Na/H and k/h antiport in root membrane vesicles isolated from the halophyte atriplex and the glycophyte cotton

Proton fluxes have been followed into and out of membrane vesicles isolated from the roots of the halophyte Atriplex nummularia and the glycophyte Gossypium hirsutum, with the aid of the ΔpH probe [¹⁴C]methylamine. Evidence is presented for the operation of Na⁺/H⁺ and K⁺/H⁺ antiporters in the membranes of both plants. Cation supply after a pH gradient has been set up across the vesicle membrane (either as a result of providing ATP to the H⁺-ATPase or by imposing an artificial pH gradient) brings about dissipation of the ΔpH, but does not depolarize the membrane potential as observed in similar experiments, but in the absence of Cl⁻, using the ΔΨ probe SCN⁻. Cation/H⁺ exchange is thus indicated. This exchange is not due to nonspecific electric coupling, nor to competition for anionic adsorption sites on the membrane, nor to inhibition of the H⁺-ATPase; coupling of the opposed cation and H⁺ fluxes by a membrane component is the most likely explanation. Saturation kinetics have been observed for both Na⁺/H⁺ and K⁺/H⁺ antiport in Atriplex. Moreover, additive effects are obtained when Na⁺ is supplied together with saturating concentrations of K⁺, and vice versa, suggesting that separate antiporters for Na⁺ and for K⁺ may be operating. In the case of both Atriplex and Gossypium evidence was obtained suggesting the presence of antiporters in both plasmalemma and tonoplast.     

Evidence for a specific glutamate/h cotransport in isolated mesophyll cells

Mechanically isolated Asparagus sprengeri Regel mesophyll cells were suspended in 1 millimolar CaSO₄. Immediate alkalinization of the medium occured on the addition of 1 millimolar concentrations of l-glutamate (Glu) and its analog l-methionine-d,l-sulfoximine (l-MSO). d-Glu and the l isomers of the protein amino acids did not elicit alkalinization. l-Glu dependent alkalinization was transient and acidification resumed after approximately 30 to 45 minutes. At pH 6.0, 5 millimolar l-Glu stimulated initial rates of alkalinization that varied between 1.3 to 4.1 nmol H⁺/10⁶ cells·minute. l-Glu dependent alkalinization was saturable, increased with decreasing pH, was inhibited by carbonyl cyanide-p-trichloromethoxyphenyl hydrazone (CCCP), and was not stimulated by light. Uptake of l-[U-¹⁴C]glutamate increased as the pH decreased from 6.5 to 5.5, and was inhibited by l-MSO. l-Glu had no influence on K⁺ efflux. Although evidence for multiple amino acid/proton cotransport systems has been found in other tissues, the present report indicates that a highly specific l-Glu/proton uptake process is present in Asparagus mesophyll cells.     

Characterization of a calcium/proton antiporter and an electrogenic calcium transporter in membrane vesicles from Azotobacter vinelandii

Ca2+ transport across the membrane of vesicles derived from Azotobacter vinelandii was studied in the absence of respiration or functioning ATPase. Two facilitated diffusion systems were found. One, an electroneutral Ca2+/2H+ antiporter, responded to an artificially imposed deltapH, was heat-labile, and was insensitive to low concentrations of ruthenium red and lanthanides. The second, an electrogenic transporter, responded to an electrical membrane potential, was heat-stable, was inhibited by ruthenium red, lanthanides, monovalent cations, and certain anions. In vivo, when coupled to the protonmotive force, the systems would provide for the cell: (i) a mechanism to keep intracellular Ca2+ concentration low (Ca2+/2H+ antiporter); (ii) a mechanism for Ca2+ entry (electrogenic transporter).     

Plasma membrane vesicles of opposite sidedness from soybean hypocotyls by preparative free-flow electrophoresis

