Effects of ethylene on the metabolism of Saccharoinyces cerevisiae K. C. THOMAS' A N D MARYSPENCER Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of British Columbia on 11/18/14 For personal use only.
I)c,prr~~/~~rc,~rr (!/'Plo~rr Sc ic.1rc.c. U~~i~,c,r.siry c!j'AIhc,rrtr. E t l ~ r r o ~ r r A o~ l ~~t .r . Ctr~rtrcltr . T(5GZiVZ
Accepted December I , 1977 -T~-iobr~zs, K. C . , and M. SI'ENCER. 1978. Effects of ethylene on the metabolism o f S t r c c ~ 1 1 t r r o t ~ r y ~ ~ ~ ~ ~ ~ c,rrc~.i.sitr~. C>ui.J . Microbiol. 24: 221-227. Supply of exogenous ethylene to lactate-grown yeast initially accelerated the rate of ethanol production from glucose, but later reduced the rate, with the overall effect being to reduce the total ethanol procluction. The rate of ethanol production by ethylene-treated yeast was not changed by removal of metabolic carbon dioxicle. However, if CO, was allowed to build up in the absence of applied ethylene, the ethanol p~.ocluctiondecreased. Ethylene increased the activities of n number of pentose phosphate and glycolytic pathway enzymes. The largest increase in (EC2.7.1. I I ) . regulatory enzyme oftheglycolytic activity was observecl for phosphofr~~ctokinase pathway. After an initial stini~~lation, glucose (and also 3-0-methyl glucose) ~ ~ p t a kwas e reduced by ethylene. Ethylene appears to inhibit non-competitively the glucose transport system. T ~ I O M AK. S .C . . ct M . S P E N C E R 1978. . Effects of ethylene on the metabolism of.Strc~c1rtrro1~1~v~'e.s c.o.rl.isitrc. Can. J . Microbiol. 24: 222-227. L'aclclition cl'ithylene exogene h des levures c ~ ~ l t i v e esur s un mi lie^^ :KI lactate accelere, au debut, le t a ~ ~dex PI-ocluctiond'ethanol i partir du glucose, mais, plus tard reduit ce taux; I'effet global aete de reduire la production totaled'ithanol. L'elimination du CO, rnetabolique n'affecte pas le taux de production cl'kthanol par les levures traitees h I'ethylkne. Cependant, en I'absence cl'ethylene exogene, le taux de production d'ethanol decroit si on permet au CO, de s'acc~~muler. L'ethylkne augmente I'activite d'un nombre d'enzymes des voies de la glycolyse et du pentose phosphate. La plus forte augmentation d'activite a ete observee avec la phospho-fr~lcto-kinase (EC 2.7.1.1I), Lln enzyme cle regulation de la voie de la glycolyse. Agres Llne stimulation initiale 1'. ab so~ption . . du glucose (ainsi que celle du 3-0-methyl glucose) est reduite par I'ethylene. L'ethylene sembleetre Lln inhibiteurnon-competitif du systkme de transport du glucose. [Traduit par le journal]
Introduction Ethylene, a normal metabolite of ( ~ b 1973) and several microorganisms (Ilga and Curtis 1968; ~~~~i~~ u [ . 1968; ~~~~h 1972) has been recognized as a n important growth regulator i n plants. Even ambient levels of ethylene have affected the physiology and metabolism of plants (Abeles p l r l / , 1971). A~~~~ the various effects of ethylene on plants are stimulation of respiration (Biale 1960), induction of certain enzymes (Ridge and Osborne 1970), alteration of membrane permeability (Irvine and Osborne 1973; Sacher and Salminen 1969), and retardation of mitotic process in the meristems of roots, shoots, and auxiliary buds (Apelbaum and Burg 1972). In contrast to the voluminous literature available metabolism, on the importance of ethylene to very little is known about its effects on rnicroorgan;sms. The fact that most microorganisms under normal growth conditions small amounts of ethylene makes the study of the effects e ~ o ~ e n o ~applied s l y ethylene conlplicated and the results difficult to interpret. However, studies of
the effects of ethylene are facilitated ifcomparisons can ~ lbe ~made ~ with a control in which the test organism does not produce any ethylene. Such a SYstern has been reported by us for the cultivation of S~~c'e.hrrror?~)~ccs c.c)re~~isiar (Thomas and Spencer 1977: Thomas and Spencer, unpublished2). his Yeast did not produce any ethylene while growing with lactate as the carbon source. Underthis condition of growth, the yeast absorbed ethylene from the ~ufloundings.It was suggested then that the absorbed ethylene might affect the metabolism of the Yeast. In the Present study we report some effects of exogenousl~supplied ethylene on the metabolismof S . c'erel'isine.
