Uptake of alpha-aminoisobutyric acid in pig spleen lymphocytes stimulated by sodium periodate.

The effect of sodium periodate on the ability of pig spleen lymphocytes to transport the nonmetabolizable amino acid, α-aminoisobutyric acid, was studied. NaIO₄-treated cells exhibited a lowered rate of uptake of α-aminoisobutyric acid in contrast to phytohemagglutinin- and concanavalin A-treated cells. However, when periodate-treated cells were preincubated with untreated cells for 2 h, the mixed cells exhibited twofold stimulation in the uptake of α-aminoisobutyric acid as compared to untreated cells. The increased uptake of α-aminoisobutyric acid in mixed cells was due to a change in the V but not in the K_m. The observed increased uptake of α-aminoisobutyric acid in mixed cells was inhibited (24%) by ouabain, although the level of uptake in untreated and NaIO₄-treated cells was not affected. Na⁺,K⁺-ATPase activity in mixed cells, which was ouabain sensitive, was stimulated 56%. Studies also showed that there was a decrease in the fluorescence polarization (P value) of diphenyl hexatriene in mixed cells (P = 0.21) as compared to untreated cells (P = 0.24). These results demonstrate that NaIO₄ treatment induces a change in the lymphocyte cell membrane and transport of α-aminoisobutyric acid. Incubation of NaIO₄-treated cells with untreated cells is required for the stimulatory effect in the uptake of α-aminoisobutyric acid, and the stimulation appears to be due to changes in Na⁺,K⁺-ATPase activity and membrane fluidity.     

Reconstitution and characterization of a sodium-stimulated active aminoisobutyric acid transport system derived from partially purified plasma membranes from mouse fibroblasts transformed by simian virus 40: comparison of reconstituted vesicles with native membrane vesicles

A Na⁺-specific and Na⁺-stimulated active α-aminoisobutyric acid transport system was reconstituted from plasma membranes isolated from mouse fibroblast BALB/c 3T3 cells transformed by simian virus 40. The plasma membranes were treated with dimethylmaleic anhydride and then extracted with 2% cholate. The cholate-solubilized supernatant proteins were combined with exogenous phospholipids and eluted through a Sephadex G-50 column. This yielded reconstituted vesicles which in the presence of Na⁺ could actively transport α-aminoisobutyric acid as shown by the transient accumulation above the equilibrium level (overshoot). The overshoot was not obtained with other monovalent cations such as K⁺, Li⁺, and choline⁺. The electrochemical effect of the lipophilic anion, SCN^?, led to greater α-aminoisobutyric acid uptake as compared to that observed with Cl^? or SO₄^2?. The Na⁺-stimulated transport of a-aminoisobutyric acid was a saturable process with an apparent K_m of 2 mm. Studies of the inhibition of α-aminoisobutyric acid transport by other amino acids showed that methylaminoisobutyric acid [specifically transported by A system (alanine preferring)]had a pronounced inhibitory effect on a-aminoisobutyric acid uptake in contrast to the slight inhibitory effect produced by phenylalanine [primarily transported by L system (leucine preferring)]. The results show that the reconstituted vesicles, prepared from partially purified membrane proteins and exogenous phospholipids, regained the same important transport properties of native membrane vesicles, i.e., Na⁺-specific and Na⁺-stimulated concentrative α-aminoisobutyric acid uptake.     

A reduction in energy-dependent amino acid transport by microtubular inhibitors in ehrlich ascites tumor cells

