Use of silicone fluids in studies of cellular amino acids

In numerous cellular studies, cells labeled with radioisotopes have been separated from the labeling medium by an aqueous solution in order to determine the quantity of internalized labels; however, the aqueous wash tends to remove significant labeling from the cells. Therefore, in order to preserve all of the internalized labels, non-aqueous medium such as silicone fluids may be used. The termination of the labeling is achieved in the silicone method when, upon centrifugation, the cells separate from the medium and enter the silicone fluid to sediment to the tube bottom. This sedimentation of cells placed above a layer of silicone fluid exhibits a critical dependence on the centrifugal force, and gives rise to an uncertainty of only 2 s in determining the time of separation of cells from the medium using General Electric F-50 silicone fluid and a modified Beckman J2-21 centrifuge. It is therefore possible to determine the kinetics of incorporation of labeled amino acids into intracellular pools and proteins. In particular, since this silicone wash method determines the size of the total pool and the aqueous wash method determines the size of the acid-extractable pool, the simultaneous measurements of the size of both pools leads to the determination of the kinetics of labeling of the free amino acid pool. Among many possible applications and extensions of these methods, the studies of formation of intracellular pools and relations among different pools of transported molecules, such as water and amino acids, appear promising.     

Chirality differences in amino acid retention and release from the acid-extractable pool of cultured mammalian cells

In previous work, no chiral differences were found between D and L enantiomers of Leu in their ability to displace one another from the acid-extractable pool in mammalian cells. Recent evidence suggested otherwise. Our aim is to examine whether, in physiological range, D-amino acids have an equivalent ability to displace L-amino acids from the acid-extractable pool of HeLa cells, and vice versa. In the millimolar range, D-Leu and L-Leu have similar uptake and displacement properties with regard to the acid-extractable pool in HeLa cells, despite only the latter isomer being incorporated into protein. Below millimolar concentrations however, a distinct difference was found in the displacement of tritium-labelled L-Leu from the pool by unlabelled D-Leu compared with unlabelled L-Leu. Thus, unlabelled L-Leu in the external medium at 10⁻⁴ or 10⁻⁵ M displaced an equivalent amount of label from the pool as D-Leu introduced at a concentration approx. one order of magnitude higher, respectively. Reciprocal experiments, in which the acid-extractable pool was preloaded with ³H-D-Leu, confirmed this finding. The chirality difference was noted whether pool prelabelling was carried out at 37 or 0°C; but in order to avoid the complications of active transport mechanisms, the competition work reported here was done at 0°C. Similar chirality differences were observed with other hydrophobic amino acids, including His, Ile and Phe, such as, preferential displacement by the L-Leu racemer compared with the D-Leu racemer below mM levels. This was also true for the D and L forms of the non-utilisable isomer of Leu, norleucine (nLeu). We conclude that D-forms of hydrophobic amino acids have lower affinity for similar or the same intracellular binding sites involved in the acid-extractable pool than their L-forms. The significance of these chirality findings to amino acid pools in cells, and to the predominance of L-forms of amino acids in the biosphere is considered.     

Accumulation of free amino acids in growing Xenopus laevis oocytes

The sizes of amino acid pools in growing Xenopus laevis oocytes have been measured. The total free amino acid content per oocyte increases nearly 25-fold during oocyte growth. Together, glutamic acid and aspartic acid account for approximately 59-75% of the total amino acid pool in Xenopus oocytes. On the other hand, methionine and cysteine are the least abundant of the amino acids detected, each accounting for less than 0.7% of the total pool in developing oocytes. It is argued that the acid-extractable amino acid pool represents the precursor pool used in protein synthesis.     

Pools and protein synthesis in mammalian cells.

From the kinetics of incorporation into protein shown by four amino acids and one amino acid analogue in suspension cultured HeLa S-3 cells, two distinctly different patterns were observed under the same experimental conditions. An initial slow exponential incorporation followed by linear kinetics was characteristic of the two non-essential amino acids, glycine and proline, whereas the two essential amino acids studied, phenylalanine and leucine, showed linear kinetics of incorporation with no detectable delay. The analogue amino acid, p-fluorophenylalanine also showed immediate linear kinetics of incorporation. There was a poor correlation between the rate of formation of acid-soluble pools and incorporation kinetics. However, the rate of formation of the freely diffusible pool of amino acids correlated more closely with incorporation kinetics. The lack of direct involvement of the acid-soluble pool in protein synthesis was also demonstrated by pre-loading of pools before treatment of cells with labelled amino acids. The results partially support the hypothesis that precursor amino acids for protein synthesis come from the external medium rather than the acid-soluble pool, but suggest that the amino acid which freely diffuses into the cell from the external medium could also be the source.     

