ABSTRACT. Glucose-induced acid extrusion, respiration and anaerobic fermentation in baker's yeast was studied with the aid of sixteen inhibitors. Uranyl(2+) ...
Folia Microbiol. 23, 409--422 (1978)
Effect of Inhibitors on Acid Production by Baker's Yeast K. SIGLER, A. KSTOTKOVXand A. KOTYK Laboratory for Cell Membrane Transport, Institute of Microbiology, Czechoslovak Academy of Sciences, 142 20 Prague 4
Received February 21, 1978
ABSTRACT. Glucose-induced acid extrusion, respiration a n d anaerobic fermentation in baker's yeast was studied with the aid of sixteen inhibitors. U r a n y l ( 2 + ) nitrate affected the acid extrusion more anaerobically t h a n aerobically; the complexing of Mg u+ a n d Ca 2+ b y E D T A a t the membrane h a d no effect. Intdbitors of glycolysis (iodoaeetamide, :N-ethylmaleimide, fluoride) suppressed acid production markedly, a n d so did the phosphorylation-blocking arsenate. Ftuoroacetate, inhibiting the citric-acid cycle, h a d no effect. Inhibition b y uncouplers depended on their pKa values: 2,4,6-trinitrophenol (pKa 0.4) < 2,4-dinitrophenol (4.1) < azide (4.7) < 3-ehlorophenylhydrazonomalononitrfle (6.0). Inhibition b y trinitrophenol was only slightly increased b y its acetylation. Cyanide and n o n p e r m e a n t oligomycin showed practically no effect; inhibition b y dieyclohexylcarbodlimlde was delayed b u t potent. The concentration profiles of inhibition of acid production differed from those of respiration a n d fermentation. Thus, t h o u g h the acid production is a metabolically dependent process, it does not reflect the intensity of metabolism, except partly in the first half of glyeolysis.
As a part of the study of proton gradients in yeast, the effect of a number of inhibitors on acid production was determined under both aerobic and anaerobic conditions and compared with their effect on respiration (Qo2) and anaerobio fermentation (Q~o,)- The inhibitors were chosen so as to affect the complex process of acid extrusion at various levels and provide information on the origin and routes of extruded protons inside the cell. MATERIALS AND METHODS
Yeast suspension. Commercial pressed baker's yeast from the Kolin distillery was used. The yeast was suspended in distilled water, washed three times by cen~rifugation, the resulting suspension was aerated under vigorous stirring for 3 h to obtain uniformly starved cells and left overnight at 4 ~ Measurements were usually done with 10 % (wet weight) suspenslon containing about 20 mg cell dry weight per ml, in distilled water at 30 ~ Chemicals. Glucose (Lachema, medicinal purity) was used as a 1 ~ solution in distilled water. Other agents included ethanol (UV-speetroscopie purity), ethylenediaminetetraacetic acid (EDTA, disodium salt), sodium fluoride, azide, arsenate, and cyanide, 2,4-dinitrophenol (DNP), all from Lachema; iodoacetamide (IAA) and N-ethylmaleimide (NEM), both from Koch-Light; nranyl(2+) nitrate (Chemapol), 2,4,6-trinitrophenol (TNP, K. ~enk Co., Czechoslovakia)., dieyclohexylcarbodiimide (DCCD, BHD Chemicals), 3-chlorophenylhydrazonomatononitrile (carbonylcyanide
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m-chlorophenylhydrazone, CCCP, Calbiochem), oligomycin (Serva), 2,4,6-trinitrophenyl acetate (Ac-TNP, prepared b y Dr. S. J a n d a of this laboratory), and sodium fluoroacetate (Sigma). Inhibitors were prepared in stock solutions (freshly before each experiment if needed) the p H of which was usually adjusted to 4.0 -- 5.0 to prevent any spurious effects of the agent's own acidity on metabolicallyinduced acidification of the yeast suspension. K C N solutions were adjusted to higher alkalinity to prevent losses b y volatilization of HCN. Measurements of p H and inhibitor application. The measurements were done on a universal digital p H - m e t e r from the Developmental Workshops (Czechoslovak A c a d e m y of Sciences), with an attached LP-EZ1 recorder, the sensor being a combined glass/calomel electrode L 15 (A.H.T., Philadelphia, USA). Suspension volume was in all cases 9 ml; to induce acidification, 1 ml 1 M glucose was added. The final glucose concentration (100 m~) ensured a prolonged measurement without the risk of glucose