Effect of hydrogen peroxide on sugar transport in - Springer Link

Folia Microbiol. 38 (2), 135 - 140 (1993)

Effect of Hydrogen Peroxide on Sugar Transport in Schizosaccharomycespombe. Absence of Membrane Lipid Peroxidation S. J A N D A a, G . G I L L E b, K . S I G L E R a a n d M . H t 3 F E R b

alnstitute of Microbiology, Academy of Sciences of tBe Czech Republic, 142 20 Prague 4, Czech Republic bInstitute of Botany Universityof Bonn, 5300Bonn, Germany Received January 14, 1993 Revised version February 16, 1993

ABSTRACT. Stationary unaerated cells of S. pombe containing endogenous substrates but not energized by any exogenous ones take up 2-deoxy-D-glucose, 6-deoxy-D-glucose, D-xylose and D-arabinose actively over diffusion equilibrium. The active uptake is inhibited by 20-100 mmol/L H202 which causes an increase in KT but has no effect on Jmax. This "competitive inhibition" indicates that H202 affects directly the sugar binding sites of the transportcrs. The ATP-binding site of the plasma membrane H+-ATPase is also affected by 100 mmol/L 11202; the KT decreases 7-fold, Jmax about 2.5-fold. These effects are not likely to be mediated by membrane lipid peroxidation which appears to be lacking in S. pombe, and this lack may be one of the reasons for the high resistance of this yeast to H202. Because of this S. pombe represents a suitable system for studying direct effects of oxidants on membrane proteins.

The potent oxidant hydrogen peroxide, which is a source of the highly reactive hydroxyl radical HO', has multiple effects on yeasts. Previous studies showed an activation of the plasma membrane H+-ATPase (Sigler and H6fer 1991) and a concentration-dependent stimulation or inhibition of amino acid uptake in Saccharomyces cerevisiae (Sigler et aL 1990), inhibition of cell reproduction and sugar uptake in the obligately aerobic yeast Rhodotomla gluthtis (Janda et al. 1990), and in both S. cerevisiae and Schizosaccharomyces pombe effects on membrane fluidity (Gille et al. 1993). The last species, a facultatively aerobic fission yeast with high and stable catalase activity and a tight control of endogenous respiration (Gille et al. 1993) was used to study the effects of H202 on sugar-H + symport in yeast cells.

MATERIALS

AND

METHODS

Growth. The yeast strain under study was Schizosaccharomyces pombe 972h- (NCYC 1824). For permanent storage it was kept on agar plates containing 1 % yeast extract, 2 % bactopeptone and 2 % glucose. For experiments it was cultured for 22 h at 30 ~ in a minimal medium (Gutz et al. 1974) containing 3 % glucose. The harvested cells were washed three times with distilled water and stored at 4 ~ for up to 1 h. Just before the experiment they were resuspended in 0.3 mol/L phosphate buffer (pH 4.5) to a concentration of 1 - 3 mg dry matter per mL. Measurement of sugar uptake. Initial rates of sugar uptake and accumulation in cells were measured as described previously (Janda 1979) using Schleicher & Schuell membrane filters with 0.8/.tm pore diameter. Consumption of U-14C-D-glucose was determined from sugar radioactivity in suspension supernatants. Concentration of sugars in cell water was calculated for cell water content of 2 mL/g dry matter. Development of ]4CO2 was determined according to Janda et al. (1990). Concentration of H202 was determined spectrophotometrically by the hosreradish peroxidase-coupled oxidation method of Frew et aL (1983). Measurement of membrane A TPase activiO,. Plasma membrane fragments isolated according to Dufour et al. (1988) were incubated at room temperature for 10 min with 100 mmol/L H202 and the ATPase activity was determined by measuring the amount of inorganic phosphate released from added ATP at 30 ~ according to Dufour et al. (1988). Measurement of endogenous respiration. The respiration was measured in 10 mL of a 2.5 % (fresh matter per volume) cell suspension in a closed plastic thermostated cuvette at 30 ~ using a Clark-type O2 electrode (Beckman). Before measurement the cells were aerated for various periods at the same temperature on a magnetic stirrer. The reduction in 02 level due to cell respiration was recorded and expressed as percentage of the O2 saturation in oxygen-supplied water (set at 100 %).