Absolute orientations (sidedness) of plasma membrane vesicles obtained in highly purified fractions by preparative free-flow electrophoresis and by aqueous two-phase partition were determined based on ATPase latency and morphological criteria. Free-flow electrophoresis yielded two plasma membrane fractions. One, the least electronegative and designated fraction `E,' was pure plasma membrane. The other, more electronegative and designated fraction `C,' was heavily contaminated by various other cellular membranes. Plasma membrane vesicles from both fraction C and fraction E partitioned into the upper phase with aqueous two-phase partitioning. Purified plasma membrane obtained from microsomes by two-phase partition (upper phase) when subjected to free-flow electrophoresis also yielded two fractions, one fraction co-migrated with fraction C and another fraction co-migrated with fraction E. Both fractions exhibited an ATPase activity sensitive to vanadate and insensitive to nitrate and azide. ATPase activity was used as a structure-linked latency marker for the inner membrane surface. Concanavalin A binding (linked to peroxidase) was used as an imposed electron microscope marker for the outer membrane surface. Fraction E vesicles showed low ATPase latency (two-fold or less) and weak reactivity with concanavalin A peroxidase. In contrast, fraction C vesicles were characterized by much greater latencies upon detergent treatment (sevenfold) and a strong reaction with concanavalin A peroxidase. Two-phase partition as the initial procedure for plasma membrane isolation, yielded mixtures of vesicles of both inside out and right-side out orientation. Free-flow electrophoresis resolved the plasma membrane isolates into vesicles from fraction C which were right-side out (cytoplasmic side in), and vesicles from fraction E which were wrong-side out (cytoplasmic side out). Therefore, the two methods used in series, provided highly purified membrane preparations of apparently homogenous vesicles of opposite known absolute orientations.     

Evidence for a zinc/proton antiporter in rat brain

The data presented in this paper are consistent with the existence of a plasma membrane zinc/proton antiport activity in rat brain. Experiments were performed using purified plasma membrane vesicles isolated from whole rat brain. Incubating vesicles in the presence of various concentrations of 65Zn2+ resulted in a rapid accumulation of 65Zn2+. Hill plot analysis demonstrated a lack of cooperativity in zinc activation of 65Zn2+ uptake. Zinc uptake was inhibited in the presence of 1 mM Ni2+, Cd2+, or CO2+. Calcium (1 mM) was less effective at inhibiting 65Zn2+ uptake and Mg2+ and Mn2+ had no effect. The initial rate of vesicular 65Zn2+ uptake was inhibited by increasing extravesicular H+ concentration. Vesicles preloaded with 65Zn2+ could be induced to release 65Zn2+ by increasing extravesicular H+ or addition of 1 mM nonradioactive Zn2+. Hill plot analysis showed a lack of cooperativity in H+ activation of 65Zn2+ release. Based on the Hill analyses, the stoichiometry of transport may include Zn2+/Zn2+ exchange and Zn2+/H+ antiport, the latter being potentially electrogenic. Zinc/proton antiport may be an important mode of zinc uptake into neurons and contribute to the reuptake of zinc to replenish presynaptic vesicle stores after stimulation.     

Active transport of Ca in membrane vesicles from pea: Evidence for a H/Ca antiport

Two non mitochondrial systems involved in ATP-dependent Ca²⁺ accumulation have been described and characterized in two membrane fractions from pea internodes purified on a metrizamide-sucrose discontinuous gradient. In the lighter membrane fraction an ATP-dependent Ca²⁺ accumulation system, which shows the characteristics of an ATP-dependent H⁺/Ca²⁺ antiport, predominates. This system is inhibited by FCCP and nigericin and stimulated by 50 mM KCl. It is saturated by 0.8–1.0 mM MgSO₄-ATP, strictly requires ATP and is severely inhibited by an excess of free Mg²⁺ or Mn²⁺. A second system of ATP-dependent Ca²⁺ accumulation, recovered mainly in the heavier membrane fraction, is insensitive to FCCP, is saturated by 8–10 mM MgSO₄-ATP, can utilize also ITP or other nucleoside triphosphates although at lower rate than ATP and is only scarcely affected by an excess of free Mg²⁺ or Mn²⁺. This system is interpreted as corresponding to the (Ca²⁺ + Mg²⁺)-ATPase described by Dieter, P. and Marmé, D. ((1980) Planta 150, 1–8).     