Identification in skeletal muscle of a distinct extracellular pool of amino acids, and its role in protein synthesis

1. The kinetics of radioactive labelling of extra- and intra-cellular amino acid pools and protein of the extensor digitorum longus muscle were studied after incubations with radioactive amino acids in vitro. 2. The results indicated that an extracellular pool could be defined, the contents of which were different from those of the incubation medium. 3. It was concluded that amino acids from the extracellular pool, as defined in this study, were incorporated directly into protein.     

Dynamic aspects of vacuolar and cytosolic amino acid pools of Saccharomyces cerevisiae.

By using the Cu2+ method (Y. Ohsumi, K. Kitamoto, and Y. Anraku, J. Bacteriol. 170:2676-2682, 1988) for differential extraction of the vacuolar and cytosolic amino acid pools from yeast cells, the amino acid compositions of the two pools extracted from Saccharomyces cerevisiae cells, grown in synthetic medium supplemented with various amino acids, were determined. Histidine and lysine in the medium expanded the vacuolar pool extremely. Glutamate also accumulated in the cells, but mainly in the cytosol. The composition of amino acids in the cytosolic pool was fairly constant, in contrast to that in the vacuolar pool. Cells grown in synthetic medium supplemented with 10 mM arginine accumulated arginine in the vacuoles at a concentration of about 430 mM. This large arginine pool was metabolically active and was effectively utilized during nitrogen starvation. Arginine efflux from the vacuoles was coupled with K+ influx, with an arginine/K+ exchange ratio of 1, as judged by the initial rate. The vacuolar arginine pool was exchangeable with lysine added to the medium and was decreased by treatment of the cells with the mating pheromone, alpha-factor.     

Kinetic analysis of amino acid pools and protein synthesis in amphibian oocytes and embryos.

Rates of protein synthesis have been measured in Rana pipiens oocytes and embryos and in Xenopus oocytes from the incorporation kinetics of two different concentrations of amino acid. This method does not require an independent measurement of the amino acid pools, since the pool size can be calculated directly from incorporation data. The effects of the concentration and diffusion of injected amino acid on the calculated values for amino acid pool size and flow rate are discussed. When the endogenous amino acid pool is appreciably expanded by the injected amino acid, the total amino acid pool in the oocytes or embryos may be considered as the precursor pool for protein synthesis. Under these circumstances, compartmentation of amino acids does not affect the results, except when lysine is used as tracer. The rates of protein synthesis in ovarian oocytes of Rana pipiens and Xenopus laevis are 18 and 50–54 ng/hr, respectively. In Rana pipiens, the rate increases 70% during maturation and another 50% before the two-cell stage. Finally, the rate approximately doubles between the two-cell and blastula stages.     

Hind leg muscle amino acid balances in cold-exposed rats

The changes in hind leg tissue (muscle and skin) amono acid pool size and arteriovenous balance were measured in rats subjected to 0–90 min of cold exposure (4°C). Tissue free amino acid pools presented a different composition pattern from protein amino acids. Muscle rapidly reacted to cold exposure by releasing small amounts of some amino acids (alanine, aspartate), with only small changes in pool size during the first 30 min. Amino acid oxidation was very limited during the whole period of cold exposure, since at all times tested there was either nil ammonia efflux or net absorption of ammonia and glutamine; i.e. the muscle was in positive nitrogen balance throughout the period studied. Thus most of the amino acid nitrogen taken up from the blood and not found in the free amino pools must have been incorporated into protein, since it was not oxidized, as shown by the glutamine and ammonia blance. The data on amino acid incorporation into proteins indicate that hind leg protein turnover is rapidly and widely modulated from a low initial setting upon cold exposure to a higher protein synthesis rate immediately afterwards, suggesting that protein turnover may have an important role in short-term events in cold-exposed muscle, in addition to its influence in long-term adaptation.     

