In vitro antileishmanial and cytotoxic activities of nerolidol are associated with changes in plasma membrane dynamics

The sesquiterpene nerolidol is a membrane-active compound that has demonstrated antitumor, antibacterial, antifungal and antiparasitic activities. In this study, we used electron paramagnetic resonance (EPR) spectroscopy and biophysical parameters determined via cell culture assays to study the mechanisms underlying the in vitro antileishmanial activity of nerolidol. The EPR spectra of a spin-labeled stearic acid indicated notable interactions of nerolidol with the cell membrane of Leishmania amazonensis amastigotes. The nerolidol IC₅₀ values in L. amazonensis amastigotes and promastigotes were found to depend on the cell concentration used in the assay. This dependence was described by an equation that considers various cell suspension parameters, such as the 50% inhibitory concentrations of nerolidol in the cell membrane (c_m50) and the aqueous phase (c_w50) and the membrane-water partition coefficient of nerolidol (K_M/W). Via cytotoxicity (CC₅₀) and hemolytic potential (HC₅₀) data, these parameters were also determined for nerolidol in macrophages and erythrocytes. With a c_w50 of 125 μM, macrophages were less sensitive to nerolidol than amastigotes and promastigotes, which had mean c_w50 values of 56 and 74 μM, respectively. The estimated c_m50 values of nerolidol for amastigotes and promastigotes and macrophages were between 2.6 and 3.0 M, indicating substantial accumulation of nerolidol in the cell membrane. In addition, the spin-label EPR data indicated that membrane dynamic changes occurred in L. amazonensis amastigotes at concentrations similar to the nerolidol IC₅₀ value.     

Terpenes Increase the Lipid Dynamics in the Leishmania Plasma Membrane at Concentrations Similar to Their IC50 Values

Although many terpenes have shown antitumor, antibacterial, antifungal, and antiparasitic activity, the mechanism of action is not well established. Electron paramagnetic resonance (EPR) spectroscopy of the spin-labeled 5-doxyl stearic acid revealed remarkable fluidity increases in the plasma membrane of terpene-treated Leishmania amazonensis promastigotes. For an antiproliferative activity assay using 5×10⁶ parasites/mL, the sesquiterpene nerolidol and the monoterpenes (+)-limonene, α-terpineol and 1,8-cineole inhibited the growth of the parasites with IC₅₀ values of 0.008, 0.549, 0.678 and 4.697 mM, respectively. The IC₅₀ values of these terpenes increased as the parasite concentration used in the cytotoxicity assay increased, and this behavior was examined using a theoretical treatment of the experimental data. Cytotoxicity tests with the same parasite concentration as in the EPR experiments revealed a correlation between the IC₅₀ values of the terpenes and the concentrations at which they altered the membrane fluidity. In addition, the terpenes induced small amounts of cell lysis (4–9%) at their respective IC₅₀ values. For assays with high cell concentrations (2×10⁹ parasites/mL), the incorporation of terpene into the cell membrane was very fast, and the IC₅₀ values observed for 24 h and 5 min-incubation periods were not significantly different. Taken together, these results suggest that terpene cytotoxicity is associated with the attack on the plasma membrane of the parasite. The in vitro cytotoxicity of nerolidol was similar to that of miltefosine, and nerolidol has high hydrophobicity; thus, nerolidol might be used in drug delivery systems, such as lipid nanoparticles to treat leishmaniasis.     

Cellular Effects of Curcumin on Plasmodium falciparum Include Disruption of Microtubules

