The carbodiimide product of diafenthiuron reacts covalently with two mitochondrial proteins, the F0-proteolipid and porin, and inhibits mitochondrial ATPase in vitro

PESTICIDE

BIOCHEMISTRY

AND

PHYSIOLOGY

42, 248-261 (1992)

The Carbodiimide Product of Diafenthiuron Reacts Covalently with Two Mitochondrial Proteins, the FO-Proteolipid and Porin, and Inhibits Mitochondrial ATPase in Vitro FRANZ J. RUDER*ANDHARTMUTKAYSER R + D Plant Protection, Agricultural

Division, CIBA-GEIGY

Ltd., CH-4002 Basel, Switzerland

Received March 12, 1991; accepted November

10, 1991

The thiourea diafenthiuron (CGA 106630), a new insecticide/acaricide, is rapidly transformed by desulfuration to the carbodiimide CGA 140408 in the presence of sunlight and singlet oxygen. There is also evidence that this conversion takes place biologically in the target organism. Diafenthiuron is thus assumed to be a propesticide acting via its carbodiimide CGA 140408. CGA 140408 affects mitochondrial respiration at the coupling site in both rat liver and Calliphora Bight muscle mitochondria in vitro. This inhibition was previously found to proceed in a time- and concentrationdependent manner. The present paper is concerned with the molecular mechanisms underlying this action. We show that the inhibition of the coupling site is paralleled by the block of FO/Fl ATPase activity. In its chemical reactivity, the carbodiimide CGA 140408 resembles dicyclohexylcarbodiimide (DCCD) in that both compounds inhibit ATPase activity, with pseudo-first order reaction constants of 0.025 (CGA 140408) and 0.020 (DCCD) (nmol carbodiimidejmg protein)-‘min-‘. Furthermore, both carbodiimides covalently bind to proteins. On incubation with CaNiphora mitochondria in vitro, both [t4C]DCCD and [t4C]CGA 140408 label an 8-kDa protein, identified as the proteolipid of the FO ATPase in the inner mitochondrial membrane, and a 32-kDa protein, the channel-forming porin of the outer mitochondrial membrane, as seen by fluorography of SDS polyacrylamide gels. In contrast, in rat liver mitochondria, [‘4C]CGA 140408 labels only the proteolipid, and not mitochondrial porin, while [‘4C]DCCD labels both, indicating that CGA 140408 reacts more selectively than DCCD in mammals. Whether the binding selectivity of CGA 140408 is related to its low mammalian toxicity is not yet known. These in vitro data suggest that the mode of action of CGA 140408 as the active product of diafenthiuron is correlated with its ability to bind covalently to proteins. Mitochondrial ATPase and pot-in are selective targets for this reaction. Several hypotheses concerning the mode of action are discussed, the focus being on partial or total disruption of mitochondrial activity. o 1992 Academic press, h.

INTRODUCTION

chondrial respiration by CGA 140408 but not, however, by diafenthiuron. It was recently shown that CGA 140408 is a potent, time-dependent inhibitor of mitochondrial respiration acting on the coupling site as oligomycin or the hydrophobic carbodiimide dicyclohexylcarbodiimide (DCCD)’ (2). DCCD reacts with the car-

There is strong evidence that the new insecticidal and acaricidal thiourea diafenthiuron, CGA 106630, is a propesticide which is converted into a carbodiimide, CGA 140408, by both abiotic factors (1) and metabolic processes (2, 3). Previous studies suggested that diafenthiuron via its active metabolite, CGA 140408, has a novel mode of action (2). Standard tests for activity at the targets of commonly used insecticides yielded no hint of how the toxic effect of CGA 140408 might be exerted. The only effect observed was the inhibition of mito-

’ Abbreviations. ATP, adenosine triphosphate; DCCD, dicyclohexylcarbodiimide; DMSO, dimethyl sulfoxide; EGTA, ethyleneglycol-bis-(2-aminoethyl)tetraacetic acid; FCCP, carbonylcyanide-4trifluoromethoxyphenylhydrazone; Hepes, 4-(2hydroxyethyl)-piperazine-I-ethane-sulfonic acid; PBO, piperonylbutoxide; Pipes, piperazine-l&bisQethane-sulfonic acid); SDS, sodium dodecyl sulfate; TEMED, N,N,N’,N’-tetramethylenediamine; Tris, tris(hydroxymethyl)-aminomethane.

* To whom reprint requests should be addressed. 248 0048-3575192 $3.00 Copyright

Au riglIt

0 1!3!Z by Academic Press, Inc. of repmduction in any form reserved.