Vincristine, other periwinkle alkaloids, and colchicine partially inhibit the energy dependent transport of α-aminoisobutyric acid in Ehrlich ascites tumor cells. The properties of this phenomenon were characterized in detail for vincristine. Maximum depression of the steady-state intracellular α-aminoisobutyric acid level was achieved with a vincristine concentration of > 0.5 m̈M. The inhibitory effect of vincristine increases as the extracellular α-aminoisobutyric acid concentration is increased reaching a maximum, however, of only ∼25% at a level of 5 mM, leaving a large gradient for α-aminoisobutyric acid across the cell membrane. Vincristine produced an asymmetrical effect on the bidirectional fluxes decreasing the initial uptake rate, while increasing the efflux of α-aminoisobutyric acid. Inhibition of net α-aminoisobutyric acid transport by vincristine was partially reversible (∼40%). Colchicine (50 m̈M) reduced the steady-state α-aminoisobutyric acid level by 30%, an effect that was not reversible. Inhibition by vinleurosine and vinrosidine was comparable to that of vincristine. Addition of glucose to the medium resulted in a small, but significant, decrease in the inhibitory effects of both vincristine and colchicine. The data indicate that these agents inhibit a small component of the uphill transport of α-aminoisobutyric acid in Ehrlich ascites tumor cells. The inhibitory effect of vincristine cannot be attributed to an increase in the passive permeability of the cell membrane to this agent. Rather, the data along with other studies from this laboratory suggest that vincristine reduces the energy-dependent transport of α-aminoisobutyric acid by either inhibiting cellular energy metabolism or by inhibiting the coupling of energy-metabolism to the transport of this amino acid and raises the possibility that cellular microtubules play a role in these processes.     

Mechanism of action of Clostridium perfringens enterotoxin. Effects on membrane permeability and amino acid transport in primary cultures of adult rat hepatocytes

Purified enterotoxin from the bacterium Clostridium perfringens rapidly decreased the hormonally induced uptake of α-aminoisobutyric acid in primary cultures of adult rat hepatocytes. At 5 min after toxin addition the decrease in α-aminoisobutyric acid uptake appeared not due to increased passive permeation (estimated with l-glucose) or to increased α-aminoisobutyric acid efflux. When short uptake assay times were employed a depression of α-aminoisobutyric acid influx was observed in toxin-treated hepatocytes. The depression of α-aminoisobutyric acid influx was correlated with a rapid increase in intracellular Na⁺ (estimated using ²²Na⁺) apparently effected by membrane damage. In contrast, the uptake of cycloleucine in the presence of unlabeled α-aminoisobutyric acid (assay for Na⁺-independent amino acid uptake) by hepatocytes treated with toxin for 5 min was decreased to only a small extent or not at all depending upon experimental design. At later times, C. perfringens enterotoxin increased the exodus of l-glucose, $\text{3-O-}\text{methylglucose}$ and α-aminoisobutyric acid from pre-loaded cells indicating that the toxin effects progressive membrane damage. When enterotoxin was removed by repeated washing after 5–20 min the decay of α-aminoisobutyric acid uptake ceased and appeared to undergo recovery towards the hormonally induced control level. The degree of recovery of α-aminoisobutyric acid uptake was inverse to the length of time of exposure to toxin. Adding at 10 min specific rabbit antiserum against C. perfringens enterotoxin without medium change also reversed the effect of toxin on increased intracellular ²²Na⁺, and on the exodus (from preloaded cells) of α-aminoisobutyric acid, L-glucose, and $\text{3-O-}\text{methylglucose}$.     

Hexose and amino acid transport by chicken embryo fibroblasts infected with temperature-sensitive mutant of rous sarcoma virus: Comparison of transport properties of whole cells and membrane vesicles