Curcumin has been widely investigated for its myriad cellular effects resulting in reduced proliferation of various eukaryotic cells including cancer cells and the human malaria parasite Plasmodium falciparum. Studies with human cancer cell lines HT-29, Caco-2, and MCF-7 suggest that curcumin can bind to tubulin and induce alterations in microtubule structure. Based on this finding, we investigated whether curcumin has any effect on P. falciparum microtubules, considering that mammalian and parasite tubulin are 83% identical. IC₅₀ of curcumin was found to be 5 µM as compared to 20 µM reported before. Immunofluorescence images of parasites treated with 5 or 20 µM curcumin showed a concentration-dependent effect on parasite microtubules resulting in diffuse staining contrasting with the discrete hemispindles and subpellicular microtubules observed in untreated parasites. The effect on P. falciparum microtubules was evident only in the second cycle for both concentrations tested. This diffuse pattern of tubulin fluorescence in curcumin treated parasites was similar to the effect of a microtubule destabilizing drug vinblastine on P. falciparum. Molecular docking predicted the binding site of curcumin at the interface of alpha and beta tubulin, similar to another destabilizing drug colchicine. Data from predicted drug binding is supported by results from drug combination assays showing antagonistic interactions between curcumin and colchicine, sharing a similar binding site, and additive/synergistic interactions of curcumin with paclitaxel and vinblastine, having different binding sites. This evidence suggests that cellular effects of curcumin are at least, in part, due to its perturbing effect on P. falciparum microtubules. The action of curcumin, both direct and indirect, on P. falciparum microtubules is discussed.     

Betulin derivatives impair Leishmania braziliensis viability and host–parasite interaction

Leishmaniasis is a public health problem in tropical and subtropical areas of the world, including Venezuela. The incidence of treatment failure and the number of cases with Leishmania-HIV co-infection underscore the importance of developing alternative, economical and effective therapies against this disease. The work presented here analyzed whether terpenoids derived from betulin are active against New World Leishmania parasites. Initially we determined the concentration that inhibits the growth of these parasites by 50% or IC₅₀, and subsequently evaluated the chemotactic effect of four compounds with leishmanicidal activity in the sub-micromolar and micromolar range. That is, we measured the migratory capacity of Leishmania (V.) braziliensis in the presence of increasing concentrations of compounds. Finally, we evaluated their cytotoxicity against the host cell and their effect on the infectivity of L. (V.) braziliensis. The results suggest that (1) compounds 14, 17, 18, 25 and 27 are active at concentrations lower than 10 μM; (2) compound 26 inhibits parasite growth with an IC₅₀ lower than 1 μM; (3) compounds 18, 26 and 27 inhibit parasite migration at pico- to nanomolar concentrations, suggesting that they impair host–parasite interaction. None of the tested compounds was cytotoxic against J774.A1 macrophages thus indicating their potential as starting points to develop compounds that might affect parasite–host cell interaction, as well as being leishmanicidal.     

Amentoflavone isolated from Selaginella sellowii Hieron induces mitochondrial dysfunction in Leishmania amazonensis promastigotes

Leishmaniasis chemotherapy is a bottleneck in disease treatment. Although available, chemotherapy is limited, toxic, painful, and does not lead to parasite clearance, with parasite resistance also being reported. Therefore, new therapeutic options are being investigated, such as plant-derived anti-parasitic compounds. Amentoflavone is the most common biflavonoid in the Selaginella genus, and its antileishmanial activity has already been described on Leishmania amazonensis intracellular amastigotes but its direct action on the parasite is controversial. In this work we demonstrate that amentoflavone is active on L. amazonensis promastigotes (IC₅₀ = 28.5 ± 2.0 μM) and amastigotes. Transmission electron microscopy of amentoflavone-treated promastigotes showed myelin-like figures, autophagosomes as well as enlarged mitochondria. Treated parasites also presented multiple lipid droplets and altered basal body organization. Similarly, intracellular amastigotes presented swollen mitochondria, membrane fragments in the lumen of the flagellar pocket as well as autophagic vacuoles. Flow cytometric analysis after TMRE staining showed that amentoflavone strongly decreased mitochondrial membrane potential. In silico analysis shows that amentoflavone physic-chemical, drug-likeness and bioavailability characteristics suggest it might be suitable for oral administration. We concluded that amentoflavone presents a direct effect on L. amazonensis parasites, causing mitochondrial dysfunction and parasite killing. Therefore, all results point for the potential of amentoflavone as a promising candidate for conducting advanced studies for the development of drugs against leishmaniasis.     

