Effects of fibrates on human organic anion-transporting polypeptide 1B1-, multidrug resistance protein 2- and P-glycoprotein-mediated transport.

The effects of different fibric acid derivatives (bezafibrate, clofibrate, clofibric acid, fenofibrate, fenofibric acid and gemfibrozil) on human organic anion transporting-polypeptide 1B1 (OATP2, OATP-C, SLC21A6), multidrug resistance protein 2 (MRP2/ABCC2) and MDR1-type P-glycoprotein (P-gp/ABCB1) were examined in vitro. Cyclosporin A (a known inhibitor of OATP1B1 and P-gp), MK-571 (a known inhibitor of MRP2) and cimetidine (an organic cation) were also tested. Bezafibrate, fenofibrate, fenofibric acid and gemfibrozil showed concentration-dependent inhibition of estradiol 17-beta-D-glucuronide uptake by OATP1B1-stably transfected HEK cells, whereas clofibrate and clofibric acid did not show any significant effects up to 100 microM. Inhibition kinetics of gemfibrozil, which exhibited the most significant inhibition on OATP1B1, was shown to be competitive with a Ki = 12.5 microM. None of the fibrates showed any significant inhibition of MRP2-mediated transport, which was evaluated by measuring the uptake of ethacrynic acid glutathione into MRP2-expressing Sf9 membrane vesicles. Only fenofibrate showed moderate P-gp inhibition as assessed by measuring cellular accumulation of vinblastine in a P-gp overexpressing cell-line. Cyclosporin A significantly inhibited OATP1B1 and P-gp, whereas only moderate inhibition was observed on MRP2. The rank order of inhibitory potency of MK-571 was determined as OATP1B1 (IC50: 0.3 microM) > MRP2 (4 microM) > P-gp (25 microM). Cimetidine did not show any effects on these transporters. In conclusion, neither MRP2- nor P-gp-mediated transport is inhibited significantly by the fibrates tested. Considering the plasma protein binding and IC50 values for OATP1B1, only gemfibrozil appeared to have a potential to cause drug-drug interactions by inhibiting OATP1B1 at clinically relevant concentrations.

Renal elimination of a novel and potent alphavbeta3 integrin antagonist in animals.

Compound A (3-{2-oxo-3-[3-(5,6,7,8-tetrahydro-[1,8]napthyridin-2-yl)propyl]-imidazolidin-1-yl}-3(S)-(6-methoxy-pyridin-3-yl)propionic acid), a hydrophilic zwitter-ion, is a potent and selective alphavbeta3 integrin antagonist currently under clinical development for the treatment of osteoporosis. The mechanism of renal excretion of compound A was investigated using a combination of in vivo and in vitro approaches. In rats, renal excretion of compound A involved tubular secretion; ratios between renal clearance, corrected for unbound fraction in plasma (CLr,u) and glomerular filtration rate (GFR) were greater than unity (2-5). The tubular secretion of compound A was saturable at high plasma levels (> 26 microM), and was inhibited significantly, although modestly (about twofold) by relatively high plasma concentrations of the organic anion PAH (160 microM) and the cation cimetidine (about 400 microM), but not by the P-gp inhibitor quinidine (about 50 microM). However, compound A (about 100 microM) had a minimal effect on CLr/GFRs for cimetidine and PAH. In rhesus monkeys, renal elimination of compound A also involved tubular secretion, with a CLr,u/GFR ratio of about 30. The renal secretion of compound A was not affected by either cimetidine (about 120 microM) or PAH (about 80 microM). Similarly, compound A (about 40 microM) had a minimal effect on the renal tubular secretion of both cimetidine and PAH. At the doses studied, neither rat nor monkey plasma protein binding of compound A, cimetidine or PAH was affected in the presence of each other. In vitro transport studies showed that compound A was not a substrate for P-gp in the Caco-2, human MDR1 and mouse mdr1a transfected LLC-PK1 cell lines. In an uptake study using rOAT1 and rOAT3 transfected HEK cell lines, compound A was shown to be a substrate for rat OAT3 (Km= 15 microM), but not rat OAT1. The results suggest that the tubular secretion of compound A is not mediated by P-gp, but rather is mediated, at least in part, via the organic anion transporter OAT3, the renal transporter shown to be capable of transporting both the organic anion PAH and the organic cation cimetidine. Although there is a possibility for pharmacokinetic interactions between compound A and substrates or inhibitors of OAT3, at the renal excretion level, the magnitude of interaction would likely be modest in humans at clinically relevant doses.