136

S. JANI)A ct al.

Vol. 38

Measttrcment of lipid peroxidation. Lipid pcroxidation was measured using the thiobarbituric acid assay described by Buege and Aust (1978). A samplc of isolated membranes contained 0.30-0.75 mg membrane protcin in a volume of 1 mL. For induction of lipid peroxidation the sample was incubated for 20 min at room temperature with 20, 50 or 80 mmol/L H 2 0 2 . Since the formation of the red complex arising in the reaction between thiobarbituric acid and the end product of lipid peroxidation, viz. malondialdehyde, is inhibited by H202 the sample was preincubated before the measurement for 5 rain with beef liver catalase with a final activity of 108 nkat to destroy the added H202. Peroxidation of arachidonic acid was carried in the same way with 1 mg arachidonic acid in i mL sample volume. 1,1,3,3-Tetraethoxypropane was used as external standard. Chemicals. 6-Deoxy-D-glucose and 4-aminoantipyrine were from Sigma, 2-deoxy-D-glucose and horseradish peroxidase from Serva, D-xylose was purchased from Koch-Light and phenol from Merck. Catalase was purchased from Boehringer (Mannheim), arachidonic acid and 1,1,3,3-tetraethoxypropane from Sigma. All other chemicals were from Lachema (Czech Republic) and were of reagent grade purity. 3H-2-Deoxy-D-glucose was from Amersham, 14C-labelled D-xylese, D-arabinose and D-glucose were from the blstitute of Research, Production and Application of Radioisotopes, Prague. 14C-6-Deoxy-D-glucose was prepared at the bzstitute of Nuclear Biology and Radiochemistry, Prague (cf. Kotyk et al. 1975).

RESULTS

AND

DISCUSSION

When H202 is added to cell suspension the catalase(s) localized within the cells, on their surface and also extruded into the medium (Gille et al. 1993) split it to water and oxygen. A relative peroxide-to-c~ll concentration ratio had to be found that would ensure sufficient peroxide concentration to be present but would not cause a complete respiration induced depletion of endogenous energy sources during the experimental interval (mostly 40-60 min). A suitable suspension density was found to be 0.75-2.0 mg dry matter per mL (Fig. 1) at which both 20 and 100 mmol/L H202 remained in the suspension for sufficiently long intervals whereas the endogenous cell reserves were still sufficient to support respiration and transport (Fig. 2).

I

100'

I

!

II

i

10

'

I

R 60

,

60 5 40

20

I

.5

~

10

15

II rain

^

60

Fig. 1. Decomposition of added H202 in S. pombe suspensions of different density; A, H202 concentration (circles, 100 m m o l / L triangles, 20 mmol/L); open symbols, 3.5 mg dry matter per mL, full symbols, 0.75 mg dry matter per mL.

0

2.5

5.0

Fig. 2. Initial rates of endogenous respiration of

S. pombe after various times of aeration without substrates; R, respiration rate (nmol 02 per mg dry matter per min). Individual symbols represent results of five independent experiments.

In the few available studies sugar uptake in a S. pombe respiratory mutant COB5 was found to be saturable and dependent on both ATP and the transmembrane pH gradient (Foury and Goffeau 1975; Goffeau et al. 1975). H6fer and Nassar (1987) demonstrated the existence in this yeast of a glucose transport system (GTS) which transports D-glucose, 2-deoxy-D-glucose and glucosamine by H+-symport and causes their intracellular accumulation (15-25-fold). A facilitated diffusion system

EFFECI ~ OF 1t202 ON SUGAR 'I'I~.ANSPOI~.T IN S.t~md~c

1993

I

I

I 15

[ 30

I

I

I

l 60

2.0

A

1.5

1.0 0

A;S

rain

Fig. 3. Effect of H202, antimycin A and CCCP on the consumption of 2.5mmol/L p-glucose by S.pombe; A, activity of 14C in the medium (initial activity 2 x 104 cpm per 50/zL medium); fidl circles, control; open circles, 20 mmol/L antimycin A; squares, 3mmol/L CCCP; triangles, 50mmol/L H202. Specific activity of 14C-D-glucose 162 cpm/nmol, suspension density 1.19 mg dry matter per mL.

