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ATP production and catalatic-type activities in etiolated or green Euglena gracilis
Comp. Biochem. Physiol. Vol. 81B, No. 4, pp. 1009-1017, 1985 Printed in Great Britain
0305-0491/85 $3.00 +0.00 © 1985 Pergamon Press Ltd
ATP P R O D U C T I O N AND CATALATIC-TYPE ACTIVITIES IN ETIOLATED OR G R E E N EUGLENA GRACILIS RI~GIS CALVAYRAC,* DANIELLE LAVAL-MARTIN,* MICHELLE HUBERT,t MARTIN BEST-BELPOMME~ a n d MONIQUE VUILLAUMEt
*Laboratoire des Membranes Biologiques, Universit~ Paris VII, 2, place Jussieu, 75005 Paris Cedex 05, France; tLaboratoire de Zoologie, ERA CNRS 1048, Ecole Normale Sup6rieure, 46, rue d'Ulm, 75230 Paris Cedex 05, France; and ~Laboratoire de Zoologie, ERA CNRS 615, Universit6 Paris VI, 7, quai Saint-Bernard, 75230 Paris Cedex 05, France
(Received 21 January 1985) Abstract--1. Whole Euglena cells, as well as mitochondrial and chloroplastic fractions (12,000-100,000 and 200,000g supernatants), displayed a H202-mediated ATP production with ADP and PO43 as necessary substrates. 2. The adenylate kinase activity could not solely account for such a reaction since APsA did not affect it. 3. Inhibitors of oxidative phosphorylation did not inhibit but sometimes enhanced the phenomenon. 4. Irradiation of the fractions could produce an ATP increase which suggested the participation of superoxide ions. 5. We conclude that the energy of H202, photoproduced or experimentally added, could be converted by a catalatic-type system, normally integrated into organelle membranes, in energy-rich ATP bonds.
INTRODUCTION
The phytoflagellate Euglena gracilis Z is an organism remarkably adaptable to various growth conditions and considered as being at the frontier of animal and plant kingdoms. For example, under darkness the cells, devoid of chloroplasts, can be grown heterotrophicaUy in virtually all possible sources, except citrate, with pHs ranging from 2 to 11 (Cook, 1968; Calvayrac, 1972; Kempner, 1982). When they are illuminated, the cells differentiate chloroplasts and become able to photosynthesize and grow autotrophically on a mineral medium only supplemented with B1 and Bt2 vitamins (Schiff and Epstein, 1968; Schiff and Schwartzbach, 1982). Lackey (1968) indicated that Euglena is resistant to deleterious agents like the heating or organic pollution of the water, thus "if Euglena species existed there before the catastrophe they quickly return". This eukaryote also resists the very same UV doses causing bacterial mutagenesis, without other damage for the cell population than an hereditary bleaching leading to obligatory heterotrophy (De Denken-Grenson, 1959; Cook and Hunt, 1965; Michaels and Gibor, 1973; Diamond and Schiff, 1970). Lackey and Bennett (1962) noticed that another property of Euglena was to exhibit unimpaired reproductive rates when exposed to massive doses of radionuclides (for example, 10% from 6°CO); this means that Euglena are able to resist highly energetic ionizing radiations which reduce oxygen in superoxide ions. Although it is known that free radicals or their terminal product of reactions, H202, are dangerous reactants for the cells (Fridovich, 1975; Epstein, 1977). Buvet (1981) and Buvet and Le Port (1983) recently suggested that they could be beneficial for aerobic organisms, because of their role in the direct or indirect production of cellular energy. In previous papers, our group has shown that: (a)
UV irradiation stimulated oxygen consumption leading to H20_, formation in mitochondrial and chloroplastic fractions from Euglena (Calvayrac and LavalMartin, 1980); this phenomenon, sensitive to 2-salicyl hydroxamic acid (SHAM) could be reinforced by 6.6 mM C N - and stayed unaffected by deoxycholate treatment (1 mg/mg of membranal proteins); (b) molecules of biological interest such as ubiquinone, plastoquinone and pheophytin a were able to exhibit similar UV-dependent H202 production (Vuillaume et al., 1982a, b); (c) the H202 integumentary photoproduction by the pterobilin of Pieris brassieae L5-1arvae was able to lead to a production of ATP in the presence of catalatic activity and did not require membrane entegrity (which is a non-classical and non-admitted physiological behavior) (Vuillaume et al., 1979, 1982b; Vallot et al., 1982); (d) an aspect of the resistance to highly energetic ionizing radiations by scorpions (Androctonus australis) could be explained by the catalatic activity of their blood pigment, the hemocyanin; this activity could be characterized in the purified native pigment as well as in its heavy dissociated subunits (i.e. dodecamers and hexamers) (Huyart et al., 1983). Such findings, involving different biological systems have lead us to the Mehler's (1951) concept and to examine the possible bond between the ATP production and the redox processes involving H202. In this paper we present data concerning different types of Euglena cells and their subcellular fractions, and we show that radiations can enhance the catalase-like reactions (catalatic reactions) producing ATP from H202. MATERIAL AND METHODS
Conditions of cultures All cultures were at 26"C in three different mediums, pH 3.5, containing 33 mM lactate as sole carbon sources.
1009
1010
RI~GIS CALVAYRACet al.
The standard medium, indicated "L", contained among other mineral salts, 173mEq of PO] and 68.4mEq of NH2; the second medium, indicated "L, NH +'', was comparable for all components but ammonium at the final concentration of 205 mEq; the third medium, indicated "L, N H ; , PO 3 ", contained 205 mEq of NH + and 370 mEq of
PO~-. Two different strains of E. gracilis were studied: the control strain, Z, which is either etiolated when cultured in darkness (heterotrophy) or which contains chloroplasts when cultured under white light (30W/m 2) (photoheterotrophy) and an apoplastidial strain ZC which has been initially obtained from the adaptation of control Z cells in a diuron containing lactate medium under light and in micro-aerophyllic conditions (Calvayrac and Ledoigt, 1976; Calvayrac et al., 1979a, b). We have tested different strains of Euglena gathered at different physiological stages during the exponential phase of growth of a culture when the cells actively divide with generation time approximating 10 hr, phase I, or when the culture had reached the stationary phase and the cells were in infradian mode of population increase, phase III; the intermediary phase, phase II, characterizes the moment when cells still divide but with long generation times due to limiting factors in the medium.
Preparation of the subcellular fractions Cells from the culture medium were gathered by a 500 g centrifugation for 10rain, rinsed in distilled water and repelleted by an identical centrifugation. The pellet was weighed and a pH 7.2, Tris-NaC1 50 mM buffer (B) containing 0.25 M sucrose, 2.5 mM CaCI 2 and 2.5 mM MgCl 2 was added in the w/v ratio of 1/4. An aliquot of the cell suspension in buffer was kept for assays on whole cells, the rest was transferred in a "Braun Broyeur" with 2.5 g of 2 m m glass beads per ml of cell suspension. The cell breakage was done at maximal speed, under carbonic snow cooling, 3 times per 10 sec separated by 2 times per 20 sec. The resulting suspension was centrifuged for 15 min at 150 g (SS 34 rotor, Sorval centrifuge), the pellet was discarded and the supernatant (S) recentrifuged for 15 min at 12,000g; the pellet then obtained constituted the mitochondrial fraction and the supernatant, S12", was partly recentrifuged at 100,000g to obtain a second supernatant, S~00, itself recentrifuged at 200,000g to give $200. In the case of green Euglena with chloroplasts the first supernatant (S) was centrifuged for 15min at 1500g to obtain a pellet essentially composed of the chloroplasts. Pellets of either chloroplasts or mitochondria were washed once in the extraction buffer and recentrifuged, respectively, at 1500 and 12,000g for 15 min. All fractions tested (pellet of mitochondria, pellet of chloroplasts, S~2, $200) were diluted in the extraction buffer at the moment of the assay to test fractions containing about 0.5mg as total proteins per ml. Protein measurement The protein content was assayed as described by Lowry et al. (1951) on fractions previously diluted 2 times in NaOH 5 N and kept for 30 min at 37'~C. A TP measurements The ATP measurements were performed in a "Nucleotimeter 107 CLV-Interbio" with lucioline (IB 20 IC CVLInterbio) dissolved in a buffer, pH 7.75 (IB 25 50 CVLInterbio) on fractions of 150#1 total volume containing 25 #1 of nucleoline (IB 40 50 CVL-Interbio) directly added to the fraction after sonication (30 K cycles, 2 x 30 sec at 0°C). Nucleoline is a complex detergent which induces the *Abbreviations: cel., whole cells; 812, 12,000g supernatant; Sl00, 10,000g supernatant; $200, 200,000g supernatant; Mit., mitochondrial fraction; NAN3, sodium azide; C N - , cyanide; DNP, dinitrophenol; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone.
total permeability of cellular membranes to nucleotides. Calculations of the ATP quantities were performed using the "peak condition" mode and 30 sec of preselection. The results were expressed either in ATP molarities per mg of total protein or in ATP increase as percent of ATP basis. At first, the initial ATP contents of each pellet and supernatant studied were individually determined in absence of all reactives to determine their "ATP basis"; then their eventual ATP productions were tested when either HzO z (10 13to 10 ~M) or white light (15 W/m2/sec) were applied. The ATP backgrounds of all reactives (ADP, PO]-, H202, nucleoline, e t c . . . ) were separately determined against lucioline as well as those of their mixture two by two, three by three or all together. This procedure permitted correction of the result obtained in each of the individual assays from the corresponding background due to the specific reactant(s) then used. Supernatants Sl00 and $200 did not display spontaneous ATP production after either addition of H20 2 (5 x 10 -6 to 5 x 10 ~ M) or illumination. They were then assayed successively: (a) in the presence of PO 3- (5 x 10 -4M) alone; (b) in the presence of ADP (5 x 10 6 M) alone; (c) with both products. This last condition ( A D P + P O ] - ) showed an increase in ATP when either H202 or light were applied to the supernatant.