P-glycoprotein-mediated efflux of indinavir metabolites in Caco-2 cells expressing cytochrome P450 3A4.

The role of P-glycoprotein in secretion of indinavir metabolites produced by CYP3A4 was evaluated in Caco-2 cells expressing CYP3A4. Metabolism of indinavir by CYP3A4 expressing Caco-2 cells grown on filters resulted in the formation of N-dealkylation products (M5 and M6) and hydroxylation of indinavir, which were preferentially secreted into the apical compartment. Apical secretion of the metabolites was inhibited by cyclosporin A (CsA) with all three classes of metabolites showing similar sensitivity to CsA, suggesting that they are all secreted by the same pathway. M6 stimulated P-glycoprotein (Pgp)-ATPase activity in a concentration-dependent manner. This stimulation was inhibited by the Pgp-specific monoclonal antibody C219. A method was developed to specifically inhibit Pgp using the monoclonal antibody UIC2 to determine whether Pgp efflux accounts for a significant proportion of the apical secretion of indinavir metabolites. UIC2 recognizes an extracellular transient conformational epitope that is stabilized by some Pgp substrates or by ATP depletion. Incubation of Caco-2 cells with UIC2 in the presence of 1 microM CsA resulted in 50 to 80% inhibition of Pgp-mediated vinblastine efflux, with no significant inhibition observed by UIC2 or CsA alone. Inhibition of Pgp in CYP3A4-expressing Caco-2 cells by UIC2 and 1 microM CsA resulted in a significant decrease in the apical secretion of M6, M5, and OH-indinavir and an increase in the amount of the metabolites secreted in the basolateral compartment and retained in the cytosol. These results are consistent with a role of Pgp in elimination of CYP3A4-generated metabolites and indicate that even relatively polar metabolites may be secreted from the cell by Pgp.

In vitro substrate identification studies for p-glycoprotein-mediated transport: species difference and predictability of in vivo results.

Two different cellular assay models were assessed as in vitro systems for P-glycoprotein (P-gp) substrate identification: cellular accumulation studies with KB-V1, a human MDR1 P-gp-overexpressing multidrug-resistant human epidermoid carcinoma cell line; and transcellular transport studies with L-MDR1 (or L-mdr1a), a human MDR1 (or mouse mdr1a)-transfected porcine renal epithelial cell line. The in vitro-in vivo correlation for P-gp-mediated transport activity was also examined by comparing in vitro data obtained from L-mdr1a cell studies and in vivo data from mdr1a (-/-)/(+/+) CF-1 mice studies for several compounds. The results are summarized as follows: 1) two in vitro assay systems routinely identified the substrate for human MDR1 P-gp-mediated transport with similar quantitative results; 2) in vitro studies with L-MDR1 and L-mdr1a cells demonstrated that the P-gp substrate susceptibility is different between human and mouse for certain compounds (species difference); and 3) in vivo brain concentration ratios of mdr1a (-/-) to (+/+) CF-1 mice, either at a certain time point or up to 60 min, correlated well with the in vitro transcellular transport ratios from L-mdr1a cells (r(2) = 0.968 and 0.926, respectively). This indicates that, at least in mice, the in vitro data are valid predictors of the in vivo contribution of P-gp: the contribution of P-gp to the distribution of the compound to the brain up to 60 min post i.v. administration. These results provide a rationale for predicting in vivo relevance of P-gp in human from in vitro data using human P-gp-expressing cells.

Regeneration and maintenance of bile canalicular networks in collagen-sandwiched hepatocytes.

