Carbonyl Cyanide 4-trifluoromethoxyphenylhydrazone Research Articles

Amiloride inhibits hydrogen peroxide-induced Ca 2+ responses in human CNS pericytes

The aims of the present study were to investigate the mechanisms of Ca 2+ signaling caused by hydrogen peroxide in CNS pericytes. In cultured human brain microvascular pericytes, cytosolic Ca 2+ concentration was measured by means of fura-2 fluorescence. Reverse transcription and polymerase chain reaction was performed to examine the expression of mRNA. Knockdown of Na +/H + exchanger (NHE) was done by transfecting the cells with specific double-strand siRNAs for NHE. Externally applied hydrogen peroxide dose-dependently (100 μM–10 mM) increased cytosolic Ca 2+ in human CNS pericytes. Cytosolic Ca 2+ remained high after wash-out of hydrogen peroxide. However, the addition of dithiothreitol rapidly reversed cytosolic Ca 2+ to the resting level. The hydrogen peroxide-induced Ca 2+ increase was not inhibited by nicardipine, Gd 3+, La 3+, or omission of external Ca 2+. Neither thapsigargin nor carbonyl cyanide 4-trifluoromethoxyphenylhydrazone attenuated the hydrogen peroxide-induced Ca 2+ rise. Amiloride and its derivatives, benzamil and hexamethylene amiloride reversed the hydrogen peroxide-induced Ca 2+ increase. Human CNS pericytes expressed acid sensing ion channel (ASIC) 1a, Na +/Ca 2+ exchanger (NCX) 1, Na +/H + exchanger (NHE) 1, and NHE7. However, the removal of external Na +, treatment with KB-R 7943 and mibefradil, or knockdown of NHE1 and NHE7 did not affect the hydrogen peroxide-induced Ca 2+ increase. Hydrogen peroxide releases Ca 2+ from intracellular Ca 2+ pool via an amiloride-sensitive protein, which is controlled by oxidation of thiol group in human CNS pericytes.

Passive potassium transport by symbiosomes from broad bean root nodules

Passive potassium release from symbiosomes isolated from broad bean root nodules and placed into K +-free assay medium was investigated by following acidification of the symbiosome interior and tetraphenylphosphonium (TPP +)-induced change in turbidity of symbiosome suspension observed under the chosen experimental conditions. The kinetics of acid pH shift inside the symbiosomes was substantially accelerated by the K +-specific ionophore valinomycin, the protonophore carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP), but rapidly reversed by centimolar concentrations of K + as well as by TPP + added to the assay medium. In addition, the acidification process was shown to be markedly inhibited by tetraethylammonium (TEA), the known blocker of K +-channels in cell membranes, and to be strongly suppressed by removal of free calcium ions from the assay medium with the Ca 2+ chelator EGTA. TPP + was found to rapidly trigger the increase in turbidity of symbiosome suspension in the same assay medium. The kinetics of the TPP +-triggered process appeared to be markedly inhibited by TEA as well. Taken together, these results indicate relatively high potassium permeability of the peribacteroid membrane (PBM) of broad bean root nodules and provide evidence that the responses tested are due to K + release from the symbiosomes through TEA-sensitive K +-permeable channel in the PBM down transmembrane electrochemical gradient of K + ions. The data obtained suggest that such a channel is voltage-gated, and Ca 2+ ions on the cytosolic side of the PBM are required for functioning of this transporter.

A novel signalling pathway originating in mitochondria modulates rat skeletal muscle membrane excitability.

Single skeletal muscle fibres from rat and cane toad were mechanically skinned and stimulated either electrically by initiating action potentials in the sealed transverse (t-) tubular system or by ion substitution causing depolarisation of the t-system to pre-determined levels. Depression of mitochondrial ATP-producing function with three diverse mitochondrial function antagonists (azide: 1-10 mM; oligomycin 1 microg ml-1 and carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP) 1 microM), under conditions in which the cytosolic ATP was maintained high and constant, invariably reduced the excitability of rat fibres but had no obvious effect on the excitability of toad fibres, where mitochondria are less abundant and differently located. The reduction in excitability linked to mitochondria in rat fibres appears to be caused by depolarisation of the sealed t-system membrane. These observations suggest that mitochondria can regulate the functional state of mammalian muscle cells and have important implications for understanding how the balance between ATP utilisation and ATP production is regulated at the cellular level in general and in mammalian skeletal muscle fibres in particular.

