Effect of H+ on the kinetics of Na+-dependent amino acid transport in ehrlich ascites tumor cells: Evidence for H+ as an alternative substrate

The effects of H⁺ on the kinetics of α-aminoisobutyric acid (AIB) influx in Ehrlich ascites tumor cells have been investigated at different external Na⁺ concentrations. Elevation of [H⁺] in the presence of both high (154 mEq/l) and low (10 mEq/l) external Na⁺ leads to decreases in the maximum influx (J) and increases in the apparent Michaleis-Menten constant (K) for influx of AIB. In the virtual absence of external Na⁺ (0.96 ± 0.04 mEq/l), alterations in [H⁺] are without measurable effect on AIB flux. Furthermore, addition of AIB (10 mM) to cell suspensions (pH 5.90) stimulates H⁺ uptake by the cells in either the presence or absence of Na⁺. The data are consistent with two kinetic models for Na⁺-dependent amino acid transport: an order bireactant (Na⁺-binding necessary before AIB binding) system or a random bireactant system. Both models require that H⁺ serve as an alternative substrate for Na⁺. The consistency of the models was tested by fit to data from the present study (not used to evaluate the kinetic parameters) and by prediction of the pH dependence of Na⁺-dependent amino acid transport compared to earlier studies.     

Amino acid and22Na+ uptake in membrane vesicles from confluent simian virus 40 transformed Balb/c3T3 and Balb/c3T3

Summary The studies reported here were carried out to characterize further previously described changes in membrane localized amino acid transport associated with simian virus 40 transformation of the mammalian cell line, Balb/c3T3. Membrane vesicles were prepared from confluent cultures of both simian virus 40 transformed Balb/c3T3 (SV3T3) and the untransformed parent line, Balb/c3T3 (3T3). An initial, externally imposed out>in, 100mm Na⁺ gradient produces acceleration of early ingress of -aminoisobutyric acid (AIB) in vesicles from both cell lines, but transient, concentrative uptake (overshooting) only in SV3T3 vesicles. Early ingress ofl-leucine is also accelerated in SV3T3 vesicles by a Na⁺ gradient, and overshooting is also demonstrable.Na⁺-gradient independent AIB permeability of SV3T3 and 3T3 membranes was estimated using uptake data, a first order rate equation and measurements of vesicle size derived from quasi-elastic light-scattering studies. AIB permeability of SV3T3 membranes is greater than that of 3T3 membranes (113 Å/min and 43 Å/min, respectively), suggesting that overshooting in 3T3 vesicles is not attenuated by a Na⁺-independent AIB leak. Na⁺ permeability of the two membranes is similar, ruling out the possibility that a slower rate of Na⁺ equilibration across the SV3T3 membrane allows development of the overshoot.In SV3T3 vesicles the height of a Na⁺-gradient dependent overshoot varies with the initial [Na⁺] _o /[Na⁺] _i ratio, and [Na⁺] _o /[Na⁺] _i is linearly related to ln AIB uptake at overshoot peak/AIB uptake at equilibrium, consistent with the possibility that for [Na⁺] _o /[Na⁺] _i ratios in the range studied, AIB overshoot is energized by a constant proportion of the energy available from the initial electrochemical gradient for Na⁺.These results are consistent with the possibility that Na⁺-gradient dependent overshooting in SV3T3 vesicles is produced by Na⁺-amino acid carrier interactions resulting in either an increase in maximum transport velocity or an incrase in carrier affinity for AIB.Abbreviations used 3T3 Balb/c3T3 - SV3T3 simian virus 40 transformed Balb/c3T3 - AIB -aminoisobutyric acid     

Amino acid active transport and stimulation by substrates in the absence of a Na+ electrochemical potential gradient

Summary Uptake of -aminoisobutyric acid (AIB) was examined in Ehrlich ascites tumor cells treated with the cation-exchange ionophore nigericin (20 g/ml). Membrane voltages were measured using the voltage-sensitive dye diethyloxadicarbocyanine (DOCC). In normal phosphate-buffered media, nigericin changed the distribution ratios of Na⁺ and K⁺ (the ratio of intra- to extracellular concentrations) nearly to unity, but AIB was still accumulated to a distribution ratio of 9.0. When all but 40mm Na⁺ in the medium was replaced by choline, nigericin resulted in K⁺ loss and Na⁺ gain and both cation distribution ratios approached 2.8–3.4, as would be expected if both ions were distributing near electrochemical equilibrium with a membrane voltage in the range of –28 to –33 mV. This conclusion was supported by the observation that the addition of 5×10^–7 m valinomycin to the nigericin-treated cell suspension produced no change in DOCC absorbance. In spite of the apparent zero electrochemical potential gradients for Na⁺ and K⁺, AIB was accumulated to a distribution ratio of 5.4 in the low-Na⁺ medium. Addition of 0.1mm oubain or 50 m vanadate did not alter the extent of AIB accumulation as would have been expected if a large component of the membrane voltage were due to electrogenic operation of the (Na⁺+K⁺)-ATPase. Addition of lactate, pyruvate or glucose increased the AIB distribution ratios to 11.9, 9.4 and 15.3, respectively. The effect of glucose could be explained, at least in part, by an enhanced Na⁺ electrochemical potential gradient. However, neither lactate nor pyruvate produced any change either in membrane voltage or the intracellular Na⁺ concentration. Therefore, these results confirm the existence of a metabolic energy source which is coupled to AIB accumulation and operates in addition to the Na⁺ co-transport mechanism, and which is augmented by metabolic substrates such as lactate and pyruvate.     

