly at
5 X 10-4 M (Figure 27)
Mycoderma cerevisiae.
Upon the oxidation of glucose, 5 x l0-4 M of monoiodoacetic acid has an extremely
severe inhibition effect. The respiration was inhibited perfectly at I x l0-3 M. Endoge-
nous respiration was also inhibited perfectly by I x 10-8 M (Figure 28) . C02 evolution
was extremely inhibited at 5 X 10-4 M (Figure 29) .
Torula candida.
Monoiodoacetic acid of 5 X 10-4 M exerted serious in_hibition upon both O 2 uptake and
C02 output (Figures 30, 31).
(2) Inhibition by Sodium Fluoride
Among the eight kinds of yeasts mentioned above, Rhodotorula glutinis which was most
insensitive to monoiodoacetic a, cid and Saccharomyces cerevisiae which was most sensitive
to monoiodoacetic acid were investigated for -*odium fluoride inhibition on their 02
uptake
Rhodotorula glutileis.
Respiration on 'exogenously supplied glucose was inhibited a little by I X l0-2 M of
sodium fluoride. The respiratory activity was left practically intact even by 2 x l0-2 M
of sodium fluoride (Figure 32) .
Sacchoromyces cerevisia~.
By I >< 10-2 M of sodium fluoride the respiration on exo*"enously upplied glucose
suffered only slight inhibition, whereas it was perfectly inhibited after 30 minutes of the
addition of 2 x 10-2 M of sodium fluoride (Figure 33)
32
60
Kazuyoshi NISHIGAM1
o E 40 ¥ ::L
x ~2 OL ::, 20
O
~ MIA addition
~ '
endog.
cf f r
o 20 4 o 60 8 o time in min,
Fig. 20. The effect of monoiodoacetic
acid on 02 uptake by washed resting cells of Schizosaccharomyces pombe. The
reaction conditions were the same as those for Fig. 16.
200
o, E 150
¥ ~ c
~ =i
'iIOO ~ 3 o (~ O
50
~
MIA
~
addition
/ //"I/i'
///
'." '-s: "":'~~_" -
endog.
o 20 40 60 eo time in min
Fig. 21. The effect of monoiodoacetic
acid on C02 Output by washed resting cells of Schizosaccharomyces pombe. The
reaction conditions were the same as those for Fig. 17 .
60 a) E
¥ ~ .S 40 ,a)
x o cl
O 20
~ MIA
~
~
addition
endog,
.l
o 20 40 60 80 time in min
Fig. 22 . The effect of monoiodoacetic
acid on 02 uptake by washed resting cells of Saccharomyces cerevisiae. The
reaction conditions were the same as those for Fig. 16.
250
200 ~ E
¥ :L c: 150
= cl ~ =1
o 100
O O 50
~ MIA addition
'~ '~ '~ . CI" 'P _, d-~ d~' " ' t I~~・Au・・'~~P""aR:"&,"B~~'・~ --~"" ' d" Il' dl' l"I-t
d,~"
~
endog.
o
Fig. 23
acid on cells of re a ction
those
20 40 60 80 time in min.
The effect of monoiodoacetic
C02 Output by washed resting SaccharQmyces cerevisiae. The
conditions were the same as for Fig. 17.
RESPIRATION OF VARIOUS YEASTS 33
50
cD40 E
¥ :~
c 30 Q)
o ~ cl 20
O
IO
~ MIA addition
.r'
~ .・ t'l /
endog,
~ ---
o 40 60 80 20
time in min,
Fig. 24. The effect of monoiodoacetic
acid on 02 uptake by wd"shed resting cells of Saccharomyces sake. The reaction
conditions were the same as those for Fig. 16 .
120
lOO
a)
~ 80 =L
c
- 60 ::i
~ s o C~ 40
O
20
~ MIA addition
~
~ endog.
o 20 60 80 40
time in min
Fig. 25. The effect of monoiodoacetic
acid on C02 Output by washed resting cells of Saccharomyces sake. The reaction
conditions were the same as those for Fig. 17.
300
c) E 200
¥ ~
o ~ ~ CL :D 100
O
20
~ MIA addition
~ endog.
20 60 8 o time in min.
Fig. 26 . The effect of monoiodoacetic
acid on 02 uptake by washed resting cells of Saccharomyces carlsbergensis. The
reaction conditions were the same as those for Fig. 16 .