'Present address: Prairie Regional Laboratory, National Research Council of Canada, University Campus, Saskatoon, Sask., Canada S7N OW9.
'Thomas. K. C.,and M. Spencer. 1978. Evolutionofethylene ~?i.cs as influenced by the carbon by S o ~ ~ . l ~ o r o ~ ~co.cl.isitrc, source for growth and the presence of air. Unpublished.
Materials and Methods Yc,trsr
A haploid strain of S. c,c,rc,~,i.sitrc, X-2180-1B was ~ l s e d throLrghoUt the study. C ~ l l r r f r Co~~tlirio/r.s c
The yeast was grown ;tt 27°C with continuous shaking in a medium of the following composition per litre: potassium lactate, 20.0g; (NH,)ZHPO,. 6.0g; MgS0,.7HZ0, 2.0g: and yeast extract (Difco), 5.0 g. The p~ of the medium was adjusted with KOH or H,PO, to 5.0 at 2YC. Cultivation of the yeast in the presence of exogenously supplied ethylene was done by bub-
223
THOMAS A N D SPENCER
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of British Columbia on 11/18/14 For personal use only.
bling 5terile 100 ppm ethylene in air through the culture at the rate of 100 ml/rnin. I)~,/(,~I~I~II(I~~OII (~/'/~/II(IIIo/ Suitable volumes (0.4 to 4 ml) o f yeast culture were chilletl in ice. and the cells removetl by centrifugation. The supernatant layer was dilutetl with double-tlistilled water and the ethanol content determined enzymirtically by the method tlescribed by Bonnischsen (1965).
I ) r l e , r o ~ i ~ r t ~ lc!f/lrr i o ~ ~ f#i,c./ (!fklrltrl,olic. COz 011 E l l r t r ~ ~l'rool c111c,/io11 Yeast cells obtainetl from the exponenti;~l phase o f growth were washetl twice with clistilled water end suspended in 0.1 .\,1 potassinm phosphate buffer(pH 5.0 at 25°C) containing 5%(w/v) glucose. The suspension was clistributed in 20-ml quantities into twelve 50-ml Hat-bottomed flasks fitted with tight-fitting rubber stoppers that had 2.5-1111plastic syringesatt;lchetl to them. This allowcd snniplingofliquitl from the flasks without opening them. I n some flasks the CO, produced during fermentation of the glucose was trapped by I mi of20% K O H placecl inside the flasks in a scintillation vial. Each flask was flushed for5 min with sterile ethylene in air at the rate o f 200 nll per minute and then tightly closed. The fl;~sks were incubated at room temperature on a reciprocal shaker (60 strokcslmin). At regular intcrvals samples (0.4-0.5 nil) were withdrawn, and the ethanol content in them cleterrninetl enzymatically. I ~ c ~ l ~ ~ t ~ l t l i~l l ~ / ' l~/ ;i /of /l ~ l ~u/)l(l/,c, o,sc~ Exponential phase cells obtained by growing the yeast in lactate medium were washed and suspended in 0. I :\,I potassium phosphate buffer(pH 5.0). Suspensions were aerated for I 0 min by bubbling with air or 100 ppm ethylene in air, at the rate o f 100 nil per minute. Glucose was added in the dry form to the suspension to give a final concentration o f 0.5% (w/v) and the aeration with the gas continued. At definite time intervals aliquots o f the suspension were withd~xwnant1 the amount of glucose reniaining in the suspending medium was determined with glucose oxidase and peroxidase as described by Bergmeyer and Bernt (1965). D r l r r ~ t ~ i ~ ~ c0f3-0-klrrlryl-[Uriio~r IJC] G111c~o.srLll,rtrXr Procedures were the same as those for glucose uptake except that 3-0-methyl L U - ' T I glucose (2.24 pCi/mmol ) ( I Ci = 37 GBq)) was used at concentrations of 1-50 mA4. After 20 niin. 2-ml portions o f the suspension were removed and filtered through a Millipore nienibnlne filter (0.8 pm). The cells on the filter were washed three times with 5-ml portions o f 0.1 M potassium phosphate buffer (pH 5.0 at 2YC) containing 50 m.ld unlabelled 3-0-methyl glucose. The filter was transferred to a scintillation vial, 10 ml of Aquasol was added, ant1 the radioactivity determined in a Nuclear Chicago scintillation counter (model Unilux 11). Pt.c~/~irt.o/io~r (!/' Yi'osl E.\-irc1e.1 t r ~ r iAsset! l c~/'hr;,ytrrc,.v The method described by Maitra and Lobo (1971) for the preparation o f yeast extract and assay ofenzymes was used with some modification. (In the preparation o f the extract the wushcd cell pellet (suspendetl in their medium) was treated with 3 tlrops of toluene and incuba~edfor 30 niin in a37" C water bath. After a preliminary clarification at low speed the suspension was centrifuged at I5 000 for 10 min. The supernatant layer thus obtainod served 21s the enzyme solution.) A l l enzymes were assayed by coupling the particular step to the appropriate N A D or NADP-linked reaction with the use of commercially available coupling enzymes. The rate o f production or disappearance of educed pyridine nucleotides was followed continuously on a Turner model Ill fluorometer fitted with a high sensitivity door. The enzyme solutions were diluted so that the reaction rates followed first-order kinetics.
