BRCA1 degradation in response to mitochondrial damage in breast cancer cells.

BRCA1 is a well-studied tumor suppressor involved in the homologous repair of DNA damage, whereas PINK1, a mitochondrial serine/threonine kinase, is known to be involved in mitochondrial quality control. Genetic mutations of PINK1 and Parkin cause autosomal recessive early-onset Parkinson's disease. We found that in breast cancer cells, the mitochondrial targeting reagents, which all induce mitochondrial depolarization along with PINK1 upregulation, induced proteasomal BRCA1 degradation. This BRCA1 degradation was dependent on PINK1, and BRCA1 downregulation upon mitochondrial damage caused DNA double-strand breaks. BRCA1 degradation was mediated through the direct interaction with the E3 ligase Parkin. Strikingly, BRCA1 and PINK1/Parkin expression were inversely correlated in cancerous mammary glands from breast cancer patients. BRCA1 knockdown repressed cancer cell growth, and high BRCA1 expression predicted poor relapse-free survival in breast cancer patients. These observations indicate a novel mechanism by which mitochondrial damage is transmitted to the nucleus, leading to BRCA1 degradation.

Inhibition of multi-drug resistant Klebsiella pneumoniae: Nanoparticles induced photoinactivation in presence of efflux pump inhibitor.

In the current scenario, frontline antibiotics are losing effectiveness against multidrug-resistant (MDR) bacteria because of the single mode of action. The accumulation of mutations and spread of antibiotic resistance markers among the bacteria results into the severe threat to community health. Now, there is an urgent requirement for the development of an alternate and as well as multiple-targeted action of drugs to stop the spread of resistance in bacteria. Here, we showed an alternative nanoparticle based photodynamic therapy (PDT) targeting the bacterial efflux pumps and its cell wall. The dextran capped gold nanoparticles (GNPDEX) were localized to the bacterial surface by nanoparticle attached Concanavalin-A (ConA), where GNPDEX attached methylene blue (MB) photosensitizer as an MB@GNPDEX-ConA formulation induced the killing of MDR Klebsiella pneumoniae clinical isolates in no time. The intervention of efflux pump inhibitor (EPI) further improved the MB@GNPDEX-ConA treatment modality and displayed the maximum bactericidal cytoplasmic phototoxicity. The CCCP EPI (carbonyl cyanide m-chlorophenylhydrazone) with the PDT increased the bacterial killing by>3 log10 as compared with or without EPI intervention. Further, the fractionated (two light treatment after long dark phase) PDT treatment modality decreased the bacterial biofilm growth up to ~90%. The microscopic as well as ROS fluorescent probes showed the singlet oxygen mediated cytotoxicity. The mode of interactions and genomic DNA photo-toxicity confirmed that EPI enhanced the killing mediated by singlet oxygen generation. The multi-targeted (Cell wall, DNA and efflux pump) modality of MB@GNPDEX-ConA in presence of EPI is an effective and alternative therapeutic approach against most potent Klebsiella MDR infections.

The Ionophores CCCP and Gramicidin but Not Nigericin Inhibit Trypanosoma brucei Aquaglyceroporins at Neutral pH.

Human African trypanosomiasis (HAT) is caused by Trypanosoma brucei parasites. The T. brucei aquaglyceroporin isoform 2, TbAQP2, has been linked to the uptake of pentamidine. Negative membrane potentials and transmembrane pH gradients were suggested to promote transport of the dicationic antitrypanosomal drug. Application of ionophores to trypanosomes further hinted at direct inhibition of TbAQP2 by carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Here, we tested for direct effects of three classical ionophores (CCCP, nigericin, gramicidin) on the functionality of TbAQP2 and the related TbAQP3 at conditions that are independent from the membrane potential or a proton gradient. We expressed TbAQP2 and TbAQP3 in yeast, and determined permeability of uncharged glycerol at neutral pH using stopped-flow light scattering. The mobile proton carrier CCCP directly inhibited TbAQP2 glycerol permeability at an IC50 of 2 µM, and TbAQP3 to a much lesser extent (IC50 around 1 mM) likely due to different selectivity filter layouts. Nigericin, another mobile carrier, left both isoforms unaffected. The membrane-integral pore-forming gramicidin evenly inhibited TbAQP2 and TbAQP2 in the double-digit micromolar range. Our data exemplify the need for suitable controls to detect unwanted ionophore side effects even when used at concentrations that are typically recommended to disturb the transmembrane ion distribution.