Plant electrophysiology: FCCP induces action potentials and excitation waves in soybean

Carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP) induces ultrafast action potentials and decreases the variation potential in a soybean. The speed of propagation of action potentials in a soybean induced by FCCP reaches up to 40 m/s. The duration of a single action potential after treatment by FCCP is 0.3 ms. Adding FCCP to soil reduces the variation potential from 80 mV to zero. The spatial distribution of protons around the soybean root was measured using pH indicators. All experiments showed that proton concentration around the plant surface was higher than in the rest of the bulk solution. When the proton distribution was measured with a Universal Indicator, three zones were observed around the plant in FCCP and control culturing solutions: orange, yellow, and green, which correspond to pH values of 5, 6, and 7, respectively. The color intensity of the orange zone was slightly lighter around FCCP-treated plants than around control plants. Two zones were observed around the soybean with Bromocresol Purple: yellow and purple, which correspond to pH values of 5 and 7, respectively. FCCP does not influence turgor pressure, H + efflux, or K +/H + antiporter in the root of a soybean; however, it was found to reduce the rate of plant maturation. The mechanism by which FCCP decreases plant maturation most likely includes depolarization of the plasma membrane, retardation of photosynthetic water oxidation and respiratory electron transfer.

Response Kinetics of Tethered Rhodobacter sphaeroides to Changes in Light Intensity

Rhodobacter sphaeroides can swim toward a wide range of attractants (a process known as taxis), propelled by a single rotating flagellum. The reversals of motor direction that cause tumbles in Eschericia coli taxis are replaced by brief motor stops, and taxis is controlled by a complex sensory system with multiple homologues of the E. coli sensory proteins. We tethered photosynthetically grown cells of R. sphaeroides by their flagella and measured the response of the flagellar motor to changes in light intensity. The unstimulated bias (probability of not being stopped) was significantly larger than the bias of tethered E. coli but similar to the probability of not tumbling in swimming E. coli. Otherwise, the step and impulse responses were the same as those of tethered E. coli to chemical attractants. This indicates that the single motor and multiple sensory signaling pathways in R. sphaeroides generate the same swimming response as several motors and a single pathway in E. coli, and that the response of the single motor is directly observable in the swimming pattern. Photo-responses were larger in the presence of cyanide or the uncoupler carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP), consistent with the photo-response being detected via changes in the rate of electron transport.

Barriers to Translocation of Organic Ions in Phospholipid Membranes

The hypothesis that a dominant barrier to the translocation of organic ions (such as carbonyl cyanide m-chlorophenylhydrazone (CCCP)) across phospholipid bilayer membranes is located between the “polar region” and “nonpolar region” of the membrane layer has been confirmed by the following observation: The rate of decay of the pH difference across soybean phospholipid vesicular membrane (ΔpH) increased substantially when the above substep was specifically catalyzed. In our strategy, the formation of the electroneutral ternary complex valinomycin−M+−CCCP- (M+ = K+, Cs+) affects this substep. Also, CCCP- ion translocation is facilitated by having an alternate path for the compensating charge flux of alkali metal ions through gramicidin channels. The observed enhancement behaviors on changing the lipid composition from soybean phospholipid (SBPL) to a mixture of PC + 6% PA, weak acid from CCCP to carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP), and the metal ion from K+ to Cs+ are consistent with the predictions of the suggested mechanisms. The increase in the ΔpH decay rate on increasing the ionic strength confirms the Coulombic nature of a dominant interaction between the CCCP- ion and the lipid contributing to the kinetic barrier. Even though the gross features of the ion translocation barrier profile inferred by us are similar to that given in the literature, our results suggest the need to consider ion translocation across the phospholipid membrane to involve at least three substeps. This requires a refinement of the barrier profile given in the literature. In our experiments, the ΔpH was created by temperature jump, and observations on the ΔpH decay were made by monitoring the fluorescence from the pyranine entrapped inside the vesicles.