Adenylate kinase activiO, ADP, 1 mM, was solubilized in a 0.5 M Tris buffer, pH 7.5. The assay was run at room temperature by mixing volume to volume the cell extract (Mit. or S~00) containing 2.5mM MgCI> and the ADP solution. To follow the kinetic, every 5 min, 10/~1 aliquots of the assay mixture were diluted 104-fold in 0.25 m M Tris buffer, pH 7.5, and the ATP contents rapidly determined with the Nucleotimeter. The 104 fold dilution stopped the adenylate kinase reaction without any risk of reversion since the reaction then tended to an equilibrium of which the constant had no more dimension since: (ATP)(AMP) (ATP) 2 K (ADP) 2 (ADP) 2" In case of the presence of an adenylate kinase activity, such an assay gives for the first hour a linear ATP production. In addition, and in another set of assays we have inhibited the adenylate kinase activity by its specific inhibitor, APsA (Lienhard and Secemeski, 1973), used at a concentration 10-100-fold the ADP concentration present in each assay.
Drug effects We tested drugs known to have inhibitory actions on either the oxidative phosphorylations or on phosphorylations resulting from the pathway insensitive to cyanide (Calvayrac and Butow, 1971), i.e. DNP, 10 -s to 10-4 M; NaN3,10 4 t o 3 x 10 2 M ; C N 1 0 - 4 t o 3 x 10 3M and FCCP, 3 x 10 3M. RESULTS P r i o r to each e x p e r i m e n t , the luciferine luciferase m i x t u r e was tested to m e a s u r e the units o f relative l u m i n e s c e n c e ( U R L ) w h i c h c o u l d be o b t a i n e d with s t a n d a r d A T P solutions. W h e n set in p e a k c o n d i t i o n s the a p p a r a t u s i n d i c a t e d linearity for the r e a c t i o n s " A T P s t a n d a r d . . . . l u c i f e r i n e - l u c i f e r a s e " b e t w e e n 500 a n d 80,000 U R L . C o n s e q u e n t l y we diluted with the buffer (B) the different fractions to be tested to p e r f o r m differently in this interval.
Choice o f extract preparation .for A T P measurements T h e m o s t r e p e a t a b l e d a t a were o b t a i n e d by o m i t ting the s o n i c a t i o n a n d utilizing only the a c t i o n o f
ATP production in Euglena
1011
Table 1. Example of experimentaldata from a typical experiment
Mixture tested
URL
10#IPO~-*+25#IN?+ II5/~IB~ or H 2 0 20 ,al A D P § + 25 .ul N + 105/.d B or H 2 0
ATP (10 -6 M)
20 7500
20 #1H~O:II + 25 #1N + 105 #1B or H20 10 #1PO43- + 25 #1N + 20 # 1 A D P + 95 #1B or H20 50 #1 Cel. + 25 #1N + 75 #1B 50 #1 Cel. + 25 #1N + 25 # 1 H : O 2 + 50/zl B 50 #1Mit. + 25 #1N + 75 #1B 50 # 1 M i t . + 25 .ul N + 25/.tl H20: + 50 # 1 B 50 #1SL00+ 25 #1N + 75 #1B or H20 50 #1 St00 + 25 #1N + 20 # 1 A D P + 55 #1B or H20 50 #1Si0o + 25 #1N + 20 # 1 A D P + 20 ~1H202 + 35 #1 b or H20 50 #1S~00 + 25 # 1 N + 20 # 1 A D P + 10 #1PO~- + 45 #1 B or H20 50 #1S~00 + 25 # 1 N + 20 # 1 A D P + 10 #1PO~- + 20 #1H~O~ + 25 #1 B or H~O
Increase in ATP due to H202 as percent of ATP basis
---
0 7600 3000 12,210 3000 8000 3700 11,000 13,200 13,500 22,600
--0.35 1.42 0.35 0.93 0.40 1.20 1.30 1.50 2.50
307 166
67
*PO43-, 5 x 10 -4 M; i'N + nucleoline; SB = buffer (50 mM Tris, 250 mM sucrose, 2.5 mM CaCI 2, 2.5 mM MgCI2); §ADP, 5 x 10 -6 M; tlH202, 5 X 10 -7 to 5 X 1 0 - 4 M depending upon the fraction tested, the maximal result obtained with the optimal concentration being the only one retained.
nucleoline which induced the total permeability of the membranes to nucleotides.
Example of raw data and expression of the results We present in Table 1 the results of a typical experiment. In such an example we retain two values: the ATP basis (line 5 for whole cells, Cel.,; line 7 for mitochondrial fraction, Mit. and line 12 for St00) and the maximum ATP in the presence of optimal H202 concentration (lines 6, 8 and 13, respectively). For a given fraction, the difference between these two results expressed as percent of the ATP basis indicates the increase of ATP production provoked by H202 addition. In the given example (Table 1) the increment of ATP production in the presence of H202 would be 307~ for whole cells, 166~o for the mitochondrial fraction and 67~ for Si00.
sonication (plateau from 5 x 10 -~2 to 5 x 10-SMH202), the absolute value of H202mediated ATP formation was six-fold increased on a protein basis but was only equal to about 180Y/owhen expressed as percent of the ATP basis, which indicated the damaging of some of the enzymatic sites.
Adenylate kinase act&ity In the supernatants the H202-mediated ATP production was maximal in the presence of both PO43- and ADP; it was possible to hypothesize either a phosphorylation, i.e. a reaction energetically important for the cellular metabolism, or that the PO43- could be an effector for the adenylate kinase activity. For this reason we tested the adenylate kinase activity in pellets and supernatants from Z cells grown heterotrophically in the darkness.
Influence of the H202 molarity on the A TP production We studied the evolution of the ATP production as function of the H202 added on Z Euglena grown in " L " medium in darkness. The addition of H202 from 10 -z3 to 10-1M increased the ATP production per reference to the fraction tested without H202 and taken as basis (i.e. 0 ~ of ATP increase). This H202-mediated ATP productior1 characterized a suspension of whole cells (5.9 mg of total proteins per ml) treated with nucleoline vol. to vol., just prior to H202 addition (Fig. 1, curve A), as well as the same cells sonicated at 0°C (3 x 10see, 8 Kcycles) and subsequently diluted 10 times with the buffer (Fig. 1 curve B). The results would be comparable for mitochondrial and $12 fractions, the curves being translated to a lower range of H202 concentrations. Both curves A and B (Fig. 1) showed a first portion corresponding to H202 concentrations limiting the phenomenon, then a plateau of optimal H202 concentrations for a maximal production of ATP (situation systematically selected for all subsequent experiments), followed by a toxic effect on the highest H202 concentrations. The ATP increase expressed as percent of ATP basis was maximal, equal to 300~, when whole cells were tested in spite of the limited diffusion of H202 towards the sites of disproportionation (plateau between 10 _4 and 10-3M H202). When the sites were exposed after
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Fig. 1. ATP increase, expressed as percent of the ATP basis, as a function of H202 concentrations. The phenomenon was tested on strain grown in "lactate" medium in darkness. Curve A corresponded to whole cells rendered permeable to nucleotides by addition, at the moment of the assay, of nucleoline; curve B corresponded to the same cells after sonication.
1012
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Fig. 2. Test of the adenylate kinase activity present in either mitochondrial fraction (curve A) or in S~2 supernatant (curve B) of dark grown Z strain cultivated in "lactate" medium and tested as described in Materials and Methods.
The adenylate kinase activities found in this fraction were reinforced by the addition of PO]- (10 -2 M) to the ADP (5 x 10-4M). The effect of the PO4~- was remarkable on the mitochondrial fraction where it could increase the ATP concentration by a factor 3 (after 18 min). These data suggested more than an effector role on the adenylate kinase activity but probably the occurrence of phosphorylation which could be due to a certain amount of H202 present in the mitochondria and able to promote H202mediated ATP production. Such an hypothesis seemed to be supported by the assays testing simultaneously the addition of H202. The addition of H202 to a mitochondrial fraction containing both ADP and PO]- in excess indicated a very strong, though = 5 . 1 0 "~ , transient, H202-mediated ATP production. It was interesting to notice in Fig. 3 the same profiles of the curves relative to the mitochondrial fraction tested ~ AS with PO]-. We know that Euglena contained, on average, 4 nmol ATP per 106 cells (in phase I, for nucleotide measurements and energy charge deter6 A A mination, data not shown). We could then measure the adenylate kinase activity, using the ATP basis =. (Table 1), in the mitochondrial fraction and the corresponding $12 supernatant (Fig. 2). Such a calculation indicated that the total content in ATP was r., 1 . 1 0 ~ Ionly about 2 ~ of the expected one (with the following distribution: 23~o in the mitochondrial fraction 5.10" 10 20 3O against 77~o in the $12 supernatant) and that consequently ATPases actively hydrolyzed the cellular DURATION_ minute_ Fig. 3. Test of the possible effector role of PO~- in the ATP during the preparation of the fractions and also mitochondrial fraction treated with nucleoline (1 vol. to when nucleoline was added. 5 vol.). This fraction proceeded from dark grown Z strain During the assays, while H2Oz-mediated ATP forcultivated in "lactate" medium and tested as described in mation reached 5 x 10 6 to 10 -5 M, addition of 10 -4 Materials and Methods. Some of the assays were done in the to 10 3 M of APsA did not decrease these ATP levels. presence of PO3 (10 2M). Simultaneously the H202- Consequently, the H202-dependent ATP production mediated ATP production was tested by adding HzO2 (8 x 10-~ M) to aliquots of the reaction mixture, prior to the did not originate from adenylate kinase activity. Such results allow us to exclude the intervention of final 10a-fold dilution. Curve A, Mit. fraction; curve B, Mit. fraction + H202; curve C, Mit. fraction + PO] ; curve D, adenylate kinase reaction in the H2Oz-mediated ATP production that we present in this paper. Mit. fraction + P043 + H202.
The first experiment done on mitochondrial fraction and S~2 without addition of either nucleoline or PO 3- (Fig. 2) showed a strong adenylate kinase activity in S~2 while the mitochondrial fraction seemed to present in addition to the adenylate kinase activity an even more rapid hydrolysis of the ATP formed, leading after 10 min to ATP concentrations lower than the one at time zero. The second experiment was designed to test the possible effector role of the PO43- on the adenylate kinase activity and also to test, in the conditions of the assay, the effect of H202. The results concerning a mitochondrial fraction are summarized in Fig. 3.
