Comparative transport activity of intact cells, membrane vesicles, and mesosomes of Bacillus licheniformis

Sodium ion was shown to stimulate strongly the transport of l-glutamic acid into cells of Bacillus licheniformis 6346 His(-). Lithium ion had a slight capacity to replace Na(+) in this capacity, but K(+) was without effect. Three of five amino acids tested. l-glutamic acid, l-aspartic acid, and l-alanine, were concentrated against a gradient in the cells. Intracellular pools of these amino acids were extractable with 5% trichloroacetic acid. Pools of l-histidine and l-lysine could not be detected. No evidence of active transport of lysine into cells could be detected, and histidine was taken up in the absence of chloramphenicol but not in its presence. The uptake of glutamic acid by membrane vesicle preparations was strongly stimulated by reduced nicotinamide adenine dinucleotide (NADH) and to a lesser extent by succinate. The presence of phenazine methosulfate increased uptake in the presence of succinate. Either l- or d-lactate and adenosine triphosphate were without effect. None of these compounds stimulated the uptake of glutamic acid by mesosomes, although some mesosome preparations contained separable membrane which was very active. NADH strongly stimulated the uptake of aspartic acid and alanine by membrane vesicles but had only a slight effect on the uptake of histidine and lysine. No evidence of active transport of any of the amino acids into mesosomes could be detected either in the presence or absence of NADH. NADH stimulation of the uptake of glutamic acid by membrane vesicles was destroyed by exposure to light of 360 nm; this inactivation was reversible by vitamin K(2(5)) or K(2(10)). Sodium ion stimulated transport of glutamic acid by membrane vesicles.     

Selective separation of amino acids by reactive extraction

The method of reactive extraction with di-(2-ethylhexyl)phosphoric acid (D2EHPA) for the separation of a range of amino acids is studied. The results obtained on the individual reactive extraction indicated the possibility of the amino acids selective separation as a function of the pH value of aqueous solution and the acidic or basic character of each amino acid. Thus, using multistage extraction, the total separation of the following amino acids groups has been performed: neutral amino acids (l-glycine, l-alanine, l-tryptophan) at pH 5–5.5 (nine extraction stages), basic amino acids (l-lysine, l-arginine) and l-cysteine at pH 4–4.5 (ten extraction stages), l-histidine at pH 3–3.5 (five extraction stages), and acidic amino acids (l-aspartic acid, l-glutamic acid) at pH 2–2.5 (three extraction stages).     

Uptake and Incorporation of Amino Acids and Peptides by Bacteroides amylophilus

Bacteroides amylophilus H-18 demonstrated a higher growth yield, a slightly higher growth rate, and a diminished lag period when Tryptose was added to the basal medium. This uptake of labeled amino acids was concentration-dependent, as the contribution of exogenous amino acid to the cell protein increased from 15.4 to 24.1% when the concentration of Casamino Acids in the medium was increased from 1.4 to 2.8 mg/ml. There was considerable redistribution of (14)C-label to other amino acids. Tryptic peptides of casein competed effectively with the amino acids for uptake. The (14)C-label from a protein was incorporated into B. amylophilus H-18 cells presumably after breakdown of the protein by the B. amylophilus H-18 protease.     

Nitrification of Aspartate by Aspergillus flavus

Heterotrophic conversion of l-aspartic acid to nitrification products by Aspergillus flavus was studied in a replacement incubation system. Numerous amino acids supported nitrification; aspartate and glutamate were about equivalent as the best sources of nitrate. Addition of sodium bicarbonate to the incubation system substantially enhanced nitrate formation for all nitrifiable amino acids except aspartic acid, but the basis for the bicarbonate effect is obscure. The yield of nitrate from l-aspartate was not approached by forms of aspartic acid resulting from substitution on the beta carbon, the amino nitrogen, or the gamma carboxyl group or by aspartate presented as the d-configuration. There was no relationship between nitrate formation and the occurrence of such possible intermediates as nitrite, bound hydroxylamine, ammonia, aspergillic acid, and beta-nitropropionic acid. Uniformly labeled (14)C-l-aspartate that was nitrified in replacement incubation led to no accumulation of label in possible nitrification products in the culture filtrate. Label was found in components of the mycelium after acid hydrolysis, with heaviest accumulation in what appeared to be glucosamine and an unidentified compound, possibly acetylglucosamine. Detectable label was redistributed into serine, glycine, and threonine.     

