Intracellular Amino Acid Pool Research Articles

A source of solute for volume regulation in marine mussels

1.1. The ionic concentrations in the blood and pericardial fluid of intertidal and subtidal species of Modiolus, acclimated to various non-lethal salinities, were examined. Variation in the intracellular free amino acid pools in response to salinity change were also studied.2.2. The ionic concentrations of the body fluids of Modiolus are not regulated. Rather, a Donnan equilibrium seems to exist between the body fluids and the sea water, and between different body fluid compartments.3.3. Free amino acid regulation, identified by a greater decrease in a portion of the intracellular free amino acid pool, with decreasing salinity, than can be accounted for on the basis of changes in amounts of tissue water, occurs in Modiolus.4.4. The amino acid pool is a likely source of solute for the volume regulating response. Further, differences in the pools of the various mussels studied seem to account for the differences in salinity tolerance.

Further studies on the kinetics of incorporation of valine in the sea urchin embryo

The kinetics of incorporation of 14C-valine in the sea urchin embryo have been studied particularly with respect to the time course of incorporation and the effect of exogenous valine on incorporation of 14C-valine from the amino acid pool. The data are interpreted as reflecting a rapid and continual expansion of the valine pool in the presence of exogenous valine. All kinetic aspects, the apparent linearity of the time course of incorporation, the nonlinear relationship of uptake and incorporation at different concentrations of exogenous valine, and the apparent cessation of incorporation of pool valine in the presence of exogenous valine, can be satisfactorily interpreted by the concept of a readily expansible valine pool. An earlier interpretation that the intracellular amino acid pool is compartmented as regards protein synthesis is untenable, and it is concluded that the total amino acid pool is an obligatory intermediate for incorporation of exogenous amino acids.

Cystine metabolism in the dog

35S- l-cystine was injected intravenously into anaesthetised dogs, and the rate of disappearance of radioactive cystine was measured. Cystine was isolated from the plasma by two different techniques before determination of the radioactivity. It was shown that there was an almost instantaneous equilibration of the isotope into a volume greater than the plasma and extracellular space. The rate of decrease of radioactivity of plasma cystine was expressed for two dogs as the sum of two exponential terms. The first term may represent uptake of extracellular cystine into the intracellular space, and the second term may more closely represent the rate of turnover of radio-active cystine. The values corresponded to a turnover rate of 16 μmole/kg/hr in one dog and 38 μmole/kg/hr in another dog. The fate of the 35S- l-cystine could not be determined, but it is believed that most of the disappearance of radioactivity could be explained by the uptake of the amino acid by the tissues and equilibration with the intracellular amino acid pool.

Protein synthesis in Ehrlich ascites cells.

ABSTRACTRapidly dividing Ehrlich ascites cells have a higher rate of protein synthesis than slowly‐growing cells. This is true whether protein synthesis is measured in vivo or in a cell‐free amino acid incorporating system. A lack of endogenous messenger RNA or a ribosomal defect in stationary cells is suggested as a likely cause of the decreased rate of protein synthesis.The soluble proteins synthesized by rapidly‐dividing cells are qualitatively different from those synthesized by slowly‐growing cells. The latter also have a larger intracellular pool of several amino acids and a slower protein turnover than rapidly‐dividing cells.

Degradation of cell constituents by starved Streptococcus lactis in relation to survival.

SUMMARY Streptococcus lactis organisms were grown in lactose-limited batch culture and transferred, after washing, to phosphate buffer at the growth temperature. Soluble protein was released from viable organisms into the suspending buffer and the intracellular free amino acid pool declined steadily with the components appearing in the suspending buffer; a net increase in the total amount of free amino acid indicated some protein hydrolysis. RNA was hydrolysed, resulting in the release of u.v.-absorbing bases and ribose from the organisms. Conditions which promoted rapid RNA breakdown also produced rapid death rates and long cell division lags in surviving organisms. After 28 hr starvation in buffer containing Mg2+, the bacterial dry wt decreased by 26 %; loss of RNA, protein and free amino acids accounted for 10·3 %, 7·3 % and 2·7 % of the total bacterial mass loss. The products of polymer hydrolysis appeared to be released in an undegraded form into the external buffer and there was no appreciable formation of lactate, ammonia or volatile fatty acids, possibly indicating the absence of any important endogenous energy sources. There was no appreciable degradation of DNA or carbohydrate but phospholipid was broken down on prolonged starvation. No polyglucose or poly-β-hydroxybutyrate was detected in the organisms.

Changes in permeability and ultrastructure of starved Streptococcus lactis in relation to survival.