ATP production in Euglena
1013
Table 2. Comparison of the H202-mediated ATP production in fractions isolated from Z strain Euglena grown in " L " medium in darkness. The beginning of the exponential phase of growth (phase I characterized by H202 optimal 2.5 x 10-4M) is compared to the stationary phase (phase III, H202 optimal 5 × 10 7M) Growth phase I
Growth phase III
Fraction
ATP basis (nmole per mg of protein)
Increase in ATP with H20 z optimal (% vs ATP basis)
ATP basis (nmole per mg of protein)
Increase in ATP with H202 optimal ( ~ vs ATP basis)
Mitochondria S~2
47 111
149 89
40 522
70 21
Table 3. Ubiquity of H202-mediated ATP production in different fractions isolated from ZC and Z strains of Euglena grown in different mediums either in darkness or under illumination. The photoorganotrophic medium in which Z strain cells were grown contained diuron 25/~M to modify the chloroplastic compartment which when keeping unmodified volume, loses 9 0 ~ of its chlorophyll content (Calvayrac et al., 1979b) Culture in darkness
Culture under illumination
Fraction
ATP basis (nmole per mg of protein)
Increase in ATP with H202 optimal (% vs ATP basis)
ATP basis (nmole per mg of protein)
Increase on ATP with H202 optimal ( ~ vs ATP bazis)
ZC strain in " L "
Mitochondria Si2
13 28
102 34
62 63
83 66
Z strain in " L "
Chloroplasts Mitochondria S~2
-47 111
-149 89
21 28 66
95 135 54
Z strain in "L, NH~-"
Chloroplasts Mitochondria Si2
-12.5 --
-168 --
Il 12 24
104 109 60
Z strain in "L, N H t , P O ~ - "
Mitochondria
28
215
26
187
Type of cell and medium
Ubiquity of the H202-mediated ATP production In an attempt to generalize the existence of an H202-mediated ATP production, we have tested Euglena grown in mediums containing different amounts of NHg and/or PO 3- in the dark and under light and gathered at different physiological stages. In the case of white cells (Z strain cultured in the dark, or ZC strain cultured either in the dark or under light), we studied the mitochondrial fraction, the S~: supernatant and sometimes St00 and $200; in the case of chlorophyllian cells (Z cells cultured under light) a chloroplastic fraction was also tested. In this latter condition of culture we modified the chloroplastic compartment by adding 25/~M D C M U to the medium; such a treatment which did not affect the volume of chloroplasts (Calvayrac and Ledoigt, 1976) not only depressed the chlorophyll content by 90~o and totally inhibited oxygen evolution (Calvayrac et al., 1979b) but favored light-induced oxygen consumption (Calvayrac and Laval-Martin, unpublished results).
Influence of the physiological stage of the cells We compared white Z strain cells, grown in darkness in the same lactate medium. Table 2 summarizes the results and indicates that the ATP basis (nmole of ATP per mg of protein) as well as the increase in ATP when 10 -5 M H202 was added, were higher in both fractions extracted from cells actively dividing (phase I) than in the corresponding fractions obtained from stationary cells (phase III). Moreover, if the ATP basis seemed depressed in mitochondrial fractions, due probably to their richness in membranal proteins, the H202-mediated ATP production (expressed as percent of the ATP basis) was important in this fraction and still important in S~2. We
then tested, in the rest of the study, cells gathered in phase I.
Influence of illumination or darkness during the cell growth Cells gathered in phase I and grown in different organotrophic mediums either in darkness or under light have been compared (Table 3). Whatever the type of cell and/or the medium and light condition, the H202-mediated ATP production (expressed as percent vs the ATP basis) is higher in mitochondrial fraction than in $12. When cells were green (Z cell cultured under illumination), the phenomenon was found in the chloroplast fraction as well as in the mitochondria. It seemed that mitochondria from etiolated cells (Z strain cultured in darkness) were more active for H202-mediated ATP production than mitochondria from the corresponding green cells (Z strain under illumination). Such an enhancement of ATP production by darkness can be observed also in apoplastidial ZC strain (never containing any chlorophyll whatever the light regimen). Thus the phenomenon, perhaps favored in heterotrophic cultures grown in darkness, would be very active in organelles but still present in Sl2 with a depressed efficiency.
Light-enhancement production
of the H202-mediated A TP
We have tested the effect of short time light exposures on the ATP production in ZC apoplastidial whole cells grown either in darkness or under illumination. Such experiments were done without any H202 addition, the cell walls being rendered permeable to ATP by the nucleoline (cf. Table I). The results in Fig. 4 showed ATP increases in both types of cells but with a faster kinetic in the
1014
P~GlS CALVAYRACet al.
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DURATION second _ Fig. 4. ATP increase, expressed vs percent of the ATP basis, in function of the 580 W/m 2 white illumination, in whole apoplastidial ZC strain cells grown in lactate medium either in darkness ( + +) or under light (Q 0 ) were tested. _
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case of dark grown cells than in the case of light grown ones. The same type of experiment done after breakage of the dark grown cells, either on the mitochondrial fraction (Mit.) or on $12 supplemented or not with ADP + PO43-, indicated (Fig. 5) rapid increase in ATP after the first minute quickly hydrolyzed in the mitochondria and a slow linear increase of ATP in Sn for 6 min.
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We tested the effects, on the H202-mediated ATP production, of some drugs applied on the fractions at the moment of the assays. Figure 6A and B indicate that not only did the drugs not inhibit the phenomenon but they provoked an increase in the ATP production: the DNP (Fig. 6A) promoting a strong activation when applied at very low concentration (10 -5 to 10 -4 M) and both C N - and NaN3 enhancing the ATP production (Fig. 6B) at concentrations from
10 -4 to 3 x 10 3M f o r C N - and to 5 x 1 0 -2M for NAN3. The FCCP 3 x 10-3M did not inhibit the H202-mediated ATP production either (data not shown). For strong concentrations (3 x 10 3 for CN and 5 x 10 -2 for NAN3) we noted a total ATP-H202 inhibition.
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DURATION_ m i n u t e _ Fig. 5. ATP increase, expressed vs percent of the ATP basis, in function of the 580 W/m 2 white illumination, in mitochondrial fraction (curve A) and 12,000g supernatant (curve B), extracted from apoplastidial ZC ceils grown in "lactate" medium under light. An experiment with supernatant was carried out in the presence of 8 x l0 -4 M PO ]and 2 × l0 -4 M ADP (U II). It gave identical results to the experiment without substrate ( + +).
Effects of drugs affecting the oxidative pathways on the H202-mediated A TP production
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Fig. 6. Effect of different drugs on the H202-mediated ATP increase, expressed vs percent of the ATP basis, in a mitochondrial fraction. This fraction was extracted from Z cells, dark grown on "lactate, NH~" medium and assayed in the presence of 5 × 10-s M H202 (optimal concentration). Curve A: effect of DNP (10 -4 to 10-SM). Curve B: effect of CN- (10 _4 to 10-2M) and of NaN~ (5 × l0 -3 to 5 × 10-1).
ATP production in Euglena
1015
Table 4. H202-mediatedATP production in mitochondrialfractions from Z cells grown in darkness in "L, NH~-" mediumcontaining or not 10-6M antimycin Culture with 10-6 M antimycin Culture without antimycin ATP basis Increase in ATP ATP basis Increase in ATP (nmole per with H202 optimal (nmole per with H202 optimal mg of protein) (~o vs ATP basis) mg of protein) (~ vs ATP basis) Control 12.5 168 41 133 Assay with 2 mM CN- or 2 mM NaN3 20 190 38 165
Intensity of the H202-mediated ATP production in mitochondrial fraction from cultures grown in presence of an antibiotic; antimyein Antimycin was known to favor, in Euglena, the shunt insensitive to cyanide (Calvayrac and Butow, 1971). We then grew Z strain cells on "lactate, NH +'' medium, in darkness, in the presence or not of 10-6M antimycin and tested the H202-mediated ATP production of their mitochondrial fractions. In addition we have, at the moment of the assays, treated fractions by 2 mM C N - or NaN 3. Table 4 shows the results obtained. They indicate that the phenomenon of ATP production was virtually unaffected in cells grown in the presence of antimycin, i.e. in cells in which the insensitivity to C N shunt was strengthened (Calvayrac and Butow, 1971); moreover the addition of either CN or NaN3 to the reactive medium did not affect the reactions but surprisingly enhanced them (cf. Fig. 6B). DISCUSSION AND CONCLUSION Whatever the conditions of culture (under light or in darkness) in lactate medium supplemented or not with NH2- and/or PO 3-, the two Euglena strains studied, i.e. the control green or etiolated Z strain and the apoplastidial ZC strain, as well as the derived cellular fractions (mitochondrial, chloroplastic, the 12,000, 100,000 and 200,000g supernatants), all presented the same phenomenon of an H202mediated ATP production measured in the presence of nucleoline by the luciferinv-luciferase method. These same strains and cellular fractions were able to consume oxygen when illuminated, especially with UV and red light (Calvayrac and Laval-Martin, 1980), as shown by observations done as soon as 1951 by Mehler. Recent results obtained by some of us had shown that integuments of Pieris brassicae larvae presented a natural H202 and/or light production of ATP (Vuillaume et al., 1979, 1982a, b) and reviews (Chance et al., 1979; Oberley, 1982) reported that some authors had found that H202 could be formed either in the mitochondria or in the cytosol (Boveris, 1978); we thought that the H202-mediated ATP production observed could be a natural process. Keeping in mind this idea of a possible ubiquitous reaction of ATP production we will discuss the data obtained in this work.
Whole cells White Z and ZC cells, illuminated by strong (580W/m 2) white light, for very short durations, displayed (Fig. 3) ATP production which increased by a factor 2.2 after 45 sec when the cells were grown in darkness, vs 1.2 after 5 sec when the cells were
illuminated during their growth. This suggested a more active regulation of the equilibrium ATP/ADP in cells perhaps able to use photons for ATP production during their growth than in cells unable to use light since grown in darkness. Such results were only obtained with intact cells treated at the moment of the assay with nucleoline which rendered the membranes permeable to nucleotides. When cells were sonicated the phenomenon was not measurable, probably because of the hydrolysis of the ATP photoproduced by ATPases released by the bursting of the membranes. We showed that growth of Euglena cultures was not affected by the addition of antimycin (Calvayrae and Butow, 1971) and proposed that the metabolism in the mitochondria was unperturbed because of the functioning of a secondary pathway insensitive to cyanide. We show in this paper that treated cells (Table 4), as control cells, could produce ATP by H202-mediated processes and were richer in ATP content if we consider the protein basis.