Photoresponses in Rhodobacter sphaeroides: role of photosynthetic electron transport

Rhodobacter sphaeroides responds to a decrease in light intensity by a transient stop followed by adaptation. There is no measurable response to increases in light intensity. We confirmed that photosynthetic electron transport is essential for a photoresponse, as (i) inhibitors of photosynthetic electron transport inhibit photoresponses, (ii) electron transport to oxidases in the presence of oxygen reduces the photoresponse, and (iii) the magnitude of the response is dependent on the photopigment content of the cells. The photoresponses of cells grown in high light, which have lower concentrations of light-harvesting photopigment and reaction centers, saturated at much higher light intensities than the photoresponses of cells grown in low light, which have high concentrations of light-harvesting pigments and reaction centers. We examined whether the primary sensory signal from the photosynthetic electron transport chain was a change in the electrochemical proton gradient or a change in the rate of electron transport itself (probably reflecting redox sensing). R. sphaeroides showed no response to the addition of the proton ionophore carbonyl cyanide 4-trifluoromethoxyphenylhydrazone, which decreased the electrochemical proton gradient, although a behavioral response was seen to a reduction in light intensity that caused an equivalent reduction in proton gradient. These results strongly suggest that (i) the photosynthetic apparatus is the primary photoreceptor, (ii) the primary signal is generated by a change in the rate of electron transport, (iii) the change in the electrochemical proton gradient is not the primary photosensory signal, and (iv) stimuli affecting electron transport rates integrate via the electron transport chain.

Detoxification of xenobiotics by plant cells: characterisation of vacuolar amphiphilic organic anion transporters

The transport activities of two primary ATP-dependent organic-anion transporters in the tonoplast of isolated barley (Hordeum vulgare L. cv. Klaxon) vacuoles have been characterised with N-ethylmaleimide glutathione (NEM-SG) and taurocholate as substrates. The transporters showed different sensitivities to organic anions and a variety of transport inhibitors and drugs. The vacuolar uptake of NEM-SG was inhibited by carbonylcyanide 4-trifluoromethoxyphenylhydrazone, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), S-(2,4-dinitrophenyl)glutathione, alkyl-S-glutathione derivatives and taurocholate but stimulated by probenecid. The uptake of taurocholate was inhibited by vinblastine, DIDS and probenecid. Both transporters were unaffected by verapamil. The kinetic properties of the transporters indicate a general preference for amphiphilic anions with some substrate overlap. These characteristics of the transporters are similar to those displayed by the multidrug resistance protein of mammalian drug-resistant cells. We suggest that these vacuolar transporters be described as plant multispecific organic anion transporters (pMOATs).

Functional expression and characterization of the Trypanosoma brucei procyclic glucose transporter, THT2