SUMMARY When Streptococcus lactis was starved in phosphate buffer the intracellular amino acid pool was rapidly released into the external medium from the onset of starvation whereas lactic dehydrogenase and DNA appeared in the suspending buffer only as organisms began to lose viability. Addition of spermine enhanced survival and suppressed the release of ultraviolet-absorbing material. Thin sections of Streptococcus lactis were examined by electron microscopy at intervals during starvation in a number of environments. Ribosome particles rapidly disappeared from organisms starved in the absence of Mg2+ and non-viable organisms remained structurally intact; in the presence of Mg2+ ribo-somes were retained but, after prolonged starvation, some organisms autolysed soon after the death rate increased. Addition of a suitable energy source maintained cell structures intact for a much longer period but again lysis occurred as the death rate increased. Starvation led to unfolding or extrusion of the mesosomes and dislocation of the nuclear material. The death rate of starved S. lactis organisms in phosphate buffer partly depended on the presence of Mg2+, which probably acted by promoting polymer stability, particularly RNA. In this environment, a suitable exogenous energy source further enhanced survival which may ultimately be a function of cell wall and/or membrane stability.

Isosmotic intracellular regulation as a mechanism of volume control in crab muscle fibers

1. 1. Muscle fibers swollen in hypotonic sodium salines undergo spontaneous decreases in volumes (volume-readjustment) towards he initial volumes of the cells found in isotonic salines. 2. 2. This volume-readjustment is due to the elimination of osmotically active particles from the intracellular space of the muscle fibre. 3. 3. The only major intracellular solutes which were significantly lost from blue crab muscles as a result of volume-readjustment were the non-protein ninhydrin-positive substances. 4. 4. It is proposed that this component represents a fraction of the large intracellular pool of free amino acids found in crab muscle, and that free amino acids are being actively transported out of the muscles during volume-readjustment. 5. 5. Results of preliminary studies on the efflux of 2-aminoisobutyric acid-1- 14C from crab muscles are consistent with the view that amino acids are being actively transported out of the muscle cells during volume readjustment.

Polyribosomes of Hamster Cells: Transit Time Measurements

The amount of radioactivity incorporated into completed polypeptides and into nascent polypeptides bound to polyribosomes was measured for rapidly dividing, cultured hamster embryo cells incubated in the presence of labelled amino acids. Using a mathematical analysis which took into account variations in the specific activity of the intracellular amino acid pool, these measurements yielded a value for the length of time required for the synthesis of a primary polypeptide of average length, the "transit time." Subject to a number of reasonable assumptions in the mathematical analysis which were, however, only approximately verified, the transit time was found to be 23 +/- 2 sec in four independent experiments. Regardless of the absolute accuracy of this measurement, the method is considered a useful measure of translation independent of transcription.

Origins and metabolism of the intracellular amino acid pools in rat liver and muscle

1. 1. When [ 14C]amino acids were continuously administered to a rat by intravenous infusion, the specific activity of the liver and muscle intracellular free amino acid soon increased but even when the intracellular pool was maximally labeled, the specific activity was lower than the plasma specific activity. This observed intracellular dilution has been interpreted as being due to intracellular unlabeled protein breakdown. 2. 2. Protein catabolism in the normal fed rats contributed approx. 50% of the liver's and 30% of the muscle's intracellular amino acid pool. 3. 3. During the early stages of fasting as much as 90% of liver amino acid was derived from protein degradation, with a major fraction entering the plasma to supply amino acids for other tissues. After depletion of the labile liver protein, muscle protein breakdown has increased and supplied 65% of muscle intracellular amino acid pool, which in time maintained the plasma and tissue amino acid levels.

Glucose-induced Release of Amino Acids from Saccharomyces carlsbergensis by Action on the Cytoplasmic Membrane

When washed yeast cells grown under appropriate conditions were suspended in glucose solution there was a sudden release of alpha-amino nitrogen into the medium. This released material was of low molecular weight, and its composition was closely similar to that of the intracellular free amino acid pool. During the leakage of amino acids, the yeast did not efficiently absorb labeled amino acids added to the test medium, despite the rapid uptake and metabolism of glucose. Uptake of a labeled amino acid and reabsorption of the released alpha-amino nitrogen occurred almost simultaneously. When these yeast cells were exposed to glucose in the presence of calcium ions, leakage was strongly inhibited. Butanol under the same conditions increased glucose-induced leakage of cell contents. The adenosine triphosphatase activity of intact yeast cells exposed to glucose was greater than that of cells exposed to water. Yeast cells treated with glucose prior to equilibration with sorbose exhibited less ability to retain the sorbose when washed at 0 C than did cells pretreated with water. It was concluded that glucose-induced leakage of amino acids was the result of two factors acting together. These were (i) a change in membrane permeability associated with glucose uptake, and (ii) a temporary shortage of energy for amino acid uptake or retention.

