Intracellular Free Amino Acid Research Articles

The time course of intracellular free amino acid accumulation in tissues of Modiolus demissus during high salinity adaptations

1. 1. The total amino acid pool increased in ventricles and gills removed from M. demissus during adaptation to high salinity. However, each major amino acid had a unique time course of accumulation. 2. 2. Initially, alanine concentration increased rapidly followed by slower accumulations of glycine and taurine. Alanine declined as the other amino acids increased. Thus the pool size remained constant following the initial elevation. 3. 3. Proline, undetected at first, increased after 1 day in the high salinity. Eventually, proline concentrations peaked, declined and disappeared again. 4. 4. Mantle tissue showed a different pattern of amino acid accumulation from the other tissues. Although alanine accumulated first, followed by glycine and proline, taurine did not increase. 5. 5. A pattern of amino acid accumulation similar to that in tissues from the whole animal was found in isolated gills and ventricles during high salinity stress.

Effects of salinity and osmolarity of the medium on amino acid metabolism in Cyclotella cryptica

The amino acid metabolism of Cyclotella cryptica is strongly influenced by changes in salinity of the medium. A sudden increase in salinity (water stress) reduces the total uptake of amino acids by the cells and also their assimilation into proteins. Under water-stress conditions, proline is readily synthesized from glutamate, arginine, and ornithine. and proline synthesized under these conditions turns over very slowly and accumulates.Increases in osmolarity of the medium, whether caused by salts, mannitol, or sucrose (but not by glycerol), is followed immediately by rapid synthesis of proline. Proteolysis is not observed under water-stress conditions. The high level of proline accumulated under these conditions is rapidly reduced when the cells are transferred back to low osmolarity media. The loss of 14C from proline is accompanied by rapid incorporation into proteins and also conversion of proline into other cell constituents.The proline level is relatively low in high-salinity-adapted cells, and proline turns over rapidly in such cells. This indicated that the accumulation of proline occurs mainly in response to abrupt changes of intracellular ionic strength during adaptation to increased salinities. Additions of salts and organic substances to cell suspensions in low-salinity media cause plasmolysis followed by rapid deplasmolysis. However, when cells are transferred to high-osmolarity solutions of single salts or organic substances, deplasmolysis is only observed in media containing KCl or glycerol. Increases of external osmolarity cause increases in concentrations of intracellular potassium and free amino acids.

Regulation of amino acid transport in growing cells of Streptomyces hydrogenans. I. Modulation of transport capacity and amino acid pool composition during the growth cycle.

(1) The active uptake of different amino acids by growing cells of Streptomyces hydrogenans was shown to be correlated with the physiological age of the cells. During the lag phase of growth the transport capacity increased and attained its highest level when the growth rate was maximum. During further growth the transport capacity declined progressively. The lowest transport activity was observed when the culture shifted into the stationary growth phase. (2) Such modulation of transport capacity was independent on the presence or absence of amino acids in the growth medium of the cells. (3) The size and the composition of the pool of free intracellular amino acids was also undergoing substantial variations during the growth cycle of the culture. In the lag phase, the levels of all amino acids decreased markedly and attained their lowest values at the end of this phase. During further growth the pool size was slowly replenished. (4) Removal of the pool resulted in a considerable gain of transport capacity. Therefore, it was concluded that active amino acid transport in growing Streptomyces hydrogenans is under feedback control by intracellular amino acids. (5) Quantitatively, the modulation of the pool size could not fully account for the variation of the transport capacity. Since a pool-independent stimulation of transport was found to be correlated with the increase of the growth rate of the cells, the possibility is discussed that the stimulation of transport is either due to increased levels of distinct RNA species, which might provide positive feedback signals for transport, or by increased rates of de novo synthesis of transport limiting proteins.