Induction of pigmentation in nonproliferating cells of Serratia marcescens by addition of single amino acids

Addition of casein hydrolysate to suspensions of washed, nonpigmented, nonproliferating Serratia marcescens incubating at 27 C induced biosynthesis of prodigiosin. Four amino acids of casein hydrolysate, dl-aspartic acid, l-glutamic acid, l-proline, and l-alanine caused formation of pigment when added individually. dl-Ornithine also was effective. Optimal concentrations for maximal pigmentation were 5 to 10 mg/ml; at these high concentrations, d-serine also induced biosynthesis of some prodigiosin. dl-Alanine and -ornithine were as effective as the l-iosomers, but l-glutamic acid and l-proline gave better responses than their racemic mixtures. Kinetics of prodigiosin biosynthesis after addition of dl-alanine (20 mg/ml) were similar to those of cells suspended in 0.2% casein hydrolysate. The other amino acids were less effective. Addition of 5 mg of dl-alanine or casein hydrolysate per ml to minimal medium increased by 30% the amount of prodigiosin formed by growing cells after incubation for 7 days at 27 C. Cultures grown for 7 days at 27 C in 0.2% casein hydrolsate formed more prodigiosin than did suspensions of nonproliferating cells containing individual amino acids or casein hydrolysate. However, more pigment was produced by cells suspended in l-alanine (5 mg/ml) or l-proline (10 mg/ml) than when suspended in 0.4% natural or synthetic casein hydrolysate. Filtrates from suspensions of nonproliferating cells forming pigment in l-proline induced more rapid formation of prodigiosin, but filtrates from suspensions in dl-alanine did not. The data supported the hypothesis that pyrrole groups of prodigiosin may be synthesized from 5-carbon amino acids such as proline, ornithine, aspartic, and glutamic acids, but the role of alanine is unknown.     

Manufacture of l-amino acids with bioreactors

Several l-amino acids are currently manufactured by using enzymes or whole-cells contained within bioreactors. Production of l-aspartic acid in Japan is one of the earliest examples of this technology. A bioreactor method recently scaled-up for manufacture of l-phenylalanine from cinnamic acid and ammonia achieves product titers of over 60 g/l and a raw material conversion of about 90%. A bioreactor process in development for making l-serine from glycine and formaldehyde exploits rDNA technology and reaches an l-serine titer of over 400 g/l. The l-serine can be enzymatically converted to L-tryptophan to achieve a bioreactor titer of about 200 g/l.     

Umami evaluation in taste epithelium on microelectrode array by extracellular electrophysiological recording

Umami is one of the basic tastes along with sweet, bitter, sour and salty. It is often elicited by amino acids and can provide a palatable flavor for food. With taste epithelium as the sensing element, microelectrodes can be used to evaluate umami taste by biological responses of the tissue. The electrophysiological activities to umami stimuli are measured with a 60-channel microelectrode array (MEA). Local field potential (LFP) recorded by a MEA system showed different temporal characteristics respectively with l-glutamic acid (l-Glu), l-aspartic acid (l-Asp), l-monosodium glutamate (l-MSG) and l-monosodium aspartate (l-MSA), while remarkable differences were observed between amino acids and their sodium salts. Wealso found that a dose-dependent behavior in the increasing concentrations of umami stimulations and a synergistic enhancement between amino acids and purine nucleotides can be detected. The investigation of this evaluation for umami represents a promising approach for distinguishing and evaluating umami tastants.     

