Ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline Research Articles

Inhibition of Reversed Electron Transfer and Proton Transport in the Beef Heart Cytochrome bc1 Complex by Chemical Modification

Chemical modification of the bovine heart cytochrome bc1 complex with N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) has been reported to inhibit the proton pumping activity without affecting the rate of electron transfer to ferricytochrome c. This study aims to examine the effect of EEDQ on energy-linked reversed electron transfer in the bc1 complex reconstituted into potassium-loaded phospholipid vesicles. Generation of a valinomycin-mediated potassium-diffusion potential induced the reduction of cytochrome b in the reconstituted bc1 complex in the presence of sodium ascorbate. The time course of the cytochrome b reduction was well correlated with that of the absorbance change of safranine, an optical probe for measuring membrane potential. Treatment of the bc1 complex with EEDQ caused a decrease in the potential-induced reduction of cytochrome b as well as in the proton translocation activity. But a significant loss in the ubiquinol-cytochrome c reducing activity was not observed in the EEDQ-treated bc1 complex. The time- and concentration-dependent effect of EEDQ on the reversed electron transfer was well correlated with that of the proton translocation activity of the bc1 complex. These findings strongly support the idea that the potential-induced reversal of electron transfer is coupled to the reverse flow of protons in the cytochrome bc1 complex.

Carboxyl residues in the iron-sulfur protein are involved in the proton pumping activity of P. denitrificans bc(1) complex.

A study is presented on chemical modification of the three subunit Paracoccus denitrificans bc(1) complex. N-(Ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) treatment caused a loss of the proton pumping activity of liposome-reconstituted bc(1) complex. A similar effect, which is referred to as the decoupling effect, resulted upon reaction of N,N'-dicyclohexylcarbodiimide (DCCD) with the complex. Direct measurement of the binding of EEDQ to the complex subunits, performed in the presence of the fluorescent hydrophobic nucleophile 4'-[(aminoacetamido)methyl]fluorescein (AMF), showed that the iron-sulfur protein (ISP) and cytochrome c(1) were labeled by EEDQ, whereas cytochrome b was not. Tryptic digestion and sequencing analysis of the fluorescent fragment of the ISP revealed this to consist of a segment with six acidic residues, among which the highly conserved aspartate 160 is present. Analogous experiments on DCCD binding showed that all the three subunits of the complex were labeled. However, DCCD concentration dependence of carboxyl residue modification in the individual subunits and of proton pumping activity showed that the decrease of the H(+)/e(-) ratio correlated only with the modification of the ISP. Tryptic digestion of labeled ISP and sequencing analysis of the fluorescent fragment gave results superimposable upon those obtained with EEDQ. Chymotryptic digestion and sequencing analysis of the single fluorescent fragment of cytochrome b showed that this fragment contained glutamate 174 and aspartate 187. We conclude that, in the P. denitrificans bc(1) complex, carboxyl residues in cytochrome b do not appear to be critically involved in the proton pump mechanism of the complex.

Localization of acidic residues involved in the proton pumping activity of the bovine heart mitochondrial bc 1 complex

Chemical modification of carboxyl residues in polypeptide subunits of the mitochondrial bc 1 complex causes a decoupling effect, that is inhibition of the proton pumping activity, without affecting the rate of electron transfer to ferricytochrome c. The study presented here is aimed at localizing and identifying the residues whose modification results in decoupling of the complex. Glutamate-53 in subunit IX (the DCCD-binding protein) and aspartate-166 in the Rieske iron-sulfur protein are the residues modified by N, N′-dicyclohexylcarbodiimide (DCCD) and N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ), respectively. The results obtained also suggest that the carboxy-terminal sequence of the Core protein II, which is fairly rich in acidic residues, may also play a role in the vectorial proton translocation activity of the complex.

Chemical modification of the bovine mitochondrial bc1 complex reveals critical acidic residues involved in the proton pumping activity.