Subcellular Jkactions Mitoehondrial and ehloroplastic fractions. These were the fractions exhibiting the highest potential for H202-mediated ATP production. Nevertheless, when strongly illuminated, they reacted (Fig. 5) with at first a rapid ATP increase, followed by a continuous drop in ATP, a secondary reaction probably due to hydrolysis by the free ATPases. Supernatants 12,000 and 100,000 g. When strongly illuminated these supernatants exhibited a strong ATP increase (cf. Fig. 5) for 6 min. For 100,000g we obtained a kinetic comparable to St2 only when ADP and PO 3- were added prior to the illumination. Previous observations indicated that the systems, present in both mitochondria and chloroplasts, which are responsible for a light-mediated ATP production, were labile and able to work from an integrated membrane with both ADP and PO 3- at their disposal.
Adenylate kinase activity We thought that we could reject the simple intervention, in the H2Oz-mediated ATP production, of the adenylate kinase activity that we had found in each of the tested fractions, alone or accompanied by an ATP hydrolysis (case of the mitochondrial fraction, Fig. 2) for the following reasons: (a) The addition of PO 3- increased the ATP formation in crude mitochondrial fractions more than in the measure adenylate kinase activity (Fig. 3A and C). We found the same data in all fractions but with less yield, indicating that PO]- is a normal substrate for mitochondrial ATP production. (b) The addition of H202 to the assay mixtures was efficient for ATP
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P-~GIS CALVAYRACel al.
production in a mitochondrial fraction containing PO~ , but was still able to increase A T P production by 50-30% in a crude mitochondrial fraction. This indicated that in the membranes, even if the adenylate kinase exists, the H2Oz-mediated A T P production was possible and favored if both substrates, PO] and A D P , were present. (c) We noted for all other fractions the same result as below, i.e. H20 2 addition increased the A T P formation but less than in the mitochondrial fraction, only when PO] is added. (d) Finally the addition of concentrations of APsA (a specific inhibitor of adenylate kinase activity, according to Lienhard, 1973) 10 to 100-fold the one of A D P , never affected the H2Oz-mediated A T P production. Catalatic activity In all the fractions studied in this work, we could always detect an intense catalatic activity. The C N and azide, already known to have an inhibitory action on the H202, did not inhibit but promoted the H202-mediated A T P production in Euglena material studied here (Fig. 6B), as was the case in Pieris brassicae (Vuillaume et al., 1979, 1982a). The remarkable A T P production by a fraction tested in the presence of D N P (Fig. 6A), known for its uncoupling effect on oxidative phosphorylation, could be explained by the peculiarity of D N P to be able to form H202, as is the case of molecules containing phenol rings (Vuillaume et al., 1982b). Thus all the drugs inhibiting phosphorylation according to Mitchell's scheme, as well as the F F C P dissipating the proton gradient (Wagner and Lin, 1982), had no action or promoted the H202-mediated A T P formation. In conclusion we could then interpret the ubiquitous phenomenon, here observed either in whole cells or in mitochondrial and/or chloroplastic fractions and even in 12,000 or 100,000g supernatants, ,~s being the result of the catalatic activity of a soluble protein able to gather in a high energy bond in ATP, the energy being provided by H202 either photoproduced or experimentally added. At p H 7 disproportionation of a mole H202 was known to produce 25kcal (Buret, 1981), energy theoretically sufficient to permit the synthesis of 1 or 2 A T P molecules, according to the following proposed scheme:
H20~.\
/"-~O~+ mO Protein with catalatic properties
A D P + PO4-3
ATP
According to our first calculations of the molecules of A T P produced vs the molecules of H202 experimentally furnished, we found the following yields for assays run without A D P and PO] : 30% for mitochondrial fractions, 50% for SL2 supernatants, 2.5~o for $100 supernatants. The yield could reach 125% for a 200,000g supernatant, $200, when it was assayed with both substrates A D P and PO] . Consequently both results are compatible with Buvet's data. We propose the isolation, analysis and characterization of such a catalatic system that we think as being, on the one hand integrated in the mito-
chondrial and thylakoidal membranes, though labile, and on the other hand on active part of the Mitchell's proposal. REFERENCES Boveris A. (1978) Production of superoxide anion and hydrogen peroxide in yeast mitochondria. In Biochemistry and Genetics o/' Yeasts (Edited by Bacila M. et al.), pp. 65 80. Academic Press, New York. Buvet R. ( 1981 ) Energetics of the chemical coupling between the ATP production and 02 reduction. Comparison with electrochemical data. Bioelectrochem. Bioenergetics 9, 299-305. Buvet R. and Le Port L. (1983) Chemical coupling between oxygen reduction and ATP production. Bioelectrochern. Bioenergetics 12, 167-172. Calvayrac R. (1972) Le cycle des mitochondries chez Eug/ena gracilis en cultures synchrones. Th6se de Doctorat d'Etat, Paris XII, CNRS. AO 7183. Calvayrac R. and Butow R. A. (1971) Action de l'antimycine A sur la respiration et la structure des mitochondries d'Euglena gracilis Z. Arch. Mikrobiol. 80, 62-69. Calvayrac R. and Ledoigt G. (1976) Croissance des eugl~nes en pr6sence de DCMU: 6volution du plastidome en fonction de la tension en oxyg~ne. Plant Sci. Lett. 7, 24%263. Calvayrac R. and Laval-Martin D. (1980) Oxygen uptake by Euglena gracilis mitochondria and chloroplasts, stimulated by ultraviolet irradiation. Plant Sci. Lett. 19, 129-141. Calvayrac R., Bomsel J. L. and Laval-Martin D. (1979a) Analysis and characterization of 3-(3,4-dichlorophenyl)1,l-dimethylurea (DCMU) resistant Euglena : I--growth, metabolic and ultrastructural modifications during adaptation to different doses of DCMU. Plant Physiol. 63, 857-865. Calvayrac R., Laval-Martin D., Dubertret G. and Bomsel J. L. (1979b) Analysis and characterization of 3-(3,4-dichlorophenyl)-l,l-dimethylurea (DCMU) resistant Euglena: II--modifications affecting photosynthesis during adaptation to different doses of DCMU. Plant Physiol. 63, 866 872. Cook J. R. (1968) The cultivation and growth of Euglena. In The Biology o/' Euglena (Edited by Buetow D. E,), Vol. 1, pp. 244~314. Academic Press, New York. Cook J. R. and Hunt W. (1965) Ultraviolet bleaching of synchronized Euglena. Photochem. Photobiol. 4, 877-880. De Denken-Grenson D. (1959) Lqnduction en masse de lign6es blanches par la lumi~re ultraviolette chez des flagell6s verts. Archs int. Physiol. Biochim. 67, 506 507. Diamond J. and Schiff J. A. (1970) Insensitivity of development of photosynthetic competence to doses of ultraviolet light which block green colony formation in Euglena. Can. J. Bol. 48, 1277 1283. Epstein H. T. (1977) The pathological effects of light on the skin. In Free Radicals in Biology (Edited by Pryor). Academic Press, New York. Fridovich I. (1975) Superoxide dismutases. A. Rev. Biochem. 44, 147 159. Huyart N., Calvayrac R., Briand J., Goyffon M. and Vuillaume M. (1983) Catalatic properties of hemocyanin in helping to account for the scorpion's radioresistance. Comp. Biochem. Physiol. 76, 153 159. Kempner E. S. (1982) Stimulation and inhibition of metabolism and growth of Euglena gracilis. In The Biology of Euglena (Edited by Buetow D. E.), Vol. 3, pp. 197-252. Academic Press, New York. Lackey J. B. (1968) Ecology of Euglena. In The Biology of Euglena (Edited by Buetow D. E.), Vol. 1, pp. 28-44. Academic Press, New York. Lackey J. A. and Bennett C. (1962) Proc. Natl. S}mp. Radioelecology, lst., Denver, Colorado.
ATP production in Euglena Lienhard G. E. and Secemeski I. I. (1973) PI,PS-di(adenosine 5') pentaphosphate a potent multisubstrate inhibitor of adenylate kinase. J. biol. Chem. 248, 1121-1123.
Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Mehler A. H. (1951a) Studies on reactions of illuminated chloroplasts. I--Mechanism of the reduction of oxygen and other Hill reagents. Archs Biochem. Biophys. 33, 65-77. Mehler A. H. (1951b) Studies on reactions of illuminated chloroplasts, lI--Stimulation and inhibition of the reaction with molecular oxygen, Archs Biochem. Biophys. 34, 339 351. Michaels A. and Gibor A. (1973) Ultrastructural changes in Euglena after ultraviolet irradiation. J. Cell Sci. 13, 799-809. Oberley L. W. (1982) In Superoxide Dismutase (Edited by Oberley C. R. C.), Vols 1 and 2. Academic Press, New York. Ringe D., Petsko G. A., Yamakura F. K., Susuki K. and Ohmori D. (1983) Structure of iron superoxide dismutase from Pseudomonas ovalis at 2.9 ~. resolution. Proc. natn. Acad. Sci., U.S.A. 80, 3879-3884. Ropp M., Courgeon A. M., Calvayrac R. and BestBelpomme M. (1983) The possible role of the superoxide ion in the induction of heat-shock and specific proteins in aerobic Drosophila cells during return to normoxia after
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a period of anaerobiosis. Can. J. Biochem. cell. Biol. 61, 456-461. Schiff J. A. and Epstein H. T. (1968) The continuity of the chloroplast in Euglena. In The Biology of Euglena (Edited by Buetow D. E.), Vol. 1, pp. 286-334. Academic Press, New York. Schiff J. A. and Schwartzbach S. D. (1982) Photocontrol of chloroplast development in Euglena. In The Biology of Euglena (Edited by Buetow D. E.), Vol. 3, pp. 313-352. Academic Press, New York. Vallot R., Vuillaume M., Bona X., Calvayrac R. and Buvet R. (1982) Electrochemical behavior of pterobilin, an insect tetrapyrolic pigment on graphite paste electrode. Bioelectrochem. Bioenergetics 9, 103-115. Vuillaume M., Calvayrac R. and Best-Belpomme M. (1979) Photoconditioning in the integument of Pieris brassicae: ATP production in vivo and in vitro. Properties of the system. Biol. Cell. 35, 71-80. Vuillaume M., Calvayrac R. and Best-Belpomme M. (1982a) Biological model for the possible importance of hydrogen peroxide. Biochem. Bioenergetics 9, 527-534. Vuillaume M., Calvayrac R., Hubert M., Briand J. and Best-Belpomme M. (1982b) Hydrogen peroxide and catalase activity in the integument of an insect (Pieris brassicae). Biol. Cell. 43, 189-194. Wagner G. J. and Lin W. (1982) An active proton pump of intact vacuoles isolated from Tulipa petals. Biochem. biophys. Acta 689, 261-266.