A mathematical model for membrane transport of amino acid and Na+ in vesicles

Summary A model with a carrier having sites for both amino acid and Na⁺ can account for AIB (-aminoisobutyric acid) transport kinetics observed in membrane vesicles from SV3T3 (simian virus 40-tranformed Balb/c3T3 cells) and 3T3 (the parent cell line). The main feature of this cotransport model is that Na⁺ binding to carrier decreases the effectiveK _m for AIB transport, Na⁺ transport kinetics observed in both vesicle systems can be described by passive (possibly facilitated) diffusion. The lag of Na⁺ transport across the membrane compared to that for AIB, coupled to the Na⁺-dependent decrease in theK _m for AIB, accounts for the overshoot in intravesicular AIB observed for SV3T3 in the presence of an initial Na⁺ gradient. Extra-vesicular Na⁺ maintains a derease in theK _m for AIB influx before intra-vesicular Na⁺ has accumulated to balance it with a comparable decrease in theK _m for AIB efflux. 3T3 vesicles display little overshoot, and this finding can be explained mostly by a lower carrier affinity for Na⁺.     

Energetic basis of development of salt-tolerant transport in a moderately halophilic bacterium,Vibrio costicola

Active transport of -aminoisobutyric acid (AIB) in Vibrio costicola utilizes a system with affinity for glycine, alanine and, to some extent, methionine. AIB transport was more tolerant of high salt concentrations (3–4 M NaCl) in cells grown in the presence of 1.0 M NaCl than in those grown in the presence of 0.5 M NaCl. The former cells could also maintain much higher ATP contents than the latter in high salt concentrations.Transport kinetic studies performed with bacteria grown in 1.0 M NaCl revealed three effects of the Na⁺ ion: the first effect is to increase the apparent affinity (K _t) of the transport system for AIB at Na⁺ concentrations <0.2 M, the second to increase the maximum velocity (V _max) of transport (Na⁺ concentrations between 0.2 and 1.0 M), and the third to decrease the V _max without affectig K _t (Na⁺ concentrations >1.0 M). Cells grown in the presence of 0.5 M or 1.0 M NaCl had similar affinity for AIV. Thus, the differences in salt response of transport in these cells do not seem due to differences in AIB binding. Large, transport-inhibitory concentrations of NaCl resulted in efflux of AIB from cells preloaded in 0.5 M or 1.0 M NaCl, with most dramatic efflux occurring from the cells whose AIB transport was more salt-sensitive. Our results suggest that the degree to which high salt concentrations affect the transmembrane electrochemical energy source used for transport and ATP synthesis is an important determinant of salt tolerance.Abbreviations AIB -aminoisobutyric acid - pmf proton motive force     

Regulation of spermidine transport in l1210 cells

• 1.1. 1 mM 2-amino isobutyric add (AIB), glutamine or asparagine when preincubated for 3 hr with L1210 cells promoted a marked increase in the rate of spermidine uptake.
• 2.2. Cycloheximide also increased the transport rate and completely prevented the increase due to AIB.
• 3.3. Trifluoperazine and iso-H7 inhibited the uptake of spermidine, much less the uptake of AIB.
• 4.4. Adenosine promoted an increase in the uptake of AIB, a decrease in that of spermidine.
• 5.5. Hypotonic stress also increased the rate of spermidine transport. This modification was only partially prevented by cycloheximide.
• 6.6. Okadaic arid had no effect on this increase, whereas it prevented the increase of ODC activity.

