Glutamate uptake into synaptic vesicles of bovine cerebral cortex and electrochemical potential difference of proton across the membrane.

Measurements have been made of the ATP-dependent membrane potential (delta psi) and pH gradient (delta pH) across the membranes of the synaptic vesicles purified from bovine cerebral cortex, using the voltage-sensitive dye bis[3-propyl-5-oxoisoxazol-4-yl]pentamethine oxanol and the delta pH-sensitive fluorescent dye 9-aminoacridine respectively. A pre-existing small delta pH (inside acidic) was detected in the synaptic vesicles, but no additional significant contribution by MgATP to delta pH was observed. In contrast, delta psi (inside positive) increased substantially upon addition of MgATP. This ATP-dependent delta psi was reduced by thiocyanate anion (SCN-), a delta psi dissipator, or carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), a protonmotive-force dissipator. Correspondingly, a substantially larger glutamate uptake occurred in the presence of MgATP, which was inhibited by SCN- and FCCP. A nonhydrolysable analogue of ATP, adenosine 5'-[beta gamma-methylene]triphosphate, did not substitute for ATP in either delta psi generation or glutamate uptake. The results support the hypothesis that a H+-pumping ATPase generates a protonmotive force in the synaptic vesicles at the expense of ATP hydrolysis, and the protonmotive force thus formed provides a driving force for the vesicular glutamate uptake. The delta psi generation by ATP hydrolysis was not affected by orthovanadate, ouabain or oligomycin, but was inhibited by N-ethylmaleimide, quercetin, trimethyltin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid. These results indicate that the H+-pumping ATPase in the synaptic vesicle is similar to that in the chromaffin granule, platelet granule and lysosome.     

Characterization of a H+-ATPase in rat brain synaptic vesicles. Coupling to L-glutamate transport

Synaptic vesicles contain a H+-ATPase that generates a proton electrochemical gradient (delta mu H+) required for the uptake of neurotransmitters into the organelles. In this study, the synaptic vesicle H+-ATPase was examined for structural and functional similarities with other identified ATPases that generate a delta mu H+ across membranes. The synaptic vesicle H+-ATPase displayed immunological similarity with the 115-, 72-, and 39-kDa subunits of a vacuolar-type H+-ATPase purified from chromaffin granules. Functionally, the ATP-dependent H+ pumping across synaptic vesicles and ATP hydrolysis were sensitive to the sulfhydryl-modifying reagents, N-ethylmaleimide and 4-chloro-7-nitrobenz-2-oxa-1,3-diazole, at concentrations known to affect vacuolar-type H+-ATPases. In addition, as with vacuolar-type H+-ATPases, the presence of NO3-, SO4(2-), or F- inhibited the generation of a delta mu H+, but addition of vanadate or oligomycin had no effect. The delta mu H+ is a function of the pH gradient (delta pH) and membrane potential (delta psi sv) across the synaptic vesicle. Acidification (delta pH) of the synaptic vesicle interior was enhanced in the presence of permeant anions, such as Cl-, or the K+ ionophore, valinomycin. In the absence of permeant anions, the H+-ATPase generated a delta psi sv that effected the transport of L-glutamate into the synaptic vesicles. Dissipation of delta psi sv by incubation with increased external Cl- or nigericin resulted in the abolition of glutamate uptake, despite the continued maintenance of a delta mu H+ across the synaptic vesicle as a substantial delta pH. The results suggest that the synaptic vesicle H+-ATPase is of a vacuolar type and energizes the uptake of anionic glutamate by virtue of the delta psi sv component of the delta mu H+ it generates.     

Electrochemical potential of protons in vesicles reconstituted from purified, proton-translocating adenosine triphosphatase.

