differential inhibition of respiration and its dependent h+ extrusion by fluorescamine in rat liver...

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 201, No. 1, April 15, pp. 330-338, 1980

Differential Inhibition of Respiration and its Dependent H+ Extrusion

by Fluorescamine in Rat Liver Mitochondrial

ERIC LAM, DAVID SHIUAN, AND SHU-I TU

Department of Chemistry, State University of New York, Stony Brook, New York 11794

Received July 20, 1979, revised November 19, 1979

Rat liver mitochondria were treated with varying amounts of fluorescamine ranging from 0 to 30 nmol/mg of protein. The biochemical activities of the modified mitochondria were analyzed. It was found that the respiration rate in the absence of ADP was not significantly affected, but that the state 3 respiration rate and the accompanying Pi0 ratio decreased as the labeling extent increased. It was also observed that the treatment inhibited the stimulation of respiration induced by the presence of uncouplers. However, the modification has no effect on the discharging rate of proton gradient by uncouplers. The intrinsic activities of NADH-cytochrome c reductase, succinate-cytochrome c reductase, and cytochrome oxidase of the inner membrane were not affected by the modification. Measurement of the respiration-dependent proton extrusion (in the presence of valinomycin and potassium ion) with secondary ion movements inhibited, showed that the initial extrusion rate was reduced progressively. However, the observed amounts of proton extruded (AH+) and Apa + were not affected. The observed reduction of the oxygen consumption rate was much less than that of the proton extrusion rate with increased labeling. These results suggest that some fluorescamine titratable primary amino groups may be involved in the controlling of the proton extrusion process. The implications on the mechanism of coupling in respiration- dependent proton extrusion are discussed.

Fluorescamine reacts specifically with primary amino groups to form intensely fluorescent products (1). This labeling reagent has been used to show that the conformational changes of chloroplast coupling factor 1 may involve free amino groups (2). The functions associated with chloroplast coupling factor 1 are also quite sensitive to fluorescamine treatment (3).

We have shown that fluorescamine is relatively impermeable to the inner membrane of mitochondria and that the extent of labeling is sensitive to the energization state (4). Recently, we also observed that fluorescamine treatment stimulates mitochondrial ATPase activity (5). This enhancement of ATP hydrolysis is accom- panied by a decreased rate of proton extrusion associated with ATPase action. However, the effects of fluorescamine

’ This work was supported by a grant from the National Science Foundation (PCM 77-09365).

modification on the coupling process of oxidative phosphorylation were not examined.

It has been proposed that respiration and proton translocation are linked directly by redox “loops” in the inner membrane (6). Among the consequences of such a mechanism are a required H+/site ratio of 2.0 and a direct interdependence between proton and electron transport processes. The first of these consequences has been challenged by the works of Lehninger’s group (7-9). They have found that the H+/site ratio approaches 4.0 under strictly controlled conditions. Wikstrom and co-workers (10) and Skulachev and co-workers (11) have found evidence for the existence of a proton pump associated with cytochrome oxidase. A recent report by Casey et al. (12) showed that DCCD*

2 Abbreviations used: FCCP, carbonyl cyaniade p-trifluoro-methoxyphenyl hydrazone; fluorescamine, 4-phenyl Spiro (furan-2(3H), 1’-phthalan)-3,3’-dione; Hepes, 4-(2hydroxyethyl)-l-piperazineethanesulfonic

0003-9861/80/050330-09$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

330

inhibits proton extrusion but not the elec- Measurements of reductase activity. Mitochondria

tron transport of cytochrome oxidase. were fractionated into soluble, outer membrane,

These findings are in contradiction with the and inner membrane fractions by the swelling-con-

“loop” mechanism. traction-sonication method (14), except that un-

In this report, the effects of fluorescamine fragmented mitochondria were removed before su-

modification on the coupled events are de- crose density centrifugation. The total and specific

scribed. Our results show that the electron activities of the NADH-cytochrome c reductase

transfer and proton extrusion processes (rotenone sensitive), succinate-cytochrome c reductase, and cytochrome oxidase present in the

respond differently to the treatment while sonicated suspension (unfragmented mitochondria re- the apparent amount of proton extruded moved) and purified inner membrane were determined.

is not affected. These results are consistent The absorbance change of cytochrome c associated

with an indirect coupling mechanism. with the redox reaction was monitored using an Aminco DW-2 spectrophotometer.