*,*~
120
IOO
o) E ¥ 80 ~ c
~ eo :5
Q.
~ :3
o (5 40
O
20
~ MIA addition
~ "'
~
endog,
o 20 40 6 o 8 o time in min
Fig. 27 . The effect of monoiodoacetic
acid on C02 output by washed resting cells of Saccharomyces carlsbergensis. The
reaction conditions were the same as those for Fig. 17
34 Kazuyoshi- NISHIGAMI
150
o) E
¥ :~.
.S iOO
.**'
CL
O so
~ MIA addition
"'--1-/' d- 'P 11' ,., ,.,
'// lll _"" ~, -.'-""" .f. .,. .p,-~ "t""___' dlP
,d.
,
~ en(!(~'gt
20 40 6 o tirne in m'n
Fig. 28 . The effect of monoiodoacetic
acid on 02 uptake by washed restin~cr
cells of Mycoderma cerevisiae. The reaction conditions were the same as those for ~Fig. 16.
200
60
cF,
E
¥ :~120 c
:s
CL
~ :50 60 (~
O 40
~ MIA addition
~
~ endog,
o 20 40 60 80 fime in min
,Fig. 29 . The effect of monoiodoacetic
acid on C02 output by washed restirL5a
cells of Mycpd.erma cerevisiae. The reaction conditions were the same as those for Fig. 17.
60 ~ MIA
CD E
¥ 40 ~ (L)
o CL
C~f 20
addition
'I-l':" .1-':':'P"I-'1-
~ " ,, ,,,:;(~' 11"~':;l ,,. ,~:3;
('~'
endOg, ~p'_'-1-""'-d'~' id.1
.,9-b:: ':: ~ ..11'.'.1~".'.
o 20 40 60 80 time in min
Fig. 30 . The ,effect of monoiodoacetic
acid on 02 uptake by washed resting cells of Torula ca7rdida. The ,reaction
conditions were the same as those for Fig. 16.
60
CD E
¥ ~:~40
a :;o
O o 20
~ MIA additlon
rt' '-" e" I I' (
ll"""""
~ "'
l 'Jr'l-' endOgl 'e'~c'a' "d'l"P llo'-" '1"P' J!dld" ' r" ';'
:'s;1:""
60 20
time in min
Fig. 31 . The effect of monoiodoacetic
scid on C02 Output by washed resting cells of Torula candida. The rear_tion
conditions were the same as those for Fig. 17 .
RESPIRATION OF VARIOUS YEASTS 35
lOO
0180 E
¥ ~ (: 60
a)
o ~ ~ :' 40
O
20
~ NaF addition
~
~ endog
/
/
80 ~ NaF addition
o) E 60 ¥ :i.
'a)
~ 40 ~ c~ C~
20
~
endog.
~~
o 40 60 80 20
time in m'n.
Fig. 32 . The effect of NaF on glucose
oxidizing activity of washed resting cells of Rhodotorula glutinis. The reaction
conditions were the same as those for
Fig. 16, except that NaF was used instead of monoiodoacetic acid.
o 20 40 6 o 8 o time in min.
Fig. 33. The A-ffect of NaF on glucose
oxidizing activity of wash~ ed restin*a
cells of Saccharomyces cerevisiae. The
reaction conditions were the same as those for Fig. 32
Concentration of added NaF (from Fig. 32 to Fig. 33) : non l x 14-2
2 x 10-2
300
~ c 200
o x o a :,
IOO
O
~ arseni~e addi~ion
controLl;'
~
400
:L
C 300 G)
X O - 200 GL :,
O 100
~ arsenife ariudi~ion
~
contrc, l
/
l
50 time in min.
Fig. 34 . The effect of arsenite on
glucose oxidizing activity of washed resting cells of Rhodotorula glutinis. The
reaction conditions were the same as those for Fig. 32, except that 5 x lO-4 M
of arsenite was used instead of NaF
50 time in min
Fig. 35 . The effect of arsenite on gluc,ose oxidizing activity of washed ' resting cells of Saccharomyces sake. The
reaction conditions were t.he same as those for Fig. 32, except that 5 x 10-4 M
of arsenite was used instead of NaF
36 Kazuyoshi NISHIGAMI (3) Inhibition by Arsenite
In the test of respiratory inhibition by arsenite, RhodotoruZa &alutinis and Sacchoromyces
sake were used.