Measurements were made of the difference in the electrochemical potential of protons (delta-mu H+) across the membrane of vesicles restituted from the ATPase complex (TF0.F1) purified from a thermophilic bacterium and P-lipids. Two fluorescent dyes, anilinonaphthalene sulfonate (ANS) and 9-aminoacridine (9AA) were used as probes for measuring the membrane potential (delta psi) and pH difference across the membrane (delta pH), respectively. In the presence of Tris buffer the maximal delta psi ans no delta pH were produced, while in the presence of the permeant anion NO-3 the maximal delta pH and a low delta psi were produced by the addition of ATP. When thATP concentration was 0.24 mm, the delta psi was 140-150 mV (positive inside) in Tris buffer, and the delta pH was 2.9-3.5 units (acidic inside) in the presence of NO-3. Addition of a saturating amount of ATP produced somewhat larger delta psi and delta pH values, and the delta -muH+attained was about 310mV. By trapping pH indicators in the vesicles during their reconstitution it was found that the pH inside the vesicles was pH 4-5 during ATP hydrolysis. The effects of energy transfer inhibitors, uncouplers, ionophores, and permeant anions on these vesicles were studied.     

Ion-dependent generation of the electrochemical proton gradient delta muH+ in reconstituted plasma membrane vesicles from the yeast Metschnikowia reukaufii

Plasma membrane vesicles were reconstituted by freezing and thawing of purified plasma membrane fraction from the yeast Metschnikowia reukaufii and phosphatidylcholine (type II-S from Sigma). The reconstituted plasma membrane vesicles generated a proton gradient (acidic inside) upon addition of ATP in presence of alkali cations. delta pH generation was most efficient when K+ was present both outside and inside the plasma membrane vesicles. Both ATPase activity and proton translocation in plasma membrane vesicles were inhibited by orthovanadate (50% inhibition at 100 microM). Plasma membrane vesicles reconstituted without added phosphatidylcholine generated in addition to delta pH, also an electrical potential difference delta psi (inside positive). Delta psi generation exhibited no K+ specificity. 50 microM dicyclohexylcarbodiimide inhibited completely delta psi generation whereas the K+-channel blocker quinine (5 microM) caused an 8-fold increase of delta psi. The proton gradient was much less affected by the agents. Taking into account the K+-dependent stimulation of the plasma membrane ATPase of M. reukaufii, these results further support the conclusion that the ATPase operates as a partially electrogenic H+/K+ exchanger, as was also suggested for other yeast plasma membrane ATPases.     

Hydrogenosomes in the rumen protozoon Dasytricha ruminantium Schuberg.

The vacuo-lysosomes of Hevea brasiliensis (rubber tree) constitute a suitable model system for the study of active transport and energization at the level of the membrane of plant vacuoles. The pH gradient (delta pH) and the membrane potential (delta psi) of vacuo-lysosomes were determined by means of the weak base methylamine and the lipophilic cation tetraphenylphosphonium. The values obtained depended strongly on the experimental conditions such as medium pH or K+ concentration. Under experimental conditions, i.e., pH 7.5 outside and low K+, the delta pH amounts to about 0.9 unit, interior acid, and the delta psi to -120 mV, interior negative. The delta psi is presumably caused by the imposed K+ gradient, and the internal acidification might be a consequence of the passive proton inflow along the electric field. This explanation is sustained by the ineffectiveness of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in destroying the delta pH and delta psi, whereas higher K+ concentration decreased both. Under conditions existing in vivo, the membrane potential might be significantly lower. The presence of ATP increased the acidification of the intravesicular space by 0.5pH unit to a delta pH of up to 1.4 and shifts the membrane potential at least 60mV to a more positive value. The change of the protonmotive potential did not occur with ADP; the pH-dependence of the change was identical with the pH-dependence of a vacuo-lysosomal membrane-bound ATPase, and the effect of ATPase was prevented by the presence of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The change of protonmotive potential difference, brought about by the ATPase, was at least 90 mV. This is evidence that a vacuo-lysosomal ATPase in plants can function as an electrogenic proton pump that transfers protons into the vacuo-lysosomal space.     

Isolation of sealed plasma membrane vesicles from Phytophthora megasperma f. sp. glycinea. I. Characterization of proton pumping and ATPase activity