C'lrc~lr1ic~rti.s
All the common chemicals used in the s t ~ ~ were d y ofanalytical grade and we!-e obtained froni Fisher Scientific Co. The 3-0methyl [U-''CI g l ~ ~ c o sanil c Aquasol wer-c from New England Nuclcar Corp. Biochemicals and enzymes were purchased from Sigma Chemic;rl Co. G;rses were from Linde, and were filteretl through sterile cotton bcfore passage into thc cultures.
t?[ri>c./.~ (/I/(/
Resi~lts o [ E \ - ~ g ~ ~EoI ~ i iI,~~~ P 011I ~ GP l i i c ~ o U/>/t/lic> .~~ E / l ~ ( i i ~f o' i l~ o ( I i / c ~ / i o ~ ~
When the yeast was grown in glucose medium, exogenously applied ethylene had no effect on the rate of uptake of glucose or the total amount of ethanol produced by the organism. S ( i c ~ l r c i r o ~ ~ ~ ? , c ~ ~ ~ c.oi~c~~~i.sicic is known to produce ethylene if the growth medium contains glucose (Thomas and Spencer. i~npublished') and the apparent lack of effect of added ethylene on the glucose-grown yeast may, therefore, be related to the endogenous production of ethylene by the yeast. Since yeast growing with lactate as the carbon source has no measurable ethylene production (Thomas and Spencer, ~~npublished') all other experiments were conducted with lactate-grown yeast. There was no detectable production of ethanol by lactate-grown starved yeast either in the presence or absence of exogenously applied ethylene. However, if glucose (2% w/v) was added to yeast cells suspended in potassium phosphate buffer, measurable amounts of ethanol were detected. The rate of ethanol production from glucose was affected by ethylene treatment (Fig. I ) . The initial rate of production of ethanol was faster in ethylene-treated samples than in the control, but the rate reached a steady value within 60 rnin after the addition of glucose. In the control, on the other hand. the rate continued to increase with time, and by the end of 2 h the rate of production of ethanol in the control was 32% higher than in the ethylenetreated sample. Therefore, the net effect of supplying the yeast with ethylene for a longer term was to decrease ethanol production froni glucose. This is shown clearly in Fig. 2. where results are expressed as 96 of control (no ethylene treatment). &/ji.c.t.s (!/' M o t r r h o l i c C O , t i ~ l t E l .rogo~ori.slj~ Siipp I i o ( 1 E / / ~ ~ ~ l c011 ~ i E~/I(IIIO/ ~c~ P ~ ~ o ( I i i c / i ojb01j1 ~r
Gl//c~o.sc~
Table I shows that the rate ofethanol production by the ethylene-treated yeast was not changed by I-emoval of metabolic COz. On the other hand, if COz was allowed to build up in the absence of applied ethylene, the ethanol production decreased. In either the presence or absence of CO,, treat-
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of British Columbia on 11/18/14 For personal use only.