Rhodotdrula glutinis.
Arsemte of 5 .. 10 4 M mhrbrted the oxldatlon of glucose only slightly (Figure 34)
Saccharomyces sake.
On the contrary, this organism received severe inhibition by 5 x 10-4 M of arsenite
(Figure 35) .
IV COMPARISON OF CYTOCHROME COMPONENTS IN YEASTS
(1) Microspectroscopic Study.
Components of cytochrome in eight kinds of yeasts were observed (Figure 36) . Four
kinds of yeasts which have comparatively low RQ values, viz., Cardida utilis, Mycoderma
cerevisiae. Pichia melnbranaefaciens and Rhodotorula glutinis showed the cytochrome a, b
and c bands. On the contrary, only two kinds of yeasts showed the cytochrome a, b
and c bands among the other four kinds of yeasts which have comparatively high RQ
values, viz., Schizosaccharomyces pombe, Saccharomyces sake, Sacchar07nyces cerevisiae and
Saccharomyces carZsbergensis. The band of cytochrome c appeared clearly in all tested
yeasts. The band of cytochrome b appeared clearly in Candida utilis, Mycoderma cerevisiae,
,Jqaccharomyces sake and L)(!accharom_vces cerevisiae. This band was shown relatively clearly
on Rhodotorula glutinis an d . . Schizosaccharomyces pombe. In Pichia membranaefaciens
and Saccharomyces carlsbergensis this band was faint. Dense bands of cytochrome a
were observed in Cardida utilis, Mycoderma cerevisiae and Saccharomyces cerevisiae. Thi**
band was comparatively clear in Rhodotorula gZutinis and Saccharonryces sake, but was
quite dim in Pichia membranaefaciens. It could not be observed in L)qchizosaccharomyces
pombe and Saccharomyces carlsbergensis. The p - bands of cytochromes were observed
only on Mycoderlna cerevisiae and Saccharofnyces cerevisiae.
(2) Spectrophotometric Study
Figure 37 - 44 show the absorption spectra of cytochromes in intact cells of eight kinds
of yeasts. Among these yeasts there were some variations in types of spectra. However,
all cytochromes a, b and c were found in each yeast
In the case of Candida utilis the peaks at 604, 563 and 551 m/1 are considered to be
due to the cytochromes a, b and c respectively (Figure 37) . It is suspected that the
peak at 522 m/e is caused by the ~-band of the cytochrome c. No peak caused by the
P - band of the cytochrome b was found
In Mycoderma cerevisiae cytochromes a, b and c were observed at 603, 562 and 548 m/1'
Peaks at 525 and 520 m/1 are considered to belong to the ~ - bands of the cytochromes cl
and c respectively (Figure 38) . In Pichia mefnbranaefaciens the peaks caused by
cytochromes a, b and c appeared at 603, 563 and 557 m/1 respectively. Peaks at 525 and
521 m/1 seem to be due to the ~-bands of the cytochromes cl and c (Figure 39)
RESPIRATION OF VARIO'US YEASTS 37
yeasts absorption bands of cytochromes
Candida utilis
Mycoderma cerevisiae
Pichia membranaefaciens
600
Rhodotorvla g lutinis
Schizosaccharom ycepomsbe
Saccharomyces sake
S. cerevisiae
S. carlsbergensis
550 500 (m~)
600 550 Fig. 36. Comparison of absorption
bands of cytochrornes. A11 observations
~l~7ere performed in anaerobic state on
resting cell suspensions.
500 (m p)
38 Kazuyosh1NISHIGAMI
O,35
>
ω1=
Φ℃
oOQ・O.3
o
O.25 500 50 600
wove Ienqfh in m u
Fig-37.Absorption spectra of resting
ce11suspens1on of C倣励伽〃〃z3 The
suspens1on conta1ns 34μ9 of n1trogen
per m1.Sodium dithionite was used as
a reductant.
O.9
>
ω⊆Φ一◎
io O.8
Q-
O
O.7
500 50 600
wove lenφh in mμ
Fig.38.Absorption spectra of resting
ce11suspension of吻60∂〃榊■oθ7θoたクα6・
The suspens1on conta11s 95μg of
mtrogen per m1 Sod1um d1th1on1te wasused as a reductant.