Cyanine dye mediated mitochondrial targeting enhances the anti-cancer activity of small-molecule cargoes.

Organelle-specific delivery systems are of significant clinical interest. We demonstrate the use of common cyanine dyes Cy3 and Cy5 as vectors for targeting and delivering cargoes to mitochondria in cancer cells. Specifically, conjugation to the dyes can increase cytotoxicity by up to 1000-fold.

Current mechanistic insights into the CCCP-induced cell survival response.

The ring-substituted derivatives of carbonyl cyanide phenylhydrazone, CCCP and FCCP, are routinely used for the analysis of the mitochondrial function in living cells, tissues, and isolated mitochondrial preparations. CCCP and FCCP are now being increasingly used for investigating the mechanisms of autophagy by inducing mitochondrial degradation through the disruption of the mitochondrial membrane potential (ΔΨm). Sustained perturbation of ΔΨm, which is normally tightly controlled to ensure cell proliferation and survival, triggers various stress pathways as part of the cellular adaptive response, the main components of which are mitophagy and autophagy. We here review current mechanistic insights into the induction of mitophagy and autophagy by CCCP and FCCP. In particular, we analyze the cellular modifications produced by the activation of two major pathways involving the signaling of the nuclear factor erythroid 2-related factor 2 (Nrf2) and the transcription factor EB (TFEB), and discuss the contribution of these pathways to the integrated cellular stress response.

An inhibitory role of Arg-84 in anion channelrhodopsin-2 expressed in Escherichia coli.

Anion channelrhodopsin-2 (ACR2) was recently identified from the cryptophyte algae Guillardia theta and has become a focus of interest in part because of its novel light-gated anion channel activity and its extremely high neural silencing activity. In this study, we tried to express ACR2 in Escherichia coli cells as a recombinant protein. The E. coli cells expressing ACR2 showed an increase in pH upon blue-light illumination in the presence of monovalent anions and the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), indicating an inward anion channel activity. Then, taking advantage of the E. coli expression system, we performed alanine-scanning mutagenesis on conserved basic amino acid residues. One of them, R84A, showed strong signals compared with the wild-type, indicating an inhibitory role of R84 on Cl- transportation. The signal was strongly enhanced in R84E, whereas R84K was less effective than the wild-type (i.e., R84). These results suggest that the positive charge at position 84 is critical for the inhibition. Thus we succeeded in functional expression of ACR2 in E. coli and found the inhibitory role of R84 during the anion transportation.

Mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone induces vasorelaxation without involving KATP channel activation in smooth muscle cells of arteries.

BACKGROUND AND PURPOSE: The effects and mechanisms of chemical mitochondrial uncouplers on vascular function have never been identified. Here, we characterized the effects of the typical mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) on vascular function in rat mesenteric arteries and aorta and elucidated the potential mechanisms. EXPERIMENTAL APPROACH: Isometric tension of mesenteric artery and thoracic aorta was recorded by using a multiwire myograph system. Protein levels were measured by western blot analyses. Cytosolic [Ca2+ ]i , mitochondrial ROS (mitoROS) and mitochondrial membrane potential of smooth muscle cells (A10) were measured by laser scanning confocal microscopy. KEY RESULTS: Acute treatment with CCCP relaxed phenylephrine (PE)- and high K+ (KPSS)-induced constriction of rat mesenteric arteries with intact and denuded endothelium. Pretreatment with CCCP prevented PE- and KPSS-induced constriction of rat mesenteric arteries with intact and denuded endothelium. Similarly, CCCP prevented PE- and KPSS-induced constriction of rat thoracic aorta. CCCP increased the cellular ADP/ATP ratio in vascular smooth muscle cells (A10) and activated AMPK in A10 cells and rat thoracic aorta tissues. CCCP-induced aorta relaxation was attenuated in AMPK α1 knockout (-/-) mice. SERCA inhibitors thapsigargin and cyclopiazonic acid (CPA) but not the KATP channel blocker glibenclamide partially inhibited CCCP-induced vasorelaxation in endothelium-denuded rat mesenteric arteries. CCCP increased cytosolic [Ca2+ ]i , mitoROS production and depolarized mitochondrial membrane potential in A10 cells. FCCP, the analogue of CCCP, had similar vasoactivity as CCCP in rat mesenteric arteries. CONCLUSIONS AND IMPLICATIONS: CCCP induces vasorelaxation by a mechanism that does not involve KATP channel activation in smooth muscle cells of arteries.