In vitro screening of compounds from the Food and Drug Administration-approved library identifies anti-Babesia gibsoni activity of idarubicin hydrochloride and vorinostat

Babesia gibsoni is mainly transmitted by hard ticks of the genus Rhipicephalus (R. sanguineus) and Haemaphysalis (H. longicornis), and causes canine babesiosis. Clinical manifestations of B. gibsoni infection include fever, hemoglobinemia, hemoglobinuria, and progressive anemia. Traditional antibabesial therapy, such as imidocarb dipropionate or diminazene aceturate, can only alleviate severe clinical manifestations and cannot eliminate parasites in the host. Food and Drug Administration (FDA)-approved drugs are a solid starting point for researching novel therapy strategies for canine babesiosis. In this work, we screened 640 FDA-approved drugs against the growth of B. gibsoni in vitro. Among them, 13 compounds (at 10 μM) exhibited high growth inhibition (>60%), and two compounds, namely idarubicin hydrochloride (idamycin) and vorinostat, were chosen for further investigation. The half-maximal inhibitory concentration (IC₅₀) values of idamycin and vorinostat were determined to be 0.044 ± 0.008 μM and 0.591 ± 0.107 μM, respectively. Viability results indicated that a concentration of 4 × IC₅₀ of vorinostat prevented the regrowth of treated B. gibsoni, whereas parasites treated with 4 × IC₅₀ concentration of idamycin remained viable. The B. gibsoni parasites treated with vorinostat exhibited degeneration within erythrocytes and merozoites, in contrast to the oval or signet-ring shape of normal B. gibsoni parasites. In conclusion, FDA-approved drugs offer a valuable platform for drug repositioning in antibabesiosis research. Particularly, vorinostat demonstrated promising inhibitory effects against B. gibsoni in vitro, and further studies on vorinostat are necessary to elucidate its mechanism as a novel treatment in infected animal models.     

Curcumin inhibits bTREK-1 K+ channels and stimulates cortisol secretion from adrenocortical cells

Bovine adrenal zona fasciculata (AZF) cells express bTREK-1 K⁺ channels that set the resting membrane potential. Inhibition of these channels by adrenocorticotropic hormone (ACTH) is coupled to membrane depolarization and cortisol secretion. Curcumin, a phytochemical with medicinal properties extracted from the spice turmeric, was found to modulate both bTREK-1 K⁺ currents and cortisol secretion from AZF cells. In whole-cell patch clamp experiments, curcumin inhibited bTREK-1 with an IC₅₀ of 0.93 μM by a mechanism that was voltage-independent. bTREK-1 inhibition by curcumin occurred through interaction with an external binding site and was independent of ATP hydrolysis. Curcumin produced a concentration-dependent increase in cortisol secretion that persisted for up to 24 h. At a maximally effective concentration of 50 μM, curcumin increased secretion as much as 10-fold. These results demonstrate that curcumin potently inhibits bTREK-1 K⁺ channels and stimulates cortisol secretion from bovine AZF cells. The inhibition of bTREK-1 by curcumin may be linked to cortisol secretion through membrane depolarization. Since TREK-1 is widely expressed in a variety of cells, it is likely that some of the biological actions of curcumin, including its therapeutic effects, may be mediated through inhibition of these K⁺ channels.     

Antiparasitic activity and effect of casearins isolated from Casearia sylvestris on Leishmania and Trypanosoma cruzi plasma membrane

Leishmaniasis and Chagas disease are infectious diseases caused by parasite Leishmania sp. and Trypanosoma cruzi, respectively, and are included among the most neglected diseases in several underdeveloped and developing countries, with an urgent demand for new drugs. Considering the antiparasitic potential of MeOH extract from leaves of Casearia sylvestris Sw. (Salicaceae), a bioguided fractionation was conducted and afforded four active clerodane diterpenes (casearins A, B, G, and J). The obtained results indicated a superior efficacy of tested casearins against trypomastigotes of T. cruzi, with IC₅₀ values ranging from 0.53 to 2.77 μg/ml. Leishmania infantum promastigotes were also susceptible to casearins, with IC₅₀ values in a range between 4.45 and 9.48 μg/ml. These substances were also evaluated for mammalian cytotoxicity against NCTC cells resulting in 50% cytotoxic concentrations (CC₅₀) ranging from 1.46 to 13.76 μg/ml. Additionally, the action of casearins on parasite membranes was investigated using the fluorescent probe SYTOX Green. The obtained results demonstrated a strong interaction of casearins A and B to the plasma membrane of T. cruzi parasites, corroborating their higher efficacy against these parasites. In contrast, the tested casearins induced no alteration in the permeability of plasma membrane of Leishmania parasites, suggesting that biochemical differences between Leishmania and T. cruzi plasma membrane might have contributed to the target effect of casearins on trypomastigotes. Thus, considering the importance of studying novel and selective drug candidates against protozoans, casearins A, B, G, and J could be used as tools to future drug design studies.     