O.2
>
⊂ O.15型
Oo-
O
0.1
O.5
>
ω仁Φ
’o
O.4
i.9
盲O
0.3
500 50 600
wove 1帥φh in m-
F1g 39 Absorpt1on spectra of rest1ng
cen suspens1on of Pκ〃α榊θ〃67α掘oθノζoα一
倣s The suspens1on conta1ns60μg of
mtrogen per m1 Sod1um dlthlomte was
used as a reductant
500 50 600
wove Ienφh in m〃
Fig-40.Absorption spectra of resting
ce11 suspens1on of R%o∂o〃07〃2αg1〃〃榊5
The suspens1on contams30μg of mtrogen
per m1.Sodium dithionite was used-as
a reductant.
RESPIRAT1ON OF VARIOUS YEASTS 39
O.35
>
0D仁Φ’o
i.90・3
盲O
O.25
ヘヘ \
500 50 600
蜘velen帥in叩
F1g41 Absorpt1on spectra of restmg
ce11 suspension of 86〃20∫αc6ゐαグo〃リ6θ8
クo榊ろθ. The suspension contains1.9μg
of n1trogen per m1 Sod1um d1th1on1te
was used as a reductant
05「> 甘
(o
50・4’◎
oOQ-
O
O.3
500 50 600
wove ,eng怖 in mμ
F1g 42 Absorpt1on spectra of rest1ng
ce11suspens1on of8oc6免αグo物ツ6θ8 8α后θ
The suspensユon conta1ns32μg of n1tro-
gen per m1 Sod1um d1th1on1te wasused as a reductant.
O.5
>
ω仁Φ
P8o.4Q-
o
O.3
500 50 600
wove leng干h in m〃
F1g43 Absorpt1on spectra of restmg
ce11suspens1on of Sα6c乃αグo〃リ6θ∫
6〃ωz5zoθThe suspens1on conta1ns128μg
of n1trogen per nl1 Sod1um d1th1on1te
was used as a reductant
O.3
>
ω⊂Φで O.25
i.9
αO
O.2
500 50 600
wclve 1eng+h in mμ
F1g 44 Absorpt1on spectra of restmg
ce11 suspens1on of8060免αクo〃リcθ8
ω〃8加グg伽8z8 The suspens1on conta1ns
8.0μg of nitrogen per m1. Sodiumd1th1on1te was used as a reductant
40 Kazuyoshi NISHIGAMI In Rhodotorula gZutinis the peaks of the cytochromes a, b and c appeared at 603, 563
and 553 m/1 respectively (Figure 40) . A peal*- of p - band of the cytochrome b was obs-
erved clearly at 530 m/1' However, the peak of the ~ - band of cytochrome c was not
detected. It is considered that the shoulder at 540 m/1 is caused by rosy pigment~ inc
luded in this organism. This peak at 540 m/1 was not observed in any other tested
yeasts.
In Schizosaccharomyces cerevisiae the peaks of cytochromes a, b and c were found at
603, 559 and 550 m/1 respectively (Figure 41). The ~ - bands of cytochromes b, cl and
c were detected at 530, 525 and 520 m/1 respectively.
In Saccharomyces sake the peaks at 600, 562 and 552 m/t are considered to have been
induced by cytochromes a, b and c respectively (Figure 42) . The peaks at 530, 525 and
520 m/e represent the p-bands of the cytochromes b, cl and c respectively.
In Saccharomyces cerevisiae the peaks at 600 and 564 m/1 and the shoulder at 550 m/e
are considered to be caused by cytochromes a, b and c respectiyely (Figure 43) . The
peaks and shoulder of the ~ - bands of cytochromes b, cl and c were observed at 530,
525 and 520 m/e respectively.
In Saccharomyces carl.・bergensis the peaks at 605, 561 and 549 m/1 and the shoulder at
552 m/1 are considered to be due to the cytochromes a, b, c and cl (Figure 44) . The pealc
at 525 m/1 is considered to represent the ~-band of the cytochrome cl'
DISCCUSSION
A. OXIDATION OF GLUCOSE, ARABINOSE, ETHANOL,, PYRUVATE, G LUCONATE AND THE TF~ICARBOXYLIC ACID CYcLE INTERJ:~'EDIATES
In the past few years it was demonstrated by numerous investigators that various
organisms utilize the hexose monophosphate (HMP) cycle in carbohydrate metabolism23~33)
Holzer and Witt27) , Karasevich28) and Imsenetskii29) demonstrated the utilization of the
HMP cycle by baker's yeast Cardida tropicalis and genus ToruZopsis respectively. Hence,
it is evident that the yeasts possess the system of the HMP cycle for the catabolism of
car bohy d rate.