137

was postulated to be operative h)r other sugars, such as 6-deoxy-D-glucose, 3-O-methyl-D-glucose, l)-xylosc and D-arabinose, which were found to bc taken up only to diffusion equilibrium. Thcse cxpcrimcnts wcrc done on ceils aerated for 1-4 h after harvest and then energized with glucose or ethanol, at suspension densities of 5 - 8 mg dry matter per mL. Since after aeration periods over 1 h the endogenous respiration rate in S. pombe was strongly reduced due to depletion of endogenous substrates (Fig. 2), and since higher suspension densities may bring about oxygen limitation, we used in our experiments cells that were transferred immediately after harvest to 4 ~ for a maximum of 1 h and then used for experiments without aeration. They had thus a practically intact complement of endogenous substrates and it was unnecessary to energize them with ethanoi or glucose. In addition, low suspension densities prevented oxygen limitation during the experiments. The results on these cells differed substantially from those on starved and reenergized cells.

siS01 I I~

I

I

I

10

20

30

I

'oF / '~

0

10

20

30

t, 0

0

~0 rain

Fig. 4. Effect of antimycin A and CCCP (left) and H202 (right) on the uptake of 5 mmol/L 2-deoxy-o-glucose in S. pombe; Si, mmol 2-deoxy-o-glucose per L cell water; fidl circles, control; open circles, 20 mmol/L antimycin A; squares, 3/zmol/L CCCP; diamonds, 20 mmol/L H202; hzverted triangles, 50 rnmol/L H202; triangles, 100 mmol/L H202. Suspension density 1.4 mg dry matter per mL. Dashed lines, diffusion equilibrium.

Measurement of glucose consumption from the medium (Fig. 3) showed that the consumption was inhibited by 20/~mol/L antimycin A and 3/zmol/L CCCP. The effect of CCCP was surprisingly strong since the usual concentration used by H6fer and Nassar was 50/zmol/L, i.e. the CCCP-to-cell ratio was 1.5 to 2 times higher than in the present experiments. A similar effect was found, as expected, also with 2-deoxy-D-glucose (Fig. 4).

138 S, JANDA et al.

Vol. 38

150

I

I

I

1

I

I

5i

-4

100

A

A

50

0

I 20

..

I 40

I 60

0

I 20

I 40

I 60 min

Fig. 5. Effect of 11202, antimycinA and CCCP on the uptake of 40 mmol/L D-arabinose (/eft) and 40 mmol/L D-xylose(right) by S. pombe; Si, mmol sugar per L cell water; fidl circles, control; triangles, 50 mmol/L 11202; open circles, 20 mmol/L antimycin A; squares, 3~umol/L CCCP. Suspensiondensity 2 mg dry matter per mL (left) and 1.30 mg dry matter per mL (right). Dashed lines, diffusionequilibrium.

Somewhat unexpected were the rcsults obtained with D-arabinose and D-xylose (Fig. 5). Both sugars were found to be accumulated over the diffusion equilibrium (about 3-fold) and the accumulation was strongly inhibited by CCCP and antimycin A. This indicates that in cells with a sufficient level of endogenous substrates and sufficient supply of oxygen both these sugars are accumulated actively and their accumulation depends on the proton gradient across thc membrane. Passive or activc transport of sugars can therefore be observed in S. p o m b e , depending on the way of energization (cf. Sims and Barnett 1978). Another interesting feature is the relatively small extent of inhibition of D-xylose uptake by CCCP and antimycin A. In order to optimize the determination of the effcct of H202 on the intial rates of uptake ot individual sugars, and to avoid the cffect of initial fluctuations that sometimes arose (Fig. 4) on the assayed uptake rate the cells were prcincubated for up to 1 h with a low, respiration-~timulating H202 concentration (1.5 mmol/L) and with 20-100 mmol/L H202 which is certain to cause transport inhibition. 1.5 m m o l / L H202 had either no effect on transport (2-deoxy-D-glucose and D-xylose) or caused a stronger or weaker stimulation which never exceeded 10 % (D-glucose and 6-deoxy-D-glucose1 whereas 20 - 100 m m o l / L hydrogen peroxide caused a clear reduction of initial uptake rate after on b 5 - 10 rain of preincubation. Fifty m m o l / L H202 reduced the consumption of 2.5 m m o l / L D-glucose after 1 h by about 35 % (Fig. 3) and its effects on the intracellular accumulation of 2-deoxy-D-glucose D-arabinose and D-xylose resembled those of 3 ttmol/L CCCP (Figs 4, 5; analogous data for 6-deoxyD-glucose not s h o w n ) . Table I summarizes the effect of a 5-min preincubation with 100 m m o l / L H202 on the KT ant Jmax values for the four sugars under study. The maximum uptake rate remained practically unaffectec while the affinity of the transporters for their substrates dropped to 3 0 - 5 0 %. This effect, which corre. sponds phenomenologically to compctitive inhibition, resembles the effect of H202 on other plasm~ membrane proteins (ATPases) in that it is not a nonspecific process affecting the protein structure ir general, but rather a a specific effect on the binding site(s). In the case of the Na+,K+-ATPase of brait cells lipid peroxidation induced by ascorbate and FeCI2 was found to increase the affinity of the ATt binding site and decrease the affinity of the Na + and K + binding sites (Mishra et al. 1989). We foum that the effect of 100 m m o l / L H202 on the H + -ATPase of S. p o m b e plasma membranes involves like wise an about 7-fold increase in the affinity of the ATP-binding site for its substrate (KT drop from 22 to 0.3 mmol/L) and a 2 - 2.5-fold decrease in Jmax (from 6.3 to 2.7/xmol Pi per mg protein per min).