exhaustion. The typical acidification curve after glucose was evalm-~ed as described in Fig. 1. The apparent total a m o u n t of extruded protons (A[H*]), obtained from the difference between initial and final p H (cf. Fig. 1), the initial acidification rate (ARI) and the acidification rate at the moment of the steepest acidification (ARII) were calculated from recorder traces, referred to 1 1 suspension, and their values in the presence of inhibitors expressed in percent of the inhibitor-free control. I f inhibitor addition caused a transient p H drop or rise (especially in phase II), the altered stope of the acidification curve was determined after the fluctuation. Aerobic and anaerobic conditions. Aeration of the suspension was ensured b y magnetic stirring throughout the experiment; anaerobic conditions were imposed b y 1-min thorough flushing of the suspension with argon from a cylinder prior to measurement and then a continuous blowing of the gas (containing about 0.4 ppm 0~) on the surface of the suspension. The measurements in air and in the argon atmosphere were carried out in a given suspension in close succession to ensure adequate comparison. Manometric measurements. Glucose respiration or anaerobic fermentation was determined manometrically on a Warburg apparatus at 30 ~ The thickness of the suspension was about half that used for p H measurements. Inhibitors were added simultaneously with glucose from a manometric vessel side-arm and the measurement was carried out for 60 rain. Anaerobic conditions were ensured b y flushing the vessels with argon and keeping a stick of white phosphorus in the central well. I~ESULTS
Tables I - - I V and Figs. 2 -- 5 summarize our results. It should be noted that in most cases the concentrations of inhibitors affecting perceptibly acid extrusion were higher than those reported as effective in metabolic studies (cf. e.g. Stickland I956a, b; Rothstein 1954; V a m b u t a s and Racker 1965). The high concentrations were used to achieve effective intracellular levels, especially with acidic agents of a low p K (picrate, fluoroacetate). Further, if the pKa of the agent was below the p H usually attained in the suspension after glucose addition (about 3.6), the inhibition was negligible. Pertinent pKa values are therefore given when possible to provide information on the actual concentration of the membrane-penetrating undissociated form at experimental p H values. The inhibitors included agents which do not penetrate into the cell and are active only at the membrane (uranyl ions, EDTA), agents affecting chiefly the glycolytie p a t h w a y (fluoride, iodoacetamide, N-ethylmaleimide, arsenate), and substances act-
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F~o. I. E v a l u a t i o n o f acidification c u r v e ; a t y p i c a l acidification c u r v e w a s d i v i d e d into p h a s e s I (spont a n e o u s p i t r e n d before s u b s $ r a t e a d d i t i o n , p H 5.76 -V 0.22, 30 m e a s u r e m e n t s ) , IX (surge o f p r o t o n s f r o m t h e cells a f t e r s u b s t r a t e addition) a n d I i I (levelling-off a t u l t i m a t e p H level m a i n t a i n e d u n t i l s u b s t r a f e e x h a u s t i o n ) . A[I-I§ = [ I t + I r a - [H+]I, a p p a r e n t t o t a l a m o u n t of p r o t o n s e x t r u d e d ( ~ 0 1 / 1 suspension); ai/bi = A R i - a c i d i f i c a t i o n r a t e d e t e r m i n e d i m m e d i a t e l y a f t e r c u r v e " l i n e a x i z a t i o n " ( ~ m o l 1-1 rain -1 o f H+); aii/biz = ARii-acidification r a t e m e a s u r e d (only in t h e p r e s e n c e o f inhibitor) f r o m t h e inflection p o i n t on (~mol 1-1 rain -1 of H+); I, II, Ill i n circles, a d d i t i o n o f inhibitor. FIG. 2. E f f e c t of i n h i b i t o r s of ARk a n d ARII; t h i n solid line, line, i n h i b i t o r a d d e d in p h a s e fluoroacetate, K C N p o t a s s i u m
g r o u p A o n acidification. T h e a g e n t s h a v e n o effect on A [ H +] b u t r e d u c e inhibitor a d d e d in p h a s e I; dashed line, i n h i b i t o r a d d e d in p h a s e I I ; dotted XII; EDTA e t h y l e n e d i a m i n e t e t r a a c e t i c acid, T N P 2,4,6-trinitrophenol; FAC cyanide.