Convergence of Parkin, PINK1, and α-Synuclein on Stress-induced Mitochondrial Morphological Remodeling.

Mutations in PARKIN (PARK2), an ubiquitin ligase, cause early onset Parkinson disease. Parkin was shown to bind, ubiquitinate, and target depolarized mitochondria for destruction by autophagy. This process, mitophagy, is considered crucial for maintaining mitochondrial integrity and suppressing Parkinsonism. Here, we report that under moderate mitochondrial stress, parkin does not translocate to mitochondria to induce mitophagy; rather, it stimulates mitochondrial connectivity. Mitochondrial stress-induced fusion requires PINK1 (PARK6), mitofusins, and parkin ubiquitin ligase activity. Upon exposure to mitochondrial toxins, parkin binds α-synuclein (PARK1), and in conjunction with the ubiquitin-conjugating enzyme Ubc13, stimulates K63-linked ubiquitination. Importantly, α-synuclein inactivation phenocopies parkin overexpression and suppresses stress-induced mitochondria fission, whereas Ubc13 inactivation abrogates parkin-dependent mitochondrial fusion. The convergence of parkin, PINK1, and α-synuclein on mitochondrial dynamics uncovers a common function of these PARK genes in the mitochondrial stress response and provides a potential physiological basis for the prevalence of α-synuclein pathology in Parkinson disease.

Heart mitochondrial TTP synthesis and the compartmentalization of TMP.

The primary pathway of TTP synthesis in the heart requires thymidine salvage by mitochondrial thymidine kinase 2 (TK2). However, the compartmentalization of this pathway and the transport of thymidine nucleotides are not well understood. We investigated the metabolism of [(3)H]thymidine or [(3)H]TMP as precursors of [(3)H]TTP in isolated intact or broken mitochondria from the rat heart. The results demonstrated that [(3)H]thymidine was readily metabolized by the mitochondrial salvage enzymes to TTP in intact mitochondria. The equivalent addition of [(3)H]TMP produced far less [(3)H]TTP than the amount observed with [(3)H]thymidine as the precursor. Using zidovudine to inhibit TK2, the synthesis of [(3)H]TTP from [(3)H]TMP was effectively blocked, demonstrating that synthesis of [(3)H]TTP from [(3)H]TMP arose solely from the dephosphorysynthase pathway that includes deoxyuridine triphosphatelation of [(3)H]TMP to [(3)H]thymidine. To determine the role of the membrane in TMP metabolism, mitochondrial membranes were disrupted by freezing and thawing. In broken mitochondria, [(3)H]thymidine was readily converted to [(3)H]TMP, but further phosphorylation was prevented even though the energy charge was well maintained by addition of oligomycin A, phosphocreatine, and creatine phosphokinase. The failure to synthesize TTP in broken mitochondria was not related to a loss of membrane potential or inhibition of the electron transport chain, as confirmed by addition of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone and potassium cyanide, respectively, in intact mitochondria. In summary, these data, taken together, suggest that the thymidine salvage pathway is compartmentalized so that TMP kinase prefers TMP synthesized by TK2 over medium TMP and that this is disrupted in broken mitochondria.

Transmembrane potential of red blood cells under low ionic strength conditions.

BACKGROUND/AIMS: In a variety of investigations described in the literature it was not clear to what extent the transmembrane potential red blood cells (RBCs) was changed after the cells have been transferred into low ionic strength (LIS) solutions. Another open question was to find out how fast the transmembrane potential of RBCs in LIS solution will change and which final new equilibrium value will be reached. METHODS: The transmembrane potential of human and bovine RBCs was investigated using the potential-sensitive fluorescent dye DIBAC4(3) (bis(1,3-dibutylbarbituric acid) trimethine oxonol) as well as the CCCP (carbonylcyanide-m-chlorophenylhydrazone) method. RESULTS: Under physiological conditions the transmembrane potential was about -10 mV in agreement with literature data. However, when the RBCs were transferred into an isosmotic low ionic strength medium containing sucrose the transmembrane potential increased to +73 mV and +81 mV for human and bovine RBCs, respectively. In case of human RBCs it continuously decreased reaching finally an equilibrium state of -10 mV again after 30 - 60 min. For bovine RBCs the transmembrane potential declined more slowly reaching a value of +72 mV after 30 min. CONCLUSIONS: Investigations of parameters of RBCs depending on transmembrane potential cannot be performed with human RBCs in LIS media.