Sealed vesicles were isolated from a plant pathogenic fungus Phytophthora megasperma f. sp. glycinea using a modification of a method previously developed for plant plasma membrane vesicle isolation. Vanadate-sensitive, proton pumping microsomal membrane vesicles were resolved on a linear sucrose density gradient and found to comigrate with a vanadate-sensitive ATPase. Both the proton pumping and ATPase activity of these vesicles had a pH optimum of 6.5 and demonstrated similar properties with respect to substrate specificity and inhibitor sensitivity. These properties were in agreement with previously published data on the Phytophthora plasma membrane ATPase. In contrast with previous reports there was no K+ stimulation of the plasma membrane ATPase and the Km for Mg:ATP (1:1 concentration ratio) was higher (2.5 mM). A comparison of anion (potassium salts) effects upon delta pH and delta psi formation in sealed Phytophthora plasma membrane vesicles revealed a correspondence between the relative ability of anions to stimulate proton transport and to reduce delta psi. The relative order for this effect was KCl greater than KBr much greater than KMes, KNO3, KClO3, K2SO4. This study presents a method for the isolation of sealed vesicles from Phytophthora hyphae. It also provides basic information on the plasma membrane H+-ATPase and its associated proton pumping activity.     

Electrogenicity of the Na+-ATPase from the marine microalga Tetraselmis (Platymonas) viridis and associated H+ countertransport

Sodium accumulation by the Na+-ATPase in the plasma membrane (PM) vesicles isolated from the marine alga Tetraselmis (Platymonas) viridis was shown to be accompanied by deltapsi generation across the vesicle membrane (positive inside) and H+ efflux from the vesicle lumen. Na+ accumulation was assayed with 22Na+; deltapsi generation was detected by recording absorption changes of oxonol VI; H+ efflux was monitored as an increase in fluorescence intensity of the pH indicator pyranine loaded into the vesicles. Both ATP-dependent Na+ uptake and H+ ejection were increased by the H+ ionophore carbonyl cyanide m-chlorophenylhydrazone (CICCP) while deltapsi was collapsed. The lipophilic anion tetraphenylboron ion (TPB-) inhibited H+ ejection from the vesicles and abolished deltapsi. Based on the effects of CICCP and TPB- on H+ ejection and deltapsi generation, the conclusion was drawn that H+ countertransport observed during Na+-ATPase operation is a secondary event energized by the electric potential which is generated in the course of Na+ translocation across the vesicle membrane. Increasing Na+ concentrations stimulated H+ efflux and caused the decrease in the deltapsi observed, thus indicating that Na+ is likely a factor controlling H+ permeability of the vesicle membrane.     

Kinetic properties of electrogenic Na+/H+ antiport in membrane vesicles from an alkalophilic Bacillus sp.

The effects of imposed proton motive force on the kinetic properties of the alkalophilic Bacillus sp. strain N-6 Na+/H+ antiport system have been studied by looking at the effect of delta psi (membrane potential, interior negative) and/or delta pH (proton gradient, interior alkaline) on Na+ efflux or H+ influx in right-side-out membrane vesicles. Imposed delta psi increased the Na+ efflux rate (V) linearly, and the slope of V versus delta psi was higher at pH 9 than at pH 8. Kinetic experiments indicated that the delta psi caused a pronounced increase in the Vmax for Na+ efflux, whereas the Km values for Na+ were unaffected by the delta psi. As the internal H+ concentration increased, the Na+ efflux reaction was inhibited. This inhibition resulted in an increase in the apparent Km of the Na+ efflux reaction. These results have also been observed in delta pH-driven Na+ efflux experiments. When Na(+)-loaded membrane vesicles were energized by means of a valinomycin-induced inside-negative K+ diffusion potential, the generated acidic-interior pH gradients could be detected by changes in 9-aminoacridine fluorescence. The results of H+ influx experiments showed a good coincidence with those of Na+ efflux. H+ influx was enhanced by an increase of delta psi or internal Na+ concentration and inhibited by high internal H+ concentration. These results are consistent with our previous contentions that the Na+/H+ antiport system of this strain operates electrogenically and plays a central role in pH homeostasis at the alkaline pH range.     