Kinetic constants for the inhibition of camel retinal acetylcholinesterase by the carbamate insecticide lannate

We have designed this study to determine various kinetic parameters of camel retinal membrane‐bound acetylcholinesterase (AChE; EC 3.1.1.7) inhibition by carbamate insecticide lannate [methyl N‐{{(methylamino)carbonyl}oxy} ethanimidothioate]. All these kinetic constants were derived by simple graphical methods. The value of kinetic parameters was estimated as follows: 0.061 (μM)⁻¹, 1.14 (μM)⁻¹, 0.216 μM, 0.016 min⁻¹, 0.0741 (μM min)⁻¹, 0.746 μM, and 4.42 μM for velocity constant (K_v), new inhibition constant (K_nic), dissociation constant (K_d), carbamylation rate constant (k_2c), overall carbamylation rate constant (k′2 ), 50% inhibition constant (K_I50), and 99% inhibition constant (K_I99), respectively. These unique methods may be used to estimate such kinetic parameters for time‐dependent inhibition of enzymes by variety of chemicals, insecticides, herbicides, and drugs. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 41–46, 1999     

Ivermectin-dependent release of IL-1beta in response to ATP by peritoneal macrophages from P2X<Subscript>7</Subscript>-KO mice

The response to ATP of peritoneal macrophages from wild-type (WT) and P2X₇-invalidated (KO) mice was tested. Low concentrations (1–100 μM) of ATP transiently increased the intracellular concentration of calcium ([Ca²⁺]_i) in cells from both mice. The inhibition of the polyphosphoinositide-specific phospholipase C with U73122 inhibited this response especially in WT mice suggesting that the responses coupled to P2Y receptors were potentiated by the expression of P2X₇ receptors. One millimolar ATP provoked a sustained increase in the [Ca²⁺]_i only in WT mice. The response to 10 μM ATP was potentiated and prolonged by ivermectin in both mice. One millimolar ATP increased the influx of extracellular calcium, decreased the intracellular concentration of potassium ([K⁺]_i) and stimulated the secretion of interleukin-1β (IL-1β) only in cells from WT mice. Ten micromolar ATP in combination with 3 μM ivermectin reproduced these responses both in WT and KO mice. The secretion of IL-1β was also increased by nigericin in WT mice and the secretory effect of a combination of ivermectin with ATP in KO mice was suppressed in a medium containing a high concentration of potassium. In WT mice, 150 μM BzATP stimulated the uptake of YOPRO-1. Incubation of macrophages from WT and KO mice with 10 μM ATP resulted in a small increase of YOPRO-1 uptake, which was potentiated by addition of 3 μM ivermectin. The uptake of this dye was unaffected by pannexin-1 blockers. In conclusion, prolonged stimulation of P2X₄ receptors by a combination of low concentrations of ATP plus ivermectin produced a sustained activation of the non-selective cation channel coupled to this receptor. The ensuing variations of the [K⁺]_i triggered the secretion of IL-1β. Pore formation was also triggered by activation of P2X₄ receptors. Higher concentrations of ATP elicited similar responses after binding to P2X₇ receptors. The expression of the P2X₇ receptors was also coupled to a better response to P2Y receptors.     

Cryptosporidium Lactate Dehydrogenase Is Associated with the Parasitophorous Vacuole Membrane and Is a Potential Target for Developing Therapeutics

The apicomplexan, Cryptosporidium parvum, possesses a bacterial-type lactate dehydrogenase (CpLDH). This is considered to be an essential enzyme, as this parasite lacks the Krebs cycle and cytochrome-based respiration, and mainly–if not solely, relies on glycolysis to produce ATP. Here, we provide evidence that in extracellular parasites (e.g., sporozoites and merozoites), CpLDH is localized in the cytosol. However, it becomes associated with the parasitophorous vacuole membrane (PVM) during the intracellular developmental stages, suggesting involvement of the PVM in parasite energy metabolism. We characterized the biochemical features of CpLDH and observed that, at lower micromolar levels, the LDH inhibitors gossypol and FX11 could inhibit both CpLDH activity (K _i = 14.8 μM and 55.6 μM, respectively), as well as parasite growth in vitro (IC₅₀ = 11.8 μM and 39.5 μM, respectively). These observations not only reveal a new function for the poorly understood PVM structure in hosting the intracellular development of C. parvum, but also suggest LDH as a potential target for developing therapeutics against this opportunistic pathogen, for which fully effective treatments are not yet available.     