Inorganic nitrogen metabolism in marine bacteria: The intracellular free amino acid pools of a marine pseudomonad

The marine pseudomonad bacterium PL1 contains an intracellular pool of free amino acids which consist mainly of glutamate with small amounts of glutamine and aspartate when grown in a nutrient medium containing 0.2 M NaCl. When the NaCl concentration of the growth medium is increased to 0.8 M, proline becomes a major component of the intracellular pool together with glutamate—at this molarity and under suitable nutrient conditions these amino acids comprise 20% of total bacterial amino acid nitrogen. When grown in a nutrient growth medium containing a constant level of NaCl, the intracellular pool size can vary by a factor of 4 depending on the concentration of carbon and nitrogen in the medium. Experiments show that the amino acid pool can act as a nitrogen reserve but has little function as a carbon reserve. At high NaCl concentrations there is a marked dependence for growth on the presence of sufficient potassium in the medium. However, no correlation between K+ and glutamate concentration in either nitrogen or K+-limited cultures has been found. None of the enzymes associated with glutamate biosynthesis was influenced by NaCl levels between 0.2 and 0.5 M. Neither Na+ or K+ stimulated the activity of these enzymes when tested in vitro.

Influence of the postoperative state on the intracellular free amino acids in human muscle tissue.

Five patients were studied before and two to three days after major, uncomplicated abdominal operation. Muscle tissue was obtained by needle biopsy from m. quadriceps femoris after eight hours overnight fast. Plasma free amino acids were analyzed in simultaneously obtained samples. In the homogenized muscle samples the intracellular concentration of each amino acid (IC) was calculated by subtracting the free extracellular part from the total amount, assuming the plasma concentration to be equal to the concentration in the interstitial fluid. Their relationships have also been calculated (IC/EC gradient). The extra- and intracellular water distribution was estimated using a modified chloride method. In similarity to the findings in normal subjects the majority of the amino acids showed much higher concentration in intracellular water than in plasma. Preoperatively all amino acids examined in muscle biopsies were formed within normal limits. Postoperatively the total amount of free amino acids in plasma and muscle was decreased, and the amino acid profiles differed from those observed in normal subjects. In plasma, as compared with normal controls, the most significant changes were an increase in phenylalanine and tyrosine and a decrease in serine, proline, histidine and isoleucine. In muscle the greatest decrease occurred in the concentrations of glutamine, arginine and lysine followed by proline and glutamic acid. The increase in taurine, valine and phenylalanine were all highly significant and in serin, glycine, alanine and leucine significant, whereas tyrosine showed only a moderate rise. Compared with normal values there were marked increases in the gradient between intracellular and plasma concentrations which were highly significant for glycine and valine and significant for serine, alanine, isoleucine and leucine. The shift in the methionine gradient was somewhat less. We confirm that alterations in the muscle free amino acid pool are not reflected in the values found in plasma. Further work is required to explore the clinical significance of the observed variations in individual amino acids.

Anaerobiosis: A Possible Source of Osmotic Solute for High-salinity Acclimation in Marine Molluscs*

ABSTRACT When stressed with high-salinity exposure, cell volume is restored in ventricles of Modiolus demissus demissus by a rapid accumulation of intracellular free amino acids. Although the total amino acid pool increases and remains at a constant high level thereafter, the pattern and time course of accumulation is different for each major amino acid (glycine, alanine, taurine, and proline). Initially, cell volume is restored by a rapid accumulation of alanine, but later its concentration decreases while glycine and taurine accumulate. Although at first not detected, the proline concentration increases, peaks and subsequently disappears again. Isolated ventricles recover normal activity after large environmental salinity increases. During recovery the intracellular free amino acid changes in isolated ventricles are similar to the initial pattern of accumulation in whole animals, i.e., alanine, and to a lesser extent, proline and glycine accumulate. Finally, isolated ventricles undergo a period of decreased oxygen consumption on exposure to an increased salinity. These results suggest that the initial stages of high-salinity acclimation in molluscs depends upon the synthesis of amino acids via a known anaerobic biochemical pathway.