The gene encoding THT2, one of two hexose-transporter isoforms present in Trypanosoma brucei, has been expressed in both Xenopus laevis oocytes and a stably transfected line of Chinese hamster ovary (CHO) cells. The heterologously expressed gene encodes a protein with pharmacological and kinetic parameters similar to those of the hexose transporter measured in procyclic-culture-form trypanosomes. The substrate recognition of the THT2 transporter differed from that of the THT1 isoform, which is expressed only in bloodstream forms, in that: (i) it has a relatively high affinity for substrate with a Km of 59 microM for 2-deoxy-D-glucose (2-DOG) and a similar high affinity for D-glucose (compared with Km of 0.5 mM for 2-DOG in bloodstream forms); (ii) the affinity for 6-deoxy-D-glucose (6-DOG) is two orders of magnitude lower than that for D-glucose, whereas the bloodstream-form transporter recognizes D-glucose and its 6-DOG analogue with similar affinity; (iii) the bloodstream-form transporter, but not THT2, recognizes 3-fluoro-3-deoxy-D-glucose. D-Fructose-transport capacity and insensitivity to D-galactose was also found in THT2-expressing CHO cells and procyclic trypanosomes. We conclude from these cumulative results that the THT2 gene encodes the transporter responsible for hexose transport in procyclic trypanosomes. The transport of 2-DOG in procyclic organisms was inhibited by both the protonophore, carbonyl cyanide 4-trifluoromethoxy phenylhydrazone (FCCP), and KCN, suggesting a requirement for a protonmotive force. However, sensitivity to these reagents depended on the external substrate concentration, with uptake being unaffected at substrate concentrations higher than 2 mM. THT2 expressed in CHO cells behaved as a facilitated transporter, and was unaffected by FCCP or KCN over the whole substrate concentration range tested.

Uptake of 5-Formyltetrahydrofolate in Isolated Rat Liver Mitochondria Is Carrier-Mediated ,

We have characterized the uptake of 5-formyltetrahydrofolate into isolated rat liver mitochondria. Uptake of 5-formyltetrahydrofolate, 5-methyltetrahydrofolate and folic acid was linear for the first 2 min of incubation. 5-Methyltetrahydrofolate and 5-formyltetrahydrofolate were accumulated in mitochondria to 3.7- and 4.3-fold, respectively. 5-Formyltetrahydrofolate transport showed a pH optimum at 5.5 and was saturable with an apparent Km = 2.80 μmol·L-1 and Vmax = 3.49 pmol·min-1·mg protein-1. Uptake of 5-formyltetrahydrofolate was inhibited by the structural analog 5-methyltetrahydrofolate and, to a much lesser extent, folic acid and methotrexate. Uptake was not inhibited by various substrates of known mitochondrial transporters, e.g., succinate, malate, citrate, glutamate and phosphate, nor was uptake inhibited by the sulfhydryl reagent, N-ethylmaleimide. Uptake was not stimulated by preloading of mitochondria with malate or aspartate. Uptake of 5-formyltetrahydrofolate was not affected by the proton ionophore carbonyl cyanide 4-trifluoromethoxyphenylhydrazone nor by collapsing the electrical gradient with valinomycin. These results indicate that 5-formyltetrahydrofolate uptake into mitochondria via a carrier-mediated system specific for the reduced, naturally occurring folates. The folates, once in the mitochondria, may be converted to the polyglutamylated forms, which will not readily diffuse out of the mitochondria.

Alcohols Protect Escherichia coli against Cold Shock

Alcohols protect Escherichia coli against cold shock, and the concentration of alcohol which provided optimal protection declined with increasing hydrophobicity of the alcohol. The rate of loss of viability after the chilling transition was decreased by n-octanol, even when it was added after that chilling transition. Cold-shocked cells exhibited a sensitivity toward dioxygen, seen as greater enumeration on anaerobic, rather than on aerobic, trypticase-yeast extract agar plates, and addition of catalase or antioxidants, such as alpha-tocopherol or probucol, to the agar plates did not lessen this dioxygen sensitivity. Respiratory capacity was diminished by cold shock, and cyanide-sensitive respiration was more affected than was cyanide-resistant respiration. Discharging the proton gradient, with the uncoupler carbonyl cyanide trifluoromethoxy-phenylhydrazone, did not change sensitivity to cold shock. There was no evidence for minimal medium recovery after cold shock. The data presented, as well as that already in the literature, are explained on the basis of membrane damage caused by patches of ordering transitions in one membrane leaflet, unmatched by comparable transitions in the mating leaflet.