Influence of amino acid supply on ribosomes and protein synthesis of perfused rat liver

1. The livers of rats were perfused in situ with medium containing mixtures of amino acids in multiples of their concentration in normal rat plasma. The incorporation of labelled amino acid into protein of the liver and of the perfusing medium increased with increasing amino acid concentration. During 60min. perfusions, labelling of liver protein reached a plateau, and labelling of medium protein was inhibited when the initial concentration of the amino acid mixture was more than ten times the normal plasma value. 2. Examination of polysome profiles derived from livers perfused without amino acids in the medium showed that the number of large aggregates was decreased and the number of small aggregates, particularly monomers and dimers, was increased with time of perfusion. The addition of amino acids to the perfusion medium reversed this polysome shift to an extent that was dependent on the initial concentration of amino acids. Polysome profiles derived from livers perfused for 60min. with ten times the normal plasma concentration of amino acids were essentially the same as the polysome profiles of normal non-perfused livers. 3. The ability of ribosome preparations from perfused livers to incorporate amino acids into protein in vitro decreased with increasing time of perfusion when no amino acids were added to the medium, but increased as the concentration of amino acids in the perfusion medium was increased. 4. The ability of cell sap from perfused livers to support protein synthesis in vitro was not influenced by the amino acid concentration of the perfusion medium. 5. Livers were perfused for 60min. with medium containing amino acid mixtures at ten times the normal plasma concentration but deficient in one amino acid. Maximal incorporation of labelled amino acid into liver protein, the stability of the polysome profile and the ability of ribosome preparations to incorporate amino acids into protein were found to depend on the presence of 11 amino acids: arginine, asparagine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan and valine. A mixture of these 11 amino acids, at ten times their normal plasma concentration, stimulated the incorporation of labelled amino acid into liver protein, stabilized the polysome profile and increased the ability of ribosome preparations to incorporate amino acids into protein to the same extent as the complete mixture. 6. It is concluded that the availability of certain amino acids plays an important role in the control of protein synthesis, possibly by stimulating the ability of ribosomes to become, and to remain, attached to messenger RNA.     

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.     

Effects of acetate and other short-chain fatty acids on sugar and amino acid uptake of Bacillus subtilis

Acetate and other short chain n-fatty acids (C(1)-C(6)) inhibit strongly the uptake of l-serine or other l-amino acids but inhibit only weakly that of alpha-methylglucoside or fructose, whether measured in whole cells of Bacillus subtilis or in membrane vesicles that have been energized with reduced nicotinamide adenine dinucleotide (NADH), l-alpha-glycerol phosphate, or ascorbate plus phenazine methosulfate. The acetate inhibition is noncompetitive, as was shown for l-alpha-aminoisobutyric acid uptake by whole cells and for l-serine uptake by membrane vesicles. In membrane preparations, neither NADH oxidation nor the reduction of cytochromes by NADH are affected by fatty acids. All of these effects are similar to those of 2, 4-dinitrophenol. It is concluded that the fatty acids "uncouple" the amino acid carrier proteins from the cytochrome-linked electron transport system (to which they may be coupled via protein interaction or via a cation gradient).     

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 α-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 α-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.     

Isolation of cDNA clones coding for spinach nitrite reductase: Complete sequence and nitrate induction

Summary The main nitrogen source for most higher plants is soil nitrate. Prior to its incorporation into amino acids, plants reduce nitrate to ammonia in two enzymatic steps. Nitrate is reduced by nitrate reductase to nitrite, which is further reduced to ammonia by nitrite reductase. In this paper, the complete primary sequence of the precursor protein for spinach nitrite reductase has been deduced from cloned cDNAs. The cDNA clones were isolated from a nitrate-induced cDNA library in two ways: through the use of oligonucleotide probes based on partial amino acid sequences of nitrite reductase and through the use of antibodies raised against purified nitrite reductase. The precursor protein for nitrite reductase is 594 amino acids long and has a 32 amino acid extension at the N-terminal end of the mature protein. These 32 amino acids most likely serve as a transit peptide involved in directing this nuclearencoded protein into the chloroplast. The cDNA hybridizes to a 2.3 kb RNA whose steady-state level is markedly increased upon induction with nitrate.     