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boxy group of a specific glutamic acid side chain of the proteolipid in the FO (membrane) part of mitochondrial ATPase as fust shownin the fungalenzyme(4). Proton influx through the inner mitochondrial membraneis therebyblockedand ATP synthesis at the Fl part of the ATPase is inhibited (for reviews, see (5, 6)). It has been proposed that covalent binding of DCCD results in a conformationalchangein the FO portion which is transmitted to the Fl part of the ATPase thereby blocking both ATP synthesis and ATPase activity (7). We thereforeinvestigatedthe inhibition of Culliphora mitochondrial ATPase by CGA 140408and DCCD in thorax homogenates. The data indicate that CGA 140408is an inhibitor of mitochondrial ATPase which is at least as potent as DCCD. Furthermore, the covalent binding of CGA 140408to mitochondrial membrane proteins matches that of DCCD. We also investigatedinhibition of Na/K- and Ca-ATPasewhich are related putative biochemical targetsfor CGA 140408that might contribute to its in vivo toxicity. MATERIALS

AND

METHODS

Chemicals

Unless otherwise indicated, the chemicals were obtained from Fluka, Switzerland. Acrylamide, NjV-methylenebisacrylamide, ammonium persulfate, mercaptoethanol and TEMED were obtained from Bio-Rad. CGA 140408,N-(2,6-diisopropyl4-phenoxyphenyl)-N’-tert-butylcarbodiimide, is a CIBA-GEIGY product. [14C]CGA 140408with a radiochemical purity of 95% and a specific activity of 1.85MBq/mg was labeled at the C2 of one of the isopropyl groups.N,N’-dicyclohexyl-[‘4C]carbodiimide with a radiochemical purity of 97.1% and a specific activity of 10 MBq/mg was obtained from Amersham. 14C-Labeled marker proteins were obtained from BioRad. Insects

Adults of Calliphora

erythrocephala

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from an in-housestrain were usedthroughout these studies. Preparation of Flight Muscle Homogenates

The thoracic musclesfrom l- to 2-weekold adult Calliphora were carefully separated from the cuticle. The flight muscles were homogenizedon ice in a buffer consisting of 250mM sucrose,5 m&f Tris/HCl, 1 mM EGTA, at pH 7.7. Homogenates(510 mg protein/ml) were aliquoted and frozen at -70°C. Immediately after thawing, the aliquots were usedfor determinationof mitochondrial, Na/K-, and Ca-ATPase activity. Preparation of Mitochondria Protein Determination

and

Rat liver mitochondria were preparedas described by Gazotti et al. (8). Thoracic flight muscle mitochondriafrom Calliphora were isolated according to the method of Slack and Bursell (9). Protein content was determinedaccordingto Bradford (10). ATPase Assays

Mitochondrial ATPase activity was determined essentially as describedby Bowman and Bowman (11). Culliphora thorax homogenatecontaining 5-15 kg of protein (ca. l-3 ~1)was addedto 300l.~lof ATPase assaymedium (5 mM ATP, 5 mM MgCl,, 15 mM NH,Cl, 3 m&f phosphoenolpyruvate, 15pg pyruvate kinase, 10mM Pipes, at pH 8.3). The ATPase reaction was carried out for 10 min at 30°Cand stoppedby addition of 750 ~1 of Fiske-Subbarow reagent(12) containing 0.5% SDS. For the zero time control, the reaction was stopped immediately after addition of the homogenate.Mitochondrial ATPase is measuredpreferentially becauseof the high pH of the ATPase assaymedium. Oligomycin (0.3 l&ml final test concentrationobtainedby addition of 1 ~1 of a methanolic stock solution of 1 mg/ ml) inhibited total ATPase activity by more than 95%. Ouabain-sensitive Na/K-ATPase and

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EGTA-sensitive Ca-ATPase were determined according to the method of Seiler and Fleischer (13). The Na/K-ATPase assay medium consisted of 120 mM NaCl, 20 mil4 KCl, 3 mM MgCl,, 3 mM ATP, 0.5 n&f EGTA, 5 rniV sodium azide, 30 mM imidazole, at pH 7.5. Na/K-ATPase activity is abolished in presence of 1 mM ouabain. Ouabain was added as a DMSO stock solution. The Ca-ATPase medium consisted of 100 m&f KCl, 5 mM MgCI,, 100 uLV CaC12, 5 n&f Hepes, 60 l&f EGTA, 1 m&f ATP, at pH 7.0. Ca-ATPase is sensitive to the presence of 4 mM EGTA. For 300 ~1 ATPase assay medium, flight muscle homogenates containing 50 pg (Na/K-ATPase) and 25 p,g (Ca-ATPase) of protein were used. Care was taken that the solvent concentrations (DMSO or methanol) did not exceed 0.7% of the final test solution. The solvent controls had no effect on ATPase activity. Inhibition of Calliphora by Carbodiimides

Thorax ATPases

For determination of the kinetics of inhibition of the mitochondrial ATPase by CGA 140408 and DCCD, series of different concentrations of the corresponding carbodiimide (3-20 @V) were incubated with thorax homogenates at varying protein concentrations (2-10 mg/ml) in homogenization buffer on ice. The carbodiimides were added as a methanolic solution not exceeding 1% methanol in the incubation medium after dilution. Since hydrophobic carbodiimides tend to react with proteins in the hydrophobic membrane rather than in the aqueous phase, the velocity of reaction of the carbodiimide is not dependent on the total concentration of carbodiimide but on the concentration in the lipid phase (14). The velocity of reaction of DCCD with the proteolipid subunit is therefore inversely related to the concentration of protein in the incubation medium (15). As expected, in our experiments, in the range of 0.25 to 3 nmol carbodiimide/mg protein, the velocity of the reaction of the carbodiimides with