Valinomycin: a very effective inhibitor of the cyanide-insensitive alternative pathway in plant mitochondria

The effect of valinomycin on the respiration of potato tuber callus mitochondria ( Solanum tuberosum L. cv. Bintje) was investigated. The rate of electron transfer via the cytochrome pathway can be stimulated by abolishing membrane potential with valinomycin. However, valinomycin was found to inhibit CN-resistant respiration in potato tuber callus mitochondria. The inhibition is independent of the substrate used (external NADH, succinate and malate). The inhibition was enhanced by increasing the K + concentration in the medium from 0 to 125 mM and was also observed when valinomycin was added in the presence of carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP). In the presence of 125 mM K + 1 μM valinomycin inhibited electron flow, mediated by the alternative pathway, for approx. 65% and 5 μM valinomycin for approx. 85%. In mitochondria prepared from sweet potato ( Ipomea batatas ) valinomycin also inhibited CN-resistant respiration.

Measurement of Na-K-ATPase-mediated rubidium influx in single segments of rat nephron

To determine the functioning rate of Na-K-ATPase in the rat nephron, a micromethod was developed to measure the rate of rubidium uptake in single nephron segments microdissected from collagenase-treated kidneys. Because the hydrolytic activity of Na-K-ATPase displayed the same apparent affinity for K and Rb ions, whereas the Vmax elicited by K was higher than that in the presence of Rb, experiments were performed in the presence of cold Rb plus 86Rb. Before the assay, tubules were preincubated for 10 min at 37 degrees C to restore the normal transmembrane cation gradients. 86Rb uptake was measured after washing out extracellular cations by rinsing the tubules in ice-cold choline chloride solution containing Ba2+. Rb uptake increased quasi-linearly as a function of incubation time up to 30 s in the thick ascending limb, 1 min in the proximal convoluted tubule, and 5 min in the collecting tubule, and reached an equilibrium after 5-30 min. The initial rates of Rb uptake increased in a saturable fashion as Rb concentration in the medium rose from 0.25 to 5 mM. In medullary thick ascending limb, the initial rate of Rb uptake was inhibited by greater than 90% by 2.5 mM ouabain and by 10(-5) M of the metabolic inhibitor carbonyl cyanide trifluoromethoxyphenylhydrazone. Correlation of Na-K-ATPase hydrolytic activity at Vmax and initial rates of ouabain-sensitive Rb uptake in the successive segments of nephron indicates that in intact cells the pump works at approximately 20-30% of its Vmax. Increasing intracellular Na concentration by tubule preincubation in a Rb- and K-free medium increased the initial rates of Rb intake up to the Vmax of the hydrolytic activity of the pump.

Combination of the Chorionic Gonadotropin Free β-Subunit with α*

The rate-limiting event for combination of hCG alpha- and beta-subunits in JAR choriocarcinoma cells is the rate of disulfide bond formation in the beta-subunit. This is accompanied by a conformational change that produces a combination-competent form of the beta-subunit. The combination reaction, however, is incomplete, and 50% of the synthesized beta molecules remain uncombined (free). In addition, 70% of biosynthetically labeled free beta is degraded in the cell. Possible explanations for incomplete dimer formation include 1) biochemical differences between the free and combined beta-subunits that limit combination of free beta, and 2) an inefficient combination reaction due to low intersubunit affinities or limiting concentrations of combination-competent subunits within the cell. To examine whether the biochemical differences between free and combined beta-subunits that we have previously observed affect the combined beta-subunits that we have previously observed affect the combination competence of free beta, free and dimer beta-subunits were purified from the culture medium and lysates of JAR cells and examined for their ability to combine with alpha purified from pregnancy urine in an in vitro combination assay. Secreted free and dimer beta obtained from culture medium combined to the same extent with urinary alpha. Although the combination efficiencies were lower for the intracellular forms, the free and dimer beta-subunits purified from cell lysates also combined to the same extent with urinary alpha. Thus, biochemical differences that exist between the beta forms do not prevent combination of free beta with alpha in an in vitro combination assay. To examine the second possibility, we speculated that if high concentrations of hCG subunits remained in the rough endoplasmic reticulum (ER) for extended periods of time, the extent of dimer formation would increase in the cell. To increase the residence time of hCG subunits in the ER, JAR cells were treated with carbonyl cyanide trifluoromethoxyphenylhydrazone, an agent that inhibits the translocation of hCG subunits from the ER to the Golgi. Treatment of cells with trifluoromethoxyphenylhydrazone in long and short term pulse-chase labeling studies did not result in an increase in the extent of dimer formation. Thus, the subunit combination reaction in JAR cells may be incomplete due to subtle conformational differences in the free beta-subunit; however, these differences do not inhibit the combination of the free beta-subunit in vitro.

