Uptake Of Α-aminoisobutyric Acid Research Articles

Uptake of α-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. NaIO 4-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 NaIO 4-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 NaIO 4 treatment induces a change in the lymphocyte cell membrane and transport of α-aminoisobutyric acid. Incubation of NaIO 4-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.

Principal substrates of fetal metabolism.

Principal substrates of fetal metabolism.

The effects of bioregulators upon amino acid transport and protein synthesis in isolated rat hepatocytes

Isolated rat hepatocytes prepared by an enzyme perfusion technique possess a functional amino acid transport system and retain the capacity to synthesize protein. Amino acid transport was studied using the non-metabolizable amino acid analog α-aminoisobutyric acid. The transport process was time, temperature and concentration dependent. Similarly,leucine incorporation into protein was time and temperature dependent being optimal at 37°C. Amino acids, fetal calf serum, growth hormone and glucose all produced small, reproducible increases in protein synthesis rates. Bovine serum albumin diminished the uptake of α-aminoisobutyric acid and leucine incorporation into protein. The amino acid content on either side of the cell membrane was found to affect transport into or out of the cellular compartment (transconcentration effects). High cell concentrations decreased transport and protein synthesis as a result of isotopic dilution of labelled amino acids with those released by the hepatocytes. This was consistent with the capacity of naturally occurring amino acids to compete with α-aminoisobutyric acid for uptake into the hepatocyte. Inorder to define more precisely the effects of bioregulators on transport and protein synthesis it will be necessary to define and subfractionate cellular compartments and proteins which are the specific targets of cellular regulation.

Transport of naturally occurring amino acids and alpha-amino isobutyric acid by normal and diapausing mouse blastocysts

Uptake of 14C-labelled α-amino isobutyric acid and a mixture of naturally occurring amino acids by normal mouse preimplantation blastocysts was found to be twice that of delayed implantation blastocysts. With both groups of embryos concentration of the amino acids against a gradient occurred. The results may explain, in part, the lower amino acid incorporation rates found with diapausing vs. normal blastocysts and open the question of “metabolic dormancy” of the diapausing embryo.

Effects of wheat germ agglutinin on membrane transport

1. (1) Low concentrations of wheat germ agglutinin are cytotoxic toward several tissue culture lines, including Chinese hamster ovary cells, Swiss 3T3 cells, mouse L cells and baby hamster kidney cells. The LD 50 ranged from 1 to 5 μg wheat germ agglutinin per ml. Similar concentrations of the lectin inhibited the transport of the non-utilizable amino acids α-aminoisobutyric acid and cyclo-leucine and inhibited the uptake of thymidine. In contrast, 2-deoxy- d-glucose uptake was not altered and colchicine uptake was enhanced. 2. (2) The inhibition of α-aminoisobutyric acid uptake occurred within minutes after lectin addition and was maximal by 1 h. Maximal inhibition ranged from 50 to 70% of control values. Studies of the kinetics of the uptake demonstrated that wheat germ agglutinin decreased the V of the uptake by 70% without affecting the apparent K m . Ovomucoid, a haptene inhibitor of wheat germ agglutinin-binding to cell surface receptors, prevented the wheat germ agglutinin-induced inhibition of α-aminoisobutyric acid transport. Three other lectins (Concanavalin A, Phaseols vulgaris E-phytohemagglutinin and l-phytohemagglutinin) inhibited the uptake by 20% or less at doses up to 50 μg/ml. 3. (3) We propose that the cytotoxicity of wheat germ agglutinin probably results in part, if not totally, from membrane alterations which impair multiple membrane transport systems

Glucagon stimulates the A system for neutral amino acid transport in isolated hepatocytes of adult rat

Summary The effect of glucagon on neutral amino acid transport systems was studied in freshly-prepared suspensions of isolated hepatocytes by measuring basal and hormone-stimulated α-aminoisobutyric acid (AIB) and cycloleucine uptake. Glucagon stimulated AIB uptake against a concentration gradient. Half-maximal stimulation occured with 0.4 nM glucagon. Glucagon stimulation was restricted to the A mediation of transport. The effect was rapid, occured without time lag, was abolished by cycloheximide and mimicked by dibutyryl cyclic AMP.