KAYSER

the mitochondrial ATPase was inversely related to the concentration of the protein. During preincubation, mitochondrial ATPase activity was determined in aliquots taken after 0,2,4,6, 10,20, 30,40,60, and 240 min and compared to untreated controls following the same schedule. The percentage of ATPase proteolipids which had been covalently modified by the carbodiimide, as evidenced by decrease of ATPase activity, was calculated. From these data, assuming constant concentrations of carbodiimide, pseudo-first order inactivation rate constants were obtained from plots of ln(v,/ v,,) of ATPase activity vs time. Carbodiimides tend to add water in aqueous solution to form the corresponding urea, thereby changing the actual concentration of the carbodiimide. Furthermore, at low carbodiimide concentrations, a significant percentage of the carbodiimide reacts with the ATPase, also reducing free carbodiimide concentration. Hence, only the early time points (O-30 min) could be used for calculating reaction constants. When inhibition of Na/K- and CaATPase by CGA 140408 or DCCD was studied, Calliphora flight muscle homogenates were preincubated with the corresponding carbodiimide for 2 hr on ice. Labeling

of Proteins

Rat mitochondria (20 mg protein/ml) were incubated with either [14C]DCCD or [ 14C]CGA 140408 at 1.5 nmol carbodiimide/ mg protein for 2 hr on ice. In some experiments, venturicidine (10 l&ml final concentration) was added. In competition experiments, unlabeled carbodiimide was added in IO-fold amount as compared to the labeled form. Under all incubation conditions, mitochondrial ATPase was blocked by the carbodiimide. To remove unreacted carbodiimide from the protein solution, 9 volumes of acetone were added and incubated for several hours on ice. Precipitated proteins were collected by centrifugation (Eppendorf centrifuge; 5 min; lO,OOOg), the supernatant was discarded and the protein

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pellet was processedfor gel electrophoresis. For in vitro labeling of Calliphora mitochondrial proteins, a freshly preparedhomogenateof thoracesor isolated mitochondria (5 mg protein/ml) was incubatedwith 3 nmol carbodiimide/mgprotein, unlessotherwise indicated, and processed as described for rat mitochondria, except that beforegel electrophoresisthe sampleswere heatedto 117°Cin a steamboiler for 3 min. Oligomycin-sensitive ATPase was blockedto 95% by incubationwith 1.5nmol carbodiimide/mg protein. Hence the incubation conditions of carbodiimidewith both rat liver mitochondria and Calliphora homogenateswerejust sufficient to block mitochondrial ATPase, but were not strong enough to block uncoupled mitochondrial respiration. Subfractionation of Calliphora Mitochondria Calliphora mitochondria were isolated and labeled with [14C]CGA140408,as described above, and subfractionatedby the swell-shrink-sonicate procedure as describedby Hovius et al. (16).The sonicated mitochondria were layered on a discontinuous sucrose gradient (25.3, 37.7, and 51.3% sucrose)and separatedby centrifugation for 3 hr at 21O,OOOg, and individual fractions of the gradient were analyzedby gel electrophoresisand fluorography. Isolation

of the Proteolipid

Fraction

Calliphora flight muscle homogenates were labeledwith [14C]DCCDor [14C]CGA 140408as describedin the previous section. Labeled homogenateswere extracted with chloroform/methanol(2:1; v/v) asdescribed by Beechey et al. (17). The proteolipid extract was divided into two halves. From one half all the solvent was removed by evaporation in a Speed Vat Concentrator (Savant),and the residuewas processedfor gel electrophoresisand fluorography.From the other half, the proteolipid was precipitated by addition of 5 volumes of diethyl-

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251

ether. The precipitatewas usedfor electrophoresis. Gel Electrophoresis

and Fluorography

Two discontinuousSDS-polyacrylamide gel electrophoresissystems were used (18, 19).Following the tricine method of Schiigger and von Jagow (19),a 4% stacking gel, a 10% spacergel, and a 16.5% separating gel were used. In the Laemmli gel system (18)a 4% stackinggel and a 15%separating gel were applied. Each lane was loaded with 50-100 Kg of protein. Identical amounts of proteins from the sameextract were usedfor eachlane on the samegel in experiments where the intensities of labeled protein bands had to be compared. The gels were processedfor fluorography as described by Ruder et al. (20) using En3hance (NEN, Boston) and a Kodak X-OMAT AR film according to the NEN protocol. RESULTS

Znhibition of ATPases in Vitro

The observationof inhibition of the coupling site in mitochondria by CGA 140408 (2), and the fact that CGA 140408is a carbodiimide, raised the hypothesis that the new insecticidal compoundinhibited mitochondrial ATPase by the same mechanism as DCCD. The ATPase activity of Calliphora thorax homogenateswas therefore investigatedand, after reaction with either DCCD or CGA 140408,the residual enzyme activity was recorded. In untreated homogenates, ATPase activity is in the range of 2 pmol phosphategeneratedper minute and milligram of protein at 30°C. Both carbodiimidesinhibit the ATPase in a time- and concentration-dependentmanner. They decreasethe activity of ohgomytin-sensitive ATPase in Calliphora mitochondriawith reaction constantssimilar to the ones describedfor bovine heart mitochondria(Table I), andthey inhibit rat liver mitochondrial ATPase in a similar manner (data not shown).