Generation of Na+ electrochemical potential by the Na+-motive NADH oxidase and Na+/H+ antiport system of a moderately halophilic Vibrio costicola

Cells of Vibrio costicola at pH 8.5 generate both membrane potential (inside negative) and delta pH (inside acidic) in the presence of a proton conductor, carbonyl cyanide m-chlorophenylhydrazone (CCCP). The generation of CCCP-resistant membrane potential was inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide that is known to inhibit the Na+-motive NADH oxidase of Vibrio alginolyticus. NADH oxidase, but not lactate oxidase, of inverted membrane vesicles prepared from V. costicola required Na+ for a maximum activity and was inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide. By the oxidation of NADH, inverted membrane vesicles generated concentration gradients of Na+ across the membrane, whose magnitude was always larger than that of delta pH by about 50 mV. In contrast, magnitudes of delta pH and Na+ concentration gradients generated by the oxidation of lactate were similar. Na+ translocation in the presence of lactate was inhibited by CCCP but little affected by valinomycin. On the other hand, Na+ translocation in the presence of NADH was resistant to CCCP and stimulated by valinomycin. Amiloride, an inhibitor for a eucaryotic Na+/H+ antiport system, inhibited the lactate-dependent Na+ translocation but had little effect on the NADH-dependent Na+ translocation. These results indicate that a primary event of lactate oxidation is the translocation of H+, which then causes the generation of Na+ concentration gradients via the secondary Na+/H+ antiport system. We conclude that the NADH oxidase of V. costicola translocates Na+ as an immediate result of respiration, leading to the generation of Na+ electrochemical potential.     

Active electrogenic transport H+ in plasma membrane vesicles of cow parsnip phloem cells

Generation of electric (delta psi) and chemical (delta pH) components of electrochemical proton gradient delta muH+, in plasma membrane vesicles of Heracleum sosnovskyi phloem cells was investigated. ATP-dependent generation of delta psi at pH 6.0 in the presence of Mg2+ and K+ was established with the help of fluorescent probes AU+ and ANS-. Protonophore CCCP and proton ATPase inhibitor DCCD suppressed generation, whereas oligomycin, the inhibitor of mitochondrial ATPases did not affect it. Measurings of delta psi value indicated its oscillations within the limits from 10 to 60 mV. ATP-dependent generation of delta pH was established by means of fluorescent probe 9-AA. The effect was eliminated by CCCP and stimulated by K+, that may testify to the transformation of a part of delta psi into delta pH at antiport H+/K+. Existence of H+-ATPase in the plasma membranes of higher plant cells insuring generation of delta muH+ is supposed.     

On the subunit composition of the Neurospora plasma membrane H+-ATPase

The resolution-reconstitution approach has been employed in order to gain information as to the subunit composition of the Neurospora plasma membrane H+-ATPase. Proteoliposomes prepared from sonicated asolectin and a highly purified, radiolabeled preparation of the 105,000-dalton hydrolytic moiety of the H+-ATPase by a freeze-thaw procedure catalyze ATP hydrolysis-dependent proton translocation as indicated by the extensive 9-amino-6-chloro-2-methoxyacridine fluorescence quenching that occurs upon the addition of MgATP to the proteoliposomes, and the reversal of this quenching induced by the H+-ATPase inhibitor, vanadate, and the proton conductors, carbonyl cyanide m-chlorophenylhydrazone and nigericin plus K+. ATP hydrolysis is tightly coupled to proton translocation into the liposomes as indicated by the marked stimulation of ATP hydrolysis by carbonyl cyanide m-chlorophenylhydrazone and nigericin plus K+. The maximum stimulation of ATPase activity by proton conductors is about 3-fold, which indicates that at least two-thirds of the hydrolytically active ATPase molecules present in the reconstituted preparation are capable of translocating protons into the liposomes. Furthermore, as estimated by the extent of protection of the reconstituted 105,000-dalton hydrolytic moiety against tryptic degradation by vanadate in the presence of Mg2+ and ATP, the fraction of the total population of ATPase molecules that are hydrolytically active is at least 91%. Taken together, these data indicate that at least 61% of the ATPase molecules present in the reconstituted preparation are able to catalyze proton translocation. This information allows an estimation of the amount of any polypeptide in the preparation that must be present in order for that polypeptide to qualify as a subunit that is required for proton translocation in addition to the 105,000-dalton hydrolytic moiety, and an analysis of the radiolabeled ATPase preparation by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea rules out the involvement of any such polypeptides larger than 2,500 daltons. This indicates that the Neurospora plasma membrane H+-ATPase has no subunits even vaguely resembling any that have been found to be associated with other transport ATPases and that if this enzyme has any subunits at all other than the 105,000-dalton hydrolytic moiety, they must be very small.     

