Proton Transport In Vesicles Research Articles

Expanding the π-system of Fatty Acid-Anion Transporter Conjugates Modulates Their Mechanism of Proton Transport and Mitochondrial Uncoupling Activity.

Mitochondrial uncoupling by small molecule protonophores is a promising strategy for developing novel anticancer agents. Recently, aryl urea substituted fatty acids (aryl ureas) were identified as a new class of protonophoric anticancer agents. To mediate proton transport these molecules self-assemble into membrane-permeable anionic dimers in which intermolecular hydrogen bonds between the carboxylate and aryl-urea anion receptor delocalise the negative charge across the aromatic π-system. In this work, we extend the aromatic π-system by introducing a second phenyl substituent to the aryl urea scaffold and compare the proton transport mechanisms and mitochondrial uncoupling actions of these compounds to their monoaryl analogues. It was found that incorporation of meta-linked phenyl substituents into the aryl urea scaffold enhanced proton transport in vesicles and demonstrated superior capacity to depolarise mitochondria, inhibit ATP production and reduce the viability of MDA-MB-231 breast cancer cells. In contrast, diphenyl ureas linked through a 1,4-distribution across the phenyl ring displayed diminished proton transport activity, despite both diphenyl urea isomers possessing similar binding affinities for carboxylates. Mechanistic studies suggest that inclusion of a second aryl ring changes the proton transport mechanism, presumably due to steric factors that impose higher energy penalties for dimer formation.

Nitrate transport across the tonoplast of Cucumis sativus L. root cells

Nitrate transport across the tonoplast has been studied using vacuole membranes isolated from cucumber roots grown in nitrate. The addition of NO3- ions into the tonoplast with ATP-generated transmembrane proton gradient caused the dissipation of delta pH, indicating the NO3(-)-induced proton efflux from vesicles. NO3(-)-dependent H+ efflux was almost insensitive to the transmembrane electrical potential difference, suggesting the presence of an electroneutral NO3-/H+ antiporter in the tonoplast. Apart from saturation kinetics, with respect to nitrate ions, NO3(-)-linked H+ efflux from the tonoplast of cucumber roots showed other characteristics expected of substrate-specific transporters. Experiments employing protein modifying reagents (NEM, pCMBS, PGO and SITS) indicated that a crucial role in the activity of tonoplast nitrate/proton antiporter is played by lysine residues (strong inhibition of NO3-/H+ antiport by SITS). None of the ion-channel inhibitors (NIF, ZnSO4 and TEA-Cl) used in the experiments had a direct effect on the nitrate transport into tonoplast membranes. On the other hand, every protein reagent, as well as NIF and ZnSO4, significantly affected the ATP-dependent proton transport in vesicles. Only TEA-Cl, the potassium channel blocker, had no effect on the vacuolar proton pumping activity.

Characterization of the nitrate transporter in root plasma membranes of Cucumis sativus L

Nitrate uptake in right-side out plasma membrane vesicles isolated from cucumber roots was characterized. Nitrate uptake into vesicles was driven by an artificially imposed pH gradient. The uptake was strongly inhibited by phenylglyoxal, an arginyl residue modificator. Only a slight repression of NO 3 − transport in vesicles was observed in the presence of NEM, a thiol group reagent. pCMBS, an other thiol reagent and DEPC, an effector of histidine residue, had no effect on the nitrate transport in plasma membranes. ATP-driven proton transport in vesicles was not significantly affected in the presence of both, phenylglyoxal and DEPC, whereas pCMBS and NEM abolished it almost completely. The possible role of the particular amino acids residues in the active nitrate transport is discussed. NO 3 − uptake into vesicles isolated from both, nitrate-induced and nitrate-depleted plant material was higher than that observed in the vesicles obtained from uninduced plants. Thus, isolated vesicles reflect the well-known in vivo response of intact plants on the exogenous nitrogen regime.

The catalytic cycle of the vacuolar H(+)-ATPase. Comparison of proton transport in kidney- and osteoclast-derived vesicles.