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TABLE 1 Pseudo-First Order Rate Constants ((nmoNmg)-‘mm-‘) for the Inhibition of Mitochondrial ATPases by Carbodiimides (Mean Value from Nine Independent Determinations of the Inhibition Constant at DiRerent Concentrations of Thorax Protein) ATPase Calliphora flight muscle Bovine heart”

DCCD (*SD)

CGA 140408 (&SD)

0.020 (0.005)

0.025 (0.005)

0.027

a Recalculated from Kopecky et al. (15).

By extrapolationto maximal inhibition of ATPase activity, it can be estimated that binding of 0.45 nmol CGA 1404OWmg protein would be necessaryto completely inhibit Culliphoru flight muscle mitochondrial ATPase in vitro (Fig. 1). This value is higher than the amount of carbodiimidere% inhibition

60604020 0

0

0.2 nmole

0.4 CGA

0.6

0.8

140408/rng

protein

FIG. 1. Titration of mitochondrial ATPase by CGA 140408. Calliphora flight muscle homogenates were incubated with the amounts of CGA 140408 indicated, for 4 hr on ice to assure complete reaction of the carbodiimide with the ATPase, and mitochondrial ATPase activity (solid line) was determined as described under Materials and Methods. Extrapolation to 100% inhibition, by using the concentrations of CGA 140408 up to 0.27 nmollmg protein, yielded a quantity of 0.45 nmol CGA 140408lmg protein (dotted line). At carbodiimide concentrations higher than 0.27 nmollmg protein, competing reactions (hydration of the carbodiimide to the urea or reaction of the carbodiimide with other proteins) become more prominent, so that in this concentration range more than just one molecule of carbodiimide is needed to react with one ATPase molecule.

quired to inhibit ADP-dependentrespiration in isolated Calliphora mitochondria (0.18 nmoYmg; 2). The reasonis probably that for the respirationexperimentsisolated mitochondria were used, whereas the kinetics of inhibition of the mitochondrial ATPase was studiedin whole thorax flight muscle homogenates, which contain a lower specific activity of mitochondrial ATPase. For comparison,in beef heart mitochondria 0.45 nmol DCCD/mg protein are necessaryfor complete inhibition of ATPase activity (21). From data on the in vitro inhibition of Calliphora mitochondrial ATPase, it can be calculatedthat binding of about 1.5-2.5 nmol (0.5-0.85 Fg) of CGA 140408per thorax (3-5 mg total protein) is necessaryto completely inhibit ATPase in the insect thorax. Na/K-ATPase is not inhibited in Calliphoru flight muscles by the carbodiimides DCCD and CGA 140408,evenwhen the homogenateis incubatedwith 30 nmol carbodiimide/mg protein (Table 2). Ca-ATPaseis inhibited to about 50% by CGA 140408and to about 20% by DCCD under the same conditions. However, no significant inhibition of Ca-ATPasecan be detectedat low carbodiimide concentrations(<5 nmol/mg protein). SarcolemmalCa-ATPaseis not inhibited, as evidencedby analysisof the microsomal fraction (data not shown). The TABLE 2 Maximal Inhibition of NaIK-, Ca-, and Mitochondrial ATPase in Calliphora after Incubation with the Indicated Amount of Carbodiimide; Average from Two Experiments Not Deviating More Than 5% from Each Other

ATPase Mitochondrial ATPase Na/K-ATPase Na/K-ATPase Ca-ATPase Ca-ATPase

nmol carbodiimide/mg protein 2 2 30 2 30

Percent inhibition by DCCD

CGA 140408

94.5 0 1.3 0 19.5

93.5 0 1.3 0 48.6

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Ca-ATPaseinhibited by CGA 140408sediments with cell debris, nuclei, and mitochondria thus might representactomyosine ATPase. Covalent Labeling of Rat Liver Mitochondrial Proteins in Vitro

If CGA 140408binds to the sameproteolipid of the FO/Fl ATPase as DCCD, thereby inhibiting ATPase activity, it should be possible to identify labeled proteins. Rat liver mitochondria were therefore incubated with either [‘4C]DCCD or [14C]CGA 140408(1.5 nmol/mg protein each), subjected to SDS-gel electrophoresis, and the radiolabeledproteins were visualizedby fluorography(Fig. 2A). Incuba-

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tion with [‘4CJDCCDyields three major radiolabeledprotein bands correspondingto 9, 19, and 32 kDa. There are also some minor labeledprotein bandsespeciallywhen a higher carbodiimide/proteinratio is applied (seealso Fig. 5). These data closely coincide with results reported for beef heart mitochondria (2224), where three proteins of 8, 16, and 35 kDa were labeled by [‘4C]DCCD at low concentration (2 nmol DCCDlmg protein). The 8-kDa DCCD-binding peptide is identical to the proteolipid of the mitochondrial ATPase representinga subunit of the proton channel-formingmoiety of the FO part of the FO/Fl ATPase. There is general agreementthat the DCCD-binding 16-kDa protein is simply a dimer of the 8-kDa pro-