The morphological and cytoskeletal reorganization of collagen-sandwiched rat hepatocytes during the de novo formation of complete canalicular networks was examined by phase, fluorescence and electron microscopy. During the initial stages of membrane repolarization, there was a marked accumulation of both microfilaments and microtubules at the sites of canalicular generation. Microtubule-disrupting agents (colchicine, nocodazole) inhibited the localization of actin filaments at cell margins and the initiation and branching of canalicular networks. After removal of microtubule-disrupting agents, microfilaments relocalized to the canalicular borders and microtubules nucleated along the margins of the bile canaliculi at sites distinct from the peri-canalicular actin networks. Microfilament-perturbing agents (cytochalasin D, phalloidin) did not affect the de novo initiation of bile canaliculi and only slightly impaired the development of canalicular lumina into networks. In established cultures with complete canalicular networks, subsequent treatment with microtubule-disrupting agents did not acutely affect the integrity of preformed canalicular networks. In contrast, treatment with microfilament-perturbing agents caused a marked dilation of most canaliculi. These results illustrate the differential role of the cytoskeleton in the regeneration and maintenance of bile canalicular networks by collagen-sandwiched hepatocytes. Moreover, this study shows the utility of this system as an in vitro model for examining the regulation of cell and membrane polarity.

Comparison of imidazole- and 2-methyl imidazole-containing farnesyl-protein transferase inhibitors: interaction with and metabolism by rat hepatic cytochrome P450s.

Methylation at the 2-position of the imidazole ring of IBN (I), a 1, 5-substituted imidazole-containing compound, was carried out to minimize its inhibition of rat cytochrome P450 (CYP)3A activity. The resulting analog 2-MIBN (II) exhibited an inhibitory potency 70-fold weaker (K(i) = 25 microM) than that of I (K(i) = 0.3 microM) toward CYP3A, the major rat liver microsomal P450 isoform(s) for the metabolism of I and II by rat liver microsomes in the presence of NADPH. The structural modification did not switch the major metabolic pathways for I and II, but significantly decreased the affinity of II to the metabolizing enzyme(s) as reflected by the difference in their K(i) values for CYP3A. Enzyme kinetic studies also demonstrated that I had a lower apparent K(m) (0.3 microM) than than II (18 microM), but an apparent V(max) 14 times lower than II. This finding indicates that methylation at the imidazole ring reduced the affinity of the compound to CYP3A, but increased the catalytic capacity, turning I as a substrate of low K(m) value but low capacity into a compound of high K(m) but high capacity for the metabolism. Our results suggest the significance of substrate concentration in comparing the metabolic stability of compounds with different kinetic parameters. Although higher intrinsic clearance is implied for I when the substrate concentration is below or close to its K(m) value, higher metabolic rate was constantly seen with II over micromolar range. The different kinetic parameters of I and II may also explain the observation that no significant difference in pharmacokinetic behavior was seen after an i.v. administration of I and II to the rat.

Influence of P-glycoprotein on the transport and metabolism of indinavir in Caco-2 cells expressing cytochrome P-450 3A4.

Caco-2 cells grown in the presence of 1alpha,25-di-OH vitamin D(3) (di-OH vit D(3)) were used as a model to evaluate the effects of P-glycoprotein (Pgp) efflux on CYP3A4-mediated metabolism of indinavir during intestinal absorption. Caco-2 cells grown under these conditions demonstrated significant CYP3A4 activity and maintained Pgp-mediated directional transport of indinavir. Metabolism of indinavir in the di-OH vit D(3)-treated cells correlated with the level of CYP3A activity and generated metabolites consistent with CYP3A4-mediated metabolism. During transport experiments, indinavir metabolites are selectively secreted into the apical compartment, consistent with Pgp-mediated efflux. Using formation of the most abundant metabolite, M6, as a marker for indinavir metabolism, we observed that the extent of indinavir metabolism is not significantly affected by the direction of indinavir transport or by inhibition of Pgp with cyclosporin A. However, because Pgp efflux results in higher indinavir transport in the basolateral-to-apical direction than in the apical-to-basolateral direction, the ratio of M6 produced normalized to the amount of drug transported across the monolayer was higher for apical-to-basolateral transport. Thus, Pgp efflux in a direction opposite to absorptive transport results in more metabolite produced per mole of drug that is absorbed. In summary, the results support a role of Pgp in increasing intestinal presystemic metabolism and in removal of CYP3A4-generated metabolites from the intracellular compartment.

Species and organ differences in first-pass metabolism of the ester prodrug L-751,164 in dogs and monkeys. In vivo and in vitro studies.