MATERIALS AND METHODS Assaying of respiration-dependent proton extrusion.

The proton movement associated with succinate oxi- Modi$cation of rat liver mitochondria. Rat liver dation was monitored by the procedure of Reynafarje

mitochondria were isolated from male rats weighing and Lehninger (15). The assaying medium, with a from 140 to 200 g, as previously described (4). volume of 2.2 ml, contained 120 mM LiCl, 10 mM KCl, Fluorescamine-treated mitochondria were prepared by 2 ELM rotenone, 500 ng valinomycin, mitochondria rapid injection of a small volume of the reagent in (3-4 mg protein), and 3 mA4 Hepes, pH 7.3. Secondary acetone to mitochondria suspended in the buffered ion movements were inhibited by additions of 3 m&f solutions. After the reaction reached completion (2 min EGTA, 40 nmol NEM/mg protein, and 1 pg oligomyciti in the medium of pH 7.4 at room temperature), mg of protein. For coupling site III, the assaying modified mitochondria were collected by centrifugation. medium contained 140 mM LiCl, 10 mM KCl, 1 mM

Treated mitochondria were then resuspended to 50 Hepes, 4 mg protein, pH 7.4 and the following ad- mg/ml in the same medium as used for modification (5). ditions were added after 1 min incubation at 24°C: Since the hydrolysis products of fluorescamine have 500 ng of valinomycin, 3 pM rotenone, 4.5 x IO-’ M no effect on all the activities mentioned in this antimycin A, 1 pg oligomycinimg protein, 40 nmol NEMi report (mitochondria added to assaying media 2 min mg protein, and 1 mM EGTA. The oxygen consumption after the addition of fluorescamine), the centrifugation and proton movement were measured simultaneously step was omitted for some measurements (see figure in a modified Gilson Medical oxygen chamber. The legends for detail). The protein concentration was reaction was started with the addition of succinate determined by biuret method. (in the case of coupling site III, ferrocyanide was added

Determination of oxidative phosphorylation activity. instead) and proton extrusion was followed by a Mark- The respiration rates were determined polarographi- son Model .J-445 combined electrode connected to a tally, using a Yellow Spring oxygen monitor (Model Corning Model 112 pH meter. The change of H+ con- 53), at 24°C in a 3-ml solution containing 0.25 M centration was recorded by a Varian A-25 recorder. sucrose, 2 mM MgCl,, 10 mM KCI, 5 mM phosphate Oxygen consumption rate was measured as described (pH 7.4), about 3 mg mitochondrial protein, and other before. The buffering capacity of the system was solutes as indicated in the figures. Respiration-coupled measured before the addition of succinate and at the ATP synthesis was measured in the same medium end of the experiment by injecting a series of 2~1 (including 2-3 &i 32P) but on a separate aliquot of aliquots of standardized HCI solution. A similar pro- mitochondria. The yield of ATP was determined from the incorporation of labeled inorganic phosphate, by

cedure was employed by using durohydroquinone as the substrate. Durohydroquinone was prepared by the

the procedure described before (13). The radioactivity reduction of duroquinone with sodium borohydride. was measured by an Intertechnique SL 30 liquid scintillation spectrometer using Fisher Scinti Verse

Estimation of proton electrochemical potential

as the counting fluid. ( &.+,+j. The apparent &.L”+ associated with succinate oxidation was estimated from the distribution of radioactive tracers by the procedure of Rottenberg (16). For the estimation of A$, RbCl (HfiRb) was used to

acid; DCCD, N,N’-dicyclohexylcarbodimide; EGTA, replace KC1 in the medium described for assaying of ethylene glycol bis (P-aminoethyl ether) N,N’- proton extrusion. For the estimation of ApH, tetraacetic acid; NEM, N-ethylmaleimide; S-13, 5- acetate ions (H,-“C-COO-) were added to the same chloro-3-tert-butyl-Z’-chloro-4’-nitrosalicylanilide; medium (with valinomycin omitted). The matrix DTFB, difluoromethyltetrachlorobenzimidazole; AH+, volume was estimated from the difference of water apparent amount of proton extruded/mg protein; c3H,0) space and sucrose (‘“C labeled) permeable A/.+,-, proton electrochemical potential. space of mitochondrial pellet.