Among numerous strains of yeasts, however, some differences seem to exist in respect to
the degree of utilization of the HMP cycle. The investigation reported here was performed
with intact cells being employed for the most part, therefore, it will be necessary to
study cell extract also in order to make the matter more certain. As for the results ob-
tained here, it was observed that there are some differences alnong the yeasts in oxidative
activity on various substrates as carbon source. Especially some interesting relationships
were found between the oxidations of exogenously supplied *・lucose, gluconate, ethanol
and the TCA cycle intermediates and the RQ values of yeast. More specifically, the
intact cells of Rhodotorula glutinis, Candida utilis and Schizosacchoromyces pombe oxi d ized
RESPIRATION OF VARIOUS YEASTS 41 glucose more actively than ethanol. On the contrary Saccharomyces cerevisiae and Torula
candida oxidized ethanol more actively than glucose. Gluconate was utilized actively by
the former, viz. Rhodotorula glutinis and Candida utilis and by Mycoderma cerevisiae
v7hich oxidized both glucose and ethanol at similar rate.
On the other hand, concerning oxidations of the TCA cycle intermediates, different
activities were seen according to the strain of yeast. The oxidation of the TCA cycle
intermediates, without citrate, was more active in Caedida utilis, Mycoderma cerevisiae,
Pichia membranaefaciens and Rhodotorula glutinis. However, in case of other yeasts,
Schizosaccharomyces pombe, D"accharomyces sake, Sacchoromyces cerevisiae and Saccharomyces
carlsbergensis, oxygen uptake increased only slightly as a result of the addition of the
TCA cycle intermediates. The former four kinds of yeasts have comparatively low RQ
values. Contrarily, the latter four kinds of yeasts have comparatively high RQ values
Citrate was oxidized actively only by Cardida utilis. Consequently, the yeast which
oxidized gluconate actively also utilized the HMP cycle intermediates. On the contrary
the added TCA cycle intermediates were oxidized fairly well by the cell-free extract of
Saccharomyces cerevisiae compared to endogenous oxidation. Concerning this problem,
Barnett and Kornberg have expressed their opinion in a recent paper 6 ) . Probably the
differences in oxidation between these yeasts may not be due only to the differences of
enzyme activities in the yeast cells. Cell permeability may be concerned in this problem
The oxidative activity on the TCA cycle intermediates in various concentrations was
tested by washed resting cells of Cardida utilis which utilized citrate most markedly
among the ei*・ht strains of yeasts tested. The optimum concentration of oxidation of
glucose by Candida utilis was comparatively low. This observation coincides with the result
obtained by use of baker's yeast by Aldous48) . However, on oxidation of four kinds of
substrates of the TCA cycle intermediates, higher concentrations had to be used necessary
to obtain active oxidation. These phenomena also support the opinion mentioned above
that oxidative activity of the intact cells on the TCA cycle intermediates is restricted by
the permeability of the cell surface
B. PREINCUBATION EFFECTS
Non-treated Rhodotorula glutinis could scarcely oxidize ethanol, acetaldehyde and acetate
However, this ability was recovered by preincubation for about four hours. Ethanol,
acetaldehyde and acetate were all effective as inducers. According to Haboucha and
Masschelein49) the causes of the lack of ability have been explained by the inactivity of
aceto-CoA-kinase, cytochrome oxidase and succinic dehydrogenase. They have explaned
this problem by mutant "petite colonie" of Saccharomyces carlsbergensis. Rhodotorula
glutinis emplo,yed here is able to oxidize succinate. For this reason, the lack of ability
of Rhodotorula glutinis in the oxidations of ethanol, acetaldehyde and acetate is considered
to be caused by the decreased activity of aceto-CoA-kinase but not by lowering of activities
of cytochrome oxidase and succinic dehydrogenase. For the restoration of the activity,
the use of ethanol, acetaldehyde and acetate were all effective. Even in the presence of
42 Kazuyoshi NISHIGAMI chloramphenicol, at 200 T Per ml., together with the inducer, ethanoJ, the induced synthesis
of the oxidation system of ethanol was not inhibited. This concentration of chloram-