The pharmacokinetics and bioavailability of L-751,164, an ethyl ester prodrug of a potent fibrinogen receptor antagonist, L-742,998, were studied in beagle dogs and rhesus monkeys. In both species, L-751,164 exhibited high clearance. After an intravenous dose, L-751,164 was converted to the parent L-742,998 to the extent of approximately 20% in dogs and 90% in monkeys. After oral administration of the prodrug, however, the bioavailability, measured either as the prodrug or as the active parent, was < 5% in both species. Several experiments were conducted subsequently to investigate possible causes for the observed similarities in the low oral bioavailability of the prodrug between species despite its differences in the in vivo conversion. In vitro metabolism studies using dog liver subcellular fractions indicated extensive metabolism of L-751,164 to metabolites other than L-742,998. Kinetically, L-742,998 formation accounted only for approximately 25% of the prodrug disappearance. In contrast, monkey liver preparations converted L-751,164 exclusively and rapidly to L-742,998. Good agreement between the in vitro hepatic metabolism and the in vivo observations suggests that liver was the major eliminating organ after intravenous administration of the prodrug in both species. In dogs, this suggestion was further supported by low bioavailability of the prodrug (20%) and the parent (below detection limit) after intraportal administration of the prodrug. In vitro metabolism of L-751,164 using intestinal S9 fractions revealed substantial metabolism in monkeys, but not in dogs. Several NADPH-dependent metabolites were observed with monkey intestinal preparation, with the parent L-742,998 being the minor product (approximately 25-30%). Furthermore, L-751,164 was shown, by means of an in vitro Caco-2 cell, and in situ rat intestinal loop models, to be highly permeable to intestinal barriers. Collectively, these results suggest that the apparent species differences in the prodrug conversion observed in vivo likely were due to species differences in the hepatic metabolism of the prodrug. In both species, the high first-pass metabolism of the prodrug, and the extensive conversion of the prodrug to metabolic products other than the parent contributed, at least in part, to the low bioavailability of the prodrug and active parent, respectively, obtained after an oral dose of the prodrug. The latter process was species-dependent, involving primarily the hepatic first-pass elimination in dogs and the intestinal first-pass metabolism in monkeys.

Comparative studies of drug-metabolizing enzymes in dog, monkey, and human small intestines, and in Caco-2 cells.

Drug-metabolizing enzymes were studied in subcellular fractions of dog, monkey, and human small intestines, and in the human adenocarcinoma cell line Caco-2, a commonly used in vitro absorption model. Immunoblot analysis indicated the presence of enzymes related to cytochrome P450 (CYP) 1A1/CYP1A2, CYP2D6, CYP3A, and carboxylesterases (ESs) in human and monkey intestines, and of CYP3A and ES in dog intestines. Catalytically, human and monkey intestines exhibited significant and comparable testosterone 6 beta-hydroxylase, (+)-bufuralol 1'-hydroxylase, and ES activities. In contrast, dog intestine possessed moderate testosterone 6 beta-hydroxylase, much lower ES, and undetectable bufuralol hydroxylase activities. In addition, low tolbutamide methylhydroxylase activity was observed in human and monkey intestines, but not in dog intestines. Of the phase I enzymes investigated, only ES was detected immunologically and functionally in Caco-2 cells. With respect to phase II enzymes, human and monkey intestines contained relatively high intestinal glucuronyltransferase, N-acetyltransferase (NAT), sulfotransferase, and glutathione S-transferase activities. Except for NAT, all phase II enzymes studied were detectable in dog intestines. In Caco-2 cells, acetaminophen sulfation activity was below the limit of detection, whereas all other conjugating activities were evident. Studies of enzyme kinetics and inhibition by known inhibitors of testosterone 6 beta-hydroxylase activity, the major intestinal mono-oxygenase in all species, revealed some similarities between the responsible enzymes. Comparative studies with human liver microsomes suggested the possible involvement of CYP3A enzymes in the intestinal catalysis of testosterone 6 beta-hydroxylation similar to those observed with human hepatic CYP3A. Further studies on ESs, however, revealed multiplicity and species and/or tissue differences in the microsomal and cytosolic enzymes. Based on kinetic studies, monkey intestines and Caco-2 cells possessed NAT activities, with properties similar to those in human intestine and liver. Overall, the results demonstrated that both the preparations of small intestines and Caco-2 cells exhibited significant drug-metabolizing enzyme activities, although several differences were noted between the intestinal enzymes in the animals or in the Caco-2 cells and those found in humans.

Formation of extensive canalicular networks by rat hepatocytes cultured in collagen-sandwich configuration.