DIFFERENTIAL INHIBITION OF COUPLING PROCESS 331

332 LAM. SHIUAN, AND TU

Uptake of succinate and phosphate by mitochondria. The uptake rates of anions were followed qualitatively by the swelling of mitochondria as described by Mitchell and Moyle (17). For succinate, modified mitochondria in 250 mM sucrose (containing 3 mM Hepes, pH 7.4) were further incubated with NEM (40 nmolimg), rotenone (1.5 cLgimg), and oligomycin (1 pg/mg) before rapidly being added to isotonic media containing various concentrations of sucrose and ammonium succinate (1 to 50 mM), 0.2 mM potassium cyanide, and 1 mM EGTA. The absorbance changes at 610 nm were followed. For phosphate, modified mitochondria were incubated with rotenone (1.5 pgimg) and oligomycin (lFg/mg) before being rapidly added to isotonic medium of potassium phosphate at pH 7.4 as described in Ref. (17). The uptake of phosphate was initiated by the addition of valinomycin (100 ng/mg) and FCCP (2.0 x lo-’ M). To test the effect of NEM on the inhibition of phosphate transport of modified mitochondria, 40 nmolimg of NEM was added to mitochondrial suspension in phosphate and allowed to incubate for 2 min before the addition of valinomycin and FCCP.

Materials. Cytochromec (Type III), NADH, sodium succinate, ADP, valinomycin, antimycin A, rotenone, Hepes, sucrose, EGTA, and biuret solution were obtained from Sigma Chemical Company. Fluorescamine and FCCP were purchased from Pierce Chemical. N-Ethylmaleimide (gold label) was from Aldrich Company. Radioactive phosphoric acid (3’P) and other radioactive labeled materials were obtained from New England Nuclear. All other reagents used were of analytical grade.

RESULTS AND DISCUSSION

Effects on Oxidative Phosphorylation

Rat liver mitochondria were treated with fluorescamine in amounts ranging from 0 to 35 nmol/mg of protein. The respiration rates and phosphorylation activity were determined by polarographic method and 32P incorporation, respectively. The amount of acetone used in the treatment of mitochondria never exceeded 0.5% of the total volume of the suspension and was found to have no apparent effect on all measured activities (redox activity, respiration, ATP synthesis, and proton extrusion) described in the present work.

In Fig. 1, the effects of the treatment on respiration rates and ATP synthesis are summarized. It can be seen that fluorescamine modification causes a slight increase of the succinate oxidation rate in the absence of ADP and with the conditions as

---a- _ 5 15 25 5.5

nmol fluorescamlne/mg

FIG. 1. Effect of fluorescamine treatment on oxidative phosphorylation. Mitochondria treated with fluorescamine were suspended in a 3-ml solution containing 0.25 M sucrose, 10 mM KCl, 2 mM MgCl,, and 5 mM Pi, pH 7.4, at 22°C. A 30-p] aliquot of 0.4 M

succinate (pH adjusted to 7.4) was added to establish the state 4 respiration rate. After that, 20 ~1 of 0.3 M

ADP, pH 7.4, was added for the determination of respiratory control. The synthesis of ATP was measured in the same medium but containing 2-3 FCi of 32P in a separate aliquot of mitochondrial suspension. Five minutes after the addition of ADP, a l.O-ml aliquot of ice-cold 2OY0 perchloric acid was added to terminate the incorporation of inorganic phosphate. The amount of oxygen consumed in that 5-min period was used to calculate the P/O ratio. (0) Respiration rates after addition of succinate but before the addition of ADP. (B) Respiration rate after the addition of ADP. (0) P/O ratios calculated from the specific radioactivity of ?‘P incorporated and the amount of oxygen consumed in the 5-min time period after the addition of ADP. (A) Respiration rate after addition of 4.0 x lo-* M FCCP to mitochondria respiring on succinate in the presence of 1 pg oligomycinimg protein.

described in the figure legend, a decrease of respiration rate after the addition of ADP, and a decrease in P/O ratio. The results described above provide no hint on the origin of the effects of fluorescamine modification since only the terminal activities (net oxygen consumption and ATP synthesis) were assayed. Thus, the following experiments were performed.