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2. Nuorograms of carbodiimide-labeled rat liver mitochondrial proteins separated by SDS gel electrophoresis. Rat liver mitochondria were incubated with either [‘4cJDCCD or [‘4C)CGA 140408, separated by SDS gel electrophoresis, and label was visualized by fluorography, as described under Materials and Methods. The positions of marker proteins are indicated. Heavily labeled protein bands are indicated by their molecular weights, as derived from their migration behavior. In all lanes, identical amounts of protein from the same mitochondrial preparation were used. Therefore all lanes within A and within B, respectively, can be compared with one another directly. (A) Standard Ltimmli system. Lanes l-4: labeling by [‘4C)DCCD (lane 3) was inhibited by unlabeled DCCD (lane I), unlabeled CGA 140408 (lane 2), and by venturicidine (lane 4). Lanes 5 and 6: labeling by [‘4c]CGA 140408 (lane 5) and when venturicidine was included (lane 6). (B) Tricine system. Lanes l-4: labeling by [14C)DCCD (lane 3) was inhibited by unlabeled DCCD (lane I), unlabeled CGA 140408 (lane 2) and venturicidine (lane 4). Lanes 5-7: labeling by [‘4CJCGA 140408 (lane 6) was inhibited by unlabeled DCCD (lane 5) and venturicidine (lane 7). FIG.

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teolipid, which is not disaggregatedprior to and during gel electrophoresis(22-24).The 35-kDaspecieshas recently been shown to be identical with the pore-forming protein of the outer mitochondrial membrane, porin, in pig heart mitochondria (25). The minor labeledprotein bands seenin Fig. 2 could not be correlated with known proteins. For comparison,in our experimentswith rat liver mitochondria (Fig. 2A, lane 4), labeling by DCCD of both the 9- and 1PkDa proteins, but not of the 32-kDa protein, is sensitiveto venturicidin, an inhibitor of mitochondrial ATPase acting on the FO proteolipid, as describedfor mitochondria for beef heart and red beet (26, 27). Further evidence that the 1PkDa DCCD-binding protein from our experiments (Fig. 2A) is the dimer of the proteolipid comesfrom the observation that, after labeling with [14C]DCCD and using different conditions of gel electrophoresis (tricine gel electrophoresis), we observeonly an 8-kDa(proteolipid) and a 32-kDa(porin) band(Fig. 2B, lane3), insteadof 9-, 19-,and 32-kDaproteins(Fig. 2A, lane 3). When [i4C]CGA 140408is used as labeling agent with rat liver mitochondria, two major labeled bands are observed in the Lgmmli system (Fig. 2A, lane5; 10.5and 19 kDa) andjust onemajor bandin a tricine gel system (Fig. 2B, lane 6; 8 kDa). Comparison of [14C]CGA 140408-labeledproteins detected using either gel system reveals that the 10.5-and 1PkDa proteins seenin the Lammli system (Fig. 2A) correspondto just one 8-kDa band in the tricine system (Fig. 2B), as has already been described abovefor the correspondingDCCD-labeled proteins. Labeling of the 32-kDa band by CGA 140408is only weak. There is strong evidencethat the proteolipid of rat liver mitochondriaFOATPase is labeled by both DCCD and CGA 140408. The small differencein the apparentmolecular weights of the [‘4C]DCCD-labeled(9 kDa) and the [‘4C]CGA 140408-labeled (10.5kDa) protein after Lglmmli electropho-

KAYSER

resis can be satisfactorily accountedfor by different migration behavior resulting from the different hydrophobicities of the two carbodiimides.Labelingof the proteinsof 8 kDa (tricine system), and 10.5kDa and 19 kDa (Liimmli system) by CGA 140408is sensitive to venturicidine, as is labeling of the correspondingproteins by DCCD, indicating that the sameproteins arelabeledby both carbodiimides. A further argument that both DCCD and CGA 140408bind not only to the FO proteolipid but at the same site comesfrom the observationthat radioactive labeling by one compound can be competitively inhibited by an excessof the other, unlabeled, carbodiimide (Figs. 2A, lane 2 and 2B, lanes 2, 5). Generally, DCCD seemsto be a better competitor than CGA 140408against labeling by both labeledcarbodiimides.In rat liver mitochondria, unlabeledCGA 140408can compete with labeling of the 32-kDa protein by [14ClDCCD only weakly, as might be expectedfrom the observationthat [14C]CGA 140408does not significantly bind to this protein. Covalent Labeling of Calliphora Mitochondrial Proteins in Vitro

When Calliphora thorax homogenates are incubated with either radiolabeled DCCD or CGA 140408(Fig. 3), two major bands (8 and 30 kDa) and one minor band (band X, approx. 70 kDa, indicated by an asterisk in Fig. 3, lane 5) can be seenafter tricine gel electrophoresis and fluorography. In addition, in sampleswhich havenot beenautoclavedbeforegel electrophoresis, there is a considerablenumber of labeled protein bands in the 40- to 60-kDa region (Fig. 3, lane 15).These proteins obviously represent oligomeric forms of the 8-kDa peptide, since the high molecular weight bands disappear by autoclaving (Fig. 3, lanes5 and 12)or chloroform/methanolextraction of the [14C]CGA 140408-labeled proteins (Fig. 5, lanes 2, 3, and 4); both procedures concomitantly increase the amount of labeled 8-kDa protein. The