Plasmodium berghei: Uptake and distribution of chloroquine in infected mouse erythrocytes

The capacity of mouse erythrocytes infected with Plasmodium berghei to accumulate chloroquine is developed with maturation of the parasites. This is shown by direct comparison of the early and mature stages, which are separated by density difference. After drug accumulation, infected cells were fractionated by saponin lysis or nitrogen decompression to study the drug distribution. Effectiveness of isolating intact parasites and host components was checked by SDS-polyacrylamide gel electrophoresis and by low leakage of parasite-specific lactate dehydrogenase used as a marker enzyme. At low external drug concentration (~10^?7M), chloroquine is principally accumulated in the parasites. However, at higher drug concentrations (~10^?5and ~10^?3M), the proportion of the drug found in the host cytosol fraction is increased. A small but significant proportion of the drug (<20%) is associated with the host cell membrane. The pellet fraction of the freed parasites, further fractionated by freeze-thaw lysis, contains a major proportion of the drug at low external concentrations. However, the pellet fraction obtained from prolonged sonication of the parasites, which contains the bulk of hemozoin pigment, carries only a small proportion of the drug. This indicates that parasite membrane components may bind most of the drug. As external chloroquine concentration is increased, the proportion of drug in the parasite supernatant increases, some or most of which is probably bound by soluble hemecontaining compounds. However, the presence of chloroquine in the parasite does not affect the partition of heme in particulate and soluble forms.     

Accommodation of NO in the active site of mammalian and bacterial cytochrome c oxidase aa3

Following different reports on the stoichiometry and configuration of NO binding to mammalian and bacterial reduced cytochrome c oxidase aa₃ (CcO), we investigated NO binding and dynamics in the active site of beef heart CcO as a function of NO concentration, using ultrafast transient absorption and EPR spectroscopy. We find that in the physiological range only one NO molecule binds to heme a₃, and time-resolved experiments indicate that even transient binding to Cu_B does not occur. Only at very high (∼ 2 mM) concentrations a second NO is accommodated in the active site, although in a different configuration than previously observed for CcO from Paracoccus denitrificans [E. Pilet, W. Nitschke, F. Rappaport, T. Soulimane, J.-C. Lambry, U. Liebl and M.H. Vos. Biochemistry 43 (2004) 14118-14127], where we proposed that a second NO does bind to Cu_B. In addition, in the bacterial enzyme two NO molecules can bind already at NO concentrations of ∼ 1 μM. The unexpected differences highlighted in this study may relate to differences in the physiological relevance of the CcO-NO interactions in both species.     

Metacytofilin has potent anti-malarial activity

Metacytofilin (MCF) was isolated from the fungus Metarhizium sp. TA2759. Although MCF possesses anti-Toxoplasma activity, the effects of this compound against other parasites are unknown. Here, we evaluated the in vitro anti-malarial activity of MCF against the 3D7 strain and the chloroquine-resistant K1 strain of Plasmodium falciparum. The half maximal inhibitory concentrations (IC₅₀) of MCF against the 3D7 and K-1 strains following culture for 48 h were 666 nM and 605 nM, respectively. Artemisinin was more potent than MCF against both strains (3D7 IC₅₀: 17.4 nM; K-1 IC₅₀: 18.3 nM), while chloroquine was ineffective against the chloroquine-resistant strain (3D7 IC₅₀: 39.1 nM; K-1 IC₅₀: 1.62 μM). MCF affected the ring stage of the parasites, resulting in their death as shown by spots within red blood cells. MCF also inhibited parasite growth following culture for 72 h (3D7 IC₅₀, 285 nM). Four optical isomers of cyclo[Leu-Phe]-diketopiperazine derivatives with modified methoxy and/or hydroxyl groups lost anti-malarial activity, suggesting that the spatial positions of the methoxy and hydroxyl groups in MCF play an important role in its anti-malarial effects. Together, these data suggest that MCF may represent a promising lead compound for treatment of drug-resistant malarial parasites.     