The effect of amino acids on the temperature sensitive phenotype of the mammalian leucyl-tRNA synthetase mutant tsHl and its revertants.

The temperature sensitive leucyl-tRNA synthetase mutant tsHl and two revertants have been compared to the parental Chinese hamster ovary cells with respect to the effects of amino acid concentrations in the medium on growth. Elevating the leucine concentration 30- or 100-fold allowed tsHl to grow exponentially at 38.5 degrees C, normally the nonpermissive temperature. Partial revertants that had recovered some enzyme activity required smaller supplements for growth. Measurements of the leucine pools indicated that they respond directly to the extracellular leucine concentration and may mediate the effect. Use of combinations of amino acids confirmed that isoleucine has a similar though weaker effect on tsHl and identified an even weaker protection by valine. The triple combination of leucine, isoleucine and valine was a much more efficient medium supplement and three times normal concentrations of these amino acids supported growth of tsHl at 38.5 degrees C. It is postulated that they are acting at their respective aminoacyl-tRNA synthetases to help stabilize a complex which also contains the mutant leucyl-tRNA synthetase. The pool size measurements also showed that the leucine pools of tsHl and a revertant increased 2-fold more in a response to increased temperature than those of WT. It is suggested that this is a regulatory response to low leucyl-tRNA synthetase activity and is important in determining growth phenotypes.     

Characterization of amino acid pools in the vacuolar compartment of Saccharomyces cerevisiae

Intact vacuoles are released from spheroplasts of Saccharomyces cerevisiae by means of a gentle mechanical disintegration method. They are purified by centrifugation in isotonic density gradients (flotation and subsequent sedimentation), and analyzed for their soluble amino acid content. The results indicate that about 60% of the total amino acid pool of spheroplasts is contained in the vacuoles. This may be an underestimate, as it presupposes no loss of amino acids from the vacuoles during the purification procedure. The amino acid concentration in the vecuoles is calculated to be approximately 5 times that in the cytoplasm if the total volumes of the two compartments are used for the calculation. The vacuolar amino acid pool is rich in basic amino acids, and in citrulline and glutamine, but contains a remarkably small amount of glutamate. Radioactive labeling experiments with spheroplasts indicate that the vacuolar amino acids are separated from the metabolically active pools located in the cytoplasm. This is particularly evident for the basic amino acids and glutamine; in contrast, the neutral amino acids and glutamate appear to exchange more rapidly between the cytoplasmic and the vacuolar compartments of the cells.     

Heterogeniety of the amino acid pool for protein synthesis in Ehrlich ascites carcinoma cells

Using a new methodological approach based on a step-wise labelling with [14C] and [3H] amino acids, it was demonstrated that the Ehrlich ascite carcinoma cells are capable of utilizing both intracellular and extracellular amino acid pools for protein synthesis. The inhibition of amino acid transport into the cells is accompanied by a more intensive utilization of the exogenous pool. The described procedure permits to calculate the specific radioactivity of the tRNA-bound amino acid and the absolute rate of protein synthesis.     

Compartmentation of Sulfur Metabolites in Tobacco Cells : USE OF EFFLUX ANALYSIS

The distribution of sulfur-containing metabolites in cultured tobacco cells was determined by analyzing efflux kinetics. Transported sulfate rapidly labeled the cytoplasmic pools of sulfate (1 hour) and sulfur amino acids (6 hours). Excess sulfate and amino acids were transported into the vacuole. The size and distribution of the amino acid pool was not affected by increasing the sulfate content of the cells.     

Amino acid pools in CHL V79 cells during induction of thermotolerance: reduction in free intracellular glutamine