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9 10 11 1213 14

15

3. Fluorograms of carbodiimide labeled Calliphora proteins separated by SDS gel electrophoresis. Calliphora flight muscle homogenates or mitochondria were incubated with either [t4C]DCCD or [14CjCGA 140408, separated by tricine gel electrophoresis, and label was visualized byjTuorography, as described under Materials and Methods. The positions of marker proteins are indicated. Before electrophoresis, proteins were either denatured by autoclaving (lanes l-14) or conventionally denatured at 95°C (lane 15). identical amounts of protein from the same thorax (lanes l-8, 15) or mitochondrial preparation (lanes 9-14) were used. Therefore, lanes l-8 and 9-14, respectively, can be compared directly with each other. Lanes l-8: total flight muscle proteins labeled by [t4C)DCCD (lanes l-4) or [‘4c]CGA 140408 (lanes 5-8). Labeling (lanes 1 and 5) was inhibited by unlabeled DCCD (lanes 2 and 6), unlabeled CGA 140408 (lanes 3 and 7), and venturicidine (lanes 4 and 8). A 70-kDa band (lane 5) labeled by CGA 140408 is indicated with an asterisk. Lanes 9-14: proteins of mitochondria isolated from flight muscles labeled by [14ClDCCD (lanes 9-11) or [‘4CjCGA 140498 (lanes 12-14). Labeling (lanes 9 and 12) was inhibited by unlabeled DCCD (lanes 10 and 13) and venturicidine (lanes 11 and 14). Lane 15: totalflight muscle proteins labeled by [‘4C’JCGA 140408 without autoclaving before gel electrophoresis (compare with lane 5). FIG.

weakly labeled70-kDaprotein (Fig. 3, lane 5) is present only if total thorax proteins have been incubated,whereasit is missing in the caseof isolatedmitochondria (Fig. 3, lane 12). It may represent nuclear lamin since monoclonalantibodiesagainstDrosophila lamin recognizean antigencomigrat-

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ing with the radiolabeledprotein (data not shown). The molecular weights of rat liver mitochondrial proteolipid (8 kDa) and porin (32 kDa) closely correspondto the labeledCalfiphoru proteins (8 and 30 kDa). In all eucaryotic mitochondria so far investigated, no proteins other than the proteolipid and porin are known to react readily with DCCD at low carbodiimide concentrations (14,25,28). Severalother lines of evidence also suggest that the labeled Calliphora proteins, except the 70-kDa species, seen after reaction with [14C]DCCDand [14C]CGA 140408,are homologouswith the correspondingproteins in rat liver mitochondria. As with the rat mitochondrial protein, labeling of the Culliphoru 8-kDa protein is sensitiveto venturicidin (Fig. 3, lanes4, 8, 11, and 14). Second,labeling of the 8-kDa peptide by [‘4C]CGA 140408can be competitively inhibited by unlabeled DCCD (Fig. 3, lanes 6 and 10) which has been shown to bind also to the mitochondrial proteolipid of cockroaches (29). Furthermore, the 8-kDa carbodiimide-bindingprotein is obtainedpredominantly from the inner mitochondrial membrane fraction, as will be shown below (Fig. 4, lane 3). The Drosophila mitochondrial porin has a molecular weight of 31 kDa (30), somewhat smaller than the correspondingmammalian protein (35 kDa), further indicating that the carbodiimide-binding30-kDa protein observedin our experimentswith Calliphora is most probably mitochondrial pot-in.Another strong argumentsupporting this identification can be derived from subfractionationof Calliphoru mitochondriaon a discontinuoussucrosegradient(Fig. 4). A membranefraction from rat liver mitochondria sedimentingbetween 25.3 and 37.7% sucrosehasbeenshown to be composedof outer mitochondrial membranes(16). Contact sites, between outer and inner membranefrom rat liver mitochondria, display a density of 1.15g/ml, correspondingto 44% sucrose (31). Inner mitochondrial membranesand matrix componentsare located

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the observation that it can be extracted by chloroform/methanol (Fig. 5, lanes 3 and 4) and precipitated by diethylether from the chloroform/methanol extract (lanes 4). These properties have been described for the mammalian mitochondrial DCCDbinding proteolipid (17,32). In contrast, the 30-kDa protein, which also binds both 14C-DCCD 1 “C-CGA140408 kDa 12

3

4

4. Fluorograms of outer and inner mitochondrial membranes separated by SDS gel electrophoresis. Isolated Calliphora mitochondria were incubated with [‘4c]CGA 140408, membrane subfractions were prepared by a discontinuous sucrose gradient centrifugation and processed for tricine gel electrophoresis and fluorography as described under Materials and Methods. Lane I: outer membrane fraction (25.3%/ 37.7% sucrose interface); lane 2: inner and outer membrane contact sites (37.7%/51.3% sucrose interface); lane 3: inner membrane fraction (pellet); lane 4: total mitochondria. The 30-kDa and 8-kDa carbodiimide binding proteins as described in the text are indicated. The dark field on lane I is an experimental artifact of this exposure. FIG.

in the pellet of the gradient (16). Ohlendieck et al. (31) showed that pot-in is especially enriched in the outer membranes, but a significant part of porin could also be demonstrated in the contact site fraction. In our experiments with Caffiphoru mitochondria, the 30-kDa carbodiimide-binding protein is most significantly enriched in the outer membrane fraction (25.3%/37.7% sucrose interface; Fig. 4, lane 1) and is also prominent in the presumed contact sites fraction (37.7%/51.3% sucrose interface; lane 2) but is almost completely missing in the inner membrane fraction (pellet; lane 3) where the 8-kDa protein is predominantly found. The 8-kDa and 30-kDa carbodiimidebinding proteins fulfill all criteria for the FO proteolipid and pot-in, respectively. Further strong support for the identification of the 8-kDa protein of Calliphora, which binds DCCD and CGA 140408, as proteolipid is