ing on mitochondria-located reactions -- the citric-acid cycle, respiratory chain, and mitochondrial ATPase (fluoroacetate, trinitophenol, trinitrophenyl acetate, dinitrophenol, azide, cyanide, CCCP, DCCD, oligomycin). The inhibitory effect of ethanol used as a solvent for some inhibitors was also studied.
Inhibition pattern According to the type of effect produced, the inhibitors can be divided into several groups (Figs. 2--4). Group A. These agents reduce the rate but not the extent of acidification (cf. Fig. 2). Group B. These inhibitors cause both a slowing-down of acidification and a suppression of its extent (Fig. 3). Inhibition occurs immediately on addition. Group C. The agents do not affect the rate but strongly inhibit the extent of acidification (Fig. 4). They act with a lag of several minutes. One should be aware t h a t the curves in Figs. 2--4 in no case reflect the magnitude of inhibition.
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SIGLER ET AL.
Vol. 23
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Fzo. 3. Effect of inhibitors of group B on acidification. Agents reduce b o t h A [ H +] and ARz a n d A R n immediately on addition; thin solid line, agent added in p h a s e I ; dashed line, agent added in phase I I ; dotted line, agent added in phase I I I ; UO2 u+ u r a n y l ( 2 + ) n i t r a t e , As-OH arsenate, NaF fluoride, Ac-TNP 2,4,6trinitrophenyl acetate, DCCD dicyclohexylearbodiimide, EtOH ethanol, DNP 2,4-dinitrophenol, CCCP 3-chlorophenylhydrazonomalononitrile, NaNs sodium azide. FIG. 4. Effect of inhibitors of group C on acidification; agents have no effect on A R I a n d ARII b u t r e d u c e A[H+] with a lag of several minutes; thin solid line, a g e n t a d d e d in phase I ; dashed line, agent added in phase I I ; dotted line, agent added in phase I I I ; NEH N-ethylmaleimide, IAA iodoacetamide.
Membrane-active inhibitors Uranyl(2+) ions. As with most other inhibitors, the pH of uranyl nitrate was adjusted to values about halfway down the expected pH drop. Its effect on acid production included both a slowing-down of the acidification rates and a reduction of A[I-I+] (Fig. 3). The extent of inhibition, in contrast to the data of Rothstein (1954), was not proportional to concentration since a two-fold concentration rise caused a shift from 0 to 100 % inhibition. U n d e r anaerobic conditions the acid production w a s inhibited completely by 0.5 m• uranyl whereas aerobically no inhibition occurred up to 1.0 m ~ uranyl, lower concentrations actually causing a 20 to 30 ~/o stimulation. This resembles the situation found with the inhibition of glucose fermentation and oxidation (l~othstein 1954) and thus seems to implicate different types of glucose binding sites in the plasma m e m b r a n e under the two sets of conditions. I f added in phase III, i.e. when a q u a n t i t y of glucose had already been taken up b y the cells, uranyl exhibited much lower effect t h a n in phases I and II. Ethylenediaminetetraacetic acid. E D T A (pKi ---- 6.27, pKe----10.95 at 25 ~ cf. Rappoport 1967) exhibited in all circumstances an effect of type A (Fig. 2), reducing ARz and A R n by 60--70 %; the extent of inhibition of A[H +] varied from experiment to experiment, being in all cases less t h a n 10 %. I f anything, E D T A stimulated Qo, and Qc~o,. Since E D T A probably does not penetrate into the cells, the
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