Simulations of substrate transport in the multidrug transporter EmrD.

EmrD is a multidrug resistance (MDR) transporter from Escherichia coli, which is involved in the efflux of amphipathic compounds from the cytoplasm, and the first MDR member of the major facilitator superfamily to be crystallized. Molecular dynamics simulation of EmrD in a phospholipid bilayer was used to characterize the conformational dynamics of the protein. Motions that support a previously proposed lateral diffusion pathway for substrate from the cytoplasmic membrane leaflet into the EmrD central cavity were observed. In addition, the translocation pathway of meta-chloro carbonylcyanide phenylhydrazone (CCCP) was probed using both standard and steered molecular dynamics simulation. In particular, interactions of a few specific residues with CCCP have been identified. Finally, a large motion of two residues, Val 45 and Leu 233, was observed with the passage of CCCP into the periplasmic space, placing a lower bound on the extent of opening required at this end of the protein for substrate transport. Overall, our simulations probe details of the transport pathway, motions of EmrD at an atomic level of detail, and offer new insights into the functioning of MDR transporters.

Interaction of carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) with lipid membrane systems: a biophysical approach with relevance to mitochondrial uncoupling.

FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone), a classical uncoupler of mitochondrial oxidative phosphorylation, is used in this study as a model to clarify how interactions of uncouplers with membrane lipid bilayers may influence membrane biophysics and their protonophoric activity itself. In order to disclose putative effects that may be important when considering using uncouplers for pharmacological purposes, an extensive characterization of FCCP membrane lipid interactions using accurate biophysical approaches and simple model lipid systems was carried out. Differential scanning calorimetry studies showed that FCCP molecules disturb lipid bilayers and favor lateral phase separation in mixed lipid systems. (31)P NMR assays indicated that FCCP alters the curvature elastic properties of membrane models containing non-bilayer lipids, favoring lamellar/H(II) transition, probably by alleviation of hydrocarbon-packing constraints in the inverted hexagonal phase. Taking advantage of FCCP quenching effects on the fluorescent probes DPH (1,6-diphenyl-1,3,5-hexatriene) and DPH-PA (3-(p-(6-phenyl)-1,3,5-hexatrienyl)phenylpropionic acid), it is demonstrated that FCCP distributes across the bilayer thickness in both a single and a ternary lipid system mimicking the inner mitochondrial membrane. This behavior is consistent with the ability of the compound to migrate through the thickness of the inner mitochondrial membrane, an event required for its protonophoric activity. Finally, the study of the membrane fluidity in different lipid systems, as reported by the rotational correlation time (θ) of DPH or DPH-PA, showed that the extension at which FCCP disturbs membrane properties associated with the dynamics and the order of lipid molecules depends on the lipid composition of the model lipid system assayed.

64Cu-labeled lissamine rhodamine B: a promising PET radiotracer targeting tumor mitochondria.

Enhanced mitochondrial potential in carcinoma cells is an important characteristic of cancer. It is of great current interest to develop a radiotracer that is sensitive to mitochondrial potential changes at the early stage of tumor growth. In this report, we present the synthesis and evaluation of (64)Cu-labeled Lissamine rhodamine B (LRB), (64)Cu(DOTA-LRB) (DOTA-LRB = 2-(6-(diethylamino)-3-(diethyliminio)-3H-xanthen-9-yl)-5-(N-(2-(2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclo-dodecan-1-yl)acetamido)ethyl)sulfamoyl)benzenesulfonate) as a new radiotracer for imaging tumors in athymic nude mice bearing U87MG human glioma xenografts by positron emission tomography (PET). We also explored its localization mechanism using Cu(DOTA-LRB) as the fluorescent probe in both the U87MG human glioma cell line and the cultured primary U87MG glioma cells. It was found that (64)Cu(DOTA-LRB) had the highest tumor uptake (6.54 ± 1.50, 6.91 ± 1.26, 5.68 ± 1.13, 7.58 ± 1.96, and 5.14 ± 1.50%ID/g at 0.5, 1, 2, 4, and 24 h postinjection, respectively) among many (64)Cu-labeled organic cations evaluated in the same animal model. The cellular staining study indicated that Cu(DOTA-LRB) was able to localize in mitochondria of U87MG glioma cells due to the enhanced negative mitochondrial potential. This statement is completely supported by the results from decoupling experiment with carbonylcyanide-m-chlorophenylhydrazone (CCCP). MicroPET data showed that the U87MG glioma tumors were clearly visualized as early as 30 min postinjection with (64)Cu(DOTA-LRB). (64)Cu(DOTA-LRB) remained stable during renal excretion, but underwent extensive degradation during hepatobiliary excretion. On the basis of the results from this study, it was concluded that (64)Cu(DOTA-LRB) represents a new class of promising PET radiotracers for noninvasive imaging of the MDR-negative tumors.