30-

a-

12 3 4 12 4 5. Fluorograms of carbodiimide binding proteins extracted by chloroformlmethanol and separated by SDS gel electrophoresis. Calliphora flight muscle proteins were labeled by 6 nmollmg protein of either [‘4c]DCCD or [14C)CGA 140408, extracted by chloroformlmethanol, denatured at 95”C, and processed for tricine gel electrophoresis and fluorography as described under Materials and Methods. The 30-kDa and 8-kDa carbodiimide binding proteins as described in the text are indicated. Total labeled proteins before chloroformlmethanol extraction (lanes l), residue after extraction (lanes 2), chloroformlmethanol extracted proteins (lane 3; DCCD-labeled proteins not shown), proteins precipitated by diethylether from the chloroform/methanol extract (lanes 4). High molecular weight bands seen in lanes I are due to incomplete denaturation of the aggregated proteolipid (heating only to 95”C, compare to Fig. 3, lane 15). Chloroform1 methanol extraction leads to complete denaturation and disappearance of the high molecular weight bands labeled by CGA 140408 (lanes 2, 3, 4). Several bands seen after DCCD-labeling were due either to a high carbodiimidelprotein ratio used for labeling so that other proteins than proteolipid and porin become labeled or incomplete denaturation of the proteolipid.

FIG.

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DCCD and CGA 140408, is not extracted by chloroform/methanol, a finding similar to that for mammalian pot-in (23). DISCUSSION

As shown and discussed in a preceding paper (2), the carbodiimide CGA 140408, the insecticidal product of the novel propesticide diafenthiuron does not exhibit the mode of action reported for any conventional insecticide. This observation, together with the carbodiimide structure of CGA 140408, which is not related to any of the conventional insecticides, suggested a novel molecular mode of action. The goal of the in vitro studies reported in this paper was to obtain more information about the possible mechanisms by which this novel insecticide and acaricide could exert its lethal effects. Previous in vitro studies revealed that CGA 140408 inhibits mitochondrial function in both rat liver and Calliphora flight muscle mitochondria (2). The carbodiimide, at concentrations of around 1 nmol/mg protein, specifically interferes with the coupling site (complex V), thereby inhibiting mitochondrial ATP synthesis. The block of mitochondrial respiration parallels the inhibition of oligomycin-sensitive ATPase activity by CGA 140408. The inhibition of ATPase activity is dependent on both the time of preincubation and the concentrations of the inhibitor and the protein, in the same way as for the chemical reaction of DCCD with mitochondrial ATPase. The pseudo-first order rate constants for the reaction with the ATPase in Calliphora flight muscle homogenates, as determined by measurement of the inhibition of ATPase activity, are 0.025 and 0.020 (nmol carbodiimidelmg protein) - ‘min- ’ for CGA 140408 and DCCD, respectively. Binding of approximately 0.45 nmol CGA 140408/mg protein is required to inhibit mitochondrial ATPase activity completely. From these data, it can be calculated that binding of 1.5-2.5 nmol (0.5-0.85 vg) of CGA 140408 per Culliphoru thorax is necessary for a to-

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tal abolition of mitochondrial activity in flight muscle. We have found that on topical application the LD50 is 3.6 pg (after 24 hr) and 1.1 pg (after 48 hr) of CGA 140408 per fly (unpublished results), although only a small part of the applied carbodiimide actually penetrates into thoracic flight muscles (2). Block of ATPase from American cockroach and house fly mitochondria by DCCD has already been shown by Dary and Cutkomp (33) and Chefurka (34). Kadir and Knowles (35) recently showed that CGA 140408 inhibits oligomycin-sensitive ATPase at the FO moiety in the bulb mite. Nonmitochondrial ATPases (e.g., Na/KATPase and Ca-ATPase) do not seem to be primary targets of CGA 140408 since in vitro they become only partially inactivated at very high carbodiimide concentration and after prolonged incubation. So far, we have no evidence that CGA 140408 at low concentrations (up to 10 nmol/mg) inhibits any of the many enzymatic reactions known to be hindered by DCCD at concentrations between 10 and 100 nmol/mg (e.g., respiratory chain enzymes (2); ATPases of the V- and P-type, unpublished and preliminary results) (for reviews on enzymes inhibited by DCCD, see Refs. (14, 28)). Analysis by gel electrophoresis and fluorography of the proteins radiolabeled by [‘4C]CGA 140408 shows that the carbodiimide covalently binds to the 8-kDa proteolipid of the FO-part of the mitochondrial ATPase in isolated mitochondria from both rat liver and Culliphoru flight muscles. Several lines of evidence show that the 8-kDa protein-binding CGA 140408 is identical with the 8-kDa proteolipid-binding DCCD. Its labeling by CGA 140408 can be inhibited by venturicidin and DCCD. Furthermore, the 8-kDa protein can be extracted by chloroform/methanol like a proteolipid (17), and it is enriched in a fraction consisting of the inner mitochondrial membrane (16). After covalent labeling with CGA 140408, the 8-kDa proteolipid displays an apparent molecular weight of approximately P-10 kDa