Integumentary Amino Acid Transport and Metabolism in the Apodous Sea Cucumber, Chiridota Rigida

ABSTRACT The apodous sea cucumber, Chiridota rigida, was found to transport exogenous amino acids to intracellular free amino acid pools in the integument, the gastrointestinal epithelium making only a minimal contribution to total animal uptake. Amino acids entering the integumentary free amino acid pool were either completely catabolized to CO2, incorporated into large molecular weight compounds, such as proteins, or left unchanged. The latter fraction was available for efflux through both apical and basal cellular membranes through saturable or non-saturable transport processes. Influx of natural (glycine) and analog (AIB) amino acids into the integumentary epithelium was achieved by a combination of saturable and non-saturable transport mechanisms: the former displaying Michaelis-Menten kinetics, being Na+-dependent, and transporting both glycine and AIB; the latter involving a simple diffusion of amino acid across the apical cell membranes and/or through the intercellular septate desmosomes. Preloaded AIB exhibited both homo-and hetero-exchange diffusion with exogenous amino acids when a relatively large internal analog pool had been established. With a small internal pool of AIB, homo-exchange diffusion persisted, but high external concentrations of glycine inhibited the washout of the analog. Transintegumentary influx of AIB to the coelomic fluid was a linear function of the exogenous amino acid concentration and was unchanged when choline chloride was used as a substitute saline. At steady-state the coelomic fluid analog concentration was maintained at a much lower level than the incubation medium or the epithelial free amino acid pool, due to apparent absorptive processes of the dermis and muscle layers of the integument, as well as by possible carrier-mediated re-uptake mechanisms on the basal membranes of the epithelial cells. A tentative model of integumentary amino acid transport and metabolism in C. rigida is presented.

Incorporation of leucine into human skeletal muscle proteins.

The in vitro incorporation of labelled leucine into human skeletal muscle proteins was studied with the aim to elucidate the relationship between the amino acid tissue pools and protein biosynthesis. The distribution volumes of leucine and cycloleucine in skeletal muscle tissue were similar but the equilibration time was shorter for leucine than for cycloleucine. The cellular uptake of leucine and cycloleucine was competitively inhibited by increased concentration of amino acids in the medium indicating an active transport. Optimal stimulation for incorporation of leucine into proteins was obtained at an amino acid concentration in the medium corresponding to 10 times that of normal human plasma. The incorporation of 14C-leucine into skeletal muscle proteins was linear before the total pool of free intracellular 14C-leucine and the incorporation rate of leucine calculated from the specific activity in the medium versus the amino acid concentration in the medium were different in the same experiment indicating a re-utilization of amino acids released at protein degradation. The results are compatible with the hypothesis that the proteolytically released amino acids have a competitive advantage for incorporation as compared with extra- and intracellular free amino acids. It is concluded that the amino acid pool which is in the immediate continuity with the protein biosynthesis sites equilibrates rapidly with the extracellular amino acid pool.

Protein metabolism in hypertrophied heart muscle.

The following aspects of protein metabolism in myocardium are discussed: (1) The kinetics of labeling of aminoacyl-transfer ribonucleic acid (tRNA) and the intracellular free amino acid pool. The specific activity of leucyl-tRNA correlates closely with that of the extracellular leucine during the first 15 minutes after intravenous injection of (/sup 3/H)leucine. Equilibrium of the leucyl-tRNA with total intracellular leucine is reached at a later time. (2) Determination of the fractional rate constant (k) of myosin synthesis with leucyl-tRNA as precursor. For a period between 15 and 30 minutes after intravenous injection, the k value was approximately 0.1 day/sup -1/(t/2 approximately = 6 days). (3) Synthesis and degradation of mitochondrial components in hypertrophied heart. Cardiac cytochrome c increases faster than the remainder of cellular proteins in the first day after aortic constriction. The results of labeling with delta-aminolevulinic acid indicate increased rate of synthesis and decreased rate of degradation of cytochrome c. (4) Role of lysosomes in regression of cardiac hypertrophy. During the period of rapid fall in cardiac mass and RNA after removal of the constricting band, the activities of cathepsin D and ribonuclease were found unchanged. (5) Properties of calcium-activated neutral protease (CAF), which specifically removes Z lines frommore » myofibrils. It was shown that CAF treatment of myofibrils results in the disappearance of a single protein, ..cap alpha..-actinin.« less

Intracellular free amino acid concentration in human muscle tissue.