Evidence for stable messenger ribonucleic acid during sporulation and enterotoxin synthesis by Clostridium perfringens type A.

Stable messenger ribonucleic acid (mRNA) was shown to be involved in both enterotoxin synthesis and synthesis of other spore coat proteins in Clostridium perfringens. When used at a concentration that inhibited [14C]uracil incorporation, rifampin, a specific inhibitor of deoxyribonucleic acid-dependent RNA polymerase, prevented incorporation of a mixture of labeled amnoo acids by 3-h sporulating cells. At that time, enterotoxin protein was first detectable and cells were primarily at stage II or III of sporulation. When rifampin or streptolydigin was added to 5-h sporulating cells, which were primarily at stage IV or V and had significant toxin levels, incorporation of labeled amino acids continued through 30 min despite its presence. Rifampin also failed to prevent the specific synthesis of enterotoxin, a structural protein of the spore coat. The half-life of enterotoxin RNA was estimated to be at least 58 min. When cell extracts from 5-h sporulating cells that had been exposed to 3H-labeled amino acids for 10 min were subjected to electrophoresis on polyacrylamide gels and the gels were subsequently analyzed for radioactivity, two major peaks of radioactivity were obtained. The two peaks corresponded to enterotoxin and another spore coat protein(s). Similar results were obtained when the cells had been preincubated for 60 min with rifampin before label addition, indicating the functioning of stable mRNA.     

Production of L-proline by Kurthia catenaforma

A number of organisms were screened for their ability to produce l-proline. Kurthia catenaforma, which we recently isolated, was selected. A serine-requiring mutant, strain 45, produced about 1.5 times the amount of this amino acid that the parent strain did. In investigations of various media, it was found that approximately 30 ml of l-proline per ml was produced in shaken culture at 30 C in a medium containing glucose, urea, corn steep liquor, casein hydrolysate, l-aspartic acid, and inorganic salts. To study the effect of l-aspartic acid on the production of l-proline, various amino or organic acids were substituted for l-aspartic acid, and the changes during fermentation were investigated. l-Aspartic acid was not replaced by the compounds tested, and this acid appeared to increase growth during the later stages of fermentation with a concurrent increase in the production of l-proline.     

Kinetic analysis of insulin action on amino acid uptake by isolated chick embryo heart cells

1. Isolated chick embryo heart cells were used to investigate the mode of action of insulin on the transport of three naturally occurring amino acids: l-proline, l-serine and glycine. Initial velocities of uptake were measured over a period of 5min with an 80-fold range of amino acid concentration. Corrections for amino acid diffusion, incorporation into protein and conversion into carbon dioxide were introduced. 2. The uptake processes approximated Michaelis-Menten kinetics within definite ranges of amino acid concentrations. A single transport system for proline and at least two transport systems for serine and glycine were detected. 3. The kinetic effects of insulin on transport systems for the amino acids tested were consistent with an acceleration of the maximal velocity of the process, without substantial changes in substrate concentration for half-maximal transport velocity. 4. These hormonal effects were not essentially altered by the corrections for amino acid incorporation into protein and conversion into carbon dioxide.     