E F F E C T O F H202 O N S U G A R T R A N S P O R T IN S.pombe

1993

139

TaMe L Effect of 5-min preincubation with 100 m m o l / L H202 on the KT and Jmax values of transport of various sugars in S. pombe a

Sugar

Concentration of H202, m m o l / L

KT mmol/L

Jmaxb

2-Deoxy-D-glucose

0 100

0.97 - 0.41 3.04 +_. 0.33

5.16 z 1.82 5,29 ~ 1.79

6-Deoxy-o-gIucose

0 100

2.q3 -+ 0.26 5.26 --+ 1.17

2.24 • 0.[4 2.t8 ~* 0.29

D-Xylose

0 100

11.91 -+ 3.19 22.56 -+ 5.58

5.78 _* 1.75 5.93 ~ 1.80

D-Arabinose

0 100

6.56 -+ 2.32 11.72 -+ 3.83

5.69 z 2.27 5.04 ~ 2.03

aAll data are means _+ SEM from three e.q;,eriments. b~,mol per mg dr), matter per mir~.

Since the damage to membrane proteins may be caused either by peroxidation of the membrane lipid MDAa matrix or by direct effect of the oxidant species on the proteins (Thomas and Reed 1990) we explored the role of lipid peroxidation in the overall effect of H202 on 2 sugar transport in S. pombe, With the model unsaturated 20:4 araeh~donic acid the lipid perox/dation was 1 I L 1 found to increase with increasing peroxide concentra0 20 t. rl 60 80 tion in a saturation-like manner, In isolated plasma H202 membranes the content of malondialdehyde decreased, if anything, with increasing H202 concentration (Fig. 6); the lipid peroxidation in the membranes was thus below the detection limit of the assay. Although some perI K II ' oxidation products can be formed that are not detected 0 Bo 2~00 by thc thiobarbituric acid assay used in our study Hz0z (Buege and Aust 1978), the absence of the final perFig, 6. Production o f ma/ondialdehyde (MOA) as oxidadou product malondialdehyde is very probably due a consequence of lipid peroxidation of arachidonic to the fact that lipid peroxidation occurs as a rule with acid (top) and purified plasma m e m b r a n e s of oligounsaturatedr i.e. at least dienoic fatty acids (Porter S.pombe (bottom) by various concentrations of 1984; Slater 1984) whereas in S. pombe cells grown H202; MDAa, nmol malondialdehyde per mg under our conditions the plasma membrane contains arachidonic acid, MDAm, nmol malondialdehyde referred to 1 mg m e m b r a n e protein, H202, hydroonly monounsaturated fatty acids (Koukou et al. 1990). gen peroxide concentration (mmol/L). As a consequence, the inhibition of sugar transport reflects obviously a net effect of H202 on the plasma membrane sugar transporters, S. pombe therefore represents an ideal model organism in which to s~:~dy the n~t oxidation of membrane proteins in their ha:ire sct~ing, wi:hou: their prcvh)~s (and potentially harmful) isolation and reconstitution into liposomes containing pcroxidati0n-insensitivc lipids. The absence of plasma membrane lipid peroxidation may be one of the main rcasons for thc extraordinarily high resistance of S. pombe to oxidative strcss. 6

We thank Dr. Vladimfr Vacata for critical reading of the manuscript and Ms. Alexandra Pokorn,~ for skilled technical assistance. T h e work was supported by a grant from the Deutsche Forschungsgenwhlschaft (I Io 555/14-1).