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during gel electrophoresisin the standard Lammli system. A modification of the standard Lammli procedureand autoclavingof the probes improves the resolution of the carbodiimide-labeledproteins in gel electrophoresis. Because binding of CGA 140408is inhibited by venturicidin (26, 27) and can be competitively blocked by DCCD, it seems very likely that CGA 140408binds to the samesite of the FOproteolipid as DCCD itself in both rat liver and Cdiphoru flight muscle mitochondria, although conclusive evidence will not be availableuntil the CGA 140408-labeled protein has been sequenced. In isolated mitochondria and total thorax proteins of Calliphora, CGA 140408covalently reactsnot only with the 8-kDa proteolipid but also with a 30-kDa protein. A correspondingprotein in rat liver mitochondria, porin (32 kDa), which is heavily labeled by [14C]DCCD,is only weakly reactive with CGA 140408.This suggeststhat CGA 140408has a protein-bindingspecificity different to DCCD. The identification of the 30-kDacarbodiimide-bindingprotein in Calliphoru with mitochondrial porin is based on a number of observations. It is present in the outer membranefraction of mitochondria, has a molecular weight very similar to that reported for Drosophila pot-inand binds DCCD like the mammalian pot-in. Binding of [‘4C]CGA 140408can be completely and competitively inhibited by excessof unlabeledDCCD and the labeled 30-kDaprotein is not extracted by chloroform/methanol. Additional evidence that the 30-kDa protein is mitochondrial pot-in comes from immunoblots showingthat antibodies against yeast porin, which crossreact with Drosophila porin (30), bind to the 30-kDabandin Calliphora (threeother, as yet unknown proteins are also recognized by the anti-yeastpot-inantibody; data not shown).In addition, weak label is found on a 70-kDa polypeptide in Calliphora which is not associatedwith mitochondria. This peptide comigrateswith nuclearlamin as seen on immunoblots using anti lamin-

KAYSER

antibodies(data not shown). No other proteins becomesignificantly labeled in Calliphora.

Labeling of mitochondrial proteins by [14C]DCCD in insects has already been shown in the American cockroach (29). There, a 1CkDa peptide,probably an overestimate of the molecular weight of the FO proteolipid, and a 40- to 4%kDa protein, perhaps an oligomer of the proteolipid, were labeledby [14C]DCCD. The in vitro datapresentedabovesuggest several possible modes of action for CGA 140408.These might act alternatively or in parallel. Most of the possibilities dependon covalent modification of proteins, thus disrupting vital cell functions. First, block of mitochondrial ATP synthesis in cells highly dependenton aerobic metabolismand lacking energyreservemetabolites such as arginine phosphatemight lead to reduction in the cellular concentration of ATP, thus affecting all energydependentfunctions. Neurones would be primary candidatesfor such a mode of action. Second, partial block of mitochondrial ATP synthesis might lead to an increased proportion of reduced components in the respiratory chain, thereby increasingproduction of active oxygen specieslike the superoxide radical or hydrogen peroxide. For example, there is strong evidencethat this is the mode of action of phosphin, an inhibitor of cytochrome oxidase (36, 37). Third, binding of the carbodiimide to porin might disrupt important functions of this protein. In rat hepatomacells, binding of DCCD to porin has been shown not to disrupt its pore-forming properties but ratherto block the binding of hexokinaseto porin (38).Hexokinaseis the first and a key regulatory enzyme in the glycolytic pathway of glucosemetabolism and its activity determinesthe velocity of glycolytic tumover. There is strongevidencethat hexokinase binding to porin is an important element in the regulationof glycolysis and the coupling of glycolysis to citric acid cycle

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turnover and oxidative phosphorylation (39, 40). Fourth, someunknown protein which appearsonly as a minor labeled protein band in our SDS gels could be disrupted in its function by binding of the carbodiimide, leadingto a lethal effect. Kadir and Knowles (41) reported that CGA 140408has an octopaminergiceffect in bulb mites. Our studies on octopamine receptors(2) and related adenylatecyclase activity in various insects and in a mite (Kayser et al., in preparation)do not support this finding. At present it is unclear which of these various mechanisms presentedabove predominatein the lethal effect of CGA 14Q408.Further in vitro and in vivo studies are necessaryto discriminate between these possibilities. The observation that, even in crude homogenatesof Cdiphora, only a few proteins are labeled by CGA 140408tends to support our view that this carbodiimide acts specifically on one or two targets only, assumingthat its activity is basedon covalent binding. Since in rat liver mitochondria, only the FO proteolipid and not porin becomessignificantly labeled by CGA 140408,this could be the basis for its relatively low mammalian toxicity. This in turn would mean that CGA 140408acts predominantly via porin, but currently we have no idea about the toxic consequencesof the binding of a carbodiimide to porin, as the function of pot-in is not yet sufficiently understood(42).

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4.

5.

6.

7.

8.

9.

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ACKNOWLEDGMENTS

The authors thank K. Leuenberger for technical assistance and J. Benson for advice on the English text. The antibodies against yeast porin were a kind gift from M. Dihanic (Friedrich Miescher Institute, Basel).

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REFERENCES

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