Sterol replacement in saccharomyces cerevisiae. Effect on cellular permeability and sensitivity to nystatin

More than 80% of the cellular ergosterol can be replaced by cholesterol in a sterol requiring mutant strain of Saccharomyces cerevisiae. The effect of this replacement, as well as the effect of sterol starvation on the uptake and exit of cytosine and α-aminoisobutyric acid (α-AIBA) was studied in an attempt to elucidate the role of sterols in cellular permeability. Neither the exit of cytosine nor the exit of α-AIBA was affected by changes in the sterol content of the cell. Cells grown on cholesterol or on ergosterol had very similar rates of cytosine uptake, but a lower rate was found for sterol-starved cells. This difference may be a consequence of the cellular growth rate. However, nystatin induces a much slower exit of α-AIBA in cells grown on cholesterol than in cells grown on ergosterol. This strongly suggests that a change in membrane structure has taken place.

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 K m but an increase in the 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.

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

The effect of triiodothyronine (T 3′) 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 T 3 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 T 3. Incorporation of leucine and alanine into tissue protein was stimulated in the liver but inhibited in tail tissues after T 3 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 T 3 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.

Stimulation of sugar transport in rat diaphragm by 8-bromoguanosine 3′,5′-monophosphate

The cyclic GMP derivative, 8-bromo cyclic GMP, increases the uptake of D-xylose and of 2-deoxy D-glucose into intact rat diaphragm incubated in vitro. 8-Bromo cyclic GMP does not stimulate the incorporation of [ 14C] glucose into glycogen in the diaphragm, or the uptake of α-amino isobutyric acid into this tissue. The effect of 8-bromo cyclic GMP on the diaphragm is consistent with the hypothesis that cyclic GMP plays a role in the regulation of sugar transport in muscle.

Early cellular responses to diverse growth stimuli independent of protein and RNA synthesis

Serum, elevated pH, excess Zn++, 9,10 dimethyl-1,2 dibenzanthracene (DMBA) and insulin accelerate the progress of growth-inhibited chick embryo cells into the S-period of DNA synthesis. A comparative study was made of their capacity to elicit other cellular responses within two hours after their application. All the agents studied stimulated the uptake of the glucose analogue 2-deoxy-D-glucose (2-dGlc). Elevated pH elicited a more striking increase than the other agents in the uptake of the amino acid analogue alpha-amino isobutyric acid (AIB). The application of subtoxic concentrations of Zn++ or DMBA did not stimulate the uptake of uridine by cells nor its incorporation into RNA when tested at 2 hours. However, it was found that the stimulation of uridine utilization did occur but was delayed several hours. Similarly, the accelerated onset of DNA synthesis was also delayed for several hours by these agents. Insulin acted like serum in stimulating the utilization of 2-dGlc, AIB and uridine. Serum and DMBA were particularly effective in stimulating the utilization of choline. It was concluded that the utilization of 2-dGlc, uridine and thymidine are affected similarly by all the agents, but that there may be differential effects in the utilization of AIB and choline. The inhibition of RNA synthesis by actinomycin D did not prevent the relative stimulation of 2-dGlc, AIB and choline utilization by serum and pH treatment. The inhibition of protein synthesis by cycloheximide did not prevent the relative stimulation of 2-dGlc and choline utilization by serum and pH treatment. It partially blocked the increased uptake of AIB and had erratic effects on the utilization of uridine. It was concluded that neither RNA nor protein synthesis is required for some, if not all, the early responses to growth stimuli measured here. The inhibited cell appears to be a poised system which carries out a programmed array of reactions characteristic of the cell type following perturbation by a variety of unrelated agents. In vivo specificity is provided by the physiological reagents available (i.e., hormones) and their capacity to interact with different cell types.

Stimulation of the Uptake of α-Aminoisobutyric Acid in Rat Thymocytes by L-Triiodothyronine: A Comparison with Insulin and Dibutyryl Cyclic AMP

L-Triiodothyronine in vitro increased the uptake of the non-metabolizable amino acid, alpha-aminoisobutyrate, into isolated rat thymocytes. This effect was evident at extracellular free hormone concentrations of 10-7 to 5 times 10-8M and was due to a marked inhibition of amino acid efflux. In contrast, insulin and dibutyryl cyclic AMP, which also increased uptake in these cells, acted to stimulate amino acid influx. When L-triiodothyronine was added to maximally effective concentrations of either insulin or dibutyryl cyclic AMP, the resultant effect was the sum of the effect of each hormone added individually. These studies extended previous observations which indicate that thyroid hormones stimulate the cellular uptake of amino acids, and they provide a convenient system for the study of thyroid hormone action in vitro.