Rat primary hepatocytes were cultured under different extracellular matrix configurations and evaluated for the acquisition and maintenance of structural and functional cell polarity. De novo repolarization of the plasma membrane was variable in rate and extent in hepatocyte cultures maintained on a conventional single layer of either gelled or ungelled collagen. However, cultures maintained in a collagen-sandwich configuration initiated uniform formation of a contiguous anastomosing network of bile canaliculi throughout the entire culture. Localization of apical membrane markers demonstrated normal distribution at the canalicular membrane. A marked rearrangement of the intracellular microfilaments to the cell periphery was observed and coincided with the development of the bile canaliculi. Acquisition of normal bile canalicular function and integrity was observed within 3-4 days postoverlay as indicated by the concentration and retention of carboxyfluorescein within the canalicular network. These results demonstrate that cultures of hepatocytes maintained in a sandwich configuration may serve as a more reliable and representative model in which to study the physiology of hepatic function as well as the morphogenesis of polarized membrane domains in vitro.

In vitro and in vivo analysis of the mechanism of absorption enhancement by palmitoylcarnitine.

Long-chain acylcarnitines (12-18 carbon fatty acid esters) dramatically enhance the absorption of hydrophilic drugs across intestinal mucosa without altering the morphology of the epithelium. The mechanism underlying these effects was studied using the colon carcinoma cell line Caco-2. Caco-2 monolayers treated with 0.2 mM palmitoylcarnitine (PCC) show dramatic increases in the transport of hydrophilic markers. This enhanced transport coincides with a rapid drop in transepithelial electrical resistance (TER). The drop in TER is initiated within the first minute after PCC addition and continues for approximately 20 min to a 70 to 85% drop of the initial TER values. This effect is reversible after removing the PCC and does not appear to involve lysis of the apical membrane. Instead PCC's effect appears to be due to loosening of tight junctions as indicated by the accumulation of fluorescent dextrans and the electron dense marker lanthanum nitrate in paracellular spaces. Moreover transmission electron microscopy and freeze fracture electron microscopy indicate that PCC produces significant structural alterations to tight junctions. In contrast to many other tight junction disrupting agents, PCC effects appear to be Ca(++)-independent and PCC does not induce significant disruption of actin filament distribution in Caco-2 cells.

Simultaneous in vitro measurement of intestinal tissue permeability and transepithelial electrical resistance (TEER) using Sweetana-Grass diffusion cells.

A simple modification of the commercially available Sweetana-Grass (S-G) side-by-side diffusion cells, allowing the simultaneous measurement of tissue permeability and transepithelial electrical resistance (TEER), has been described and validated for rat excised, muscle-free intestinal tissue. The TEER-lowering effects of a series of acylcarnitines were shown to be correlated with previously reported in vitro (i.e., membrane perturbation) and in vivo (i.e., absorption enhancement) activity. The TEER-lowering effect of palmitoyl carnitine chloride (PCC) was also shown to be reversible. The effects of PCC on TEER and the permeability of poorly absorbed compounds (cefoxitin and lucifer yellow) were simultaneously determined. Compared to controls (mannitol-treated), PCC immediately produced a rapid drop in colon TEER. By 5 min post-PCC addition, colon TEER was 50% of control; by 10 min post-PCC addition, colon TEER was 17% of control. After a lag of about 5-10 min post-PCC addition, the cefoxitin or lucifer yellow permeability coefficient increased more than 20-fold. The modified S-G cells provide a simple and reproducible method whereby flux and TEER can be simultaneously determined, providing a valuable link between the effect of absorption enhancers on TEER measurements and the increased permeability of poorly absorbed compounds.

Endocytosis and dissociation of class I MHC molecules labeled with fluorescent beta-2 microglobulin.

Membrane class I MHC molecules of Con-A activated and lymphoma murine cells have been labeled by exchange of the cell's beta 2m with soluble fl-beta 2m. It has previously been shown that this method of labeling is specific and does not affect the biologic properties of class I MHC Ag. With this labeling it has been possible to demonstrate the constitutive endocytosis of class I MHC by fluorescence microscopy and by measuring the resistance to quenching by crystal violet of the internalized fl-beta 2m molecules. We could also follow the kinetics of beta 2m dissociation from the class I molecules at different pH. At pH 5.5, that is the average pH of endosomes, there is considerable dissociation within 15 to 20 min, that is the average recycling half time of class I MHC containing endosomes in activated T cells. Inasmuch as the process is reversible it is likely that, in the recycling endosomes of T cells, class I MHC molecules undergo conformational changes with beta 2m going off and on and with consequent changes of the peptide binding site. This process might be involved in Ag presentation, but, because it is apparently limited to T cells, it would play a role in the presentation of the cell's own TCR in idiotypic interactions between T cells.