Urea-induced modification of cytochrome c flexibility as probed by cyanide binding

Cyanide binding to cytochrome c was monitored by absorption spectroscopy from neutral to acidic pH in the presence of urea. These results were compared with acid-induced unfolding at corresponding urea concentration monitored by absorption spectroscopy and circular dichroism. The association rate constant k_a increased 20-fold when the concentration of urea was raised from 0 M to 6 M at neutral pH. However, the secondary structure of the protein was not affected, i.e. there was no striking conformational change in these urea concentrations at neutral pH. At the pH that was very close to the pK of acid-induced unfolding, the k_a value reached its maximum (k_a,max) in all urea concentrations. Interestingly, the k_a,max value increased exponentially with increasing urea concentrations. These results are interpreted in terms of a change in the flexibility of the least stable part of the cyt c structure that is responsible for the Fe–S(Met80) bond disruption and for ligand binding to heme iron.     

Novel enoyl-ACP reductase (FabI) potential inhibitors of Escherichia coli from Chinese medicine monomers

By structure-based virtual screening and experimental verification, two Chinese medicine monomers, luteolin and curcumin, had been proved to be uncompetitive inhibitors of enoyl-ACP reductase from Escherichia coli (EcFabI) with the inhibition constant (K_i) of 7.1 μM and 15.0 μM, respectively. In particular, curcumin had apparent antibacterial activity against E. coli, and the minimum inhibition concentration (MIC₉₀) was 73.7 μg/mL. Importantly, fabI-overexpressing E. coli showed reduced susceptibility to the inhibitor compared with the wild-type strains, demonstrating that its antibacterial action is mediated by the inhibition of EcFabI.     

Copper chloride and copper sulphate in combination with nitroxynil against gastrointestinal nematodes of ruminants: A possible hitchhiking synergic effect at low concentrations

Infections caused by Haemonchus spp. and Trichostrongylus spp. are major health problems for sheep and cattle. The objective of this study was to determine the efficacy of copper chloride (CuCl₂), and copper sulphate (CuSO₄) at 2.0, 7.0, 30.0, 125.0, 500.0, and 2000.0 µM formulations, and nitroxynil 34% (NTX) at 0.235 mM against gastrointestinal nematodes (GINs) of ruminants. Hence, the in vitro egg hatch test (EHT), the larval development test (LDT), and the larval migration inhibition test (LMIT) were used. Haemonchus spp. (52%) and Trichostrongylus spp. (38%) were the most frequently found parasites. The data fitted a concentration-dependent shape with the highest efficacies of CuCl₂ and CuSO₄ at 95.2 and 97.3% for parasites collected from sheep, and 95.8 and 93.4% from cattle, respectively. The combination of the 50% inhibitory concentration (IC₅₀) of CuCl₂ and CuSO₄ and the IC₁₀ of NTX showed up to a 52% increase in efficacy above the expected additive results, demonstrating a synergic/drug enhancer interaction. NTX may retain Cu-II ions by complexation, in a hitchhiking mechanism carrying the salts across the parasite cell wall, causing oxidative stress as a consequence of free radical production and cell damage. Synergy data between NTX and CuCl₂, and CuSO₄ represent a viable opportunity to develop new formulations for combating parasites of ruminants (i.e., Fasciola hepatica, Haemonchus spp., and Oesophagostomum spp.).     