0305-0491/85 $3.00 +0.00 © 1985 Pergamon Press Ltd
ATP P R O D U C T I O N AND CATALATIC-TYPE ACTIVITIES IN ETIOLATED OR G R E E N EUGLENA GRACILIS RI~GIS CALVAYRAC,* DANIELLE LAVAL-MARTIN,* MICHELLE HUBERT,t MARTIN BEST-BELPOMME~ a n d MONIQUE VUILLAUMEt
*Laboratoire des Membranes Biologiques, Universit~ Paris VII, 2, place Jussieu, 75005 Paris Cedex 05, France; tLaboratoire de Zoologie, ERA CNRS 1048, Ecole Normale Sup6rieure, 46, rue d'Ulm, 75230 Paris Cedex 05, France; and ~Laboratoire de Zoologie, ERA CNRS 615, Universit6 Paris VI, 7, quai Saint-Bernard, 75230 Paris Cedex 05, France
(Received 21 January 1985) Abstract--1. Whole Euglena cells, as well as mitochondrial and chloroplastic fractions (12,000-100,000 and 200,000g supernatants), displayed a H202-mediated ATP production with ADP and PO43 as necessary substrates. 2. The adenylate kinase activity could not solely account for such a reaction since APsA did not affect it. 3. Inhibitors of oxidative phosphorylation did not inhibit but sometimes enhanced the phenomenon. 4. Irradiation of the fractions could produce an ATP increase which suggested the participation of superoxide ions. 5. We conclude that the energy of H202, photoproduced or experimentally added, could be converted by a catalatic-type system, normally integrated into organelle membranes, in energy-rich ATP bonds.
INTRODUCTION
The phytoflagellate Euglena gracilis Z is an organism remarkably adaptable to various growth conditions and considered as being at the frontier of animal and plant kingdoms. For example, under darkness the cells, devoid of chloroplasts, can be grown heterotrophicaUy in virtually all possible sources, except citrate, with pHs ranging from 2 to 11 (Cook, 1968; Calvayrac, 1972; Kempner, 1982). When they are illuminated, the cells differentiate chloroplasts and become able to photosynthesize and grow autotrophically on a mineral medium only supplemented with B1 and Bt2 vitamins (Schiff and Epstein, 1968; Schiff and Schwartzbach, 1982). Lackey (1968) indicated that Euglena is resistant to deleterious agents like the heating or organic pollution of the water, thus "if Euglena species existed there before the catastrophe they quickly return". This eukaryote also resists the very same UV doses causing bacterial mutagenesis, without other damage for the cell population than an hereditary bleaching leading to obligatory heterotrophy (De Denken-Grenson, 1959; Cook and Hunt, 1965; Michaels and Gibor, 1973; Diamond and Schiff, 1970). Lackey and Bennett (1962) noticed that another property of Euglena was to exhibit unimpaired reproductive rates when exposed to massive doses of radionuclides (for example, 10% from 6°CO); this means that Euglena are able to resist highly energetic ionizing radiations which reduce oxygen in superoxide ions. Although it is known that free radicals or their terminal product of reactions, H202, are dangerous reactants for the cells (Fridovich, 1975; Epstein, 1977). Buvet (1981) and Buvet and Le Port (1983) recently suggested that they could be beneficial for aerobic organisms, because of their role in the direct or indirect production of cellular energy. In previous papers, our group has shown that: (a)
UV irradiation stimulated oxygen consumption leading to H20_, formation in mitochondrial and chloroplastic fractions from Euglena (Calvayrac and LavalMartin, 1980); this phenomenon, sensitive to 2-salicyl hydroxamic acid (SHAM) could be reinforced by 6.6 mM C N - and stayed unaffected by deoxycholate treatment (1 mg/mg of membranal proteins); (b) molecules of biological interest such as ubiquinone, plastoquinone and pheophytin a were able to exhibit similar UV-dependent H202 production (Vuillaume et al., 1982a, b); (c) the H202 integumentary photoproduction by the pterobilin of Pieris brassieae L5-1arvae was able to lead to a production of ATP in the presence of catalatic activity and did not require membrane entegrity (which is a non-classical and non-admitted physiological behavior) (Vuillaume et al., 1979, 1982b; Vallot et al., 1982); (d) an aspect of the resistance to highly energetic ionizing radiations by scorpions (Androctonus australis) could be explained by the catalatic activity of their blood pigment, the hemocyanin; this activity could be characterized in the purified native pigment as well as in its heavy dissociated subunits (i.e. dodecamers and hexamers) (Huyart et al., 1983). Such findings, involving different biological systems have lead us to the Mehler's (1951) concept and to examine the possible bond between the ATP production and the redox processes involving H202. In this paper we present data concerning different types of Euglena cells and their subcellular fractions, and we show that radiations can enhance the catalase-like reactions (catalatic reactions) producing ATP from H202. MATERIAL AND METHODS
Conditions of cultures All cultures were at 26"C in three different mediums, pH 3.5, containing 33 mM lactate as sole carbon sources.
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RI~GIS CALVAYRACet al.
The standard medium, indicated "L", contained among other mineral salts, 173mEq of PO] and 68.4mEq of NH2; the second medium, indicated "L, NH +'', was comparable for all components but ammonium at the final concentration of 205 mEq; the third medium, indicated "L, N H ; , PO 3 ", contained 205 mEq of NH + and 370 mEq of
PO~-. Two different strains of E. gracilis were studied: the control strain, Z, which is either etiolated when cultured in darkness (heterotrophy) or which contains chloroplasts when cultured under white light (30W/m 2) (photoheterotrophy) and an apoplastidial strain ZC which has been initially obtained from the adaptation of control Z cells in a diuron containing lactate medium under light and in micro-aerophyllic conditions (Calvayrac and Ledoigt, 1976; Calvayrac et al., 1979a, b). We have tested different strains of Euglena gathered at different physiological stages during the exponential phase of growth of a culture when the cells actively divide with generation time approximating 10 hr, phase I, or when the culture had reached the stationary phase and the cells were in infradian mode of population increase, phase III; the intermediary phase, phase II, characterizes the moment when cells still divide but with long generation times due to limiting factors in the medium.
Preparation of the subcellular fractions Cells from the culture medium were gathered by a 500 g centrifugation for 10rain, rinsed in distilled water and repelleted by an identical centrifugation. The pellet was weighed and a pH 7.2, Tris-NaC1 50 mM buffer (B) containing 0.25 M sucrose, 2.5 mM CaCI 2 and 2.5 mM MgCl 2 was added in the w/v ratio of 1/4. An aliquot of the cell suspension in buffer was kept for assays on whole cells, the rest was transferred in a "Braun Broyeur" with 2.5 g of 2 m m glass beads per ml of cell suspension. The cell breakage was done at maximal speed, under carbonic snow cooling, 3 times per 10 sec separated by 2 times per 20 sec. The resulting suspension was centrifuged for 15 min at 150 g (SS 34 rotor, Sorval centrifuge), the pellet was discarded and the supernatant (S) recentrifuged for 15 min at 12,000g; the pellet then obtained constituted the mitochondrial fraction and the supernatant, S12", was partly recentrifuged at 100,000g to obtain a second supernatant, S~00, itself recentrifuged at 200,000g to give $200. In the case of green Euglena with chloroplasts the first supernatant (S) was centrifuged for 15min at 1500g to obtain a pellet essentially composed of the chloroplasts. Pellets of either chloroplasts or mitochondria were washed once in the extraction buffer and recentrifuged, respectively, at 1500 and 12,000g for 15 min. All fractions tested (pellet of mitochondria, pellet of chloroplasts, S~2, $200) were diluted in the extraction buffer at the moment of the assay to test fractions containing about 0.5mg as total proteins per ml. Protein measurement The protein content was assayed as described by Lowry et al. (1951) on fractions previously diluted 2 times in NaOH 5 N and kept for 30 min at 37'~C. A TP measurements The ATP measurements were performed in a "Nucleotimeter 107 CLV-Interbio" with lucioline (IB 20 IC CVLInterbio) dissolved in a buffer, pH 7.75 (IB 25 50 CVLInterbio) on fractions of 150#1 total volume containing 25 #1 of nucleoline (IB 40 50 CVL-Interbio) directly added to the fraction after sonication (30 K cycles, 2 x 30 sec at 0°C). Nucleoline is a complex detergent which induces the *Abbreviations: cel., whole cells; 812, 12,000g supernatant; Sl00, 10,000g supernatant; $200, 200,000g supernatant; Mit., mitochondrial fraction; NAN3, sodium azide; C N - , cyanide; DNP, dinitrophenol; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone.
total permeability of cellular membranes to nucleotides. Calculations of the ATP quantities were performed using the "peak condition" mode and 30 sec of preselection. The results were expressed either in ATP molarities per mg of total protein or in ATP increase as percent of ATP basis. At first, the initial ATP contents of each pellet and supernatant studied were individually determined in absence of all reactives to determine their "ATP basis"; then their eventual ATP productions were tested when either HzO z (10 13to 10 ~M) or white light (15 W/m2/sec) were applied. The ATP backgrounds of all reactives (ADP, PO]-, H202, nucleoline, e t c . . . ) were separately determined against lucioline as well as those of their mixture two by two, three by three or all together. This procedure permitted correction of the result obtained in each of the individual assays from the corresponding background due to the specific reactant(s) then used. Supernatants Sl00 and $200 did not display spontaneous ATP production after either addition of H20 2 (5 x 10 -6 to 5 x 10 ~ M) or illumination. They were then assayed successively: (a) in the presence of PO 3- (5 x 10 -4M) alone; (b) in the presence of ADP (5 x 10 6 M) alone; (c) with both products. This last condition ( A D P + P O ] - ) showed an increase in ATP when either H202 or light were applied to the supernatant.
Adenylate kinase activiO, ADP, 1 mM, was solubilized in a 0.5 M Tris buffer, pH 7.5. The assay was run at room temperature by mixing volume to volume the cell extract (Mit. or S~00) containing 2.5mM MgCI> and the ADP solution. To follow the kinetic, every 5 min, 10/~1 aliquots of the assay mixture were diluted 104-fold in 0.25 m M Tris buffer, pH 7.5, and the ATP contents rapidly determined with the Nucleotimeter. The 104 fold dilution stopped the adenylate kinase reaction without any risk of reversion since the reaction then tended to an equilibrium of which the constant had no more dimension since: (ATP)(AMP) (ATP) 2 K (ADP) 2 (ADP) 2" In case of the presence of an adenylate kinase activity, such an assay gives for the first hour a linear ATP production. In addition, and in another set of assays we have inhibited the adenylate kinase activity by its specific inhibitor, APsA (Lienhard and Secemeski, 1973), used at a concentration 10-100-fold the ADP concentration present in each assay.