phenicol is considerably higher than in the case of bacter'ia 50) . It has already been observed
that in case of other inhibitors higher conQentration was required for inhibiting the activity
0L enzymatic syst~ms in yeasts51)
C. EFFECTS OF RESPIRATORY INEIIBITORS
It is certain that the mechanism of enzyme inhibition by monoiodoacetic acid has relation-
ship with radical SH of apo-enzyme 52) . 3 - Phosphoglyceraldehyde dehydrogenase, alco-
hol dehydrogenase, pyruvic carboxylase and succinic dehydrogenase are known to be in-
hibited by monoiodoacetic acid 39,53). It is also widely accepted that sodium fluoride inhibits
specifically the activity of enolase. Further, the inhibition of the EMP pathway by
arsenite has been reported 23)
In general, yeast follows two courses in catabolizing glucose. These are the EMP
pathway the alcoholic fermentation or the TCA cycle system and the HMP cycle system
The degree of utiliz-ation of these systems varies according to the strain oi yeast. In the
studies here reported, examination was made of the rate of decrease of oxidib-ing activity
by monoiodoacetic acid, sodium fluoride and arsenite. Results of inhibition experiments
are summarized in Table JV. It can be tentatively concluded that the yeast, the glucose
respiration of which is severely inhibited by these substances, is utilizing the EMP pathway
considerable extents during glucose breakdown
The tested eight kinds of yeasts can be classified into two groups according to the type
of inhibition by monoiodoacetic acid : a) Rhodotorula glutinis, Cardida utilis and
Schizosaccharolnyces pombe, the respiration of which are comparatively insensitive to
monoiodoacetic acid, and b) Saccharomyces sake, Sacchoromyces cerevisiae, Saccharomyces
carlsbergensis, Mycoderma cerevisiae and Torula candida, the respiration of which are
sensitive monoiodoacetic acid inhibition.
Table IV Rate of inhtbltlan of oxidatlon of glucose by varlous InhlZltors
Inhibition rate (olo)
Yeasts
Rhodctorula glutiteis
Can,dida utilis
Schizosaccharomyces pombe
Mycoderma cerevisiae
Saccharof,'.'・yces sake
Saccharomyces cerevisiae
Saccharomvces carlsbergel'zsis
Torula candida
(Inhibitors : concentration in M)
M IA
5 x 10-4 2 x 10 5 x 10
2
l 9
4
84
46
54
60
79
67
49
77
100
57
92
lOO
93
70
81
lOO
lOO
lOO
97
lOO
95
Na F Arsenite
10-2 2 x I O 5 x 10-4
3
5
66
96
5
71
RESPIRATION OF VARIOUS YEASTS 43 In general, inhibition was intense in the cases of yeast with comparatively high RQ
values. However, the latter two genera have comparatively low RQ values and suffered
intense inhibition
The test of inhibition by sodium fluoride was done with Rhodotorula glutinis which
has comparatively low RQ values, and Saccharomyces cerevisiae which has comparatively
high RQ values. The inhibition appeared more ,severely on Saccharomyces cerevisiae
The test of inhibition by arsenite was carried out with Rhodotorula glutinis which has
comparatively low RQ values, ar~d Saccharomyces sake which has comparatively high RQ
values. The effect was heavier on Saccharomyces sake. Therefore, it seems that the
inhibition by these three enzyme inhibitors is more effective In the case of yeasts which
have comparatively high RQ values. It could be considered that probably in the cells
of these yeasts which have comparatively high RQ values the EMP pathway TCA cycle system is predominantly functioning
D. CYTOCHROME COMPONENTS
Clear absorption bands of cytochrome c were observed in the case of all tested yeasts
From these results it is considered that all these yeasts have essentially an ability of respi-
ration. This opinion was supported by the spectrophotometric study reported here. The
strengths of absorption spectra caused by cytochromes are reported to be remarkably
varied depending upon the stage and condition of growih 54) . Nevertheless, under the
90nditions reported, there were no essential differences in the type of absorption spectra
of yeast.
Experimental results obtained may be summarized as follows : the tested nine kinds of
yeasts are divided into at least two main groups. The first group includes Rhodotorula
glutinis. Mycoderma cerevisiae, Candida utilis and Pichia membranaefaciens. The second
group iucludes genus Saccharomyces, viz., Sacchoromyces sake, Saccharomyces cerevisiae and
Saccharomyces carlsbergensis and other two species, Schizosaecharomyces pombe and Torula
cand ida .