224
C A N . J . MICROBIOL. VOL. 24, 1978
2 0 0 0 I,ilrn e l h y l e n e n air
0 1 0 0 r i ~ ~e r hl y~l c n e in 88'
0
30
60
90
120
150
MINUTES AFTER T H E ADDITION O F GLUCOSE
0
0
1
2
3
5
TIME (hours)
F I G . 2. Rate of eth:unol production from glucose by Sot.FIG. I. Ethanol protluction by previously starved .Ttrc,- c ~ l r o ~ ~ o ~ i rc.rrc~.;.\ioc, ~ ~ c . c s (X-2 180- 1 B) in presence and absence o f c.lrtrr.o~~r~c~os (~c,rr,~.i.,ictc (X-2 180- 1 B) on addition of gll~cose.Mid- applied ethylene. The results expressed a s a pcrcentage of conlog phase cells oht~iinedby growing the yeast in lactate rnedil~m trol (no ethylene treatment). were starved for 21 h in 0. I .A)/ p o t a s s i ~ ~ phosphate m buffer (pH 5.0) in the presence 01-absence of 100 ppni ethylcne in air. the final rate of ethanol production increased t o Glucose (256) was added and the rate of ethanol production 161% of the initial rate in the presence of metabolic determined. CO, and 167% in its absence. Although similar
rnent of the yeast with ethylene initially increased the rate of ethanol production; then there was a gradual decrease (Table I). With ethylene concentrations of 2000 pprn and I00 ppm the total amounts of ethanol produced per millilitre of yeast suspension were 36 and 34 pmol respectively. (Metabolic CO, did not have a significant effect on total ethanol production in ethylene-treated samples.) In the absence of exogenously applied ethylene the total amounts of ethanol produced per millilitl-e of yeast suspension were 40 pmol (CO, removed) and 36 pmol (CO, not removed). The effects of C 0 2 removal and of exogenous application of ethylene on the rate of production of ethanol are made clear by comparing the ratios of final rate to initial rate (Table 1). When no ethylene was added and the me.tabolic COz was absorbed by KOH, the final rate of ethanol production increased to 683% of the initial rate. When Co, was not removed this increase was only 464% of the initial rate. With an ethylene concentration of 3,000 ppm
effects were observed at the two ethylene concentrations, they were less marked at low than at the high concentration.
E/fk~c./.sc?f'E/h~!lcile 0 1 7 Ei1zyii1~A ( . / i ~ . i / i ~ s Determinations of the activities of the enzymes prepared from yeasts grown with or without exogenously applied ethylene were made under identical conditions. Any increased specific activity is, therefore, taken as an indication of increased enzyme synthesis. (Treating the enzyme preparations with ethylene did not affect their activities.) Res~lltsin Table 3 show that ethylene treatment resulted in increased activity of all enzymes tested. Hexokinase (EC 2.7.1.1) seemed to be affected by ethylene only to a small extent. Maximum increase (60%) was observed with phosphofructokinase (EC 2.7.1.11). a regulatory enzyme of the glycolytic pathway. The specific activities of glucose-6phosphate dehydrogenase (EC 1.1.1.49) and gluconate-6-phosphate dehydrogenase (EC 1.1.1.44) in the yeast were also increased by ethylene.
225
THOMAS A N D SPENCER
TABLE1. Effects of metabolic C 0 2 and exogenously applied ethylene on rate of ethanol production from glucose by S n c c l ~ n r o n i y c e sc e r e u i s i n e (X-2180-1 B)
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of British Columbia on 11/18/14 For personal use only.
(~.lniolethanol/niin.nil-') 10' Concentration of ethylene in air, PPm
Ratio 0-60 niin (initial)
Metabolic C 0 2
120-210 min (final)
Final ratelinitial rate
0
Removed Not removed
5.9 5.8
40.4 27.1
6.83 4.64
100
Removed Not removed
9.3 8.4
25.4 22.4
2.72 2.67
2000
Removed Not removed
9.6 10.2
15.4 16.9
1.61 1.67
NOTE:Midlog phasecells obtained by growing the yeast in lactate medium \%eresuspended in 0.1 ibl pot;issium phosphate b u L r (pH 5.0) containing 5% glucosc. Flasks conta~ning20 In1 o f yeast suspension were fl~lshedwith the indicated concenlrations of ethylene for 5 min, and then sealed. Mc~abolicCO? was absorbed by K O H placed in a scintillation vilil inside the flask. Ethanol content was determined cnzynialically (see Mnterials and Melllods).