To characterize the catalytic cycle involved in the initial rate of proton transport by the vacuolar ATPases (V-ATPase), we have analyzed and compared the catalytic parameters of V-ATPase-dependent proton transport in vesicles from chicken kidney or purified osteoclasts. The relationship of acidification to ATP obeyed simple Michaelis-Menten kinetics with a single Km for ATP of 62 microM in the kidney and 191 microM in the osteoclast. The activity was dependent on the presence of Mg2+, with a single Km of 258 microM MgCl2 in the kidney and 504 microM MgCl2 in the osteoclast. In both preparations, ADP competed with ATP and inhibited the proton transport (Ki = 37 microM in kidney and 17 microM in osteoclast). Phosphate was found to be a noncompetitive inhibitor of ATP, with a calculated Ki of about 10.5 and 5.5 mM, respectively. In both preparations, the catalytic mechanism determining the V-ATPase-mediated proton transport, fits the model of a "uni-bi-ordered release" mechanism. According to this model, ATP is the single substrate, and P(i) and ADP are the two products where phosphate, being noncompetitive, is released first and ADP, being competitive, second. The findings of an elevated Km for ATP and Mg2+ and a decrease in the Ki of ADP and phosphate in the osteoclast relative to the kidney preparation suggests that the V-ATPases present in these two tissues may differ.

The Effect of Extracellular Components from Colletotrichum lindemuthianum on Membrane Transport in Vesicles Isolated from Bean Hypocotyl

Extracellular components released from mycelia of the alpha and beta races of the bean pathogen, Colletotrichum lindemuthianum, inhibited proton uptake in sealed vesicles prepared from bean hypocotyls. Differential sensitivity of ATP-driven proton transport to nitrate, vanadate, N,N'-dicyclohexylcarbodiimide, diethylstilbestrol, and oligomycin suggested the vesicles were enriched for tonoplast. Anion stimulation of proton transport, by enhancement of ATPase activity and dissipation of the membrane potential, was consistent with this conclusion. Although fungal components inhibited the formation of a pH gradient, the membrane potential was unaffected and the ATPase activity slightly stimulated. These data suggest that the fungal components produce an electroneutral proton exchange. Proton transport in Dark Red Kidney bean tonoplast vesicles was inhibited by mycelial preparations from the incompatible alpha race and compatible beta race. Elicitor activity, however, was greater in the alpha race fractions. Elicitor purified from alpha race culture filtrate did not inhibit proton transport in vesicles isolated from Dark Red Kidney bean. Consequently, elicitor activity need not be associated with an ability to impair tonoplast function.

Characterization of N-ethylmaleimide-sensitive proton pump in the rat kidney. Localization along the nephron.

This study is aimed both at characterizing an ATPase activity in rat kidney equivalent to the proton pump described in bovine kidney medulla and at localizing this enzyme along the nephron. Membrane fractions isolated from kidney homogenates by differential and density gradient centrifugations were enriched 7-fold in ATPase activity sensitive to N-ethylmaleimide (NEM). These fractions also displayed ATP-dependent proton transport. ATPase activity and proton transport in vesicles had similar pharmacological properties as both were insensitive to vanadate and ouabain and had similar sensitivities toward NEM (apparent Ki = 20 microM) and N,N'-dicyclohexylcarbodiimide (apparent Ki = 50 microM). Proton transport was dependent on chloride availability as chloride addition to the extravesicular medium stimulated proton transport in a dose-dependent fashion (apparent K 1/2 = 7 mM). NEM-sensitive ATPase activity displaying similar pharmacological properties as proton transport in vesicles was also found in single segments of nephron. It was insensitive to vanadate and ouabain, was inhibited by similar concentrations of NEM (apparent Ki = 15-20 microM) and N,N'-dicyclohexylcarbodiimide (apparent Ki = 30 microM), and is therefore likely to be a proton pump. NEM-sensitive ATPase was localized in all the segments of the rat nephron; its activity was highest in proximal convoluted tubules; intermediate in proximal straight tubules, thick ascending limbs, and cortical collecting tubules; and lowest in outer medullary collecting tubules.