Drug effects We tested drugs known to have inhibitory actions on either the oxidative phosphorylations or on phosphorylations resulting from the pathway insensitive to cyanide (Calvayrac and Butow, 1971), i.e. DNP, 10 -s to 10-4 M; NaN3,10 4 t o 3 x 10 2 M ; C N 1 0 - 4 t o 3 x 10 3M and FCCP, 3 x 10 3M. RESULTS P r i o r to each e x p e r i m e n t , the luciferine luciferase m i x t u r e was tested to m e a s u r e the units o f relative l u m i n e s c e n c e ( U R L ) w h i c h c o u l d be o b t a i n e d with s t a n d a r d A T P solutions. W h e n set in p e a k c o n d i t i o n s the a p p a r a t u s i n d i c a t e d linearity for the r e a c t i o n s " A T P s t a n d a r d . . . . l u c i f e r i n e - l u c i f e r a s e " b e t w e e n 500 a n d 80,000 U R L . C o n s e q u e n t l y we diluted with the buffer (B) the different fractions to be tested to p e r f o r m differently in this interval.
Choice o f extract preparation .for A T P measurements T h e m o s t r e p e a t a b l e d a t a were o b t a i n e d by o m i t ting the s o n i c a t i o n a n d utilizing only the a c t i o n o f
ATP production in Euglena
1011
Table 1. Example of experimentaldata from a typical experiment
Mixture tested
URL
10#IPO~-*+25#IN?+ II5/~IB~ or H 2 0 20 ,al A D P § + 25 .ul N + 105/.d B or H 2 0
ATP (10 -6 M)
20 7500
20 #1H~O:II + 25 #1N + 105 #1B or H20 10 #1PO43- + 25 #1N + 20 # 1 A D P + 95 #1B or H20 50 #1 Cel. + 25 #1N + 75 #1B 50 #1 Cel. + 25 #1N + 25 # 1 H : O 2 + 50/zl B 50 #1Mit. + 25 #1N + 75 #1B 50 # 1 M i t . + 25 .ul N + 25/.tl H20: + 50 # 1 B 50 #1SL00+ 25 #1N + 75 #1B or H20 50 #1 St00 + 25 #1N + 20 # 1 A D P + 55 #1B or H20 50 #1Si0o + 25 #1N + 20 # 1 A D P + 20 ~1H202 + 35 #1 b or H20 50 #1S~00 + 25 # 1 N + 20 # 1 A D P + 10 #1PO~- + 45 #1 B or H20 50 #1S~00 + 25 # 1 N + 20 # 1 A D P + 10 #1PO~- + 20 #1H~O~ + 25 #1 B or H~O
Increase in ATP due to H202 as percent of ATP basis
---
0 7600 3000 12,210 3000 8000 3700 11,000 13,200 13,500 22,600
--0.35 1.42 0.35 0.93 0.40 1.20 1.30 1.50 2.50
307 166
67
*PO43-, 5 x 10 -4 M; i'N + nucleoline; SB = buffer (50 mM Tris, 250 mM sucrose, 2.5 mM CaCI 2, 2.5 mM MgCI2); §ADP, 5 x 10 -6 M; tlH202, 5 X 10 -7 to 5 X 1 0 - 4 M depending upon the fraction tested, the maximal result obtained with the optimal concentration being the only one retained.
nucleoline which induced the total permeability of the membranes to nucleotides.
Example of raw data and expression of the results We present in Table 1 the results of a typical experiment. In such an example we retain two values: the ATP basis (line 5 for whole cells, Cel.,; line 7 for mitochondrial fraction, Mit. and line 12 for St00) and the maximum ATP in the presence of optimal H202 concentration (lines 6, 8 and 13, respectively). For a given fraction, the difference between these two results expressed as percent of the ATP basis indicates the increase of ATP production provoked by H202 addition. In the given example (Table 1) the increment of ATP production in the presence of H202 would be 307~ for whole cells, 166~o for the mitochondrial fraction and 67~ for Si00.
sonication (plateau from 5 x 10 -~2 to 5 x 10-SMH202), the absolute value of H202mediated ATP formation was six-fold increased on a protein basis but was only equal to about 180Y/owhen expressed as percent of the ATP basis, which indicated the damaging of some of the enzymatic sites.
Adenylate kinase act&ity In the supernatants the H202-mediated ATP production was maximal in the presence of both PO43- and ADP; it was possible to hypothesize either a phosphorylation, i.e. a reaction energetically important for the cellular metabolism, or that the PO43- could be an effector for the adenylate kinase activity. For this reason we tested the adenylate kinase activity in pellets and supernatants from Z cells grown heterotrophically in the darkness.
Influence of the H202 molarity on the A TP production We studied the evolution of the ATP production as function of the H202 added on Z Euglena grown in " L " medium in darkness. The addition of H202 from 10 -z3 to 10-1M increased the ATP production per reference to the fraction tested without H202 and taken as basis (i.e. 0 ~ of ATP increase). This H202-mediated ATP productior1 characterized a suspension of whole cells (5.9 mg of total proteins per ml) treated with nucleoline vol. to vol., just prior to H202 addition (Fig. 1, curve A), as well as the same cells sonicated at 0°C (3 x 10see, 8 Kcycles) and subsequently diluted 10 times with the buffer (Fig. 1 curve B). The results would be comparable for mitochondrial and $12 fractions, the curves being translated to a lower range of H202 concentrations. Both curves A and B (Fig. 1) showed a first portion corresponding to H202 concentrations limiting the phenomenon, then a plateau of optimal H202 concentrations for a maximal production of ATP (situation systematically selected for all subsequent experiments), followed by a toxic effect on the highest H202 concentrations. The ATP increase expressed as percent of ATP basis was maximal, equal to 300~, when whole cells were tested in spite of the limited diffusion of H202 towards the sites of disproportionation (plateau between 10 _4 and 10-3M H202). When the sites were exposed after
i
';300
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Fig. 1. ATP increase, expressed as percent of the ATP basis, as a function of H202 concentrations. The phenomenon was tested on strain grown in "lactate" medium in darkness. Curve A corresponded to whole cells rendered permeable to nucleotides by addition, at the moment of the assay, of nucleoline; curve B corresponded to the same cells after sonication.
1012
R~GIS CALVAYRACet al. I u~
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Fig. 2. Test of the adenylate kinase activity present in either mitochondrial fraction (curve A) or in S~2 supernatant (curve B) of dark grown Z strain cultivated in "lactate" medium and tested as described in Materials and Methods.
The adenylate kinase activities found in this fraction were reinforced by the addition of PO]- (10 -2 M) to the ADP (5 x 10-4M). The effect of the PO4~- was remarkable on the mitochondrial fraction where it could increase the ATP concentration by a factor 3 (after 18 min). These data suggested more than an effector role on the adenylate kinase activity but probably the occurrence of phosphorylation which could be due to a certain amount of H202 present in the mitochondria and able to promote H202mediated ATP production. Such an hypothesis seemed to be supported by the assays testing simultaneously the addition of H202. The addition of H202 to a mitochondrial fraction containing both ADP and PO]- in excess indicated a very strong, though = 5 . 1 0 "~ , transient, H202-mediated ATP production. It was interesting to notice in Fig. 3 the same profiles of the curves relative to the mitochondrial fraction tested ~ AS with PO]-. We know that Euglena contained, on average, 4 nmol ATP per 106 cells (in phase I, for nucleotide measurements and energy charge deter6 A A mination, data not shown). We could then measure the adenylate kinase activity, using the ATP basis =. (Table 1), in the mitochondrial fraction and the corresponding $12 supernatant (Fig. 2). Such a calculation indicated that the total content in ATP was r., 1 . 1 0 ~ Ionly about 2 ~ of the expected one (with the following distribution: 23~o in the mitochondrial fraction 5.10" 10 20 3O against 77~o in the $12 supernatant) and that consequently ATPases actively hydrolyzed the cellular DURATION_ minute_ Fig. 3. Test of the possible effector role of PO~- in the ATP during the preparation of the fractions and also mitochondrial fraction treated with nucleoline (1 vol. to when nucleoline was added. 5 vol.). This fraction proceeded from dark grown Z strain During the assays, while H2Oz-mediated ATP forcultivated in "lactate" medium and tested as described in mation reached 5 x 10 6 to 10 -5 M, addition of 10 -4 Materials and Methods. Some of the assays were done in the to 10 3 M of APsA did not decrease these ATP levels. presence of PO3 (10 2M). Simultaneously the H202- Consequently, the H202-dependent ATP production mediated ATP production was tested by adding HzO2 (8 x 10-~ M) to aliquots of the reaction mixture, prior to the did not originate from adenylate kinase activity. Such results allow us to exclude the intervention of final 10a-fold dilution. Curve A, Mit. fraction; curve B, Mit. fraction + H202; curve C, Mit. fraction + PO] ; curve D, adenylate kinase reaction in the H2Oz-mediated ATP production that we present in this paper. Mit. fraction + P043 + H202.
The first experiment done on mitochondrial fraction and S~2 without addition of either nucleoline or PO 3- (Fig. 2) showed a strong adenylate kinase activity in S~2 while the mitochondrial fraction seemed to present in addition to the adenylate kinase activity an even more rapid hydrolysis of the ATP formed, leading after 10 min to ATP concentrations lower than the one at time zero. The second experiment was designed to test the possible effector role of the PO43- on the adenylate kinase activity and also to test, in the conditions of the assay, the effect of H202. The results concerning a mitochondrial fraction are summarized in Fig. 3.