It is generally accepted that the redox energy released from the repiratory chain is first utilized to generate a “high energy state” which is then used to drive the ATP synthesis. Since uncouplers can stimulate the respiration even in the presence of phosphorylation inhibitors (oligomycin, DCCD), it is thought that uncouplers can discharge


the “high energy state” directly. Thus, the effect of the modification on uncoupler stimulated respiration was examined. As shown in Fig. 1, the modification decreases the ability of FCCP to stimulate the respiration (in the presence of oligomycin). This result suggests that either the coupling process is affected or the sensitivity of mitochondrial membrane to uncoupler is decreased by the modification. However, the latter is rendered very unlikely by an experiment in which mitochondria modified with varying amounts of fluorescamine were titrated with the uncoupler FCCP. If the latter were true, a shift of the optimal uncoupler concentration to a higher value would be expected as the modification extent increased. As shown in

3 6 9

[FCCP] X 10’ (Ml

FIG. 2. Inhibition of FCCP-stimulated respiration by fluorescamine modification. Mitochondria were modified with fluorescamine of various amounts in a medium containing 3.4 mg of protein, 120 ITIM LiCl, 10 mM KCl, 3 mM EGTA, pH 7.3 at a final volume of 2.2 ml. After incubation for 2 min at 22”C, 40 nmol NEM/mg, 1 fig oligomycin/mg, and 3.5 pM rotenone were added to the suspension. After further incubation for another minute, 0.1 M succinate was injected into the suspension and the oxygen consumption rate was monitored by a Yellow Spring Oxygen Monitor as described under Materials and Methods. Various amounts of FCCP were then added to the suspension to stimulate the oxygen consumption rate. The stimulation by uncoupler is measured by the ratio of oxygen consumption rates before and after the addition of FCCP. (O), (Cl), (A) Represent mitochondria treated with 0, 10, and 25 nmol fluorescamine/mg protein, respectively. Their respective oxygen consumption rates before addition of FCCP were 15.0, 15.0, and 13.4 nmol O/(min.mg).

Fig. 2, the optimal concentration of FCCP for respiration stimulation does not shift to higher concentration for modified mitochondria.

Effects on Redox Activity of Inner Membrane

Although the observed changes of state 3 respiration rates suggest that the modification may impair the coupling process, it does not provide any information as to whether the intrinsic redox activity of the electron transport chain itself is affected or not. The catalytic activities of the en- zymes NADH-cytochrome c reductase (rotenonesensitive), succinate-cytochrome c reductase, and cytochrome oxidase of the inner membrane fraction of modified mitochondria were measured according to the procedure of Sottocasa et a1.(14). The results are shown in Fig. 3. As shown, these activities are not seriously affected by the modification even at the level at which no respiratory control was observed (-30 nmol fluorescamine/mg protein). Above this concentration, the redox activities begin to ex- hibit inhibition (not shown). As mentioned before (5), under the experimental conditions employed, the inner membrane, outer membrane, and soluble fraction have about 65,15, and 20% of the total fluorescent labels, respectively. Since the reaction between fluorescamine and primary amino group is nearly quantitative (85-90%, in efficiency) (18, 19>, and since the labels distribute evenly between the protein and lipid portions of the inner membrane (4), we conclude that the redox activity of the electron transport chain is not affected when less than about 9 nmol of -NH, groups of the protein part of the inner membrane is modified for every milligram of total mitochondrial protein. Furthermore, it should be mentioned that fluorescamine treatment does not significantly change the protein distributions which are about 65, 10, and 25% of the total protein associated with the inner membrane, outer membrane, and soluble fractions, respectively, as determined by the fractionation procedure used.

Since the intrinsic redox activities of the inner membrane are not affected by the modification, the observed effects on

334 LAM, SHIUAN, AND TU

respiration rates are most likely due to the effects of fluorescamine treatment on the coupling process in whole mitochondria.