     

Search for genes influencing the maintenance of the [<Emphasis Type="Italic">ISP</Emphasis><Superscript>+</Superscript>] prion-like antisuppressor determinant in yeast with the use of an insertion gene library

The prion-like determinant [ISP ⁺] manifests itself as an antisuppressor of certain sup35 mutations. To establish that [ISP ⁺] is indeed a new yeast prion, it is necessary to identify the gene that codes for the protein whose prion form is [ISP ⁺]. Analysis of the transformants obtained by transformation of an [ISP ⁺] strain with an insertion gene library revealed three genes controlling the [ISP ⁺] maintenance: UPF1, UPF2, and SFP1. SFP1 codes for a potentially prionogenic protein, which is enriched in Asn and Gln residues, and is thereby the most likely candidate for the [ISP ⁺] structural gene. UPF1 and UPF2 code for components of nonsense-mediated mRNA decay. The [ISP ⁺] elimination caused by UPF1 and UPF2 inactivation was reversible, and Upf1p and Upf2p were not functionally related to phosphatase Ppz1p, which influences the [ISP ⁺] manifestation. Possible mechanisms sustaining the influence of UPF1 and UPF2 on [ISP ⁺] maintenance are discussed.     

Halophytic NHXs confer salt tolerance by altering cytosolic and vacuolar K<Superscript>+</Superscript> and Na<Superscript>+</Superscript> in <Emphasis Type="Italic">Arabidopsis</Emphasis> root cell

While the role of the vacuolar NHX Na⁺/H⁺ exchangers in plant salt tolerance has been demonstrated on numerous occasions, their control over cytosolic ionic relations has never been functionally analysed in the context of subcellular Na⁺ and K⁺ homeostasis. In this work, PutNHX1 and SeNHX1 were cloned from halophytes Puccinellia tenuiflora and Salicornia europaea and transiently expressed in Arabidopsis wild type Col-0 and the nhx1 mutant. Phylogentic analysis, topological prediction, analysis of evolutionary conservation, the topology structure and analysis of hydrophobic or polar regions of PutNHX1 and SeNHX1 indicated that they are unique tonoplast Na⁺/H⁺ antiporters with characteristics for salt tolerance. As a part of the functional assessment, cytosolic and vacuolar Na⁺ and K⁺ in different root tissues and ion fluxes from root mature zone of Col-0, nhx1 and their transgenic lines were measured. Transgenic lines sequestered large quantity of Na⁺ into root cell vacuoles and also promoted high cytosolic and vacuolar K⁺ accumulation. Expression of PutNHX1 and SeNHX1 led to significant transient root Na⁺ uptake in the four transgenic lines upon recovery from salt treatment. In contrast, the nhx1 mutant maintained a prolonged Na⁺ efflux and the nhx1:PutNHX1 and nhx1:SeNHX1 lines started to actively pump Na⁺ out of the cell. Overall, our findings suggest that PutNHX1 and SeNHX1 improve Na⁺ sequestration in the vacuole and K⁺ retention in the cytosol and vacuole of root cells of Arabidopsis, and that they interact with other regulatory mechanisms to provide a highly orchestrated regulation of ionic relations among intracellular cell compartments.     

Molecular characterization and expression analysis of the Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger gene family in <Emphasis Type="Italic">Medicago truncatula</Emphasis>

One important mechanism plants use to cope with salinity is keeping the cytosolic Na⁺ concentration low by sequestering Na⁺ in vacuoles, a process facilitated by Na⁺/H⁺ exchangers (NHX). There are eight NHX genes (NHX1 through NHX8) identified and characterized in Arabidopsis thaliana. Bioinformatics analyses of the known Arabidopsis genes enabled us to identify six Medicago truncatula NHX genes (MtNHX1, MtNHX2, MtNHX3, MtNHX4, MtNHX6, and MtNHX7). Twelve transmembrane domains and an amiloride binding site were conserved in five out of six MtNHX proteins. Phylogenetic analysis involving A. thaliana, Glycine max, Phaseolus vulgaris, and M. truncatula revealed that each individual MtNHX class (class I: MtNHX1 through 4; class II: MtNHX6; class III: MtNHX7) falls under a separate clade. In a salinity-stress experiment, M. truncatula exhibited ~?20% reduction in biomass. In the salinity treatment, sodium contents increased by 178 and 75% in leaves and roots, respectively, and Cl^? contents increased by 152 and 162%, respectively. Na⁺ exclusion may be responsible for the relatively smaller increase in Na⁺ concentration in roots under salt stress as compared to Cl^?. Decline in tissue K⁺ concentration under salinity was not surprising as some antiporters play an important role in transporting both Na⁺ and K ⁺ . MtNHX1, MtNHX6, and MtNHX7 display high expression in roots and leaves. MtNHX3, MtNHX6, and MtNHX7 were induced in roots under salinity stress. Expression analysis results indicate that sequestering Na⁺ into vacuoles may not be the principal component trait of the salt tolerance mechanism in M. truncatula and other component traits may be pivotal.