Characterization of the ligand and DNA binding properties of a putative archaeal regulator ST1710.

While a rich collection of bacterium-like regulating proteins has been identified in the archaeal genome, few of them have been studied at the molecular level. In this study, we characterized the ligand and DNA binding properties of a putative regulator ST1710 from the archaeon Sulfolobus tokodaii. ST1710 is homologous to the multiple-antibiotic resistance repressor (MarR) family bacterial regulators. The protein consists of a ligand binding site, partially overlapping with a winged helix-turn-helix DNA binding site. We characterized the interactions between ST1710 and three ligands, salicylate, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and ethidium, which bind to bacterial MarRs. The binding affinities of the ligands for ST1710 were comparable to their affinities for the bacterial MarRs. The ligand binding was temperature sensitive and caused conformational changes in ST1710. To investigate the effect of ligand binding on the interaction between ST1710 and DNA, we fluorescently labeled a 47mer dsDNA (ST1) containing a putative ST1710 recognition site and determined the dissociation constant between ST1 and ST1710 using the fluorescence polarization method. The binding affinity almost doubled from 10 degrees C (Kd = 618 +/- 34 nM) to 30 degreesC (Kd = 334 +/- 15 nM), and again from 30 to 50 degrees C (Kd = 189 +/- 9 nM). This result suggests that under the natural living condition (80 degrees C) of S. tokodaii, the binding affinity might increase even further. The presence of CCCP and salicylate suppressed ST1710-ST1 interaction, indicating that ST1710 functioned as a repressor.

Second harmonic studies of ions crossing liposome membranes in real time.

The transport kinetics of the positively charged triphenylmethane dye, malachite green (MG(+)), across liposome bilayers effects the transport of monovalent inorganic cations when ionophores are present in the membrane. Three different types of ionophores characterized by different transport mechanisms have been studied. The ionophores are gramicidin A (gA) (a channel former), valinomycin (VAL) (a lipophilic cyclopeptide that encloses an alkali ion), and carbonyl cyanide-m-chlorophenylhydrazone (CCCP) (a weak acid that functions as a protonophore). The effects of these ionophores on the kinetics and extent of MG(+) crossing into the liposome, investigated using the interface selective second harmonic generation method, were found to be markedly different.

Quantification of Mg2+ extrusion and cytosolic Mg2+-buffering in Xenopus oocytes.

Intracellular Mg(2+) buffering and Mg(2+) extrusion were investigated in Xenopus laevis oocytes. Mg(2+) or EDTA were pressure injected and the resulting changes in the intracellular Mg(2+) concentration were measured simultaneously with Mg(2+)-selective microelectrodes. In the presence of extracellular Na(+), injected Mg(2+) was extruded from the oocytes with an estimated v(max) and K(M) of 74 pmol cm(-2)s(-1) and 1.28 mM, respectively. To investigate genuine cytosolic Mg(2+) buffering, measurements were carried out in the nominal absence of extracellular Na(+) to block Mg(2+) extrusion, and during the application of CCCP (inhibiting mitochondrial uptake). Under these conditions, Mg(2+) buffering calculated after both MgCl(2) and EDTA injections could be described by a buffer equivalent with a concentration of 9.8mM and an apparent dissociation constant, K(d-app), of 0.6mM together with an [ATP](i) of 0.9 mM with a K(d-app) 0.12 mM. Xenopus oocytes thus possess highly efficient mechanisms to maintain their intracellular Mg(2+) concentration.

Membrane photopotential generation by interfacial differences in the turnover of a photodynamic reaction.