Kinetics of ubiquinol-1-cytochrome c reductase in bovine heart mitochondria and submitochondrial particles

A kinetic study on ubiquinol-cytochrome f reductase (EC 1.10.2.2) has been undertaken either in situ in KCN-inhibited mitochondria and submitochondrial particles, or in the isolated cytochrome b-c₁ complex using ubiquinol-1 and exogenous cytochrome c as substrates. The steady-state two-substrate kinetics of the reductase appears to follow a general sequential mechanism, allowing calculation of a K_m for ubiquinol-1 of 13.4 μM in mitochondria and of 24.6 μM in the isolated cytochrome b-c₁ complex. At low concentrations of cytochrome c, however, the titrations as a function of quinol concentration appear biphasic both in mitochondria and in submitochondrial particles containing trapped cytochrome c inside the vesicle space, fitting two apparent K_m values for ubiquinol-1. Relatively high antimycin-sensitive rates of ubiquinol-1-cytochrome c reductase have been found in submitochondrial particles: both the V_max and the K_m for ubiquinol-1 are, however, affected by the overall orientation of the particle preparation, i.e., by the reactivity of cytochrome c with its proper site. The turnover numbers corrected for particle orientation with respect to cytochrome c interaction are at least 2-fold higher in submitochondrial particles than in mitochondria. This is particularly evident using inside-out particles containing trapped cytochrome c in the vesicle space (and therefore reacting with its physiological site). A diffusion step for the quinol substrate appears to be rate limiting in mitochondria and can be removed by addition of deoxycholate, suggesting that the oxidation site of ubiquinol may be more exposed to the matrix side of the inner mitochondrial membrane.     

Sodium and potassium fluxes and compartmentation in roots of atriplex and oat

K⁺ and Na⁺ fluxes and ion content have been studied in roots of Atriplex nummularia Lindl. and Avena sativa L. cv Goodfield grown in 3 millimolar K⁺ with or without 3 or 50 millimolar NaCl. Compartmental analysis was carried out with entire root systems under steady-state conditions.

Increasing ambient Na⁺ concentrations from 0 to 50 millimolar altered K⁺, in Atriplex, as follows: slightly decreased the cytoplasmic content (Q_c), the vacuolar content (Q_v), and the plasma membrane influx and efflux. Xylem transport for K⁺ decreased by 63% in Atriplex. For oat roots, similar increases in Na⁺ altered K⁺ parameters as follows: plasma membrane influx and efflux decreased by about 80%. Q_c decreased by 65%, and xylem transport decreased by 91%. No change, however, was observed in Q_v for K⁺. Increasing ambient Na⁺ resulted in higher (3 to 5-fold) Na⁺ fluxes across the plasma membrane and in Q_c of both species. In Atriplex, Na⁺ fluxes across the tonoplast and Q_v increased as external Na⁺ was increased. In oat, however, no significant change was observed in Na⁺ flux across the tonoplast or in Q_v as external Na⁺ was increased. In oat roots, Na⁺ reduced K⁺ uptake markedly; in Atriplex, this was not as pronounced. However, even at high Na⁺ levels, the influx transport system at the plasma membrane of both species preferred K⁺ over Na⁺.

Based upon the Ussing-Teorell equation, it was concluded that active inward transport of K⁺ occurred across the plasma membrane, and passive movement of K⁺ occurred across the tonoplast in both species. Na⁺, in oat roots, was actively pumped out of the cytoplasm to the exterior, whereas, in Atriplex, Na⁺ was passively distributed between the free space, cytoplasm, and vacuole.

     

Antibody-Induced Alterations in the Kinetic Characteristics of the Na:K Pump in Goat Red Blood Cells

The kinetic characteristics of the Na:K pump in high potassium (HK) and low potassium (LK) goat red cells were investigated after altering the intracellular cation concentrations. At low concentrations of intracellular K (K_c), increasing K_c at first stimulates the active K influx in HK cells, but at higher K_c the pump is inhibited. These results suggest that in HK cells K_c acts both at a stimulatory site at the inner aspect of the pump and by competition with intracellular Na (Na_c) at the Na translocation sites. In LK cells, K_c inhibits the active K influx and the sensitivity of LK cells to inhibition is much greater than the sensitivity of HK cells. Exposure of LK cells to an antibody (anti-L), raised in an HK sheep by injection of LK sheep cells, increased the active K influx at any given K_c. The effect of the antibody was greater at higher intracellular K concentrations, and in cells with very low concentrations of K the antibody had little effect on the pump rate. The failure of anti-L to stimulate the pump in low K_c LK cells was not due to failure of the antibody to bind to the cells. Anti-L combining at the outer surface of the cell reduces the affinity of the pump at the inner surface for K at the inhibitory sites. The maximal pump rate in LK cells at optimal Na and K concentrations is less than the maximal pump rate of HK cells under the same circumstances.