The intracellular amino acid pool of Candida utilis during growth in batch and continuous flow cultures.

Abstract 1. 1. The amino acid pool extracted from cells of Candida utilis , grown on simple chemically defined media by batch and chemostat methods, was investigated. 2. 2. The spectrum of the amino acids in the pool was significant as a register of the cell metabolism. 3. 3. The composition of the pool was found to be related to the medium, to the growth rate, and to the environmental conditions. 4. 4. The relationship of the pool to cell regulation is discussed.

Action de la vératrine, de la cocaïne et de la stimulation électrique sur la synthèse et sur le pool des acides aminés de la chaîne nerveuse ventrale du homard

Resume 1. 1. The synthesis of amino acids from [ 14 G 6 ]glucose and [I- 14 C]pyruvate in the ventral nerve cord of the lobster ( Homarus vulgaris L.) has beeb studied under the following conditions: in the presence of veratrine sulfate (4 μM), cocaine hydrochloride (4 mM) and during electrical stimulation. Our results show that veratrine and electrical stimulation increase the turn-over rate of the following amino acids: alanine, glycine, glutamic acid and aspartic acid. Cocaine brings about a decrease in the turn-over rate of dicarboxylic acids (glutamic acid and aspartic acid) and an increase in the labelling of alanine and glycine. Our experimental conditions are without effect on the labelling of arginine and lysine. These results are discussed in the light of the various metabolic pathways envisaged in a previous paper. Veratrine and electrical stimulation seem to be without effect on the rate of pyruvate amination. On the contrary, they seem to affect the metabolic pathways involving hydroxypyruvate or the decarboxylation in C-1 of pyruvate. Moreover, it would appear that these experimental conditions increase the rate of entry of pyruvate into the Krebs cycle. Concerning the effects of cocaine, our results indicate that it inhibits the access of pyruvate to the Krebs cycle. 2. 2. Contrary to that observed with cocaine, veratrine sulfate and electrical stimulation affect the cationic composition of the nerve cord (increase in Na concentration, decrease in K concentration). 3. 3. The pool of intracellular free amino acids is unaffected by veratrine. Thus veratrine seems to increase the turn-over rate of various amino acids. This conclusion is substantiated by the fact that the ammoniac content increases after application of veratrine. Electrical stimulation leads to an increase in the total concentration of the pool of free amino acids. 4. 4. The results are discussed within the framework of the osmoregulatory phenomena in aquatic invertebrates. A hypothesis concerning the mechanisms used by euryhaline invertebrates to adjust the intracellular concentration to that of the internal medium is proposed.

EFFECT OF TETRACYCLINES ON THE INTRACELLULAR AMINO ACIDS OF MOLDS.

Freeman, Bob A. (University of Chicago, Chicago, Ill.) and Richard Circo. Effect of tetracyclines on the intracellular amino acids of molds. J. Bacteriol. 86:38-44. 1963.-The tetracycline antibiotics were shown to alter the amino acid metabolism of molds whose growth is not markedly affected. Eight molds were grown in the presence of these antiobiotics; four exhibited a general reduction in the concentration of the intracellular amino acids, except for glutamic acid and alanine. In most of these four cultures, the tetracyclines also caused the complete disappearance of arginine, lysine, proline, phenylalanine, and tyrosine from the intracellular amino acid pool. The significance of these observations and the usefulness of the method in the study of the mechanisms of antibiotic action are discussed.

Studies on the biochemical basis of the low maximum temperature in a psychrophilic cryptococcus.

SUMMARY: A psychrophilic species of Cryptococcus, freshly inoculated cultures of which grew well in a glucose + salts + vitamins medium at 16° but not at all at 30°, was induced to grow rapidly for a period at 30° by previous incubation at 16°. A study was made of certain aspects of the biochemical composition of the cryptococcus in cultures maintained at 16° as compared with that in cultures transferred from 16° to 30°, in order to obtain information about the biochemical basis of the low maximum growth temperature for this organism. With the cryptococcus transferred from 16° to 30°, there was rapid utilization of intracellular reserves of total amino acids and total oxo acids. Exogenous sources of amino acids were unable to replenish the intracellular amino acid pool in the cryptococcus incubated at 30°. However, addition of α-oxoglutarate, citrate, or dl-isocitrate to cultures transferred to 30° after 120 hr. at 16° enabled the cryptococcus to continue to grow at the higher temperature; stimulation of growth at 30° was most marked in cultures supplemented with α-oxoglutarate. Other tricarboxylic acid cycle and related intermediates (pyruvate, acetate, succinate, fumarate, l-malate, oxaloacetate) had no growth-promoting effect. Stimulation of growth at 30° in the presence of α-oxoglutarate was obtained only in cultures which had been incubated for 2–4 days at 16°, indicating the existence of other temperature-sensitive metabolic processes in addition to those concerned in α-oxoglutarate synthesis.