Effects of l-Glutamine Deprivation on Growth of HVJ (Sendai Virus) in BHK Cells

l-Glutamine requirement for viral maturation was found in BHK-HVJ cells, a cell line of baby hamster kidney cells persistently infected with HVJ (Sendai virus). Synthesis of envelope protein in BHK-HVJ cells was markedly suppressed by deprivation of l-glutamine, whereas development of nucleocapsid (S) antigen was less affected. More detailed examination of this phenomenon was carried out by using a cytolytic system. Growth of HVJ in BHK cells cultured in media deprived of various amino acids was investigated, and omission of l-glutamine from culture medium resulted in a marked inhibitory effect on the release of infectious virus and synthesis of envelope protein, although synthesis of virus-specific RNA and nucleocapsid antigen in the cells was readily detected. When l-glutamine was restored to the culture medium, infectious virus and envelope protein could be detected. l-Glutamic acid, l-aspartic acid, or l-alanine could be substituted for l-glutamine. Effects of l-glutamine deprivation on HVJ growth in several other cells were also investigated. The growth of HVJ in the cells other than BHK and FL cells was not suppressed by lack of l-glutamine. Growth of Sindbis virus in BHK cells was also markedly retarded in the absence of l-glutamine.     

Enhanced release of cholecystokinin by dietary amino acids in chicks (Gallus domesticus)

1. The effect of dietary amino acids and protein on cholecystokinin (CCK) release into plasma was investigated in chicks by feeding a meal through a stomach tube, followed by the CCK determination with specific CCK-8 antibody. 2. The results showed that both isolated soya protein and an amino acid mixture simulating the amino acid composition of the soya protein increased the release of CCK, though to a lesser extent with a delayed response in the former, when added to a protein-free diet. 3. Among amino acids added singly to the protein-free diet, phenylalanine was more efficient than arginine and valine, exerting a response almost identical to the complete amino acid mixture.     

Metabolic Fate of Cysteine and Methionine in Rumen Digesta

Estimates were obtained of the extent to which cysteine and methionine were incorporated into the protein of the microbes of rumen digesta without prior degradation and resynthesis. By using the amino acids labeled with both (35)S and (14)C, it was observed that a large proportion of the (35)S appeared in the sulfide pool and of the (14)C appeared in volatile fatty acids. By isolating the appropriate amino acid, obtaining the (14)C to (35)S ratio, and comparing this with the ratio in the added amino acid, the degree of direct incorporation was calculated. For cysteine it was estimated that at most 1% and for methionine, at most 11% of the amino acid in the free pool was incorporated unchanged into microbial protein. As a consequence of these findings, it is considered that the method for measuring microbial protein synthesis in rumen digesta based upon incorporation of (35)S from the free sulfide pool is not seriously affected by direct utilization of sulfur amino acids arising from dietary sources.     

Stimulation of amino acid accumulation in neuroblastoma and astrocytoma cells byl-histidine

Uptake of amino acids by cultured neuroblastoma and astrocytoma cells was studied in the presence and absence ofl-histidine. Intracellularly accumulated histidine was assumed to induce accumulation of radioactively labeled amino acids from medium by means of exchange transport. Neuroblastoma cells accumulated more histidine than astrocytoma cells and were more sensitive to the enhancement of the uptake of other large neutral amino acids by histidine. Histidine also increased glutamic acid uptake in astrocytoma cells, but reduced it in neuroblastoma cells. The greatest differences between the cell lines in amino acid uptake without histidine were found with acidic amino acids (astrocytoma cells accumulated them more than neuroblastoma cells) and with taurine (the reverse was found). The uptake and exchange mechanisms for some neutral and acidic amino acids may thus be dissimilar in the plasma membranes of cultured cells of neuronal and glial origin.     

Deamination of amino acids by Bacillus pasteurii

Resting cells of Bacillus pasteurii as employed in the treatment of distillery waste showed deamination of amino acids. The deamination of l-glutamic acid and dl-aspartic acid was found to be oxidative while that of dl-serine, dl-threonine and l-asparagine was non-oxidative. dl-Alanine and glycine were not deaminated when present individually but when incubated together showed oxidative deamination. NAD stimulated the oxidation of l-glutamic acid and α,α′-dipyridyl completely inhibited it.