The amino acid pools in Chinese hamster lung V79 cells were measured as a function of time during hyperthermic exposure at 40.5 degrees and 45.0 degrees C. Sixteen of the 20 protein amino acids were present in sufficient quantity to measure accurately. The total amino acid pool and all individual amino acids, except glutamine, remained relatively constant for at least 90 min at 40.5 degrees C and for 30 min at 45 degrees C. The glutamine pool decreased rapidly to 20% of its control value within 30 min at 40.5 degrees C with a T1/2 = 15 min. At 45 degrees C, the decrease was 36%. Thermotolerance developed at 40.5 degrees C with a T1/2 = 30 min; thus, glutamine depletion preceeds the development of thermotolerance. The depletion of glutamine is probably due to increased metabolism and oxidation of glutamine through the TCA cycle at hyperthermic temperatures. Glutamine, as is true for other amino acids, was shown to protect proteins from thermal inactivation and V79 cells from hyperthermic killing when added in excess (4-10 mM) to the medium during heat stress. However, the stability of the total amino acid pool during the development of thermotolerance indicates that resistance to heat does not result from the accumulation of amino acids which then protect against thermal damage. The effects of the large decrease in the glutamine pool are unknown, although glutamine depletion may act as a signal for part of the heat shock response.     

Inactive and protein precursor pools of amino acids in the soybean hypocotyl

There are at least 2 amino pools for leucine and for valine in the soybean hypocotyl, a small protein precursor pool and a large inactive pool. The precursor pool decreased in size during incubation of excised hypocotyls presumably because the cotyledonary sources of amino acids had been removed. The precursor pool was subject to expansion by supplying the amino acid externally at high concentrations. After the transfer of tissue to unsupplemented media, the expanded pool was rapidly depleted.     

Distribution of amino acids in the cellular fractions ofStaphylococcus aureus

WhenStaphylococcus aureus cells were labeled with a single radioactive amino acid for 20 minutes, the highest activity, except for alanine, leucine, and glycine, was found in the free pool. Significant amounts of the above amino acids and also valine and methionine were incorporated into the protein — cell wall fraction.Cells previously labeled with a single amino acid underwent a net loss of radioactivity when transferred to buffer, glucose, or complete medium. An exception was glycine. The greatest loss in activity occurred in the free pool.While some amino acids (alanine, cystine) were transferred from the free pool to the protein — cell wall fraction under all conditions tested, others (glutamic acid, proline) were transferred only under conditions of growth.Cells labeled with certain single amino acids and then transferred to a complete medium lost a significant portion of the label. The most extreme case noted was proline, but other amino acids also effluxed from the cell under these conditions.     

Neurospora crassa conidial germination: role of endogenous amino acid pools.

The levels of the endogenous amino acid pools in conidia, germinating conidia, and mycelia of wild-type Neurospora crassa were measured. Three different chromatographic procedures employing the amino acid analyzer were used to identify and quantitatively measure 28 different ninhydrin-positive compounds. All of the common amino acids were detected in conidial extracts except proline, methionine, and cystine. The levels of these three amino acid pools were also very low in mycelia. During the first hour of germination in minimal medium, the levels of most of the free amino acid pools decreased. The pool of glutamic acid, the predominant free amino acid in conidia, decreased 70% during the first hour. Very little glutamic acid or any other amino acid was excreted into the medium. During the first 20 min of germination, the decrease in the glutamic acid pool was nearly equivalent to the increase in the aspartic acid pool. The aspartic acid and lambda-aminobutyric acid pools were the only amino acid pools that increased to maximum levels within the first 20 min of germination and then decreased. It is proposed that an important metabolic event that occurs during the early stages of conidial germination is the production of reduced pyridine nucleotides. The degradation of the large glutamic acid pool existing in the conidia (2.5% of the conidial dry weight) could produce these reduced coenzymes.     

Pools and protein synthesis: studies with the D- and L-isomers of leucine.

D-leucine and L-leucine produce pools of an identical nature in HeLa cells. Both isomers noncompetitively inhibit to the same extent pool formation and incorporation of valine. In the presence of D-leucine, [3H]-L-leucine at high specific activity is avidly incorporated into protein while forming a highly radioactive pool. The development of this pool was suppressed to normal levels by the presence of cycloheximide. It therefore represented a largely catabolic pool derived from proteins which had already become labelled. Discharge of pools of D-leucine followed first order kinetics and was significantly retarded when medium contained 10(-2) M of either isomer. Discharge of catabolic pools was equally as fast but the continual flow of labelled amino acid from protein sustained its intracellular level. The presence of 10(-2) to 10(-4) M leucine in the chase medium did not apparently alter the rate of discharge of this catabolic pool. The results are discussed in terms of the specificity of amino acids for different stages of the pathway leading to and from protein synthesis, and support the intracellular perfusion mechanism described elsewhere (Wheatley and Inglis, 1979).     