Intracellular free amino acid concentration in human muscle tissue.

Inhibition of Hemoglobin Synthesis by Cyanate In Vitro

Abstract Cyanate inhibits sickling and prolongs red cell survival in sickle cell anemia. However, cyanate markedly inhibits hemoglobin synthesis in vitro. Incorporation of radioactive amino acid into hemoglobin by human sickle reticulocytes or bone marrow and by rabbit reticulocytes (whole cell or cell-free lysate) was inhibited by as little as 2 mM cyanate and abolished by 50 mM. Both alpha- and beta-S chains were equally affected. The inhibition was only partially reversible by washing the cells after exposure to cyanate. Transport of radioactive amino acid into the cell was not impaired, and free intracellular amino acid was not carbamylated. Aminoacylation of transfer RNA was not inhibited; the acylated amino acid was not carbamylated. Examination of polysome patterns by sucrose density gradient centrifugation revealed degradation of polysomes to monosomes, suggesting inhibition of initiation of protein synthesis by cyanate. In a cell-free lysate, cyanate prevented hemin stimulation of initiation. We conclude that cyanate profoundly inhibits initiation of hemoglobin synthesis in vitro.

The Initiation and Control of Free Amino Acid Regulation of Cell Volume in Salinity-Stressed Marine Bivalves*

ABSTRACT Molluscan cells maintain constant volume in decreasing salinities by releasing intracellular free amino acids as osmotic solute. During a salinity decrease both osmotic pressure and ion concentrations decline simultaneously. Either condition might initiate the amino acid release. Under constant osmotic conditions a decrease in Na+, K+ or Cl− concentration in the medium surrounding the isolated bivalve heart, did not result in an amino acid efflux. On the other hand, removal of Ca2+ and Mg2+ resulted in a vigorous amino acid efflux which continued until the divalent ion concentration was restored. The hypo-osmotically induced amino acid efflux could be partially blocked by raising the external divalent ion concentration. Similarly, lanthanum proved to be an effective blocking agent, both in low osmotic pressures and in Ca2+- and Mg2+-free SW. These results show that salinity-induced free amino acid regulation is initiated by a decrease in external osmotic pressure. The time course of the efflux is dependent on external divalent ion concentration. Finally, the site of divalent ion action is at the external membrane surface. A hypothetical membrane model is proposed.

Mechanisms of intracellular isosmotic regulation: Extracellular space of the shore crabCarcinus maenas in relation to environmental salinity

1. In shore crabsCarcinus maenas, which had been maintained for one month in 10, 20, 30, 40, and 50‰ salinity, the extracellular space was estimated by means of the dilution of the food dye amaranth and the polyglycan14C-inulin. 2. The extracellular space does not differ significantly in response to the external salinity. It varies between 29.2 and 36.0% (with a mean of 33.1%) of body weight when estimated by dilution of amaranth and between 16.7 and 18.5% (with a mean of 17.9%) of body weight when estimated by14C-inulin dilution. Differences of values in regard to the substances used for estimation are discussed. 3. The results confirm that the magnitude of the extracellular space is not involved in phenomena of osmoregulation. 4. The rapid reduction in the concentration of haemolymph proteins after transfer of freshwater and marine decapods from lower to higher salinities (Drilhon-Courtois 1934,Siebers et al. 1972) implies, on the basis of the present results, also a reduction in the total amount of haemolymph protein in the crab, since the protein-distribution volume remains unchanged within a wide variety of salinities. The assumption that haemolymph proteins participate in the increase of intracellular free amino acids during isosmotic intracellular regulation is confirmed.