Transport of guanidine in rabbit intestinal brush-border membrane vesicles.

The characteristics of guanidine uptake were studied in brush-border membrane vesicles isolated from the rabbit proximal intestine. Guanidine uptake was manyfold greater in the presence of an outward-directed H+ gradient (intracellular pH = 5.5; extracellular pH = 7.2) than in the absence of a H+ gradient (intracellular and extracellular pH = 7.2). The time course of guanidine uptake exhibited an overshoot phenomenon in the presence of the H+ gradient, indicating occurrence of uphill transport. This H+ gradient-stimulated guanidine uptake was not due to an inside-negative H+-diffusion potential because carbonyl cyanide 4-trifluoromethoxyphenylhydrazone, a protonophore, failed to have any effect on guanidine uptake. Moreover, the transient uphill transport of guanidine was observed even in voltage-clamped membrane vesicles. However, under the conditions that effectively dissipated the H+ gradient, there was no active transport of guanidine. This H+ gradient-dependent transport mechanism for guanidine is distinct from the Na+-H+ exchanger, because amiloride did not inhibit guanidine uptake even at a concentration as high as 100 microM. These data provide evidence for the presence of a guanidine-H+ antiport system in the rabbit intestinal brush-border membrane. The presence of a carrier for guanidine in these membranes is further substantiated by the trans-stimulation of the uptake of radiolabeled guanidine by unlabeled guanidine and by the inhibition of guanidine uptake by imipramine under equilibrium exchange conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of inhibitors and herbicides on the membrane potential of mung bean mitochondria

Alterations imposed by herbicides on the membrane potential (Δψ), oxygen utilization, and ATP synthesis of intact mung bean mitochondria were measured under state 3 conditions. Effects were correlated with changes imposed by classical electron transport inhibitors, energy transfer inhibitors, and uncouplers. In the dose-response studies, complete inhibition of ATP synthesis produced by electron transport inhibitors (rotenone, antimycin A, KCN), uncouplers [bis(hexafluoroacetonyl)acetone (1799) and carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP)], and the herbicides was associated with a decrease in Δψ from the state 3 value of 126 mV to between 90 and 100 mV. In contrast, the complete inhibition of phosphorylation produced by the energy transfer inhibitor N,N′-dicyclohexylcarbodimide correlated with an increase in Δψ from the state 3 to the state 4 potential (145 mV). In the titrations, the herbicides and classical uncouplers, but not the electron transport inhibitors, progressively collapsed Δψ below the potential associated with the complete inhibition of phosphorylation (to the apparent Donnan potential of 60 mV). The herbicides could be placed into two groups according to the dose-response relationships exhibited with respect to Δψ and oxygen utilization. The first group, designated as dinoseb types (dinitrophenols, benzimidazoles, benzonitriles, thiadiazoles, and bromofenoxim), uncoupled phosphorylation and collapsed Δψ to the Donnan level before oxygen utilization was inhibited. These compounds possess dissociable protons and are postulated to act as protonophores, much like 1799 and FCCP. With the second group, termed dicryl types (acylanilides, dinitroanilines, diphenylethers, bis-carbamates, and perfluidone), collapse of Δψ was paralleled by uncoupling of phosphorylation and inhibition of oxygen utilization. However, phosphorylation was inhibited to a greater extent than was respiration. The dicryl-type herbicides are not classical-type protonophores. Some of their action can be attributed to interference with the redox pumps. The complete collapse of Δψ to the Donnan potential is associated with alterations and perturbations induced in the membranes by classical uncouplers and by both types of herbicides. The perturbations are postulated to increase the permeability of the membranes to protons and other cations and to induce unfavorable conformational changes that impede interactions between redox enzymes. Conceivably, the combined responses collapse Δψ and inhibit electron transport.