The Basis of the Alkalophilic Property of a Species of Bacillus

An alkalophilic bacterium belonging to the genus Bacillus was isolated from an indigo ball. The bacterium exhibited a maximum growth rate at pH 10-0 TO 10-5. The incorporation of 14C-labelled amino acids or [14C]uracil, uptake of 14C-labelled alpha-amino isobutyric acid into the bacterium and oxygen consumption of the bacterium with amino acids as substrates were all maximum at pH 9-0 to 10-5. The uptake of [U-14C]glucose into the organism and oxygen consumption with carbohydrates, on the other hand, showed little variation of rate in the pH 8 to 10 region. The oxygen consumption of intact bacteria or protoplasts in culture medium was maximum at pH 10. The membrane of the bacterium oxidized NADH maximally at pH 7-5, and ATPase bound to the membrane exhibited maximum activity at pH 7.L-Lactate, L-alanine and malate dehydrogenases in the soluble fraction exhibited maximum activities at pH 7-4 to 8-4. The alkalophilic property of the bacterium may be due to the behaviour of the membrane towards charged substances admitted into the organisms.

Influence of the EGG formation cycle on α-aminoisobutyric acid (AIB) transport by magnum mucosa and liver slices in Japanese quails ( coturnix coturnix japonica)

1. The uptake of α-aminoisobutyric acid (AIB) by magnum mucosa and liver slices as affected by the egg formation cycle was studied in Japanese quails. 2. Much higher distribution ratios of AIB between the intracellular fluid and the incubation medium were obtained for magnum mucosa slices as compared to liver slices. 3. The presence of an egg in the magnum was associated with an increased uptake of AIB into the cells of the magnum mucosa. 4. Compared to quails with an egg in the oviduct (magnum or uterus) in quails without an egg in the oviduct lower AIB distribution ratios between the intracellular fluid and the incubation medium were obtained for liver slices. 5. The results indicate that in the Japanese quail amino acid uptake by both the magnum mucosa and liver cells is influenced by egg formation.

Distinctive alterations of nucleoside, sugar and amino acid uptake by sulfhydryl reagents in cultured mouse cells

The differential effect of several sulfhydryl reactive reagents on the initial rate of uptake of nucleoside, amino acids, and deoxyglucose in mouse embryo cells was studied. Distinctive patterns of inhibition were found for each combination of substrate and inhibitor. The hydrophilic mercurial p-chloromercuribenzenesulfonate (PCMB-S), inhibited the initial rate of l-phenylalanine, α-aminoisobutyric acid, and deoxyglucose uptake. On the contrary, the uptake of nucleosides and the amino acid, l-lysine, was slowly inhibited by PCMB-S. The hydrophobic sulfhydryl reactive chemical, dithiodipyridine (DTP), inhibited the initial rate of uridine uptake while inhibiting deoxyglucose more slowly and l-phenylalanine and l-lysine not at all. Fluorodinitrobenzene (FDNB) slowly inhibited uridine and deoxyglucose without altering amino acid uptake. Two amino reactive compounds, the hydrophilic 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS) and the hydrophobic reagent 2-methoxy-5-nitrotropone (MNT) failed to interfere with the uptake of any of these substrates. These results demonstrate that the permeation pathways for nucleosides, sugars, and several amino acids are chemically distinguishable by their characteristic response to two sulfhydryl reactive chemicals. On the basis of the inhibitory patterns, the involvement of sulfhydryl groups as well as their location in each permeation pathway is discussed.