Specific associations of fluorescent beta-2-microglobulin with cell surfaces. The affinity of different H-2 and HLA antigens for beta-2-microglobulin.

We prepared single-labeled FITC derivatives of beta-2-microglobulin (b2m) and examined their interactions with class I MHC Ag H chains on living cells. Human b2m was reacted with FITC under mild conditions and separated by hydroxylapatite chromatography into three peaks containing single labeled derivatives of b2m peaks A, B, and C, and a peak containing the unmodified protein. The three fluorescent derivatives labeled the surfaces of cells bearing class I MHC Ag. The labeling was specific for class I MHC Ag as indicated by failure to label cells in the presence of excess unlabeled b2m and failure to label the HLA-negative cell lines Daudi and 721.221. Mouse cells labeled with fluorescent human b2m were recognized by mAb to the class I MHC Ag and by virus-restricted cytotoxic T lymphocytes, suggesting that labeling with the fluorescent b2m does not significantly alter the structure of class I MHC Ag or impair their ability to present viral antigens to cytotoxic T lymphocytes. We determined the kinetic and equilibrium binding parameters for the fluorescent b2m derivatives associating with the class I H chains of mouse and human cells. Peaks B and C exhibited biphasic binding to the mouse lymphoma cells EL-4(G-CSA-) (Kd1 = 1 x 10(-9) M; K2 = 1.5 to 3.0 x 10(-8) M whereas peak A bound to a small number of low affinity binding sites. In contrast to the biphasic binding observed with EL-4(G-CSA-), only monophasic binding was observed for peak C binding to RDM4 cells. Biphasic binding was also observed with the human B cell line LCL 721. Analysis of a series of LCL 721 class I MHC loss mutants and gene transferents revealed that the heterogeneity in binding is due to differences in the affinity of different class I encoded H chains for b2m.

Lateral mobility of cytochrome c on intact mitochondrial membranes as determined by fluorescence redistribution after photobleaching.

Lateral mobility of an active fluorescent derivative of cytochrome c on the membranes of giant mitochondria was measured by fluorescence redistribution after photobleaching. A diffusion coefficient of 1.6 X 10(-10) cm2/sec was determined for the labeled cytochrome c on inner mitochondrial membranes under conditions where succinate oxidase activity was demonstrated. This relatively low rate of diffusion, together with results of other investigators, is explained in terms of a model involving a dynamic equilibrium between freely diffusing and associated forms of electron-transfer components.

An effective electron donor to cytochrome oxidase. Purification, identification, and kinetic characterization of a contaminant of ruthenium red, hexaamineruthenium II/III.

When the calcium-transport inhibitor, ruthenium red, is chromatographed on a cation exchange resin, it yields a number of colored fractions and a colorless component that absorbs in the ultraviolet. The electron transfer activity previously ascribed to ruthenium red (Schwerzmann, K., Gazzotti, P., and Carafoli, E. (1976) Biochem. Biophys. Res. Commun. 69, 812) fractionates exclusively with the UV-absorbing material. On the basis of spectral, physical, and activity studies, we have identified this compound as Ru(NH3)62+/3+. It is shown that Ru(NH3)62+/3+ is an efficient electron donor directly to cytochrome oxidase, without mediation by cytochrome c. The steady state kinetics of electron transfer from Ru(NH3)62+ to purified oxidase resembles that of cytochrome c, showing a biphasic pattern but higher apparent Km values (Km1 = 8 microM, Km2 = 88 microM). Under conditions that favor tight binding to the oxidase, cytochrome c acts as a competitive inhibitor of Ru(NH3)62+, indicating that the two electron donors interact with cytochrome oxidase at the same site(s). The efficiency of Ru(NH3)62+ as an electron mediator to cytochrome aa3 and the similarity of its kinetic behavior to that of cytochrome c, make it a potentially valuable tool for investigating the mechanism of energy conservation in the terminal segment of the mitochondrial respiratory chain.