Cimetidine transport in rabbit renal cortical brush-border membrane vesicles.

Cimetidine is an organic cation and commonly prescribed drug that is eliminated primarily by proximal renal tubular secretion. The present studies evaluated cimetidine transport in rabbit renal cortical brush-border membrane vesicles (BBMV). [3H]Cimetidine uptake varied inversely with media osmolarity and was stimulated with uphill transport above equilibrium values (overshoot) produced by an initial proton gradient directed from the vesicle interior outwardly. Uphill transport occurred earlier and was of greater magnitude at 25 degrees C than at 5 degrees C. pH-stimulated [3H]cimetidine uptake was inhibited by excess nonradiolabeled cimetidine, quinidine, and procainamide but was affected little by probenecid. Tetraethylammonium inhibited cimetidine uptake in the presence and absence of an initial proton gradient, indicating that nonionic diffusion and simple diffusion cannot totally account for cimetidine transport in BBMV. The protonophore carbonyl cyanide trifluoromethoxyphenylhydrazone (FCCP) inhibited pH-stimulated cimetidine uptake but had no effect on uptake occurring in the absence of an initial pH gradient. Preloading BBMV with an excess of procainamide enhanced cimetidine uptake. However, in the presence of FCCP, the combination of FCCP and valinomycin, or nigericin the effect of preloading with procainamide was diminished, suggesting that the apparent countertransport of cimetidine produced by procainamide was indirect and due to generation of a transvesicular proton gradient. These results are consistent with the hypothesis that cimetidine is transported across BBMV by organic cation-proton exchange.

Precursor forms of the constituent polypeptides of pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes from ox heart

The pyruvate dehydrogenase (PDC) and 2-oxoglutarate dehydrogenase (OGDC) multienzyme complexes of mitochondria are high-M, assemblies approximately the size of large and small ribosomal subunits, respectively. As all translation products of the mitochondrial genome have now been identified in higher animals (Chomyn et al . , 1985), i t is clear that the individual enzymes of the complex are nuclearcoded with a site of synthesis on cytoplasmic ribosomes (Suissa & Schatz, 1982). Thus, a complex sequence of molecular events must be involved in the biosynthesis, import, processing and assembly of these macromolecular aggregates including insertion of prosthetic groups, e.g. lipoic acid and FAD. Both these complexes are composed of multiple copies of three separate enzymes with a distinct lipoyl acyltransferase (E2) forming the structural core of each assembly. Initial decarboxylation of pyruvate or 2-oxoglutarate and reductive acylation of lipoyl groups on E2 is catalysed by a substrate-specific, thiamine diphosphate-requiring dehydrogenase, El. Production of the appropriate acyl-CoA derivative is promoted by E2 with subsequent re-oxidation of dihydrolipoamide groups by E3, lipoamide dehydrogenase (EC I .8.1.4), an FAD-linked homodimer, common to both complexes. Our approach to studying the initial events in the biosynthesis of PDC and OGDC has involved monitoring the appearance of cytoplasmic forms of these enzymes in vivo employing monospecific anitisera to the native complexes and each of the individual subunits. Immune replica analysis of a variety of cultured cells has established that three cell lines PK-15 (pig kidney), NBL-I (bovine kidney) and BRL (Buffalo rat liver) cells, exhibiting high rates of aerobic metabolism, contain substantial amounts of these complexes, approaching the levels in beef heart. In addition, immunoblotting has confirmed the monospecific nature of the antisera which can detect 5-50 ng of complex in crude SDS-extracts of whole cells. Detection of transient precursors is facilitated by inhibiting their uptake into the organelle with uncouplers of oxidative phosphorylation, e.g. 2,4-dinitrophenol (2 mM) or carbonyl cyanide trifluoromethoxyphenylhydrazone (1 0 PM) during a 4 h incubation with ['5S]methionine (Schleyer et al., 1982). Under these conditions, the mitochondrial electrochemical potential gradient (ApH+ ) is abolished completely with minimal effects (2&40%) on the overall rate of protein synthesis. Immunoprecipitations of ['5S]methionine-labelled cellular extracts were conducted in 1 O/ O (v/v) Triton X-100, 1 O/ O