Changes in amino acid permeation during sporulation

Changes in amino acid uptake in Bacillus licheniformis and in the amino acid pools of three Bacillus species were investigated, by use of cells from different stages of the life cycle. B. licheniformis contains catalytic uptake systems for all of the 10 amino acids studied. The apparent maximal velocities of uptake decreased during sporulation but did not fall below the range observed for other microorganisms. In sporulating cells, the apparent affinity constants of the uptake systems for individual amino acids remained about the same as in growing cells, i.e., from 2 x 10(-7)m to 7 x 10(-6)m, whereas, in some cases, the apparent maximal velocities decreased significantly. Because the velocity of uptake showed an atypical dependence on substrate concentration, it was postulated that these cells contain two or more uptake systems for each amino acid. Only one of these systems appeared to be operative at a substrate concentration below 10(-6)m. Working at these low substrate concentrations, catalytic activities producing a net efflux of amino acids were demonstrable in vegetative cells in the presence of chloramphenicol, but these exit systems were lost during sporulation. A pool formed by the addition of radioactive algal hydrolysate will exchange with the external medium in vegetative cells but not in sporulating cells. Glutamic acid and alanine comprise at least 60% of the amino acid pool of B. licheniformis A-5, B. subtilis 23, and B. cereus T during all stages of growth and sporulation. The concentrations of the other amino acids in the pool varied extensively, but reflected, in general, the amino acid turnover known to occur during sporulation.     

Impact of UV radiation on the production patterns and composition of dissolved free and combined amino acids in marine phytoplankton

Using the ¹³C tracer technique in conjunction with gas chromatographic-massspectro-metric (GC-MS) techniques, we examined the patternsof synthesis and the composition of dissolved free and combinedamino acids within phytoplankton photosynthesizing in the presenceand absence of natural solar ultraviolet radiation (UVR). Atlevels that still permitted the uptake of carbon assimilationinto the cells, UVR caused a marked decline in the overall rateof carbon incorporated into amino acids and a reduction in thepool size of total cellular amino acids (TCAA). In contrast,absolute concentrations of amino acids within the intracellulardissolved free amino acid (INDFAA) pool (measured using an aminoacid analyzer) were higher in the presence of UVR. An examinationof the production patterns and composition of amino acids constitutingthe INDFAA and TCAA pools revealed a marked diminution in thesynthesis and accumulation of alanine and valine in the presenceof UVR. On the other hand, the rates of synthesis and concentrationsof glutamic acid (glutamic acid + glutamine) in the INDFAA andTCAA pools of phytoplankton were higher in samples exposed toUVR. These changes are discussed with reference to the knowneffects of UVR on nitrogen and carbon assimilation within phytoplankton.     

Bimodal effects of cellular amino acids on Na+-dependent amino acid transport in ehrlich cells

Cells depleted of amino acids show lower rates of glycine or aminoisobutyric acid uptake than do freshly isolated cells. In the amino acid-depleted cells, addition of valinomycin stimulates amino acid influx at least to the level observed in freshly isolated cells. In cells containing high levels of cellular amino acids, valinomycin has little effect on influx of amino acids. It is concluded that the transport of amino acids in freshly isolated cells is elevated compared to depleted cells because the cells are hyperpolarized by the continuous loss of cellular amino acids during the transport assay. During this hyperpolarization by amino acid loss, transport of amino acids is not further stimulated by valinomycin at low external [K⁺] ($\text{10 mM ± 5 mM}$).With the exception of preloading with glycine, cells preloaded with a single amino acid to a concentration greater than 20 mM show reduced rates of glycine and aminoisobutyric acid influx at early times (less than 15 min) compared to amino acid-depleted cells. The reduction of infiux is transient and by 30 min, influx is greater in preloaded than in amino acid-depleted cells.Knowing that increases and decreases in the membrane potential are achieved by using varying external [K⁺] in the presence of valinomycin and propranolol, and using amino acid-depleted cells, it can be shown that an increased membrane potential increases the $\text{V}$ for glycine and aminoisobutyric acid influx. A decrease in the potential difference results in a decreased $\text{V}$. Changes in $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ also occur when the membrane potential is varied.