Bovine heart ubiquinol-cytochrome c reductase (bc1 complex) was modified with N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ), which is a selective reagent for buried carboxyl groups. EEDQ treatment caused a loss of the proton pumping activity of liposome-reconstituted bc1 complex, without effect on the passive proton conductivity of the proteoliposomes. Although the decoupling effect produced on proton translocation was similar to that elicited by N,N'-dicyclohexylcarbodiimide (DCCD) modification of cytochrome b and subunit IX, EEDQ modified different subunits, namely the Core protein II and the iron-sulfur protein (ISP). A time-dependent increase of the labeling of both subunits was observed which was kinetically comparable with the decrease of the H+/e- ratio. Trypsin treatment of the complex showed that the EEDQ-modified carboxyl group in the ISP belongs to the protruding moiety of the protein, holding the Fe/S cluster. The results obtained show that critical acidic residues, located in different subunits of the bc1 complex, at both sides of the membrane, contribute to its proton pumping activity.

Effect of nucleotides and actin on the intramolecular cross-linking of myosin subfragment-1.

The heavy chain of myosin subfragment-1 (S1) is cleaved by limited trypsinolysis into three fragments, 27, 50, and 20 kDa--aligned in this order from the N-terminus. The tertiary structure of the molecule is essentially not affected by trypsinolysis. The spatial relations between the various regions of the molecule and the nucleotide- and actin-induced intramolecular movements were studied by cross-linking tryptic S1 with N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), phenylenediglyoxal (PDG), and glutaraldehyde. The formation of cross-linked products was monitored by SDS-PAGE, using the fluorescent probes 9-anthronitrile and N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS), which specifically label the 27- and 20-kDa fragments, respectively. The reaction with the cross-linkers leads to the formation of 50-kDa/20-kDa, 27-kDa/20-kDa, 27-kDa/50-kDa, and 20-kDa/light chain cross-linked products. Of these, the most intensive was the formation of the 50-kDa/20-kDa products, which appeared as a doublet on the SDS-PAGE with all the cross-linkers. This indicates that the interface between the two fragments is rather extended. The presence of MgATP or MgADP promoted the formation of the 20-kDa/50-kDa cross-linked products, especially with the lower electrophoretic mobility band, when EEDQ was used as a cross-linker. With PDG as a cross-linker, MgATP also affected the cross-link formation between the 20-kDa fragment and the light chains whereas it had no influence on the formation of other products. On the other hand, the effect of actin on the cross-linking with the various cross-linkers was quite extensive, and it was manifested in the reduction of cross-link formation between the various S1 domains. It is concluded that both nucleotides and actin induce intramolecular movements in S1 and that the nucleotide-induced movements are more restricted than those induced by actin, which extend to larger regions of the molecule.

Energy-transducing nicotinamide nucleotide transhydrogenase. Nucleotide binding properties of the purified enzyme and proteolytic fragments.

The mitochondrial energy-transducing nicotinamide nucleotide transhydrogenase is a homodimer of monomer M(r) = 109,065. It is composed of a 430-residue-long N-terminal hydrophilic domain, a 400-residue-long central hydrophobic domain, and a 200-residue-long C-terminal hydrophilic domain. The N- and the C-terminal hydrophilic domains extend into the mitochondrial matrix, and the central hydrophobic domain intercalates into the inner membrane. Equilibrium binding of [14C]NADH and [14C]NADPH was carried out to the purified transhydrogenase, transhydrogenase modified with N,N'-dicyclohexylcarbodiimide (DCCD) at the NADH-protectable Glu257, and to proteolytic fragments corresponding to dimers of the N-terminal and the C-terminal hydrophilic domains. The intact enzyme bound 1 mol of [14C]NADH or [14C]NADPH per dimer with Kd values of 9.5 and 5.7 microM, respectively. The 90% DCCD-inhibited enzyme bound [14C]NADPH with undiminished capacity and [14C]NADH with 10% capacity as compared with unmodified enzyme. The Kd values were unaltered. The N-terminal fragment bound [14C]NADH at 0.76 mol/dimer with unaltered Kd but did not bind [14C]NADPH. The C-terminal fragment bound 0.32 mol of [14C]NADPH/dimer with unaltered Kd but did not bind [14C]NADH. These results are consistent with our previous assignment of the NAD(H) and the NADP(H) binding sites to the N- and the C-terminal hydrophilic domains, respectively. The binding stoichiometries of 1 mol each of [14C]NADH and [14C]NADPH/dimeric transhydrogenase suggest half-of-the-sites reactivity, which is consistent with our previous findings that 100% inhibition by either p-[3H]fluorosulfonylbenzoyl-5'-adenosine or [14C]DCCD involved the incorporation of 1 mol of inhibitor/dimeric enzyme. We had shown earlier that N-(ethoxycarbonyl)2-ethoxy-1,2-dihydroquinoline (EEDQ) inhibits the transhydrogenase, apparently at the NAD binding site, but differently from DCCD. The enzyme was protected by 5'-AMP strongly against DCCD and weakly against EEDQ, whereas it was protected by NMNH strongly against EEDQ and not at all against DCCD. We have now determined the EEDQ binding sites to be Glu232 and Glu880. The different mononucleotide protections against DCCD and EEDQ suggest that the AMP moiety of NAD binds near Glu257 and its NMN portion near Glu232. They also suggest that the NMN moiety of NADP may bind near Glu880.

