compafative physiological studies on the respiration … · gluconic acid in the presence of...

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 .


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。


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


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.


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,


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


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.


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


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


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


scid on C02 Output by washed resting cells of Torula candida. The rear_tion

conditions were the same as those for Fig. 17 .


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-


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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compafative physiological studies on the respiration … · gluconic acid in the presence of...

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