Na+-dependent transport of glycine in renal brush border membrane vesicles: Evidence for a single specific transport system

The uptake of glycine in rabbit renal brush border membrane vesicles was shown to consist of glycine transport into an intravesicular space. An Na⁺ electrochemical gradient (extravesicular>intravesicular) stimulated the initial rate of glycine uptake and effected a transient accumulation of intravesicular glycine above the steady-state value. This stimulation could not be induced by the imposition of a K⁺, Li⁺ or choline⁺ gradient and was enhanced as extravesicular Na⁺ was increased from 10 mM to 100 mM. Dissipation of the Na⁺ gradient by the ionophore gramicidin D resulted in diminished Na⁺-stimulated glycine uptake. Na⁺-stimulated uptake of glycine was electrogenic. Substrate-velocity analysis of Na⁺-dependent glycine uptake over the range of amino acid concentrations from 25 μM to 10 mM demonstrated a single saturable transport system with apparent K_m = 996 μM and V_max = 348 pmol glycine/mg protein per min. Inhibition observed when the Na⁺-dependent uptake of 25 μM glycine was inhibited by 5 mM extravesicular test amino acid segregated dibasic amino acids, which did not inhibit glycine uptake, from all other amino acid groups. The amino acids d-alanine, d-glutamic acid, and d-proline inhibited similarly to their l counterparts. Accelerative exchange of extravesicular [³H]glycine was demonstrated when brush border vesicles were preloaded with glycine, but not when they were preloaded with l-alanine, l-glutamic acid, or with l-proline. It is concluded that a single transport system exists at the level of the rabbit renal brush border membrane that functions to reabsorb glycine independently from other groups of amino acids.     

Evidence for tripeptide/H+ co-transport in rabbit renal brush-border membrane vesicles.

L-Phe-L-Pro-L-Ala is a tripeptide which is hydrolysable almost exclusively by dipeptidyl peptidase IV in rabbit renal brush-border membrane vesicles. In order to delineate the mechanism of the transport of an intact tripeptide across the brush-border membrane, we studied the characteristics of the uptake of [3H]Phe-Pro-Ala in membrane vesicles in which the activity of dipeptidylpeptidase IV was completely inhibited by treatment with di-isopropyl fluorophosphate. In these vesicles, uptake of radiolabel from the tripeptide was found to be Na(+)-independent, but was greatly stimulated by an inwardly directed H+ gradient. The H(+)-gradient-dependent radiolabel uptake appeared to be an active process, because the time course of uptake exhibited an overshoot phenomenon. The process was also electrogenic, being stimulated by an inside-negative membrane potential. Under the uptake-measurement conditions there was no detectable hydrolysis of [3H]Phe-Pro-Ala in the incubation medium when di-isopropyl fluorophosphate-treated membrane vesicles were used. Analysis of intravesicular contents revealed that the radiolabel inside the vesicles was predominantly (greater than 90%) in the form of intact tripeptide. These data indicate that the uptake of radiolabel from [3H]Phe-Pro-Ala in the presence of an inwardly directed H+ gradient represents almost exclusively uptake of intact tripeptide. Uphill transport of the tripeptide was also demonstrable in the presence of an inwardly directed Na+ or K+ gradient, but only if nigericin was added to the medium. Under these conditions, nigericin, an ionophore for Na+, K+ and H+, was expected to generate a transmembrane H+ gradient. Uptake of Phe-Pro-Ala in the presence of a H+ gradient was inhibited by di- and tri-peptides, but not by free amino acids. It is concluded that tripeptide/H+ co-transport is the mechanism of Phe-Pro-Ala uptake in rabbit renal brush-border membrane vesicles.     

Preparation of highly enriched photosystem II membrane vesicles by a non-detergent method

An improved, non-detergent, method for preparative isolation of PS II membrane vesicles from spinach chloroplasts is presented. Thylakoids (chlorophyll (Chl) a/b ratio 2.8, Chl/P700 435) were fractionated by Yeda press treatment and aqueous two-phase partition to yield inside-out vesicles (1) (chl a/b 2.2, chl/P700 700). These vesicles were subjected a sonication — phase partitioning procedure; steps of sonication of inside-out vesicles, while still present in a dextran-polyethylene glycol two-phase system were alternated by phase partition. These steps selectively removed P700-containing membrane fragments from the inside-out vesicles and yielded a membrane fraction with improved PS II purity (Chl a/b ratio 1.9, Chl/P700 1500) and retained oxygen evolving capacity (295 mol O₂ mg Chl^-1 h^-1).     