Effects on Respiration-Dependent H+ Movement

It is generally accepted that vectorial proton movement is the detectable primary

FIG. 3. Effect of fluorescamine treatment on the activities of redox catalysts. Fluorescamine-treated mitochondria were fractionated into inner membrane, outer membrane, and soluble fractions as mentioned under Materials and Methods. The total and specific activities of NADH-cytochrome c reductase (rotenone sensitive), succinate-cytochrome c reductase, and cytochrome oxidase of purified inner membrane were determined as was that of the supernatant prior to density gradient centrifugation. It was found that fluorescamine treatment did not affect the protein distributions which are 68 f 3, 9 k 2, 23 2 3% of total protein associated with inner membrane, outer membrane, and soluble fraction, respectively. The recovery of each redox enzyme activity in the inner membrane fraction was better than 95% of that of mitochondria (normal and modified). The absorbance change at 550 nm was measured in order to determined the activities of the reductases and the change at 535 nm was used to determine cytochrome oxidase activity. The assay mixture contained, in 3.0 ml: 0.1 mM NADH, 0.1 mM ferricytochrome c, 0.3 mM KCN, and 50 mM phosphate buffer, pH 7.5, in the case of NADH-cytochrome c reductase (total); the above plus 1.5 PM rotenone in the case of NADH-cytochrome c reductase (rotenone insensitive); 3 mM succinate, 0.1 mM ferricytochrome c, 0.3 mM KCN, 1.5 WM rotenone, and 50 mM phosphate buffer, in the case of succinate-cytochrome c reductase; 0.1 mM ferrocyto- chrome c, 50 nM antimycin A, and 50 mM phosphate, pH 7.5, in the case of cytochrome oxidase. The rotenone-sensitive NADH-cytcchrome c reductase activity was calculated from the difference of the total and the insensitive part. (W) Rotenone-sensitive NADH-cytochrome c reductase; (a) succinate-cytochrome c reductase; (0) cytochrome oxidase activity.

FIG. 4. Effects of fluorescamine treatment on H+ extrusion. Fluorescamine-treated mitochondria (4.2 mg of protein) were suspended in a 2.2 ml solution containing 120 mM LiC1, 10 mM KCl, 2 PM rotenone, 500 ng valinomycin, 40 nmol NEM, and 1 pg oligomycinimg protein, 3 mM EGTA, and 3 mM Hepes, pH 7.31 at 22°C. The reaction was started by the addition of 10 ~1 of succinate (S). After the outward movement of H+ passed the maximal level, 3 ~1 of 5.0 x lo-” M FCCP (U) in acetone was added. The proton movement was monitored as mentioned under Materials and Methods. Traces 1, 2, 3, and 4 represent mitochondria modified with 0, 5, 15, and 25 nmol fluorescamineimg protein, respectively. The initial proton extrusion rates are 180, 162, 144, and 90 nmol H+/min/mg protein and AH+ are 25, 26, 25, and 20 nmol H+/mg protein for traces 1, 2, 3, and 4, respectively.

event coupled to electron transport in oxidative phosphorylation (20). The effects of fluorescamine modification on the succinate oxidation-dependent proton extrusion were examined. As shown in Fig. 4, the initial proton extrusion rate (in the presence of valinomycin, K+, oligomycin, NEM, and EGTA) is decreased by the modification. However, the amount of proton extruded (AH+) is not affected even when mitochondria are treated with 20 nmol fluorescaminel mg of total protein. This observation demonstrates that the effect of fluorescamine modification is really quite different from that of an uncoupler, which increases the influx of H+ across the membrane and


TABLE I

FIRST-ORDER RATE CONSTANT OF FCCP-INDUCED DECAYOF AHi"

FCCP (M)

nmol Fl/m$ 6.8 x IO-” 2.1 x 10-n 2.1 x lo-’

0 0.19 * 0.03 0.24 t- 0.03 0.52 i 0.05 (4.0 s) (3.6 s) (1.5 s)

5 - 0.22 i- 0.03 0.56 -+ 0.05 (3.6 s) (1.4 s)

10 0.18 2 0.03 0.23 -' 0.03 0.54 + 0.05 (4.2 s) (3.4 s) (1.5 s)

20 0.18 i 0.03 0.24 -t- 0.03 0.61 '- 0.05 (4.2 s) (3.3 s) (1.3 s)

il The proton movement was measured as described in Fig. 4. After passing the maximum point of AH+ and attaining the steady alkalinization rate, uncoupler (FCCP) was added to discharge the remaining AH+ (at the same level for all the experiments). The rate con stants (s-l) were calculated from the decay curves using the least squares fitting (average of two independent experiments). In the calculation, the steady alkalinization rate was subtracted. The data shown in paren- theses were the visually determined half-lives of the decay process.