TABLE2. Effect of growlng Sncc1101o r r i j ~ c ec~e ec'/sine ~ (X-7180IB) in the plesence of 100 ppln ethylene ~nall on the actlvltles of certaln enzymes nmol substrate converted/ni~nnig-' protetn
Enzyme Hexokinase Phosphofructokinase Glyceraldehyde-3-P dehydrogenase Pyruvate kinase Alcohol dehydrogenasc Glucose-6-P dehydrogenase 6-Phosphogl~~conatc dehydrogenase
Air (control)
100 ppn1 ethylene in air
:
I)c,prr~~/~~rc,~rr (!/'Plo~rr Sc ic.1rc.c. U~~i~,c,r.siry c!j'AIhc,rrtr. E t l ~ r r o ~ r r A o~ l ~~t .r . Ctr~rtrcltr . T(5GZiVZ
Accepted December I , 1977 -T~-iobr~zs, K. C . , and M. SI'ENCER. 1978. Effects of ethylene on the metabolism o f S t r c c ~ 1 1 t r r o t ~ r y ~ ~ ~ ~ ~ ~ c,rrc~.i.sitr~. C>ui.J . Microbiol. 24: 221-227. Supply of exogenous ethylene to lactate-grown yeast initially accelerated the rate of ethanol production from glucose, but later reduced the rate, with the overall effect being to reduce the total ethanol procluction. The rate of ethanol production by ethylene-treated yeast was not changed by removal of metabolic carbon dioxicle. However, if CO, was allowed to build up in the absence of applied ethylene, the ethanol p~.ocluctiondecreased. Ethylene increased the activities of n number of pentose phosphate and glycolytic pathway enzymes. The largest increase in (EC2.7.1. I I ) . regulatory enzyme oftheglycolytic activity was observecl for phosphofr~~ctokinase pathway. After an initial stini~~lation, glucose (and also 3-0-methyl glucose) ~ ~ p t a kwas e reduced by ethylene. Ethylene appears to inhibit non-competitively the glucose transport system. T ~ I O M AK. S .C . . ct M . S P E N C E R 1978. . Effects of ethylene on the metabolism of.Strc~c1rtrro1~1~v~'e.s c.o.rl.isitrc. Can. J . Microbiol. 24: 222-227. L'aclclition cl'ithylene exogene h des levures c ~ ~ l t i v e esur s un mi lie^^ :KI lactate accelere, au debut, le t a ~ ~dex PI-ocluctiond'ethanol i partir du glucose, mais, plus tard reduit ce taux; I'effet global aete de reduire la production totaled'ithanol. L'elimination du CO, rnetabolique n'affecte pas le taux de production cl'kthanol par les levures traitees h I'ethylkne. Cependant, en I'absence cl'ethylene exogene, le taux de production d'ethanol decroit si on permet au CO, de s'acc~~muler. L'ethylkne augmente I'activite d'un nombre d'enzymes des voies de la glycolyse et du pentose phosphate. La plus forte augmentation d'activite a ete observee avec la phospho-fr~lcto-kinase (EC 2.7.1.1I), Lln enzyme cle regulation de la voie de la glycolyse. Agres Llne stimulation initiale 1'. ab so~ption . . du glucose (ainsi que celle du 3-0-methyl glucose) est reduite par I'ethylene. L'ethylene sembleetre Lln inhibiteurnon-competitif du systkme de transport du glucose. [Traduit par le journal]
Introduction Ethylene, a normal metabolite of ( ~ b 1973) and several microorganisms (Ilga and Curtis 1968; ~~~~i~~ u [ . 1968; ~~~~h 1972) has been recognized as a n important growth regulator i n plants. Even ambient levels of ethylene have affected the physiology and metabolism of plants (Abeles p l r l / , 1971). A~~~~ the various effects of ethylene on plants are stimulation of respiration (Biale 1960), induction of certain enzymes (Ridge and Osborne 1970), alteration of membrane permeability (Irvine and Osborne 1973; Sacher and Salminen 1969), and retardation of mitotic process in the meristems of roots, shoots, and auxiliary buds (Apelbaum and Burg 1972). In contrast to the voluminous literature available metabolism, on the importance of ethylene to very little is known about its effects on rnicroorgan;sms. The fact that most microorganisms under normal growth conditions small amounts of ethylene makes the study of the effects e ~ o ~ e n o ~applied s l y ethylene conlplicated and the results difficult to interpret. However, studies of
the effects of ethylene are facilitated ifcomparisons can ~ lbe ~made ~ with a control in which the test organism does not produce any ethylene. Such a SYstern has been reported by us for the cultivation of S~~c'e.hrrror?~)~ccs c.c)re~~isiar (Thomas and Spencer 1977: Thomas and Spencer, unpublished2). his Yeast did not produce any ethylene while growing with lactate as the carbon source. Underthis condition of growth, the yeast absorbed ethylene from the ~ufloundings.It was suggested then that the absorbed ethylene might affect the metabolism of the Yeast. In the Present study we report some effects of exogenousl~supplied ethylene on the metabolismof S . c'erel'isine.