Rat liver plasma membrane Ca2+- or Mg2+-activated ATPase. Evidence for proton movement in reconstituted vesicles

The Ca2+- or Mg2+-activated ATPase from rat liver plasma membrane was partly purified by treatments with sodium cholate and lysophosphatidylcholine, and by isopycnic centrifugation on sucrose gradients. The ATPase activity had high sensitivity to detergents, poor nucleotide specificity and broad tolerance for divalent cations. It was insensitive to mitochondrial ATPase inhibitors such as oligomycin and to transport ATPase inhibitors such as vanadate and ouabain. Using the cholate dialysis procedure, the partly purified enzyme was incorporated into asolectin vesicles. Upon addition of Mg2+-ATP, fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine (ACMA) was observed. The quenching was abolished by a protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Asolectin vesicles or purified ATPase alone failed to promote quenching. These data suggest that the Ca2+- or Mg2+-activated ATPase from rat liver plasma membrane is able of H+-translocation coupled to ATP hydrolysis.     

Existence of an adenosine 5'-triphosphate dependent proton translocase in bovine neurosecretory granule membrane

The addition of ATP to bovine neurohypophysial secretory granules suspended in isotonic sucrose medium induces a positive polarization, delta psi, of their interior without affecting their internal pH. In KCl-containing media, ATP failed to generate large delta psi but induced a pH gradient (delta pH; interior acidic). These observations are consistent with the existence in the neurosecretory granule membrane of an ATP-dependent inward electrogenic H+ translocase (H+ pump), capable in KCl-containing media of acidifying the granule matrix by H+-Cl- cotransport. The delta psi and delta pH generated by the H+ pump, defined as the ATP-induced changes sensitive to the H+ ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), were blocked by N,N'-dicyclohexylcarbodiimide, an inhibitor of all H+ pumps, and were insensitive to oligomycin, a mitochondrial ATPase inhibitor. In sucrose medium, measurements were complicated by a Donnan equilibrium reflecting the presence in the granule of peptide hormones and neurophysins which resulted in a CCCP-resistant resting delta pH. In KCl-containing media, the Donnan equilibrium was destroyed since the membrane is permeable to cations, but under these conditions a CCCP-resistant K+-diffusion potential was observed. The ATP-induced delta psi was also monitored by the extrinsic fluorescent probe bis(3-phenyl-5-oxoisoxazol-4-yl)pentamethine oxonol. The hypothesis of a granule H+ pump is further supported by the presence of an oligomycin-resistant ATPase in the preparation and the ultrastructural localization of such an activity on the granule membrane. The H+ pump has been found in both newly formed and aged neurosecretory granules. Its possible physiological function is discussed with reference to that of chromaffin granules, with which it has many similarities.     

Spectrophotometric measurements of transmembrane potential and pH gradients in chromaffin granules

The electrical potential (delta psi) and proton gradient (alpha pH) across the membranes of isolated bovine chromaffin granules and ghosts were simultaneously and quantitatively measured by using the membrane- permeable dyes 3,3'dipropyl-2,2'thiadicarbocyanine (diS-C3-(5)) to measure delta psi and 9-aminoacridine or atebrin to measure delta pH. Increases or decreases in the delta psi across the granular membrane could be monitored by fluorescence or transmittance changes of diS-C3- (5). Calibration of the delta psi was achieved by utilization of the endogenous K+ and H+ gradients, and valinomycin or carbonyl cyanide-p- trifluoromethoxyphenylhydrazone (FCCP), respectively, with the optical response of diS-C3-(5) varying linearly with the Nernst potential for H+ and K+ over the range -60 to +90 mV. The addition of chromaffin granules to a medium including 9-aminoacridine or atebrin resulted in a rapid quenching of the dye fluorescence, which could be reversed by agents known to cause collapse of pH gradients. From the magnitude of the quenching and the intragranular water space, it was possible to calculate the magnitude of the alpha pH across the chromaffin granule membrane. The time-course of the potential-dependent transmittance response of diS-C3-(5) and the delta pH-dependent fluorescence of the acridine dyes were studied simultaneously and quantitatively by using intact and ghost granules under a wide variety of experimental conditions. These results suggest that membrane-permeable dyes provide an accurate method for the kinetic measurement of delta pH and delta psi in an amine containing subcellular organelle.     