* Amount of fluorescamine (Fl) used to modify mitochondria.

thus decreases the observed AH+ (21). Since the modification inhibits the stimulation of respiration by FCCP, one expects that the rate of discharging AH+ by uncouplers would be decreased to the same extent. However, as shown in Table I, the modification has no effect on the rate of discharging AH+ by the presence of suboptimal, optimal, and excess concentrations of FCCP. This observation is consistent with the conclusion mentioned before that modification does not affect the sensitivity of the membrane to uncouplers. Similar results were also obtained with S-13 and DTFB (data not shown). It should be mentioned that the observed AH+ of modified mitochondria, like that of normal ones, is completely discharged by the addition of uncouplers. It is possible that modification may increase the rate of H+ leak across mitochondrial inner membrane (it is assumed that outer membrane pro-

vides no barriers to proton movement) and therefore decreases the apparent extrusion rate. However this possibility is excluded by the results shown in Fig. 5 which demonstrates the rate of H+ leak remains unaffected by the modification (up to 30 nmol fluorescamineimg protein).

The observed inhibition of proton extrusion under the experimental conditions may be due to the possible enhancement of succinate uptake rate by modification. This possibility was tested by using the sensitive but semiquantitative method of measuring the rate of swelling of modified mitochondria (up to 30 nmol fluorescaminei mg protein) in isotonic media which were made of various concentrations of sucrose and ammonium succinate. The results showed that modification has no effect on the uptake rate over a wide range of succinate concentration (l-50 mM). The

FIG. 5. The decay of AH+ under anaerobic conditions. The assaying conditions were the same as that described in Fig. 3 except mitochondria with 6.8 mg protein were used. The suspension has less than 10% of saturated oxygen content (determined from oxygen monitor). The addition of succinate caused rapid H+ extrusion and oxygen consumption. The uptake of H+ by mitochondria when the suspension became anaerobic was taken as the H+ leak. Curves A, B, C, and D represent the H+ leak kinetics of mitochondria modified with 0, 10, 20, and 30 nmol fluorescamine/ mg protein, respectively. The apparent half-time of decay for A, B, C, and D are 8.3, 8.6, 8.1, and 9.0 L 0.5 s, respectively. The J indicates the time at which the suspension became anaerobic. Identical H+ leak kinetics was obtained by oxygen-pulse method.


possible complication of succinate uptake is further discounted by the fact that similar results were also obtained by using durohydroquinone as the substrate (see Fig 6). The possibility that the modification may interfere with the inhibition action of NEM on the transport of phosphate was also tested in a similar manner. The results showed that modification does not change the inhibitory effect of NEM on phosphate transport; swelling induced by the addition of valinomycin + FCCP in isotonic potassium phosphate medium was completely blocked by pretreating modified mitochondria with 40 nmol NEM/mg of protein. The details of fluorescamine modification on secondary ion transports will be presented elsewhere.

I I I I I

IO 20 30 nmol fluorescamino/mg

FIG. 6. Effects of fluorescamine treatment on sue&ate oxidation-linked H+ movement and the concomitant oxygen consumption rate. Mitochondria were prepared and modified aa described in Fig. 4. The H+ extrusion and oxygen consumption were followed simultaneously as described under Materials and Methods. (0), (Cl), (0) Represent the observed initial H+ extrusion rate, oxygen consumption rate, and AH+ of modified mitochondria, respectively. Typical values for normal (100%) H+ extrusion rate, oxygen consumption rate, and AH+ are 180 nmol H+/ (min’mg), 20.5 ng atom O/(min.mg), and 25 nmol H+/mg, respectively. The values shown in the graph are the average of three independent experiments. When durohydroquinone was used as the substrate, data points 0 (initial H+ extrusion rate), I:! (oxygen consumption rate), and A(AH+) were obtained.