Effects of Solubilization on the Inhibition of the P-Type ATPase from Maize Roots by N-(Ethoxycarbonyl)-2-Ethoxy-1,2-Dihydroquinoline

The biochemical events utilized by transport proteins to convert the chemical energy from the hydrolysis of ATP into an electro-chemical gradient are poorly understood. The inhibition of the plasma membrane ATPase from corn (Zea mays L.) roots by N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) was compared to that of ATPase solubilized with N-tetradecyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate (3-14) to provide insight into the minimal functional unit. The chromatographic behavior of the 3-14-solubilized ATPase activity during size exclusion chromatography and glycerol gradient centrifugation indicated that the solubilized enzyme was in a monomeric form. Both plasma membrane-bound and solubilized ATPase were inhibited by EEDQ in a time- and concentration-dependent manner consistent with a first-order reaction. When the log of the reciprocal of the half-time for inhibition was plotted as a function of the log of the EEDQ concentration, straight lines were obtained with slopes of approximately 0.5 and 1.0 for membrane-bound and 3-14-solubilized ATPase, respectively, indicating a change in the number of polypeptides per functional ATPase complex induced by solubilization with 3-14.

A new, general synthetic route to multidentate N,S ligands for use in technetium-99m radiopharmaceuticals. Preparation of diamido disulfur, diamino dithiol, and tripodal N3S3 prototypes. Comparative biodistributions of [99mTcVO-DADS]- analogs which contain 5,5,5- and 5,7,5-membered chelate ring systems

A new, versatile synthetic route to a variety of tetradentate N2S2 and hexadentate N3S3 ligands for use in technetium-99m radiopharmaceuticals has been developed. The key reaction employs 1-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) for coupling an appropriate di- or triamine with S-protected thioglycolic acid. Selected DADS (diamido disulfur) and DADT (diamino dithiol) and analogues derived from ethanediamine-1,2 and butanediamine-1,4, as well as a tripodal N3S3 analogue, were synthesized in high yield. The labeling of DADS analogue ligands derived from butanediamine-1,4 (DADS-bn) with 99mTc results in a single radiochemical product, or the expected number of stereoisomeric products. FAB MS analysis, using 99Tc, indicates that DADS-bn ligands form a tetradentate 5,7,5-membered ring chelate system with the monooxo Tc(V) core: [TcO-DAD-bn]-. In rat biodistribution studies the DADS-en and DADS-bn complexes show very similar biological activity. Phenyl substituents on the 7-membered ring give rise to a 99mTc complex of increased lipophilicity, increased liver uptake, and minimal hepatobillary clearance. Thus, new classes of DADS ligand analogues which contain a 5,7,5-membered chelate ring system are readily synthesized and labeled with 99mTc to form stable complexes. The presence of the 7-membered chelate ring allows for facile introduction of pendant groups into the ligand system, and thus for a ready route to control the biodistribution of the resulting 99mTc radiopharmaceutical. The 99mTc labeling of the DADT analogues derived from butanediamine-1,4 provided to be erratic, despite the use of a variety of labeling techniques; the larger ring system of DADT-bn apparently does not favor the monooxo Tc(V) core and appears to generate a mixture of mono- and dioxo Tc(V) cores.