ATP production in Euglena
1013
Table 2. Comparison of the H202-mediated ATP production in fractions isolated from Z strain Euglena grown in " L " medium in darkness. The beginning of the exponential phase of growth (phase I characterized by H202 optimal 2.5 x 10-4M) is compared to the stationary phase (phase III, H202 optimal 5 × 10 7M) Growth phase I
Growth phase III
Fraction
ATP basis (nmole per mg of protein)
Increase in ATP with H20 z optimal (% vs ATP basis)
ATP basis (nmole per mg of protein)
Increase in ATP with H202 optimal ( ~ vs ATP basis)
Mitochondria S~2
47 111
149 89
40 522
70 21
Table 3. Ubiquity of H202-mediated ATP production in different fractions isolated from ZC and Z strains of Euglena grown in different mediums either in darkness or under illumination. The photoorganotrophic medium in which Z strain cells were grown contained diuron 25/~M to modify the chloroplastic compartment which when keeping unmodified volume, loses 9 0 ~ of its chlorophyll content (Calvayrac et al., 1979b) Culture in darkness
Culture under illumination
Fraction
ATP basis (nmole per mg of protein)
Increase in ATP with H202 optimal (% vs ATP basis)
ATP basis (nmole per mg of protein)
Increase on ATP with H202 optimal ( ~ vs ATP bazis)
ZC strain in " L "
Mitochondria Si2
13 28
102 34
62 63
83 66
Z strain in " L "
Chloroplasts Mitochondria S~2
-47 111
-149 89
21 28 66
95 135 54
Z strain in "L, NH~-"
Chloroplasts Mitochondria Si2
-12.5 --
-168 --
Il 12 24
104 109 60
Z strain in "L, N H t , P O ~ - "
Mitochondria
28
215
26
187
Type of cell and medium
Ubiquity of the H202-mediated ATP production In an attempt to generalize the existence of an H202-mediated ATP production, we have tested Euglena grown in mediums containing different amounts of NHg and/or PO 3- in the dark and under light and gathered at different physiological stages. In the case of white cells (Z strain cultured in the dark, or ZC strain cultured either in the dark or under light), we studied the mitochondrial fraction, the S~: supernatant and sometimes St00 and $200; in the case of chlorophyllian cells (Z cells cultured under light) a chloroplastic fraction was also tested. In this latter condition of culture we modified the chloroplastic compartment by adding 25/~M D C M U to the medium; such a treatment which did not affect the volume of chloroplasts (Calvayrac and Ledoigt, 1976) not only depressed the chlorophyll content by 90~o and totally inhibited oxygen evolution (Calvayrac et al., 1979b) but favored light-induced oxygen consumption (Calvayrac and Laval-Martin, unpublished results).
Influence of the physiological stage of the cells We compared white Z strain cells, grown in darkness in the same lactate medium. Table 2 summarizes the results and indicates that the ATP basis (nmole of ATP per mg of protein) as well as the increase in ATP when 10 -5 M H202 was added, were higher in both fractions extracted from cells actively dividing (phase I) than in the corresponding fractions obtained from stationary cells (phase III). Moreover, if the ATP basis seemed depressed in mitochondrial fractions, due probably to their richness in membranal proteins, the H202-mediated ATP production (expressed as percent of the ATP basis) was important in this fraction and still important in S~2. We
then tested, in the rest of the study, cells gathered in phase I.
Influence of illumination or darkness during the cell growth Cells gathered in phase I and grown in different organotrophic mediums either in darkness or under light have been compared (Table 3). Whatever the type of cell and/or the medium and light condition, the H202-mediated ATP production (expressed as percent vs the ATP basis) is higher in mitochondrial fraction than in $12. When cells were green (Z cell cultured under illumination), the phenomenon was found in the chloroplast fraction as well as in the mitochondria. It seemed that mitochondria from etiolated cells (Z strain cultured in darkness) were more active for H202-mediated ATP production than mitochondria from the corresponding green cells (Z strain under illumination). Such an enhancement of ATP production by darkness can be observed also in apoplastidial ZC strain (never containing any chlorophyll whatever the light regimen). Thus the phenomenon, perhaps favored in heterotrophic cultures grown in darkness, would be very active in organelles but still present in Sl2 with a depressed efficiency.
Light-enhancement production
of the H202-mediated A TP
We have tested the effect of short time light exposures on the ATP production in ZC apoplastidial whole cells grown either in darkness or under illumination. Such experiments were done without any H202 addition, the cell walls being rendered permeable to ATP by the nucleoline (cf. Table I). The results in Fig. 4 showed ATP increases in both types of cells but with a faster kinetic in the
1014
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DURATION second _ Fig. 4. ATP increase, expressed vs percent of the ATP basis, in function of the 580 W/m 2 white illumination, in whole apoplastidial ZC strain cells grown in lactate medium either in darkness ( + +) or under light (Q 0 ) were tested. _
i
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case of dark grown cells than in the case of light grown ones. The same type of experiment done after breakage of the dark grown cells, either on the mitochondrial fraction (Mit.) or on $12 supplemented or not with ADP + PO43-, indicated (Fig. 5) rapid increase in ATP after the first minute quickly hydrolyzed in the mitochondria and a slow linear increase of ATP in Sn for 6 min.
Q.
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We tested the effects, on the H202-mediated ATP production, of some drugs applied on the fractions at the moment of the assays. Figure 6A and B indicate that not only did the drugs not inhibit the phenomenon but they provoked an increase in the ATP production: the DNP (Fig. 6A) promoting a strong activation when applied at very low concentration (10 -5 to 10 -4 M) and both C N - and NaN3 enhancing the ATP production (Fig. 6B) at concentrations from
10 -4 to 3 x 10 3M f o r C N - and to 5 x 1 0 -2M for NAN3. The FCCP 3 x 10-3M did not inhibit the H202-mediated ATP production either (data not shown). For strong concentrations (3 x 10 3 for CN and 5 x 10 -2 for NAN3) we noted a total ATP-H202 inhibition.
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DURATION_ m i n u t e _ Fig. 5. ATP increase, expressed vs percent of the ATP basis, in function of the 580 W/m 2 white illumination, in mitochondrial fraction (curve A) and 12,000g supernatant (curve B), extracted from apoplastidial ZC ceils grown in "lactate" medium under light. An experiment with supernatant was carried out in the presence of 8 x l0 -4 M PO ]and 2 × l0 -4 M ADP (U II). It gave identical results to the experiment without substrate ( + +).
Effects of drugs affecting the oxidative pathways on the H202-mediated A TP production
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Fig. 6. Effect of different drugs on the H202-mediated ATP increase, expressed vs percent of the ATP basis, in a mitochondrial fraction. This fraction was extracted from Z cells, dark grown on "lactate, NH~" medium and assayed in the presence of 5 × 10-s M H202 (optimal concentration). Curve A: effect of DNP (10 -4 to 10-SM). Curve B: effect of CN- (10 _4 to 10-2M) and of NaN~ (5 × l0 -3 to 5 × 10-1).
ATP production in Euglena
1015
Table 4. H202-mediatedATP production in mitochondrialfractions from Z cells grown in darkness in "L, NH~-" mediumcontaining or not 10-6M antimycin Culture with 10-6 M antimycin Culture without antimycin ATP basis Increase in ATP ATP basis Increase in ATP (nmole per with H202 optimal (nmole per with H202 optimal mg of protein) (~o vs ATP basis) mg of protein) (~ vs ATP basis) Control 12.5 168 41 133 Assay with 2 mM CN- or 2 mM NaN3 20 190 38 165
Intensity of the H202-mediated ATP production in mitochondrial fraction from cultures grown in presence of an antibiotic; antimyein Antimycin was known to favor, in Euglena, the shunt insensitive to cyanide (Calvayrac and Butow, 1971). We then grew Z strain cells on "lactate, NH +'' medium, in darkness, in the presence or not of 10-6M antimycin and tested the H202-mediated ATP production of their mitochondrial fractions. In addition we have, at the moment of the assays, treated fractions by 2 mM C N - or NaN 3. Table 4 shows the results obtained. They indicate that the phenomenon of ATP production was virtually unaffected in cells grown in the presence of antimycin, i.e. in cells in which the insensitivity to C N shunt was strengthened (Calvayrac and Butow, 1971); moreover the addition of either CN or NaN3 to the reactive medium did not affect the reactions but surprisingly enhanced them (cf. Fig. 6B). DISCUSSION AND CONCLUSION Whatever the conditions of culture (under light or in darkness) in lactate medium supplemented or not with NH2- and/or PO 3-, the two Euglena strains studied, i.e. the control green or etiolated Z strain and the apoplastidial ZC strain, as well as the derived cellular fractions (mitochondrial, chloroplastic, the 12,000, 100,000 and 200,000g supernatants), all presented the same phenomenon of an H202mediated ATP production measured in the presence of nucleoline by the luciferinv-luciferase method. These same strains and cellular fractions were able to consume oxygen when illuminated, especially with UV and red light (Calvayrac and Laval-Martin, 1980), as shown by observations done as soon as 1951 by Mehler. Recent results obtained by some of us had shown that integuments of Pieris brassicae larvae presented a natural H202 and/or light production of ATP (Vuillaume et al., 1979, 1982a, b) and reviews (Chance et al., 1979; Oberley, 1982) reported that some authors had found that H202 could be formed either in the mitochondria or in the cytosol (Boveris, 1978); we thought that the H202-mediated ATP production observed could be a natural process. Keeping in mind this idea of a possible ubiquitous reaction of ATP production we will discuss the data obtained in this work.
Whole cells White Z and ZC cells, illuminated by strong (580W/m 2) white light, for very short durations, displayed (Fig. 3) ATP production which increased by a factor 2.2 after 45 sec when the cells were grown in darkness, vs 1.2 after 5 sec when the cells were
illuminated during their growth. This suggested a more active regulation of the equilibrium ATP/ADP in cells perhaps able to use photons for ATP production during their growth than in cells unable to use light since grown in darkness. Such results were only obtained with intact cells treated at the moment of the assay with nucleoline which rendered the membranes permeable to nucleotides. When cells were sonicated the phenomenon was not measurable, probably because of the hydrolysis of the ATP photoproduced by ATPases released by the bursting of the membranes. We showed that growth of Euglena cultures was not affected by the addition of antimycin (Calvayrae and Butow, 1971) and proposed that the metabolism in the mitochondria was unperturbed because of the functioning of a secondary pathway insensitive to cyanide. We show in this paper that treated cells (Table 4), as control cells, could produce ATP by H202-mediated processes and were richer in ATP content if we consider the protein basis.