The effect of transformation on hexose and amino acid transport has been studied using whole cells and membrane vesicles of chicken embryo fibroblasts infected with the temperature-sensitive mutant of the Rous sarcoma virus, TS-68. In whole cells, TS-68-infected chicken embryo fibroblasts cultured at the permissive temperature (37°C) had a 2-fold higher rate of 2-deoxy-d-glucose uptake than the same cells cultured at the non-permissive temperature (41°C). However, both the non-transformed and transformed cells had comparable rates of α-aminoisobutyric acid transport. Membrane vesicles, isolated from TS-68-infected chicken embryo fibroblasts cultured at 41°C or 37°C, displayed carrier-mediated, intravesicular uptake of d-glucose and α-aminoisobutyric acid. Membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 37°C had an approx. 50% greater initial rate of stereospecific hexose uptake than the membrane vesicles from fibroblasts cultured at 41°C. The two types of membrane vesicle had similar uptake rates of α-aminoisobutyric acid. The results of hexose and amino acid uptake by the membrane vesicles correlated well with those observed with the whole cells. K_m values for stereospecific d-glucose uptake by the membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 41 and 37°C were similar, but the V value was greater for the membrane vesicles from TS-68-infected cells cultured at 37°C. Cytochalasin B competitively inhibited stereospecific hexose uptake in both types of membrane vesicle. These findings suggest that the membrane vesicles retained many of the features of hexose and amino acid transport observed in whole cells, and that the increased rate of hexose transport seen in the virallytransformed chicken embryo fibroblasts was due to an increase in the number or availability of hexose carriers.     

Transport and metabolic effects of α-aminoisobutyric acid in saccharomyces cerevisiae

α-Aminoisobutyric acid is actively transported into yeast cells by the general amino acid transport system. The system exhibits a K_m for α-aminoisobutyric acid of 270 μM, a V_max of 24 nmol/min per mg cells (dry weight), and a pH optimum of 4.1–4.3. α-Aminoisobutyric acid is also transported by a minor system(s) with a V_max of 1.7 nmol/min per mg cells. Transport occurs against a concentration gradient with the concentration ratio reaching over 1000:1 (in/out). The α-aminoisobutyric acid is not significantly metabolized or incorporated into protein after an 18 h incubation. α-Aminoisobutyric acid inhibits cell growth when a poor nitrogen source such as proline is provided but not with good nitrogen sources such as NH₄⁺. During nitrogen starvation α-aminoisobutric acid strongly inhibits the synthesis of the nitrogen catabolite repression sensitive enzyme, asparaginase II. Studies with a mutant yeast strain (GDH-CR) suggest that α-aminoisobutyric acid inhibition of asparaginase II synthesis occurs because α-aminoisobutyric acid is an effective inhibitor of protein synthesis in nitrogen starved cells.     

Energetics of sodium-dependent α-aminoisobutyric acid transport in the moderate halophile Vibrio costicola

The energetics of α-aminoisobutyric acid transport were examined in Vibrio costicola grown in a medium containing the NaCl content (1 M) optimal for growth. Respiration rate, the membrane potential (Δψ) and α-aminoisobutyric acid transport had similar pH profiles, with optima at 8.5–9.0. Cells specifically required Na⁺ ions to transport α-aminoisobutyric acid and to maintain the highest Δψ (150–160 mV). Sodium was not required to sustain high rates of O₂-uptake. Δψ (and α-aminoisobutyric acid transport) recovered fully upon addition of Na⁺ to Na⁺-deficient cells, showing that Na⁺ is required in formation or maintenance of the transmembrane gradients of ions. Inhibitions by protonophores, monensin, nigericin and respiratory inhibitors revealed a close correlation between the magnitudes of Δψ and α-aminoisobutyric acid transport. Also, dissipation of Δψ with triphenylmethylphosphonium cation abolished α-aminoisobutyric acid transport without affecting respiration greatly. On the other hand, alcohols which stimulated respiration showed corresponding increases in α-aminoisobutyric acid transport, without affecting Δψ. Similarly, N,N′-dicyclohexylcarbodiimide (10 μM) stimulated respiration and α-aminoisobutyric acid transport and did not affect Δψ, but caused a dramatic decline in intracellular ATP content. From these, and results obtained with artificially established energy sources (Δψ and Na⁺ chemical potential), we conclude that Δψ is obligatory for α-aminoisobutyric acid transport, and that for maximum rates of transport an Na⁺ gradient is also required.     

The effect of reversal of Na and K electrochemical potential gradients on the active transport of amino acids in Ehrlich ascites tumor cells

1.