Cross-linking of the skeletal myosin subfragment 1 heavy chain to the N-terminal actin segment of residues 40-113.

Glutaraldehyde (GA) and N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ), a hydrophobic, carboxyl group directed, zero-length protein cross-linker, were employed for the chemical cross-linking of the rigor complex between F-actin and the skeletal myosin S-1. The enzymatic properties and structure of the new covalent complexes obtained with both reagents were determined and compared to those known for the EDC-acto-S-1 complex. The GA- or EEDQ-catalyzed covalent attachment of F-actin to the S-1 heavy chain induced an elevated Mg2+-ATPase activity. The turnover rates of the isolated cross-linked complexes were similar to those for EDC-acto-S-1 (30 s-1). The solution stability of the new complexes is also comparable to that exhibited by EDC-acto-S-1. The proteolytic digestion of the isolated AEDANS-labeled covalent complexes and direct cross-linking experiments between actin and various preformed proteolytic S-1 derivatives indicated that, as observed with EDC, the COOH-terminal 20K and the central 50K heavy chain fragments are involved in the cross-linking reactions of GA and EEDQ. KI-depolymerized acto-S-1 complexes cross-linked by EDC, GA, or EEDQ were digested by thrombin which cuts only actin, releasing S-1 heavy chain-actin peptide cross-linked complexes migrating on acrylamide gels with Mr 100K (EDC), 110K and 105K (GA), and 102K (EEDQ); these were fluorescent only when fluorescent S-1 was used. They were identified by immunostaining with specific antibodies directed against selected parts of he NH2-terminal actin segment of residues 1-113.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential recovery rates of rat D2 dopamine receptors as a function of aging and chronic reserpine treatment following irreversible modification: a key to receptor regulatory mechanisms

The density of D2 dopamine receptors identified by 3H-spiperone binding was significantly lower in the striatum of senescent (28-month-old) than in mature (4-month-old) Fischer 344 rats. The time course of recovery of 3H-spiperone binding to D2 dopamine receptors following irreversible receptor modification by a single injection of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was significantly slower in senescent than in mature rats. The kinetics of D2 dopamine receptor repopulation could be adequately described by a model assuming a constant rate of receptor production and a rate of degradation that was dependent on receptor concentration. Both the production rate and degradation rate constant of D2 dopamine receptors were lower in the senescent rats compared to mature rats. The changes in receptor density that occur as a function of development and aging are the result of changes in both the receptor production rate and receptor degradation rate constant, which, while both are reduced, tend to maintain receptor density. That is, receptor density decreased by 26% from 4 to 28 months of age, although the receptor production rate and degradation rate constant decreased by 40-50%. Chronic treatment of rats with reserpine produced a 21% increase in the density of striatal D2 dopamine receptors of mature, but not senescent, rats, accompanied by an increase in the initial recovery rate of 3H-spiperone binding sites following EEDQ treatment. It might have been expected that the reduced formation of agonist-bound receptors would have lead to a decrease in the receptor degradation rate constant.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions of Neurospora crassa plasma membrane H+-ATPase with N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline.

The carboxyl group activating reagent N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) interacts with the Neurospora plasma membrane H+-ATPase in at least three different ways. This reagent irreversibly inhibits ATP hydrolysis with kinetics that are pseudo-first-order at several concentrations of EEDQ, and an appropriate transform of these data suggests that 1 mol of EEDQ inactivates 1 mol of the H+-ATPase. Inhibition probably involves activation of an ATPase carboxyl group followed by a nucleophilic attack by a vicinal nucleophilic functional group in the ATPase polypeptide chain, resulting in an intramolecular cross-link. The enzyme is protected against EEDQ inhibition by MgATP in the presence of vanadate, a combination of ligands that has previously been shown to "lock" the H+-ATPase in a conformation that presumably resembles the transition states of the enzyme phosphorylation and dephosphorylation reactions, but is not protected by the substrate analogue MgADP, which is consistent with the notion that one or both of the residues involved in the EEDQ-dependent inhibitory intramolecular cross-linking reaction normally participate in the transfer of the gamma-phosphoryl group of ATP, or are near those that do. The ATPase is also labeled by the exogenous nucleophile [14C]glycine ethyl ester in an EEDQ-dependent reaction, and the labeling is diminished in the presence of MgATP plus vanadate. However, peptide maps of [14C]glycine ethyl ester labeled ATPase demonstrate that the labeling is not related to the EEDQ inhibition reaction in any simple way.(ABSTRACT TRUNCATED AT 250 WORDS)