'Present address: Prairie Regional Laboratory, National Research Council of Canada, University Campus, Saskatoon, Sask., Canada S7N OW9.
'Thomas. K. C.,and M. Spencer. 1978. Evolutionofethylene ~?i.cs as influenced by the carbon by S o ~ ~ . l ~ o r o ~ ~co.cl.isitrc, source for growth and the presence of air. Unpublished.
Materials and Methods Yc,trsr
A haploid strain of S. c,c,rc,~,i.sitrc, X-2180-1B was ~ l s e d throLrghoUt the study. C ~ l l r r f r Co~~tlirio/r.s c
The yeast was grown ;tt 27°C with continuous shaking in a medium of the following composition per litre: potassium lactate, 20.0g; (NH,)ZHPO,. 6.0g; MgS0,.7HZ0, 2.0g: and yeast extract (Difco), 5.0 g. The p~ of the medium was adjusted with KOH or H,PO, to 5.0 at 2YC. Cultivation of the yeast in the presence of exogenously supplied ethylene was done by bub-
223
THOMAS A N D SPENCER
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of British Columbia on 11/18/14 For personal use only.
bling 5terile 100 ppm ethylene in air through the culture at the rate of 100 ml/rnin. I)~,/(,~I~I~II(I~~OII (~/'/~/II(IIIo/ Suitable volumes (0.4 to 4 ml) o f yeast culture were chilletl in ice. and the cells removetl by centrifugation. The supernatant layer was dilutetl with double-tlistilled water and the ethanol content determined enzymirtically by the method tlescribed by Bonnischsen (1965).
I ) r l e , r o ~ i ~ r t ~ lc!f/lrr i o ~ ~ f#i,c./ (!fklrltrl,olic. COz 011 E l l r t r ~ ~l'rool c111c,/io11 Yeast cells obtainetl from the exponenti;~l phase o f growth were washetl twice with clistilled water end suspended in 0.1 .\,1 potassinm phosphate buffer(pH 5.0 at 25°C) containing 5%(w/v) glucose. The suspension was clistributed in 20-ml quantities into twelve 50-ml Hat-bottomed flasks fitted with tight-fitting rubber stoppers that had 2.5-1111plastic syringesatt;lchetl to them. This allowcd snniplingofliquitl from the flasks without opening them. I n some flasks the CO, produced during fermentation of the glucose was trapped by I mi of20% K O H placecl inside the flasks in a scintillation vial. Each flask was flushed for5 min with sterile ethylene in air at the rate o f 200 nll per minute and then tightly closed. The fl;~sks were incubated at room temperature on a reciprocal shaker (60 strokcslmin). At regular intcrvals samples (0.4-0.5 nil) were withdrawn, and the ethanol content in them cleterrninetl enzymatically. I ~ c ~ l ~ ~ t ~ l t l i~l l ~ / ' l~/ ;i /of /l ~ l ~u/)l(l/,c, o,sc~ Exponential phase cells obtained by growing the yeast in lactate medium were washed and suspended in 0. I :\,I potassium phosphate buffer(pH 5.0). Suspensions were aerated for I 0 min by bubbling with air or 100 ppm ethylene in air, at the rate o f 100 nil per minute. Glucose was added in the dry form to the suspension to give a final concentration o f 0.5% (w/v) and the aeration with the gas continued. At definite time intervals aliquots o f the suspension were withd~xwnant1 the amount of glucose reniaining in the suspending medium was determined with glucose oxidase and peroxidase as described by Bergmeyer and Bernt (1965). D r l r r ~ t ~ i ~ ~ c0f3-0-klrrlryl-[Uriio~r IJC] G111c~o.srLll,rtrXr Procedures were the same as those for glucose uptake except that 3-0-methyl L U - ' T I glucose (2.24 pCi/mmol ) ( I Ci = 37 GBq)) was used at concentrations of 1-50 mA4. After 20 niin. 2-ml portions o f the suspension were removed and filtered through a Millipore nienibnlne filter (0.8 pm). The cells on the filter were washed three times with 5-ml portions o f 0.1 M potassium phosphate buffer (pH 5.0 at 2YC) containing 50 m.ld unlabelled 3-0-methyl glucose. The filter was transferred to a scintillation vial, 10 ml of Aquasol was added, ant1 the radioactivity determined in a Nuclear Chicago scintillation counter (model Unilux 11). Pt.c~/~irt.o/io~r (!/' Yi'osl E.\-irc1e.1 t r ~ r iAsset! l c~/'hr;,ytrrc,.v The method described by Maitra and Lobo (1971) for the preparation o f yeast extract and assay ofenzymes was used with some modification. (In the preparation o f the extract the wushcd cell pellet (suspendetl in their medium) was treated with 3 tlrops of toluene and incuba~edfor 30 niin in a37" C water bath. After a preliminary clarification at low speed the suspension was centrifuged at I5 000 for 10 min. The supernatant layer thus obtainod served 21s the enzyme solution.) A l l enzymes were assayed by coupling the particular step to the appropriate N A D or NADP-linked reaction with the use of commercially available coupling enzymes. The rate o f production or disappearance of educed pyridine nucleotides was followed continuously on a Turner model Ill fluorometer fitted with a high sensitivity door. The enzyme solutions were diluted so that the reaction rates followed first-order kinetics.