1. The net uptake of α-aminoisobutyric acid (AIB) in Ehrlich ascites tumor cells has been studied under a variety of transmembrane concentration gradients of Na⁺, K⁺ and AIB itself.     

Discrimination of single transport systems. The Na plus-sensitive transport of neutral amino acids in the Ehrlich cell

Uptake of methionine, α-aminoisobutyric acid, and α-(methyl-amino)-isobutyric acid has been shown to occur by at least two transport systems, one sensitive and the other insensitive to the Na⁺ concentration. For α-aminoisobutyric acid and its N-methyl derivative, the Na⁺-insensitive uptake is not concentrative and its rate increases almost linearly with concentration within the range examined. In contrast, the Na⁺-insensitive uptake of methionine is concentrative and subject to inhibition by such amino acids as phenylalanine, leucine, and valine, although not in a manner to indicate that the uptake is mediated by a single agency. This component is not produced by a residual operation of the Na⁺-requiring transport system, handicapped by the absence of Na⁺ or by its having combined with α-aminoisobutyric acid. The increase in the rate of methionine uptake is linear with concentration only above about 16 mM methionine. The Na⁺-sensitive uptakes of methionine, α-aminoisobutyric, and α-(methylamino)-isobutyric acid appear to occur by the same population of transport-mediating sites. Both K_m and V _max of the Na⁺-sensitive uptake of these three amino acids change with changes in the concentration of Na⁺, an effect which is shown to have a theoretical basis. A similarity in the values of Vmax for ten amino acids entering principally by the Na⁺-sensitive agency indicates that differences in their K_m values probably measure differences in their affinities for that transport-mediating system.     

l-Phenylalanine active transport in the midgut of Bombyx mori larva

l-phenylalanine and α-aminoisobutyric acid are actively transported from the lumen to the haemolymph in the isolated midgut of Bombyx mori larva. Kinetics of l-phenylalanine fluxes as a function of concentration have been studied. The influx shows a convex relationship to the aminoacid concentration, while the outflux bears a linear relationship within the range of concentrations considered. The net flux shows a saturation kinetic, is sodium independent and is inhibited by DNP and anoxia. l-phenylalanine pools obtained by luminal or haemolymph loading are similar and no intracellular accumulation of the aminoacid takes place. Conversely the amount of α-aminoisobutyric acid entering the cells through the basolateral membrane exceeds that of luminal origin and it reaches an intracellular concentration twofold higher than that of the bathing medium.     

Activating the NaK pump with monensin increases aminoisobutyric acid uptake by mouse fibroblasts

Monensin rapidly tripled the initial rate and extent of α-aminoisobutyric acid accumulation by Swiss 3T3 cells. This ionophore catalyzes the electroneutral exchange of external Na for cellular protons and stimulates the NaK pump by suppling it with more Na. The stimulation of the NaK pump and α-aminoisobutyric acid uptake exhibited a similar dependence on monensin concentration. Ouabain prevented monensin from increasing α-aminoisobutyric acid transport. Aminoisobutryic acid transport was more than doubled at low doses of monensin that activated the NaK pump by elevating cell Na without significantly changing cell K. The rapid activation of α-aminoisobutyric acid transport is probably due to the hyperpolarizing effect of stimulating the electrogenic NaK pump. The stimulation of the NaK pump is quiescent fibroblasts by serum or growth factors may be sufficient to activate the Na-dependent amino acid transport systems.     

Regulatory characteristics of amino acid transport in newborn rat renal cortex cells

The uptake of α-aminoisobutyric acid by slices of kidney cortex from newborn rats is enhanced by a preliminary incubation of the tissue in buffer at 37 °C. This effect is abolished by anaerobiosis, the presence of dinitrophenol or the removal of Na⁺ during the preliminary incubation. Cycloheximide (50 μM) and purimycin (1 mM) as well as α-aminoisobutyric acid, glycine and proline (5 mM) in the pre- incubation buffer also abolish the effect, while actinomycin D (0.8 μM) partially inhibits the enhancement due to preliminary incubation. A kinetic examination of the phenomenon indicates that the enhanced uptake is due to an increased entry rate into the cells without a change in efflux. There is no alteration in the apparent transport $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ but an increase in the $\text{V}$ for entry. The effect is dependent on tissue age being observed between birth and 22 days, after which there is a decrease in response to preliminary incubation with no effect seen in adult tissues.     