The first group has 'the following characteristics : (1) comparatively low RQ values ; (2)
stronger activity of oxidation of glucose than that of ethanol ; (3) the activity of oxi-
dation of the TCA cycle members and gluconate ; (4) relatively low inhibition rate in glucose
oxidation by monoiodoacetic acid, sodium fluoride and arsenite compared with' the case
of the second group. The second group has the following natures : (1) ' comparatively
high RQ values cxcept Torula candida ; (2) stronger activity in respect to the oxidation
of ethanol than that of glucose ; (3) ne*'1igibly small activity of oxi dation o{ the TCA
cycle members and unavailability of, gluconate, except Torula cardida ; (4) relatively high
inhibition rate in glucose oxidation by monoiodoacetic acid, sodium fluoride and arsenite
compared with the case of the first group.
Therefore, these findings seem to indicate that the first group of yeasts utilize,s the
members of the HMP and TCA cycles and acquires the energy by aerobic system. On
the contrary, as to 'the second group of yeasts, the acquirement of energy is rather per-
44 Kazuyoshi NISHIGAMI
formed by the fermentative system
SUlV~M:ARY
Nine strains of yeasts in intact cells were investigated with respect to the oxidation of
exogenously supplied substrates : glucose, arabinose, ethanol, pyruvate, *'1uconate and the
TCA cycle intermediates and the effects of monoiodoacetic acid, sodium fluoride and
arsenite on oxygen uptake and carbon dioxide output
1. In general, yeasts which have comparatively low RQ values oxidized these above
named exogenously supplied substrates more actively than the yeasts with comparatively
high RQ 2. The sensitivity to m(~noiodoacetic acid varies with the kind of yeast. The inhibition
was small on Rhodotorula glutinis and Candida utilis which have comparatively low RQ
values and scarcely ferment. Severe inhibition was observed on genera Saccharomyces
which actively ferment alcohol. The highest inhibition, however, appeared on Mycoderma
cerevisiae which has comparatively high RQ values and on Torula candida which has
comparatively low RQ values
3. Relatively high concentrations of sodium fluoride were needed to inhibit the respi-
ration of yeast. The inhibition was less on Rhodotorula glutinis which has comparatively
low RQ values. contrary to this, Saccharomyces cerevispae which has comparatively high
RQ values is remarkably inhibited
4. Similar effect was observed as a result of the addition of arsenite to yeasts. Rho-
dotorula glutinis was insignificantly inhibited. Saccharomyces sake which has comparatively
high RQ values was greatly inhibited
5. The results obtained by means of microspectroscopic study showed some variations
of absorption bands of cytochromes. However, by the study using a spectrophotometer,
it is found that there are no great differences among cytochrome components of yeast
tested
6. The variations in permeability of the intact cells and the decrease of enzyme activi-
ties may cause the differences in oxidation rate of exogenously supplied substrates and
in sensibility against inhibitors
7. The ability in respect to oxidation of ethano] in Rhodotorula glutinis was adaptively
acquired by preincubation in either ethanol, acetaldehyde or acetate
AC~NO wr.EDGEI~:ENTS
The work reported here was done under the direction of Professor Shoichiro Usami of
the Faculty of Science of Hokkaido University. The author wishes to express his deepest
appreciation to him. To Dr. Kimiko Sasaki, Dr. Shoji Sasaki and Dr. Kazutami Wake
of the Faculty of Science of Hokkaido University, the author is indebted for much valuable
advise and discussion during the course of the study. He expresses his gratitude to Miss
Hiroko Abe and Mr. Sadayuki Sho for their invaluable assistance. Also the author de-
RESPIRATION OF VARIOUS YEASTS 45 sires to express his obligation to Professor Isamu Yokozuka of Yamanas. hi University, Dr
Minoru Yoneyama of Hiroshima University and also to the Institute for Fermentation
Osaka for kindly supplyin*a strains of yeast for this work. Further the author must make
acknowledgement to Professor Shintaro Saito, Dr. Masami Ouji and Mr. Masaru Akiyama
of our Institute for much encouragement rendered during the course of this work. The
author is also indebted to Mr. Yoshihiro Ohgami of the Institute for the preparation of
the photographs of text-figures in this paper
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