Interrelationship between amino acid pools and protein synthesis in the rat submandibular gland

The uptake and incorporation in vitro of radioactive leucine in rat submandibular gland fragments have been studied. Both uptake and incorporation were found to be influenced by the concentration of leucine in the medium. By means of a cold-wash procedure we were able to distinguish between extra- and intracellular free amino acids. We found that it takes 10 to 15 min for the radioactive leucine to reach maximal concentration in the intracellular soluble amino acid pool. Nevertheless their incorporation into protein becomes linear shortly after the start of incubation in the radioactive medium. We did not find any sign of a decreasing rate of protein synthesis during the incubation. In chase-type experiments, the incorporation of radio-active leucine stops immediately when the labelled medium is replaced by unlabelled medium, though the free, intracellular amino acid radioactivity declines only slowly. These results confirm earlier reports, that in vitro, under conditions of sufficient extracellular amino acid supply, the amino acid molecules incorporated into protein are not selected from the intracellular pool.

Osmoregulation in Acanthamoeba castellanii—I. Variations of the concentrations of free intracellular amino acids and of the water content

1. 1. The osmoregulation of Acanthamoeba castellanii, Neff strain, was studied in media having the same osmotic pressures but different salt contents. 2. 2. The amoeba survives a direct transfer from Neff's optimum growth medium, at 240 mOsmoles, to incubation media at 40, 200 and 500 mOsmoles. 3. 3. The amoeba water content is inversely proportional to the osmotic pressure of the incubation medium, but it is significantly influenced by the nature of the medium. 4. 4. The results of chromatographic analysis show that the free amino acid pool plays an active role in intracellular osmotic regulation. 5. 5. By comparing the results obtained in media having the same osmotic pressure but different salt contents, it is shown that the stimulus for increasing the concentration of free amino acids is mainly the osmolality of the medium. 6. 6. The role of the free amino acids in the osmoregulation of A. castellanii is discussed from the standpoint of comparative physiology.

The Nature of Cellular Volume Regulation in Marine Bivalves*

ABSTRACT Regulation of cell volume utilizing intracellular free amino acids has been studied in isolated ventricles from marine bivalves of the genus Modiolus. As in the intact animal, ventricles taken from Modiolus acclimated to various salinities show only a slight change in tissue hydration. This control over cell volume is accomplished by isosmotic intracellular regulation of taurine, alanine, glycine and proline concentrations. When stressed with decreased external salinities the isolated spontaneously beating ventricle becomes quiescent for a period, and then resumes activity. During the period of quiescence ninhydrin-positive substances (NPS) are released. The duration of quiscence and the amount of NPS released increase with increasing dilution of the external medium. The salinity-induced NPS efflux is composed of taurine, alanine, glycine and proline. In molluscs, the amino acids utilized for volume regulation are released from the cells unchanged and are not degraded into keto-acids and ammonia as they are in the crustaceans.

Kinetics of osmoregulation in the crab Carcinus maenas

When shore crabs Carcinus maenas are transferred from 11 to 38‰ S at 11°C, new constant levels of hemolymph freezing points and of concentrations of Na, K, Ca, and Mg in the hemolymph are accomplished within 24h. From a decrease in serum protein and in serum free amino acids and an increase in the relative amounts of individual essential free amino acids in the body fluids of whole crabs, a participation of proteolytic activities and a transport of amino acids from hemolymph to cell is deduced; the stationary concentration of total intracellular free amino acids increases up to a nearly two-fold value, compared with the concentration in crabs remaining in diluted sea water. Also, the low molecular neutral sugars increase in whole crabs after high salinity stress, reaching values of more than two-fold initial concentrations within a period of 10 days. This increase is fully accounted for by a 6.7-fold increase in trehalose levels. The processes of increasing the concentrations of low molecular organic material seem to be slower than those of establishing new osmotic pressures in the hemolymph. The oxygen consumption decreases by 30 to 45% to new constant values within 8 to 12 h after the salinity change from 11 to 38‰, reflecting similar kinetics as the establishment of new osmotic pressures in the hemolymph.