Mitochondrial nicotinamide nucleotide transhydrogenase: Nonidentical modification by N,N′-dicyclohexylcarbodiimide and n-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline at the NAD(H) binding site

The energy-linked nicotinamide nucleotide transhydrogenase (TH) purified from bovine heart mitochondria is inhibited by the carboxyl group modifiers, N,N′-dicyclohexylcarbodiimide (DCCD) and N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ). With either reagent, complete activity inhibition corresponds to modification of one carboxyl group per 2 mol (monomers) of this dimeric enzyme, suggesting half-site reactivity toward DCCD and EEDQ [ D. C. Phelps, and Y. Hatefi (1984) Biochemistry 23, 4475–4480; 6340–6344]. It has also been shown in the former reference that DCCD appears to modify TH at the NAD(H)-binding site. The present paper presents data suggesting that EEDQ also binds at or near the NAD(H)-binding domain of TH, but at a site not identical to that of DCCD: (i) TH modified with and inhibited ~85% by EEDQ could be further labeled with [ 14C]DCCD to the extent of 70% of the maximum in the same time period that unmodified TH was modified by [ 14C]DCCD to near saturation (1 mol DCCD/ TH dinier); (ii) DCCD-modified TH did not bind to NAD-agarose, while EEDQ-modified TH showed partial affinity for NAD-agarose; (iii) 5′-AMP completely protected TH against modification by DCCD, but showed only a weak protective effect against EEDQ; (iv) by contrast, NMNH, which is a TH substrate and binds to the NADH site, did not protect TH against DCCD, but completely protected the enzyme against attack by EEDQ. The results are consistent with the possibility that DCCD modifies TH where the 5′-AMP moiety of NAD(H) binds, while EEDQ modifies the enzyme where the NMN(H) moiety of NAD(H) resides.

The use of [ 3H]aniline to identify the essential carboxyl group in the bovine mitochondrial F 1-ATPase that reacts with 1-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline

The inactivation of the bovine heart mitochondrial F 1-ATPase with 1-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) in the presence of [ 3H]aniline at pH 7.0 led to the covalent incorporation of 3H into the enzyme. When the ATPase was inactivated by 94% with 0.9 m m EEDQ in the presence of 3.6 m m [ 3H]aniline in a large-scale experiment in which the protein concentration was 21 mg/ml, 4.2 mol [ 3H]anilide were formed per mol enzyme, of which 0.35 mol was incorporated per mol of the α subunit and 1.0 mol was incorporated per mol of the β subunit. Examination of a tryptic digest of the isolated α subunit revealed that the majority of the 3H was contained in a single tryptic peptide, which, when purified, was shown to contain the [ 3H]anilide of a glutamic acid residue which corresponds to α-Glu-402 of the Escherichia coli F 1-ATPase. This residue was labeled to the extent of about 1.0 mol/mol enzyme. Analysis of tryptic peptides purified from the isolated β subunit showed that 0.8 and 1.5 mol, respectively, of the [ 3H]anilides of β-Glu-341 and β-Glu-199 were formed per mol MF 1 during the inactivation of the enzyme at 21 mg/ml. When the ATPase was inactivated by 90% at a protein concentration of 1.7 mg/ml by 0.9 m m EEDQ in the presence of 1.7 m m [ 3H]aniline, 3.1 mol [ 3H]anilide were formed per mol enzyme. From the analysis of the radioactive peptides purified from a tryptic digest of the labeled ATPase from this experiment it was estimated that 0.7 mol of the [ 3H]anilide of α-Glu-402, 0.3 mol of the [ 3H]anilide of β-Glu-341, and 1.5 mol of the [ 3H]anilide of β-Glu-199 were formed per mol F 1-ATPase. Since β-Glu-199 is labeled to the same extent in the two experiments while α-Glu-402 and β-Glu-341 were not, suggests that the modification of β-Glu-199 is responsible for inactivation of the enzyme by EEDQ.