Salicylhydroxamic acid‐stimulated NADH oxidation by purified plasmalemma vesicles from wheat roots

Purified, right side‐out plasmalemma vesicles were isolated from 7‐day‐old roots of dark‐grown wheat (Triticum aestivum L. cv. Drabant) by aqueous polymer two‐phase partitioning. The oxygen consumption by these vesicles at pH 6.5 in the presence of 1 mM NADH [12–29 nmol (mg protein)−1min−1] was 66% inhibited by 1 mM KCN and ca 40% by 1 mM EDTA. It was unaffected by rotenone, antimycin A, carbonyl cyanide trifluoromethoxyphenylhydrazone (FCCP), mersalyl, chlorotetracycline + Ca2+, and EGTA. Salicylhydroxamic acid (SHAM) and its analogue, m‐chlorobenzhydroxamic acid, stimulated the rate of oxygen consumption 10–20 fold in the presence of 1 mM NAD(P)H with an apparent Km (SHAM) of ca 40 μM (with NADH). The dependence of O2 consumption on NADH concentration in the presence of SHAM (2 mM) was sigmoidal, possibly due to endogenous catalase activity, and half‐maximal rate was obtained at 1.5 mM. In the absence of SHAM the rate increased with increasing acidity and no pH optimum was detectable between pH 4.5 and 8.5. In the presence of SHAM an optimum was observed at pH 6.5 and 0.8 mol of H2O2 was produced for every 1 mol O2 consumed. Endogenous catalase converted this H2O2 to O2 and after complete conversion the stoichiometry was 2 mol NADH consumed for every mol O3. SHAM was not consumed in the reaction. The possible involvement of a cytochrome P‐450/420 system is discussed.

The effect of carbonyl cyanide trifluoromethoxyphenylhydrazone and methylamine on the processing and secretion of the glycoprotein hormone chorionic gonadotropin by human choriocarcinoma cells.

Carbonyl cyanide trifluoromethoxyphenylhydrazone (FCCP), a protonophore, and methylamine, a weak base, agents that dissipate hydrogen gradients across cellular membranes, were used to probe the coupling of hydrogen gradients to the processing and secretion of the glycoprotein hormone hCG by human choriocarcinoma cells (JAR) in culture. Both drugs disrupted the processing of asparagine-linked oligosaccharides such that the secreted hCG forms contained mostly high mannose rather than complex oligosaccharide chains. As the concentrations of FCCP were increased above 1 microgram/ml and those of methylamine above 12.5 mg/ml, the secretion of the labeled hCG dimer and free alpha-subunit was progressively inhibited. Both FCCP and methylamine also inhibited the incorporation of [35S] methionine and [3H]mannose into hCG subunits. Nevertheless, the inhibition of secretion was clearly apparent as an intracellular accumulation of the hCG subunit precursors in spite of the diminished incorporation of radioactive substrates. The intracellular hCG precursors that accumulated in the drug-treated cells contained predominantly Man8-9GlcNAc2 units, structures characteristic of glycoproteins localized in the endoplasmic reticulum. Both FCCP and methylamine inhibited hCG secretion at concentrations that did not lower the cellular content of ATP. We postulate on the basis of these results that a hydrogen gradient across the membrane either of the rough endoplasmic reticulum or the transitional vesicle is coupled to the rough endoplasmic reticulum to Golgi translocation step such that dissipation of the proton gradient blocks the secretion of hCG.