The adsorption of a membrane-impermeable photosensitizer to only one membrane leaflet is found to trigger a localized photodynamic reaction; i.e., the amount of carbonyl cyanide m-chlorophenylhydrazone (CCCP) molecules damaged in the leaflet facing the photosensitizer is roughly identical to the total amount of CCCP inactivated. Whereas the latter quantity is assessed from the drop in membrane conductivity G, the former is evaluated from the photopotential phi that is proportional to the interfacial concentration difference of the uncoupler. Localized photodestruction is encountered by CCCP diffusion to the site of photodamage. A simple model that accounts for both photoinhibition and diffusion predicts the dependence of the photopotential on light intensity, buffer capacity, and pH of the medium. It is concluded that only a limited amount of the reactive oxygen species responsible for CCCP photodamage diffuses across the membrane. If the concentration of reactive oxygen species is decreased by addition of NaN(3) or by substituting aqueous oxygen for argon, phi is inhibited. If, in contrast, their life time is increased by substitution of H(2)O for D(2)O, phi increases.

Modulation of flash-induced photosystem II fluorescence by events occurring at the water oxidizing complex.

The mechanism of flash-induced changes with a periodicity of four in photosystem II (PSII) fluorescence was investigated with the aim of further using fluorescence measurements as an approach to studying the structural and functional organization of the water-oxidizing complex (WOC). The decay of the flash-induced high fluorescence state of PSII was measured with pulse amplitude modulated fluorometry in thylakoids and PSII enriched membrane fragments. Calculated QA- decay was well described by three exponential decay components, reflecting QA- reoxidation with halftimes of 450 and 860 micros, 2 and 7.6 ms, and 111 and 135 ms in thylakoids and PSII membranes, respectively. The effect of modification of the PSII donor side by changing pH or by removal of the extrinsic 17 and 24 kDa proteins on period four oscillations in both maximum fluorescence yield and the relative contribution of QA- reoxidation reactions was compared to flash-induced oxygen yield. The four-step oxidation of the manganese cluster of the WOC was found to be necessary but not sufficient to produce modulation of PSII fluorescence. The capacity of the WOC to generate molecular oxygen was also required to observe a period four in the fluorescence; however, direct quenching by oxygen was not responsible for the modulation. Potential mechanisms responsible for the periodicity of four in both maximum fluorescence yield pattern and flash-dependent changes in proportion of centers with different QA- reoxidation rates are discussed with respect to intrinsic deprotonation events occurring at the WOC.

Carbonyl cyanide phenylhydrazones as probes of the anionic activator site of the human erythrocyte glutathione adduct transport ATPase.

We have previously shown that the ATPase activity associated with the erythrocyte glutathione adduct transporter is also stimulated by 2,4-dinitrophenol and p-trifluoromethoxy carbonylcyanide phenylhydrazone, both well-known anionic and lipophilic uncouplers of oxidative phosphorylation by mitochondria [C. G. Winter, D. C. DeLuca, and H. Szumilo (1994) Arch. Biochem. Biophys. 314, 17-22]. In this paper, we report the testing of a series of ring-substituted carbonylcyanide phenylhydrazones as activators of the ATPase. All of the compounds tested stimulated the ATPase to similar extents, based on Vmax values. The K0.5 for stimulation of the ATPase depended on the electron-withdrawing characteristics of the ring substituents, resulting in a Hammett linear free energy relationship for the m- and p-substituted derivatives. The slope of this relationship, with lower K0.5 values for electron-withdrawing substituents, suggests that an anionic residue in the active site partially discourages binding of this class of activators. ortho-Substituted carbonylcyanide phenylhydrazones do not follow this relationship, but show lower apparent affinities than expected from their pKa values. This finding suggests that steric effects in that region of the binding site negatively influence the affinity.

A distinctive effect of CCCP on the transfer of erythromycin to 1-octanol: a possible model in promoting the intracellular antibiotic-accumulation through lipid in a staphylococcal cytoplasmic membrane.

Movement of erythromycin (EM), a basic antibiotic solute (pKa 8.6), in an aqueous buffer (pH7.6) at 37 degrees C into 1-octanol was found approximately twofold greater in the presence of another acidic and lipophilic solute, carbonylcyanide m-chlorophenylhydrazone (CCCP), when compared with the EM-movement in the absence of CCCP. CCCP appears to behave just like a carrier of EM, besides being a well-known inhibitor, such as the uncoupling agent of oxidative phosphorylation. The probability is discussed that such an analogous event takes place in the lipid layers of a bacterial-cell membrane.