Maturation of membrane function: Transport of amino acid by rat erythroid cells

The membrane changes which occur during cellular maturation of erythroid cells have been investigated. The transport of α-aminoisobutyric acid, alanine, and N-methylated-α-aminoisobutyric acid have been studied in the erythroblastic leukemic cell, the reticulocyte, and the erythrocyte of the Long-Evans rat. The dependence of amino acid transport on extracellular sodium concentration was investigated. Erythrocytes were found to transport these amino acids only by Na-independent systems. The steady state distribution ratio was less than 1. Reticulocytes were found to transport α-aminoisobutyric acid and alanine by Na-dependent systems, but only small amounts of N-methylated-α-aminoisobutyric acid. Small amounts of these amino acids were transported by Na-independent systems. The steady state distribution ratio was greater than one for Na-dependent transport. The erythroblastic leukemia cell, a model immature erythroid cell, showed marked Na-dependence (>90%) for α-aminoisobutyric acid and alanine transport, and >80% for the Na-dependent transport of N-methyl-α-aminoisobutyric acid. The steady state distribution ratio for the Na-dependent transport was >4. In the erythroblastic leukemic cell, at least three Na-dependent systems are present: one includes alanine and α-aminoisobutyric acid, but excludes N-methyl-α-aminoisobutyric acid; one is for α-aminoisobutyric acid, alanine and also N-methyl-α-aminoisobutyric acid; and one is for N-methyl-α-aminoisobutyric acid alone. In the reticulocyte, the number of Na-dependent systems are reduced to two: one for α-aminoisobutyric acid and alanine; one for N-methyl-α-aminoisobutyric acid. In the erythrocytes, no Na-dependent transport was found. Therefore, maturation of the blast cell to the mature erythrocyte is characterized by a systematic loss in the specificity and number of transport systems for amino acids.     

Membrane function in differentiating skeletal muscle cells: I. Kinetic analysis of amino acid transport

Primary cultures of mononucleated myoblasts from 12-day-old chick embryos have a twofold higher rate of α-aminoisobutyric acid (AIB) transport before fusion occurs to form multinucleated myotubes. Several lines of evidence indicate that the uptake of AIB observed in both myoblasts and myotubes is primarily carrier-mediated by a membrane transport system. Increasing the temperature from 24 to 37°C results in a threefold increase in the rate of AIB uptake; both methionine and glycine inhibit AIB uptake by more than 85%; and 2,4-dinitrophenol inhibits AIB uptake by approximately 50%. In addition, the energies of activation (14.5 and 14.0 kcal/mole for myoblasts and myotubes, respectively) are characteristic of carrier-mediated transport. Resolution of AIB uptake into a saturable, carrier-mediated component and a nonsaturable, diffusion component shows that at concentrations of AIB≤1.5 mM over 97% of total AIB uptake is carriermediated in both myoblasts and myotubes. Kinetic analysis of carrier-mediated AIB uptake indicates that myoblasts and myotube membrane carriers have the same affinity for AIB (K_m values = 1.73 and 1.31 mM, respectively). However, the V_max for myoblasts is 23.7 nmole/mg/min while myotubes have a V_max of 12.6 nmole/mg/min. The twofold difference in V_max is shown to be due to a twofold difference in the quantity of membrane transport sites per milligram of protein.     