The sodium cycle. I. Na+-dependent motility and modes of membrane energization in the marine alkalotolerant vibrio Alginolyticus

Respiration, membrane potential generation and motility of the marine alkalotolerant Vibrio alginolyticus were studied. Subbacterial vesicles competent in NADH oxidation and delta psi generation were obtained. The rate of NADH oxidation by the vesicles was stimulated by Na+ in a fashion specifically sensitive to submicromolar HQNO (2-heptyl-4-hydroxyquinoline N-oxide) concentrations. The same amounts of HQNO completely suppressed the delta psi generation. Delta psi was also inhibited by cyanide, gramicidin D and by CCCP + monensin. CCCP (carbonyl cyanide m-chlorophenylhydrazone) added without monensin exerted a much weaker effect on delta psi. Na+ was required to couple NADH oxidation with delta psi generation. These findings are in agreement with the data of Tokuda and Unemoto on Na+-motive NADH oxidase in V. alginolyticus. Motility of V. alginolyticus cells was shown to be (i) Na+-dependent, (ii) sensitive to CCCP + monensin combination, whereas CCCP and monensin, added separately, failed to paralyze the cells, (iii) sensitive to combined treatment by HQNO, cyanide or anaerobiosis and arsenate, whereas inhibition of respiration without arsenate resulted only in a partial suppression of motility. Artificially imposed delta pNa, i.e., addition of NaCl to the K+ -loaded cells paralyzed by HQNO + arsenate, was shown to initiate motility which persisted for several minutes. Monensin completely abolished the NaCl effect. Under the same conditions, respiration-supported motility was only slightly lowered by monensin. The artificially-imposed delta pH, i.e., acidification of the medium from pH 8.6 to 6.5 failed to activate motility. It is concluded that delta mu Na+ produced by (i) the respiratory chain and (ii) an arsenate-sensitive anaerobic mechanism (presumably by glycolysis + Na+ ATPase) can be consumed by an Na+ -motor responsible for motility of V. alginolyticus.     

Proton motive force-dependent and -independent protein translocation revealed by an efficient in vitro assay system of Escherichia coli

Inverted membrane vesicles prepared from Escherichia coli spheroplasts were fractionated by means of sucrose gradient centrifugation, and a vesicle preparation exhibiting efficient and quantitative translocation of secretory proteins was obtained. The translocation of OmpA and an uncleavable model protein, uncleavable OmpF-Lpp, took place almost completely in 2-3 min, whereas that of OmpF-Lpp, a chimeric secretory protein, required 20 min for completion. The requirement of the proton motive force (delta muH+) for in vitro translocation was then examined with these three proteins. The translocation of all these proteins was significantly inhibited by the addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP) or when stripped membrane vesicles lacking F1-ATPase were used, suggesting that delta muH+ generally participates in the translocation reaction. The inhibition was complete with OmpF-Lpp, whereas significant amounts of uncleavable OmpF-Lpp and OmpA were translocated at a slower rate even with the stripped membrane vesicles in the presence of a high concentration of carbonyl cyanide m-chlorophenylhydrazone. The delta muH+-independent translocation was inhibited by a nonhydrolyzable ATP analogue. These results indicate that although translocation of OmpF-Lpp obligatory requires delta muH+, the latter two proteins can be translocated in not only a delta muH+-dependent manner but also a delta mu H+-independent manner.     

Role of an energized inner membrane in mitochondrial protein import. Delta psi drives the movement of presequences.

The transport of precursor proteins into mitochondria requires an energized inner membrane. We report here that the import of various precursor proteins showed a differential sensitivity to treatment of the mitochondria with the uncoupler carbonyl cyanide m-chlorophenylhydrazone. The differential inhibition by carbonyl cyanide m-chlorophenylhydrazone was not influenced by the length of the precursor, the presence of mature protein parts, or the folding state of the precursor but was specific for the presequence. Moreover, only the membrane potential delta psi and not the total proton motive force was required for the transport of precursors, indicating that protein translocation across the inner membrane is not driven by a movement of protons. We conclude that delta psi (negative inside) is needed for the translocation of the positively charged presequences, possibly via an electrophoretic effect.     