Changes in free amino acid production and intracellular amino acid pools of Bacillus licheniformis as a function of culture age and growth media.

Changes in the endogenous intracellular amino acid pool and total free amino acid production in Bacillus licheniformis grown in minimal media were investigated. The total intracellular pool increased during exponential growth and then decreased rapidly after the end of growth. Most of the amino acids were present at low concentrations, but glutamate and alanine comprised 60 to 90% of the total intracellular free amino acid at most times during the growth cycle. It was concluded that, in addition to providing monomers for protein synthesis, the intracellular amino acid pool may be maintained for the storage of energy-providing metabolic intermediates and possibly as a balance to the ionic strength of the medium. The total free amino acid production by the cell was found to be dependent upon the composition of the salts medium as well as the culture age under conditions in which the carbon and nitrogen sources were the same. A 10-fold increase in extracellular amino acid was observed as the cells changed from vegetative to sporulation metabolism, mostly due to the extrusion of intracellular amino acid. The impact of this increase upon amino acid uptake and pulse-labeling studies using unwashed cells is discussed.

Relationship between extracellular amino acids and protein synthesis in vitro in the rat pancreas.

The uptake and incorporation in vitro of radioactive leucine and lysine in rat pancreas fragments have been studied. The uptake and incorporation of both leucine and lysine were found to be influenced by the concentration of leucine in the medium. Extracellular and intracellular free amino acids can be distinguished by washing the tissue fragments at 0 °C. Though it takes some 20 min for each of both radioactive amino acids to reach maximum concentration in the intracellular soluble amino acid pool, their incorporation into protein becomes linear shortly after the start of incubation in a medium with labelled amino acids. In chase‐type experiments the incorporation of each of both radioactive amino acids stops immediately when the labelled medium is replaced by unlabelled medium, though the free, intracellular amino‐acid radioactivity declines only slowly. These results indicate that in the pancreas of the fasting rat in vitro the amino acid molecules incorporated into protein are derived from the cxtracellular pool. Other molecules enter and leave the intracellular amino acid pool but do not contribute to incorporation.

A chemostat study of the effect of fixed nitrogen sources on nitrogen fixation, membranes and free amino acids in Azotobacter chroococcum.

SUMMARY: Increasing concentrations of ammonium ions in the medium of nitrogen-fixing, sulphate-limited continuous cultures of Azotobacter chroococcum caused a proportionate repression of nitrogenase activity; free NH4 + could be detected in the extracellular culture fluid only when nitrogenase activity was wholly repressed. The NH4 + concentrations giving 50% or 100% repression were proportional to the population density. Nitrate ions repressed with similar stoichiometry; glutamate, glutamine and aspartate did not repress and were not metabolized; repressed and derepressed populations contained equal amounts and proportions of glutamate-forming enzymes. Repressed populations lacked both enzymatic components of nitrogenase. The intracellular free amino acid pools were typical of Gram-negative bacteria; an increase in the degree of repression was associated with an increase in the pool levels of ammonia, aspartate and glutamate. Nitrogen-fixing populations possessed a convoluted intracytoplasmic membrane system which was absent from ammonia-assimilating organisms, but the phospholipid contents of the two types of population were similar. All members of a half-repressed population possessed these membranes, but to a lesser extent that fully derepressed populations. When N2-fixing chemostat populations were abruptly exposed to repressive concentrations of ammonium succinate. repression occurred exponentially and nitrogenase activity disappeared from the culture faster than wash-out of stable enzyme. Repression was not alleviated by exogenous cyclic AMP. Derepression was complete, according to the acetylene test, within half a doubling time of disappearance of free ammonium ions from the culture.