Regulation of amino acid transport in chicken embryo fibroblasts by purified multiplication-stimulating activity (MSA)

Enhanced amino acid transport is observed when quiescent cultures of chicken embryo fibroblasts are stimulated to proliferate by the addition of purified multiplication-stimulating activity (MSA). This increase in amino acid transport is an early event occuring prior to the onset of DNA synthesis in stimulated cells. Results indicate that the changes in transport activity, as measured by α-aminoisobutyric acid (AIB) uptake, are due to stimulation of only the Na⁺-dependent A transport system. There is little or no change in the activities of transport systems ASC, L, or Ly⁺ upon exposure to MSA. A kinetic analysis shows this increased activity is due to a change in Vmax while K_m remains unaltered. Continuous exposure to the stimulus is required to maintain the increased level of transport activity and the presence of inhibitors of RNA and protein synthesis significantly inhibits the response. Results also indicate that a similar specific increase in the A transport system is initiated when RSV tsNY68 infected cells are shifted to the permissive temperature. It appears that the A system of mediation is emerging as a strategic regulatory site for cell function.     

Changes in amino acid uptake and protein synthesis of bullfrog tadpole liver and tail tissues during triiodothyronine-induced metamorphosis

The effect of triiodothyronine (T₃′) on the uptake of several amino acids into the amino acid pools and into proteins of Rana catesbeiana tadpole liver and tail muscle and tail fin has been studied. Labeling of the alanine and glycine pool was stimulated in the liver more than the leucine pool. After exposure to T₃ for 3 days, uptake of α-aminoisobutyric acid (a transport model substrate) into liver was stimulated about 55%. In tail tissues uptake of leucine was stimulated but uptake of alanine was depressed by T₃. Incorporation of leucine and alanine into tissue protein was stimulated in the liver but inhibited in tail tissues after T₃ injection.Changes in other macromolecules and ATP and ADP levels in liver and tail muscle were also investigated during induced metamorphosis. In the liver, the total DNA content did not change, but the RNA and protein content per liver increased significantly. The increase in RNA/DNA and protein/DNA ratios, suggested that liver cells underwent hypertrophy during induced metamorphosis. The ATP level showed a transient decrease after 3 days of T₃ treatment. In tail muscle, protein and RNA content decreased as the muscle regressed, but the DNA content and ATP level remained unchanged throughout the experimental period.     

The effect of α-aminoisobutyric acid on wood decay and wood spoilage fungi

The amino acid analogue α-aminoisobutyric acid (AIB) decreased linear extension growth in fifteen out of sixteen wood decay and wood spoilage fungi. In Serpula lacrimans inhibition of extension growth by AIB was accompanied by an increase in the frequency with which the hyphae of the fungus initiated branches. AIB was shown to have a preservative effect against Lentinus lepideus, Serpula lacrimans and Pleurotus ostreatus when wood blocks were impregnated with this chemical prior to challenge by cultures of these fungi. The effectiveness of this compound in limiting growth in a large number of different fungi suggests that competitive inhibitors of nitrogen uptake and metabolism could be used to control fungi which decay wood and similar materials, and may also have wider applications.     

UPTAKE SPECIFICITY FOR ORGANIC SUBSTRATES BY THE MARINE DIATOM MELOSIRA NUMMULOIDES1

Melosira nummuloides, clone Mel-3, shows a very high specificity with regard to its ability to take up organic substrates. Amino acids supplied in the medium at 1 X 10^-4 M are taken up at initial rates of the same order of magnitude as that of photoassirnilation of COj. However, sugars, sugar alcohols, or organic acids supplied at the same concentration are not taken up. The mechanism for uptake of amino acids appears to require energy, since tlie uptake of the amino acid analog α-aminoisobutyric acid is strongly inhibited by 2 f-dinitrophenul. The uptake mechanism does not appear to be inducible. The ability of M. numinuloides to utilize amino acids as a nitrogen source is quite restricted. Arginine, ghttamine, asparagine, proline, and glutamic acid were good nitrogen sources. Seventeen other amino acids, including α-aminoisobutyric acid, were unsatisfactory for growth, although they were rapidly taken up from the medium.