A vacuolar-type proton pump in a vesicle fraction enriched with potassium transporting plasma membranes from tobacco hornworm midgut

Mg-ATP dependent electrogenic proton transport, monitored with fluorescent acridine orange, 9-aminoacridine, and oxonol V, was investigated in a fraction enriched with potassium transporting goblet cell apical membranes of Manduca sexta larval midgut. Proton transport and the ATPase activity from the goblet cell apical membrane exhibited similar substrate specificity and inhibitor sensitivity. ATP and GTP were far better substrates than UTP, CTP, ADP, and AMP. Azide and vanadate did not inhibit proton transport, whereas 100 microM N,N'-dicyclohexylcarbodiimide and 30 microM N-ethylmaleimide were inhibitors. The pH gradient generated by ATP and limiting its hydrolysis was 2-3 pH units. Unlike the ATPase activity, proton transport was not stimulated by KCl. In the presence of 20 mM KCl, a proton gradient could not be developed or was dissipated. Monovalent cations counteracted the proton gradient in an order of efficacy like that for stimulation of the membrane-bound ATPase activity: K+ = Rb+ much greater than Li+ greater than Na+ greater than choline (chloride salts). Like proton transport, the generation of an ATP dependent and azide- and vanadate-insensitive membrane potential (vesicle interior positive) was prevented largely by 100 microM N,N'-dicyclohexylcarbodiimide and 30 microM N-ethylmaleimide. Unlike proton transport, the membrane potential was not affected by 20 mM KCl. In the presence of 150 mM choline chloride, the generation of a membrane potential was suppressed, whereas the pH gradient increased 40%, indicating an anion conductance in the vesicle membrane. Altogether, the results led to the following new hypothesis of electrogenic potassium transport in the lepidopteran midgut. A vacuolar-type electrogenic ATPase pumps protons across the apical membrane of the goblet cell, thus energizing electroneutral proton/potassium antiport. The result is a net active and electrogenic potassium flux.     

The properties of arginine transport in vacuolar membrane vesicles of Neurospora crassa

We have measured the uptake of arginine into vacuolar membrane vesicles from Neurospora crassa. Arginine transport was found to be dependent on ATP hydrolysis, Mg2+, time, and vesicle protein with transported arginine remaining unmodified after entry into the vesicles. The Mg2+ concentration required for optimal arginine transport varied with the ATP concentration so that maximal transport occurred when the MgATP2- concentration was at a maximum and the concentrations of free ATP and Mg2+ were at a minimum. Arginine transport exhibited Michaelis-Menten kinetics when the arginine concentration was varied (Km = 0.4 mM). In contrast, arginine transport did not follow Michaelis-Menten kinetics when the MgATP2-concentration was varied (S0.5 = 0.12 mM). There was no inhibition of arginine transport when glutamine, ornithine, or lysine were included in the assay mixture. In contrast, arginine transport was inhibited 43% when D-arginine was present at a concentration 16-fold higher than that of L-arginine. Measurements of the internal vesicle volume established that arginine is concentrated 14-fold relative to the external concentration. Arginine transport was inhibited by dicyclohexylcarbodiimide, carbonyl cyanide m-chlorophenyl-hydrazone, and potassium nitrate (an inhibitor of vacuolar ATPase activity). Inhibitors of the plasma membrane or mitochondrial ATPase such as sodium vanadate or sodium azide did not affect arginine transport activity. In addition, arginine transport had a nucleoside triphosphate specificity similar to that of the vacuolar ATPase. These results suggest that arginine transport is dependent on vacuolar ATPase activity and an intact proton channel and proton gradient.     

The sinusoidal domain of the plasma membrane of rat hepatocytes contains an amiloride-sensitive Na+/H+ antiport

ATP-dependent trapping of [14C]methylamine was demonstrated in vesicles selectively derived from the sinusoidal plasma membrane of rat hepatocytes; activity was lacking in vesicles prepared from the canalicular domain of the plasma membrane of rat hepatocytes. The proton movement was inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, strophanthidin, vanadate, amiloride, and absence of sodium. 22Na efflux from sinusoidal membrane vesicles increased inversely to extravesicular pH. The results indicate that the sinusoidal plasma membrane of rat hepatocytes contains a Na+/H+ antiport.