140

S. JANDA et aL

Vol. 3,S

REFERENCES BUEGE J.A., AUST S.D.: Microsomal lipid peroxidation. Meth.En~,mol. 52, 302 - 310 (1978). DUFOUR J.-P., AMORYA., GOFFEAUA.: Plasma membrane ATPase from the yeast Schizosaccharomyces pombe. Mcth.En~,mol. 157, 513- 527 (1988). FOURY F., GOFFEAU A.: Stimulation of active uptake of nucleosides and amino acids by cyclic adenosine 3,5-monophosphate m the yeast Sctu'zosaccharomycespombe. J.BioLChem. 250, 2354- 2362 (1975). FREW J., JONES P., SCHOLES G.: Spectrophotometric determination of hydrogen peroxide and organic hydroperoxides at low concentrations in aqueous solutions. AnaLChim.Acta 155, 139-150 (1983). GILLE G., SIGLERK., H~FER M.: Response of catalase activity and membrane fluidity of aerobically grown Schizosaccharomyccs pombe and Saccharomyces cerevisiae to aeration and the presence of substrates. J.Gen.MicrobioL, submitted (1993). GOFFEAU A., BRIQUET M., COLSON A.M., DELHEZ J., FOtJRY F., LABAILLE F., LANDRY Y., MoItat~ O., MRENA E.: Stable pleiotropic respiratory-deficient mutants of a"petite-negative" yeast, pp. 63-97 in Membrane Biogenesis (A. Tzagoloff, Ed.). Plenum Press, New Y o r k - London 1975. Gtrrz H., HESLOT H., LEUI'OLD U., LOPRIENO N.: Schizosaccharomyces pombe, pp. 395-446 in Handbook of Gcnetics (R.C. King, Ed.), Vol. 1. Plenum Press, London 1974. H6FER M., NASSAR F.R.: Aerobic and anaerobic uptake of sugars in Schizosaccharomyces pombe. J.Gen.Microbiol. 133. 2163 - 2172 (1987). JANDA S.: Relationship of active membrane transport systems in the yeast Rhodotorula ghainis: possibility of two respiratot'y systems. Cell.Mol.Biol. 25, 131 - 136 (1979). JANDA S., BENE~ I., OPEKAROVAM., S'fASTNAJ., TAUCttOVA R.: Effect of hydrogen peroxide on the aerobic yeast Ir glutinis. Microbios Lett. 43, 37-42 (1990). KOTYKA., MICHALIANI~:OVAD., VERF~ K., SOUKUPOVAV.: Transport of 4-deoxy- and 6-deox'y-D-glucose in baker's yeast. Folia Microbiol. 20, 496-502 (1975). KOUKOUA.I., TSOUKATOSD., DRAINASC.: Effect of ethanol on the phospholipid and fatty acid content of Schizosaccharomyces pombe membranes. J.Gen.Microbiol. 136, 1271-1277 (1990). M1SHRA O., DELIVORIA-PAPADOPOULOSM., CAHILLANEG., WAOERLE kC.: Lipid peroxidation as the mechanis,~a of modification of the affinity of the Na +,K+-ATPase active sites for ATP, K +, Na +, and strophantidin in vitro. Neurochem.Rcs. 14, 845-851 (1989). PORTER N.A.: Chemistry of lipid peroxidation. Meth.Enzymol. 105, 273- 282 (1984). SIGLER K., GILLE G., KURZWEILOVAH., VANI~KD.: Effects of hydrogen peroxide on yeasts, p. 17 in Proc. 5th Small McethTg on Yeast Transport and Energetics, Zvikovsk6 Podhradf (Czech Republic) 1990. SIGLER K., HOFER M.: Activation of the plasma membrane H+-ATPase of Saccharomyces cerevisiae by addition of hydrogen peroxide. Biochem.lnternat. 23, 861 - 873 (1991). StMS A.P., BARNETtJ.A.: The requirement of oxygen for the utilization of maltose, cellobiose and D-galactose by certain anaerobically fermenting yeasts (Kluyver effect). J.Gen.Microbiol. 106, 277- 288 (1978). SLATERT.F.: Overview of methods used for detecting lipid peroxidation. Meth.En~,mol. 105, 283-293 (1984).