TABLE II

EFFECTSOFFLUORESCAMINETREATMENT ON STEADY-STATE ApHfn

A@ A/G,+ Matrix volume nmol FYme ApH (mV) (mV) Wmg)

0 1.1 - 128 -194 0.40 + 0.05 6 1.1 - 126 -192 0.42 + 0.05

12 1.0 -119 -179 0.41 t 0.05 24 1.1 - 120 -186 0.43 2 0.05

4 The proton electrochemical potential was estimated as described under Materials and Methods. Modified mitochondria were incubated in the medium mentioned in the legend to Fig. 4 (KC1 was replaced by 0.1 mM RbCl for A$ estimation; 0.2 mM acetate was added and valinomycin omitted for ApH estimation) containing proper radiotracers for 10 min at 15°C. The energization was achieved by the addition of succinate. Two minutes later mitochondria pellets were collected by either Millipore filtration or rapid centrifugation (sep- aration completed within 2 min). The distribution of radioactivity was determined by liquid scintillation counting method.

b Amount of fluorescamine used to modify mitochondria.

Since the steady-state AH+ is not significantly affected by the modification, it is of interest to investigate whether the Apn+ at steady state is influenced by the modification or not. As shown in Table II, both the magni- tude of APT+ and the relative contribution of A$ and ApH are not significantly affected by the modification. This result suggests that the possible energy-dependent secondary ion flows probably have very little effect on the observed inhibition of proton extrusion, since these flows usually either decrease A$ or ApH of energized mitochondria (22).

Using a Gilson-Medical thermal-jacked oxygen cell, the respiration and the con- current proton movement can be determined simultaneously. With this method, the effects of fluorescamine treatment on respiration rate and proton extrusion rate were measured. The results are summarized in Fig. 6. Although both respiration and proton extrusion are inhibited, the former is less sensitive than the latter to the modification. For example, with mitochondria treated with 20 nmol fluorescaminel mg protein, the proton extrusion rate is


slowed down by about 40% but the con- current respiration remains essentially unaffected.

The differential inhibition on respiration- linked electron transfer and proton extrusion was also observed at coupling site III. As shown in Fig. 7, using ferrocyanide as the electron donor, the proton extrusion rate was reduced significantly more than the electron transport rate. Similar results were also observed by using ferricyanide as the electron acceptor for succinate- linked respiration in cyanide inhibited mitochondria (data not shown).

CONCLUSIONS

According to the loop hypothesis (6), the redox activity of some electron carriers, e.g., ubiquinone, is directly responsible for the transmembranous proton transfer during respiration. Consequently, any modification of the electron transport activity must also affect the H+ transport to the same extent, and vice versa. The fact that fluorescamine treatment decreases the proton extrusion rate more readily than that of electron transfer, without affecting the AH+ significantly, is inconsistent with proton extrusion

100

& i+ a 60

IO 20 30 nmcd flu0rescam1ne/mg

FIG. ‘7. Differential inhibitory effects of fluorescamine treatment in coupling site III. Mitochondria were modified and assayed under the conditions described under Materials and Methods for coupling site III. (A), (O), (0) Represent initial rate of proton extrusion, oxygen consumption rate, and the H+/em ratio determined from the initial proton extrusion, respectively. The initial proton extrusion rate, oxygen consumption rate, and the H+/e- ratio of unmodified mitochondria (100%) are 33 nmol H+/(min’mg), 34 nmol e-/(min.mg), and 0.97, respectively.

being by loops exclusively as suggested by the loop hypothesis at its present form.

On the other hand, the differential inhibition reported here suggests that proton extrusion and electron transfer may be indirectly coupled; in the sense that at least some intermediate steps, whether chemical or conformational, are required for the coupling between these two events. The fact that modification inhibits the stimulation of respiration but not the discharge of H+ by uncouplers, supports the above suggestion. Thus, the results presented are consistent with an indirect proton extrusion mechanism such as the vectorial Bohr effect (23, 24) or the conformational coupling model (25).

Since fluorescamine modification results are consistent with the indirect coupling mechanism, it is possible that some primary amino groups of a membrane component(s) may be involved in regulating the respiration-dependent H+-releasing process in mitochondria. This may be by facilitation of either the migration of protons in the proton pumps, or the activation of the pumps by redox events in the electron transport chain, or both. In either case, only the proton extrusion rate but not the total amount of proton released (AH+) would be affected as observed in this report.

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differential inhibition of respiration and its dependent h+ extrusion by fluorescamine in rat liver...

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