Functional heterogeneity of the intracellular amino acid pool in mammalian cells

1. 1. The kinetics of tracer amino acid appearance in the intracellular amino acid pool and in cellular protein was examined under steady-state conditions in the intact rat-diaphragm preparation and in isolated guinea pig and rabbit-lymph node cells. 2. 2. The concentration ratio of tracer amino acid between the extracellular and intracellular amino acid pools approached equilibrium exponentially whereas incorporation into cellular protein proceeded linearly from the earliest time period studied. 3. 3. A theoretical formulation is presented predicting the kinetics of amino acid incorporation into cellular protein if the total intracellular amino acid pool were an obligatory intermediate in protein synthesis. 4. 4. The marked deviation of experimental observations from those theoretically predicted has been interpreted to indicate functional heterogeneity (compartmentation) of the intracellular amino acid pool.

Some Properties of a Gram-Negative Heterotrophic Marine Bacterium

SUMMARY: Some properties of a marine bacterium are described. This organism belongs to a large, but ill-defined, group comprising Gram-negative rods with no apparent action on monosaccharides. It is a polar flagellate, has limited acid tolerance, consistent with its inability to produce acid from sugars and has no constitutive system for metabolizing glucose. A weak inducible system was available, however, which enabled adapted organisms to grow on glucose and use it as a respiratory substrate. The organism also lacks a constitutive citric acid cycle but ability to metabolize succinate was induced by growth on succinate or glucose. It cannot use inorganic nitrogen for growth. Of the compounds examined, amino acids were the most generally satisfactory sources of energy, carbon, and nitrogen. The organism accumulated a relatively large pool of intracellular amino acids which probably contributed largely to its high rate of endogenous respiration. The classification of the bacterium is discussed.

Poliovirus Protein: Source of Amino Acids and Time Course of Synthesis

The techniques previously available for the study of purified animal viruses have not permitted the production under controlled conditions of sufficient virus for experiments involving degradative chemical analysis. The utilization of suspension cell cultures (1) and the development of a simple, convenient virus purification procedure (2) have now made possible an investigation of the questions of the origin and time of synthesis of virus protein. Type 1 poliovirus was propagated in HeLa 53 cells (3) differentially labeled with L-valine-Cl”, and the isotope content of the valine of the isolated virus protein was determined. The results indicate that the virus protein is assembled from the large intracellular amino acid pool of the cells, and that the synthesis of the virus protein begins almost at the same time as the appearance of mature virus, but proceeds thereafter at a more rapid rate than maturation.

An effect of insulin on peptide synthesis independent of glucose or amino-acid transport.

INSULIN enhances incorporation of amino-acids labelled with carbon-14 into protein of isolated rat diaphragms independent of its action in facilitating entry of glucose1–3. Kipnis and Noall4 report that in similar circumstances insulin increases the accumulation of 14C-α-aminoisobutyric acid, a non-utilizable amino-acid. If the rate of penetration of the utilizable labelled amino-acids is also increased by insulin, the result would be a rise in the radioactivity of the intracellular amino-acid pool. Then an apparent increase in the rate of incorporation could occur without an actual change in the rate of peptide synthesis. This possibility has now been tested. Increased transport does not account for the insulin-mediated increase in the incorporation of utilizable amino-acids.

Observations on the amino acid pool and cellular leakage in intact cells of Rhizobium meliloti.

Cells of Rhizobium meliloti contained an intracellular pool of amino acids. The passage of C14-histidine or C14-glutamate into this pool apparently occurred against a concentration gradient and was energy dependent, being inhibited by azide and dinitrophenol. An internal bound form of C14, containing a number of radioactive amino acids, was synthesized at the expense of radioactive pool components. This bound C14consisted predominantly of several proteins whose syntheses were inhibited by chloramphenicol. Added histidine was not incorporated directly into this protein since the protein histidine was nonradioactive. When C14-labelled cells were suspended in glucose-containing basal medium there was an efflux of pool amino acids into the extracellular environment. This leakage presumably was the result of simple diffusion since it was energy independent, occurred with the concentration gradient, and was appreciable at 5 °C. Rapid leakage occurred at 30 °C in the presence and absence of dinitrophenol for an initial period of 6 hours, after which time growth occurred in the absence of this inhibitor. During growth leakage ceased, and a considerable quantity of material passed back into the cells and was incorporated into a bound form. In the presence of dinitrophenol no growth occurred, and the outflow continued after the 6th hour, but at a much reduced rate. X irradiation increased the efflux of pool constituents, but the reason for this is conjectural.