Phosphate transport in membrane vesicles from Escherichia coli

Escherichia coli strain AN710 possesses only the PIT system for phosphate transport. Membrane vesicles from this strain, which contain phosphate internally, perform exchange and active transport of phosphate. The energy for active transport is supplied by the respiratory chain with ascorbate-phenazine methosulphate as electron donor. To a lesser extent also the oxidation of d-lactate energizes phosphate transport; the oxidation of succinate is only marginally effective. Phosphate transport is driven by the proton-motive force and in particular by the pH gradient across the membrane. This view is supported by the observation that phosphate transport is stimulated by valinomycin, inhibited by nigericin and abolished by the uncoupler carbonyl cyanide m-chlorophenylhydrazone. Neither inhibitor affects phosphate exchange. The phosphate analogue arsenate inhibits both the exchange reaction and active transport. Both processes are stimulated by K⁺ and Mg²⁺, the highest activities being observed with both ions present.Membrane vesicles have also been isolated from Escherichia coli K10, a strain which possesses only a functional PST phosphate transport system. These vesicles perform neither exchange nor active transport of phosphate, although active transport of amino acids is observed in the presence of ascorbate-phenazine methosulphate or d-lactate.     

Evidence for carrier-mediated transport of glucocorticoids by human placental membrane vesicles

Glucocorticoid uptake by isolated placental membrane vesicles has been studied in an attempt to identify a membrane-mediated carrier mechanism. A preliminary communication from this laboratory has reported that uptake of the glucocorticoid corticosterone by these vesicles was a time-dependent, saturable, osmotically sensitive process (Fant, M.E., Harbison, R.D. and Harrison, R.W. (1979) J. Biol. Chem. 254, 6218-6221), but did not conclusively demonstrate a carrier mechanism. Further studies of labeled corticosterone uptake by placental vesicles are described herein which indicate that steroid uptake by these vesicles is a carrier-mediated process. We found that corticosterone uptake was temperature-sensitive, and an apparent phase-transition effect on the rate of uptake was seen to occur at approximately 16 degrees C. Treatment of the vesicles with phospholipase A2 and the sulfhydryl group attacker, p-chloromercuriphenylsulfonate, inhibited corticosterone uptake. In contrast to our previous findings in intact cells, neuraminidase treatment of membranes did not inhibit steroid uptake, perhaps indicating a species variation. Lastly, it was possible to show that corticosterone movement across the membrane exhibited countertransport, a phenomenon common only to carrier-mediated transport mechanisms. These studies show that placental vesicles accumulate corticosterone by a carrier-mediated mechanism.     

l- andd-alanine transport in brush border membrane vesicles from lepidopteran midgut: Evidence for two transport systems

Summary In brush border membrane vesicles from the midgut ofPhilosamia cynthia larvae (Lepidoptera) thel- andd-alanine uptake is dependent on a potassium gradient and on transmembrane electrical potential difference. Each isomer inhibits the uptake of the other form: inhibition ofl-alanine uptake byd-alanine is competitive, whereas inhibition ofd-alanine uptake byl-alanine is noncompetitive. Transstimulation experiments as well as the different pattern of specificity to cations suggest the existence of two transport systems. Kinetic parameters for the two transporters have been calculated both when K_out>K_in and K_out=K_in.d-alanine is actively transported also by the whole midgut, but it is not metabolized by the intestinal tissue.     

Evidence of glutathione transporter in rat brain synaptosomal membrane vesicles

Glutathione (GSH) transport was studied in synaptosomal membrane vesicles (SMV) of rat cerebral cortex. The present study shows that GSH uptake into SMV occurs very quickly in a time-dependent manner into an osmotically active intravesicular space. The initial rate of transport followed Michealis-Menten saturation kinetics with a K_m 4.5±0.8 μM that shows a high affinity of the transporter for GSH. Therefore GSH uptake in SMV occurs by a mediated transport system which can be activated by either an inward gradient of cations, like Na⁺ or K⁺, or membrane depolarization. These results, together with those obtained by valinomycin-induced K⁺ diffusion potential, indicate that GSH synaptosomal transport is electrogenic by a negative charge transfer. The increase of GSH uptake measured by trans-stimulation experiments confirms a GSH bidirectional mediated transport which seems susceptible of modulation by changes in ionic fluxes and in the membrane potential. These results may indicate a possible involvement of this transporter in the role suggested for GSH in synaptic neurotransmission; also considering that GSH precursor of neuroactive aminoacids (glyeine, glutamate), may contribute to regulate their level in synapses. Finally, a GSH transporter in synaptosomes may contribute to maintaining the GSH homeostasis in cerebral cortex, where decreases of GSH levels have been related to susceptibility to neuropathologies.