Effects of N,N-dicyclohexylcarbodiimide and N-{ethoxycarbonyl}-2-ethoxy-1,2-dihydroquinoline on hydride ion transfer and proton translocation activities of mitochondrial nicotinamide nucleotide transhydrogenase

N,N'-Dicyclohexylcarbodiimide (DCCD) inhibits the mitochondrial energy-linked nicotinamidenucleotide transhydrogenase (TH). Our studies [Phelps, D.C., & Hatefi, Y. (1981) J. Biol. Chem. 256, 8217-8221; Phelps, D.C., & Hatefi, Y. (1984) Biochemistry 23, 4475-4480] suggested that the inhibition site of DCCD is near the NAD(H) binding site, because NAD(H) and competitive inhibitors protected TH against inhibition by DCCD and, unlike the unmodified TH, the DCCD-modified TH did not bind to NAD-agarose. Others [Pennington, R.M., & Fisher, R.R. (1981) J. Biol. Chem. 256, 8963-8969] could not demonstrate protection by NADH, obtained data indicating DCCD inhibits proton translocation by TH much more than hydride ion transfer from NADPH to 3-acetylpyridine adenine dinucleotide (AcPyAD), and concluded that DCCD modifies an essential residue in the proton channel of TH. The present studies show that N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) also inhibits TH. The inhibition is pseudo first order at several EEDQ concentrations, and the reaction order with respect to [EEDQ] is unity, suggesting that inhibition involves the interaction of one molecule of EEDQ with one active unit of TH. The EEDQ-modified TH reacts covalently with [3H]aniline, suggesting that the residue modified by EEDQ is a carboxyl group. More significantly, it has been shown that the absorbance change of oxonol VI at 630 minus 603 nm is a reliable reporter of TH-induced membrane potential formation in submitochondrial particles and that TH-catalyzed hydride ion transfer from NADPH to AcPyAD and the membrane potential induced by this reaction are inhibited in parallel by either DCCD or EEDQ.

Inhibitory chemical modifications of F1-ATPase: effects on the kinetics of adenosine 5'-triphosphate synthesis and hydrolysis in reconstituted systems.

The purified, soluble F1-ATPase was modified by several covalently reacting inhibitors, either known or considered to bind to the active site bearing beta-subunit, to cause partial inhibition up to 99%. The modified enzyme was then reconstituted in the presence of OSCP (oligomycin sensitivity conferring protein) with submitochondrial particles (SMP) almost completely (greater than 99%) denuded of active F1-ATPase and was assayed for oligomycin-sensitive ATPase and oxidative phosphorylation activities. The inhibitors used were 1-fluoro-2,4-dinitrobenzene (FDNB), N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMCD), quinacrine mustard (QM), 5-(dimethylamino)-naphthalene-1-sulfonyl chloride (dansyl-Cl), 5'-[p-(fluoro-sulfonyl)benzoyl]adenosine (FSBA), and N,N'-dicyclohexylcarbodiimide (DCCD). The SMP reconstituted with unmodified F1 exhibited oxidative phosphorylation and oligomycin-sensitive ATPase (in the presence of uncouplers) activities as high as 500 nmol min-1 mg-1 and 8 mumol min-1 mg-1, respectively. The systems reconstituted with F1 modified to cause various degrees of inhibition with FDNB, EEDQ, CMCD, QM, and dansyl-Cl exhibited the same degree of inhibition of oxidative phosphorylation and oligomycin-sensitive ATPase activities as the inhibition of the ATPase activity of the modified F1 before reconstitution. The systems reconstituted with FSBA-modified F1 showed the following relative degrees of inhibition: oxidative phosphorylation greater than oligomycin-sensitive ATPase of particles greater than ATPase of soluble F1. In contrast, the systems reconstituted with DCCD-modified F1 showed much greater inhibition of oligomycin-sensitive ATPase than of oxidative phosphorylation activity.(ABSTRACT TRUNCATED AT 250 WORDS)