C'lrc~lr1ic~rti.s
All the common chemicals used in the s t ~ ~ were d y ofanalytical grade and we!-e obtained froni Fisher Scientific Co. The 3-0methyl [U-''CI g l ~ ~ c o sanil c Aquasol wer-c from New England Nuclcar Corp. Biochemicals and enzymes were purchased from Sigma Chemic;rl Co. G;rses were from Linde, and were filteretl through sterile cotton bcfore passage into thc cultures.
t?[ri>c./.~ (/I/(/
Resi~lts o [ E \ - ~ g ~ ~EoI ~ i iI,~~~ P 011I ~ GP l i i c ~ o U/>/t/lic> .~~ E / l ~ ( i i ~f o' i l~ o ( I i / c ~ / i o ~ ~
When the yeast was grown in glucose medium, exogenously applied ethylene had no effect on the rate of uptake of glucose or the total amount of ethanol produced by the organism. S ( i c ~ l r c i r o ~ ~ ~ ? , c ~ ~ ~ c.oi~c~~~i.sicic is known to produce ethylene if the growth medium contains glucose (Thomas and Spencer. i~npublished') and the apparent lack of effect of added ethylene on the glucose-grown yeast may, therefore, be related to the endogenous production of ethylene by the yeast. Since yeast growing with lactate as the carbon source has no measurable ethylene production (Thomas and Spencer, ~~npublished') all other experiments were conducted with lactate-grown yeast. There was no detectable production of ethanol by lactate-grown starved yeast either in the presence or absence of exogenously applied ethylene. However, if glucose (2% w/v) was added to yeast cells suspended in potassium phosphate buffer, measurable amounts of ethanol were detected. The rate of ethanol production from glucose was affected by ethylene treatment (Fig. I ) . The initial rate of production of ethanol was faster in ethylene-treated samples than in the control, but the rate reached a steady value within 60 rnin after the addition of glucose. In the control, on the other hand. the rate continued to increase with time, and by the end of 2 h the rate of production of ethanol in the control was 32% higher than in the ethylenetreated sample. Therefore, the net effect of supplying the yeast with ethylene for a longer term was to decrease ethanol production froni glucose. This is shown clearly in Fig. 2. where results are expressed as 96 of control (no ethylene treatment). &/ji.c.t.s (!/' M o t r r h o l i c C O , t i ~ l t E l .rogo~ori.slj~ Siipp I i o ( 1 E / / ~ ~ ~ l c011 ~ i E~/I(IIIO/ ~c~ P ~ ~ o ( I i i c / i ojb01j1 ~r
Gl//c~o.sc~
Table I shows that the rate ofethanol production by the ethylene-treated yeast was not changed by I-emoval of metabolic COz. On the other hand, if COz was allowed to build up in the absence of applied ethylene, the ethanol production decreased. In either the presence or absence of CO,, treat-
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of British Columbia on 11/18/14 For personal use only.