Subcellular Jkactions Mitoehondrial and ehloroplastic fractions. These were the fractions exhibiting the highest potential for H202-mediated ATP production. Nevertheless, when strongly illuminated, they reacted (Fig. 5) with at first a rapid ATP increase, followed by a continuous drop in ATP, a secondary reaction probably due to hydrolysis by the free ATPases. Supernatants 12,000 and 100,000 g. When strongly illuminated these supernatants exhibited a strong ATP increase (cf. Fig. 5) for 6 min. For 100,000g we obtained a kinetic comparable to St2 only when ADP and PO 3- were added prior to the illumination. Previous observations indicated that the systems, present in both mitochondria and chloroplasts, which are responsible for a light-mediated ATP production, were labile and able to work from an integrated membrane with both ADP and PO 3- at their disposal.
Adenylate kinase activity We thought that we could reject the simple intervention, in the H2Oz-mediated ATP production, of the adenylate kinase activity that we had found in each of the tested fractions, alone or accompanied by an ATP hydrolysis (case of the mitochondrial fraction, Fig. 2) for the following reasons: (a) The addition of PO 3- increased the ATP formation in crude mitochondrial fractions more than in the measure adenylate kinase activity (Fig. 3A and C). We found the same data in all fractions but with less yield, indicating that PO]- is a normal substrate for mitochondrial ATP production. (b) The addition of H202 to the assay mixtures was efficient for ATP
1016
P-~GIS CALVAYRACel al.
production in a mitochondrial fraction containing PO~ , but was still able to increase A T P production by 50-30% in a crude mitochondrial fraction. This indicated that in the membranes, even if the adenylate kinase exists, the H2Oz-mediated A T P production was possible and favored if both substrates, PO] and A D P , were present. (c) We noted for all other fractions the same result as below, i.e. H20 2 addition increased the A T P formation but less than in the mitochondrial fraction, only when PO] is added. (d) Finally the addition of concentrations of APsA (a specific inhibitor of adenylate kinase activity, according to Lienhard, 1973) 10 to 100-fold the one of A D P , never affected the H2Oz-mediated A T P production. Catalatic activity In all the fractions studied in this work, we could always detect an intense catalatic activity. The C N and azide, already known to have an inhibitory action on the H202, did not inhibit but promoted the H202-mediated A T P production in Euglena material studied here (Fig. 6B), as was the case in Pieris brassicae (Vuillaume et al., 1979, 1982a). The remarkable A T P production by a fraction tested in the presence of D N P (Fig. 6A), known for its uncoupling effect on oxidative phosphorylation, could be explained by the peculiarity of D N P to be able to form H202, as is the case of molecules containing phenol rings (Vuillaume et al., 1982b). Thus all the drugs inhibiting phosphorylation according to Mitchell's scheme, as well as the F F C P dissipating the proton gradient (Wagner and Lin, 1982), had no action or promoted the H202-mediated A T P formation. In conclusion we could then interpret the ubiquitous phenomenon, here observed either in whole cells or in mitochondrial and/or chloroplastic fractions and even in 12,000 or 100,000g supernatants, ,~s being the result of the catalatic activity of a soluble protein able to gather in a high energy bond in ATP, the energy being provided by H202 either photoproduced or experimentally added. At p H 7 disproportionation of a mole H202 was known to produce 25kcal (Buret, 1981), energy theoretically sufficient to permit the synthesis of 1 or 2 A T P molecules, according to the following proposed scheme:
H20~.\
/"-~O~+ mO Protein with catalatic properties
A D P + PO4-3
ATP
According to our first calculations of the molecules of A T P produced vs the molecules of H202 experimentally furnished, we found the following yields for assays run without A D P and PO] : 30% for mitochondrial fractions, 50% for SL2 supernatants, 2.5~o for $100 supernatants. The yield could reach 125% for a 200,000g supernatant, $200, when it was assayed with both substrates A D P and PO] . Consequently both results are compatible with Buvet's data. We propose the isolation, analysis and characterization of such a catalatic system that we think as being, on the one hand integrated in the mito-
chondrial and thylakoidal membranes, though labile, and on the other hand on active part of the Mitchell's proposal. REFERENCES Boveris A. (1978) Production of superoxide anion and hydrogen peroxide in yeast mitochondria. In Biochemistry and Genetics o/' Yeasts (Edited by Bacila M. et al.), pp. 65 80. Academic Press, New York. Buvet R. ( 1981 ) Energetics of the chemical coupling between the ATP production and 02 reduction. Comparison with electrochemical data. Bioelectrochem. Bioenergetics 9, 299-305. Buvet R. and Le Port L. (1983) Chemical coupling between oxygen reduction and ATP production. Bioelectrochern. Bioenergetics 12, 167-172. Calvayrac R. (1972) Le cycle des mitochondries chez Eug/ena gracilis en cultures synchrones. Th6se de Doctorat d'Etat, Paris XII, CNRS. AO 7183. Calvayrac R. and Butow R. A. (1971) Action de l'antimycine A sur la respiration et la structure des mitochondries d'Euglena gracilis Z. Arch. Mikrobiol. 80, 62-69. Calvayrac R. and Ledoigt G. (1976) Croissance des eugl~nes en pr6sence de DCMU: 6volution du plastidome en fonction de la tension en oxyg~ne. Plant Sci. Lett. 7, 24%263. Calvayrac R. and Laval-Martin D. (1980) Oxygen uptake by Euglena gracilis mitochondria and chloroplasts, stimulated by ultraviolet irradiation. Plant Sci. Lett. 19, 129-141. Calvayrac R., Bomsel J. L. and Laval-Martin D. (1979a) Analysis and characterization of 3-(3,4-dichlorophenyl)1,l-dimethylurea (DCMU) resistant Euglena : I--growth, metabolic and ultrastructural modifications during adaptation to different doses of DCMU. Plant Physiol. 63, 857-865. Calvayrac R., Laval-Martin D., Dubertret G. and Bomsel J. L. (1979b) Analysis and characterization of 3-(3,4-dichlorophenyl)-l,l-dimethylurea (DCMU) resistant Euglena: II--modifications affecting photosynthesis during adaptation to different doses of DCMU. Plant Physiol. 63, 866 872. Cook J. R. (1968) The cultivation and growth of Euglena. In The Biology o/' Euglena (Edited by Buetow D. E,), Vol. 1, pp. 244~314. Academic Press, New York. Cook J. R. and Hunt W. (1965) Ultraviolet bleaching of synchronized Euglena. Photochem. Photobiol. 4, 877-880. De Denken-Grenson D. (1959) Lqnduction en masse de lign6es blanches par la lumi~re ultraviolette chez des flagell6s verts. Archs int. Physiol. Biochim. 67, 506 507. Diamond J. and Schiff J. A. (1970) Insensitivity of development of photosynthetic competence to doses of ultraviolet light which block green colony formation in Euglena. Can. J. Bol. 48, 1277 1283. Epstein H. T. (1977) The pathological effects of light on the skin. In Free Radicals in Biology (Edited by Pryor). Academic Press, New York. Fridovich I. (1975) Superoxide dismutases. A. Rev. Biochem. 44, 147 159. Huyart N., Calvayrac R., Briand J., Goyffon M. and Vuillaume M. (1983) Catalatic properties of hemocyanin in helping to account for the scorpion's radioresistance. Comp. Biochem. Physiol. 76, 153 159. Kempner E. S. (1982) Stimulation and inhibition of metabolism and growth of Euglena gracilis. In The Biology of Euglena (Edited by Buetow D. E.), Vol. 3, pp. 197-252. Academic Press, New York. Lackey J. B. (1968) Ecology of Euglena. In The Biology of Euglena (Edited by Buetow D. E.), Vol. 1, pp. 28-44. Academic Press, New York. Lackey J. A. and Bennett C. (1962) Proc. Natl. S}mp. Radioelecology, lst., Denver, Colorado.
ATP production in Euglena Lienhard G. E. and Secemeski I. I. (1973) PI,PS-di(adenosine 5') pentaphosphate a potent multisubstrate inhibitor of adenylate kinase. J. biol. Chem. 248, 1121-1123.
Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Mehler A. H. (1951a) Studies on reactions of illuminated chloroplasts. I--Mechanism of the reduction of oxygen and other Hill reagents. Archs Biochem. Biophys. 33, 65-77. Mehler A. H. (1951b) Studies on reactions of illuminated chloroplasts, lI--Stimulation and inhibition of the reaction with molecular oxygen, Archs Biochem. Biophys. 34, 339 351. Michaels A. and Gibor A. (1973) Ultrastructural changes in Euglena after ultraviolet irradiation. J. Cell Sci. 13, 799-809. Oberley L. W. (1982) In Superoxide Dismutase (Edited by Oberley C. R. C.), Vols 1 and 2. Academic Press, New York. Ringe D., Petsko G. A., Yamakura F. K., Susuki K. and Ohmori D. (1983) Structure of iron superoxide dismutase from Pseudomonas ovalis at 2.9 ~. resolution. Proc. natn. Acad. Sci., U.S.A. 80, 3879-3884. Ropp M., Courgeon A. M., Calvayrac R. and BestBelpomme M. (1983) The possible role of the superoxide ion in the induction of heat-shock and specific proteins in aerobic Drosophila cells during return to normoxia after
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1017
a period of anaerobiosis. Can. J. Biochem. cell. Biol. 61, 456-461. Schiff J. A. and Epstein H. T. (1968) The continuity of the chloroplast in Euglena. In The Biology of Euglena (Edited by Buetow D. E.), Vol. 1, pp. 286-334. Academic Press, New York. Schiff J. A. and Schwartzbach S. D. (1982) Photocontrol of chloroplast development in Euglena. In The Biology of Euglena (Edited by Buetow D. E.), Vol. 3, pp. 313-352. Academic Press, New York. Vallot R., Vuillaume M., Bona X., Calvayrac R. and Buvet R. (1982) Electrochemical behavior of pterobilin, an insect tetrapyrolic pigment on graphite paste electrode. Bioelectrochem. Bioenergetics 9, 103-115. Vuillaume M., Calvayrac R. and Best-Belpomme M. (1979) Photoconditioning in the integument of Pieris brassicae: ATP production in vivo and in vitro. Properties of the system. Biol. Cell. 35, 71-80. Vuillaume M., Calvayrac R. and Best-Belpomme M. (1982a) Biological model for the possible importance of hydrogen peroxide. Biochem. Bioenergetics 9, 527-534. Vuillaume M., Calvayrac R., Hubert M., Briand J. and Best-Belpomme M. (1982b) Hydrogen peroxide and catalase activity in the integument of an insect (Pieris brassicae). Biol. Cell. 43, 189-194. Wagner G. J. and Lin W. (1982) An active proton pump of intact vacuoles isolated from Tulipa petals. Biochem. biophys. Acta 689, 261-266.