Transportprozesse in Vallisneria-Blättern und die Wirkung von Kinetin und Kolchizin: I. Aufnahme von α-Aminoisobuttersäure in Gewebe von Vallisneria und die Wirkung von Kinetin

Summary The uptake of α-aminoisobutyric acid (AIB)—a metabolically inert amino acid—into the leaf tissue of Vallisneria spiralis L. was studied. These are the main results: 1. The uptake proceeds proportional with time over a period of 100 hours from a 1 mM AIB solution reaching an accumulation ratio of about 5 during this period. The time proportional uptake phase continues for 24 hours from a 20 mM solution. 2. The concentration dependence of the uptake shows saturation kinetics with half saturation at 1 mM. 3. The efflux of the amino acid is very low and can be neglected at a first approximation in the· uptake experiments. 4. Kinetin (1 μ M) shows typical effects on uptake and leakage. After the kinetin effect has developed, the uptake of amino acid from a 1 mM AlB solution proceeds with a higher rate but a constant velocity during a period of 100 hours. 5. The capacity for amino acid accumulation is enhanced under the influence of kinetin. 6. The maximal velocity of uptake (V max ) reached at saturating concentrations is increased by kinetin. 7. The leakage rate is reduced by kinetin, however, this effect does not explain the higher uptake rate and accumulation ratio.

Characterization of neutral amino acid accumulation by human term placental slices.

Characterization of neutral amino acid accumulation by human term placental slices.

Action of insulin on amino acid uptake by the immature rat uterus in vitro

1. 1.The uptake of α-aminoisobutyric acid by the immature rat uterus was studied kinetically in vitro, and the stimulatory action of insulin on uptake was characterized. Other amino acids were also studied for comparison. 2. 2.α-Aminoisobutyric acid uptake was increased if the uteri were pretreated with insulin for 20 min or more. The V of the saturable transport was increased by 19% by insulin treatment, but no K m change was apparent. 3. 3.Puromycin eliminated the insulin action on α-aminoisobutyric acid uptake within 40 min of treatment. 4. 4.The effect of insulin on α-aminoisobutyric acid uptake was abolished either by a preliminary 10-min incubation of the uterus with a high concentration (1 mM) of N-ethylmaleimide; or by incubation, concurrent or subsequent, with a low concentration of N-ethylmaleimide (0.05 mM). 5. 5.The uptake of l-proline, l-methionine and glycine, but not that of l-leucine or l-valine, was also increased by insulin treatment. Insulin acted on a Na +-dependent uptake; it was without effect on the Na +-independent uptake of either α-aminoisobutyric acid or methionine. The evidence suggests that insulin acts on the A system of amino acid transport in the uterus.

The in-vitro and in-vivo effects of polyamines on cardiac protein biosynthesis

In cell-free systems putrescine, spermine, and spermidine at concentrations of 15 to 100 μg had no significant effect at 11 mM Mg2+ on rat myocardial aminoacyl-tRNA synthetase activity, transfer of amino acids from aminoacyl-tRNA into protein, or the incorporation of amino acid into protein using a myocardial ribosomal preparation. The polyamines stimulated cell free protein synthesis at low Mg2+ concentration. In the perfused rat heart, the addition of these polyamines to the perfusate increased the rate of incorporation of 14C-phenylalanine from the perfusate into myocardial protein. Spermine increased the uptake of α-aminoisobutyric acid into the myocardium of the perfused rat heart.

Properties of α-Aminoisobutyric Acid Transport in a Thermophilic Microorganism

Uptake of alpha-aminoisobutyric acid (AIB) by a leucine-tyrosine auxotroph of a thermophilic microorganism starved for leucine was studied. AIB was taken up by the cells against a substantial concentration gradient (300:1) and was present there in a free and unchanged form. Various energy inhibitors and sulfhydryl reagents strongly inhibited the accumulation of AIB. AIB uptake obeyed saturation kinetics, and the Lineweaver-Burk plot is characterized by a biphasic curve. AIB most probably shares a common transport system(s) with alanine, serine, and glycine. A mutant defective in l-alanine uptake was isolated by using the suicide effect due to accumulation of the tritiated substrate. The mutant also exhibited impaired transport activity towards AIB, glycine, and l-serine, but not to phenylalanine or valine. The transport of AIB, glycine, l-alanine, and l-serine was induced by d-alanine (5 x 10(-3) M) during growth in a succinate- and ammonia-containing medium. De novo protein synthesis was required for the induction of AIB transport; the induction was inhibited when growth occurred in glucose-containing media. The apparent differential rate of synthesis of the AIB transport system was decreased considerably in glucose-grown cells as compared to succinate-grown cells. A common genetic basis of either the regulatory or structural nature for the transport of AIB, alanine, glycine, and serine in a thermophilic microorganism is suggested.