224
C A N . J . MICROBIOL. VOL. 24, 1978
2 0 0 0 I,ilrn e l h y l e n e n air
0 1 0 0 r i ~ ~e r hl y~l c n e in 88'
0
30
60
90
120
150
MINUTES AFTER T H E ADDITION O F GLUCOSE
0
0
1
2
3
5
TIME (hours)
F I G . 2. Rate of eth:unol production from glucose by Sot.FIG. I. Ethanol protluction by previously starved .Ttrc,- c ~ l r o ~ ~ o ~ i rc.rrc~.;.\ioc, ~ ~ c . c s (X-2 180- 1 B) in presence and absence o f c.lrtrr.o~~r~c~os (~c,rr,~.i.,ictc (X-2 180- 1 B) on addition of gll~cose.Mid- applied ethylene. The results expressed a s a pcrcentage of conlog phase cells oht~iinedby growing the yeast in lactate rnedil~m trol (no ethylene treatment). were starved for 21 h in 0. I .A)/ p o t a s s i ~ ~ phosphate m buffer (pH 5.0) in the presence 01-absence of 100 ppni ethylcne in air. the final rate of ethanol production increased t o Glucose (256) was added and the rate of ethanol production 161% of the initial rate in the presence of metabolic determined. CO, and 167% in its absence. Although similar
rnent of the yeast with ethylene initially increased the rate of ethanol production; then there was a gradual decrease (Table I). With ethylene concentrations of 2000 pprn and I00 ppm the total amounts of ethanol produced per millilitre of yeast suspension were 36 and 34 pmol respectively. (Metabolic CO, did not have a significant effect on total ethanol production in ethylene-treated samples.) In the absence of exogenously applied ethylene the total amounts of ethanol produced per millilitl-e of yeast suspension were 40 pmol (CO, removed) and 36 pmol (CO, not removed). The effects of C 0 2 removal and of exogenous application of ethylene on the rate of production of ethanol are made clear by comparing the ratios of final rate to initial rate (Table 1). When no ethylene was added and the me.tabolic COz was absorbed by KOH, the final rate of ethanol production increased to 683% of the initial rate. When Co, was not removed this increase was only 464% of the initial rate. With an ethylene concentration of 3,000 ppm
effects were observed at the two ethylene concentrations, they were less marked at low than at the high concentration.
E/fk~c./.sc?f'E/h~!lcile 0 1 7 Ei1zyii1~A ( . / i ~ . i / i ~ s Determinations of the activities of the enzymes prepared from yeasts grown with or without exogenously applied ethylene were made under identical conditions. Any increased specific activity is, therefore, taken as an indication of increased enzyme synthesis. (Treating the enzyme preparations with ethylene did not affect their activities.) Res~lltsin Table 3 show that ethylene treatment resulted in increased activity of all enzymes tested. Hexokinase (EC 2.7.1.1) seemed to be affected by ethylene only to a small extent. Maximum increase (60%) was observed with phosphofructokinase (EC 2.7.1.11). a regulatory enzyme of the glycolytic pathway. The specific activities of glucose-6phosphate dehydrogenase (EC 1.1.1.49) and gluconate-6-phosphate dehydrogenase (EC 1.1.1.44) in the yeast were also increased by ethylene.
225
THOMAS A N D SPENCER
TABLE1. Effects of metabolic C 0 2 and exogenously applied ethylene on rate of ethanol production from glucose by S n c c l ~ n r o n i y c e sc e r e u i s i n e (X-2180-1 B)
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of British Columbia on 11/18/14 For personal use only.
(~.lniolethanol/niin.nil-') 10' Concentration of ethylene in air, PPm
Ratio 0-60 niin (initial)
Metabolic C 0 2
120-210 min (final)
Final ratelinitial rate
0
Removed Not removed
5.9 5.8
40.4 27.1
6.83 4.64
100
Removed Not removed
9.3 8.4
25.4 22.4
2.72 2.67
2000
Removed Not removed
9.6 10.2
15.4 16.9
1.61 1.67
NOTE:Midlog phasecells obtained by growing the yeast in lactate medium \%eresuspended in 0.1 ibl pot;issium phosphate b u L r (pH 5.0) containing 5% glucosc. Flasks conta~ning20 In1 o f yeast suspension were fl~lshedwith the indicated concenlrations of ethylene for 5 min, and then sealed. Mc~abolicCO? was absorbed by K O H placed in a scintillation vilil inside the flask. Ethanol content was determined cnzynialically (see Mnterials and Melllods).
TABLE2. Effect of growlng Sncc1101o r r i j ~ c ec~e ec'/sine ~ (X-7180IB) in the plesence of 100 ppln ethylene ~nall on the actlvltles of certaln enzymes nmol substrate converted/ni~nnig-' protetn
Enzyme Hexokinase Phosphofructokinase Glyceraldehyde-3-P dehydrogenase Pyruvate kinase Alcohol dehydrogenasc Glucose-6-P dehydrogenase 6-Phosphogl~~conatc dehydrogenase
Air (control)
100 ppn1 ethylene in air
:
