scholarly journals ywfE in Bacillus subtilis Codes for a Novel Enzyme, l-Amino Acid Ligase

2005 ◽  
Vol 187 (15) ◽  
pp. 5195-5202 ◽  
Author(s):  
Kazuhiko Tabata ◽  
Hajime Ikeda ◽  
Shin-ichi Hashimoto
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
High Performance ◽  
Consensus Sequence ◽  
Reaction Products ◽  
In Silico Screening ◽  
Vitro Assay ◽  
Hydrolysis Of

ABSTRACT The ATP-dependent carboxylate-amine/thiol ligase superfamily is known to contain enzymes catalyzing the formation of various types of peptide, such as d-alanyl-d-alanine, polyglutamate, and γ-peptide, but, curiously, no enzyme synthesizing α-dipeptides of l-amino acids is known. We attempted to find such an enzyme. By in silico screening based on the consensus sequence of the superfamily followed by an in vitro assay with purified enzyme to avoid the degradation of the peptide(s) synthesized, ywfE of Bacillus subtilis was found to code for the activity forming l-alanyl-l-glutamine from l-alanine and l-glutamine with hydrolysis of ATP to ADP. No AMP was formed, supporting the idea that the enzyme belongs to the superfamily. Surprisingly, the enzyme accepted a wide variety of l-amino acids. Among 231 combinations of l-amino acids tested, reaction products were obtained for 111 combinations and 44 kinds of α-dipeptides were confirmed by high-performance liquid chromatography analyses, while no tripeptide or longer peptide was detected and the d-amino acids were inert. From these results, we propose that ywfE encodes a new member of the superfamily, l-amino acid ligase.

scholarly journals QUANTITATIVE EFFECTS OF NUTRITIONAL ESSENTIAL AMINO ACID DEFICIENCY UPON IMMUNE RESPONSES TO TUMORS IN MICE

1973 ◽  
Vol 137 (1) ◽  
pp. 1-9 ◽  
Author(s):  
David G. Jose ◽  
Robert A. Good
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Immune Responses ◽  
Tumor Growth ◽  
Essential Amino Acid ◽  
Cell Mediated Immunity ◽  
Host Animal ◽  
Vitro Assay ◽  
Cytotoxic Cell

Mice were fed diets deficient in a single essential amino acid, and the primary immune responses to inoculation of allogenic tumor cells was measured by in vitro assay of cellular immunity. Moderate reduction of the amino acids phenylalanine-tyrosine, valine, threonine, methionine-cystine, isoleucine, and tryptophane in the diet produced profound depression of hemagglutinating and blocking antibody responses, although cytotoxic cell-mediated immunity remained intact. These diets had previously been shown to result in a selective depression of tumor growth in mice. Limitation of the amino acids arginine, histidine, and lysine in the diets gave rise to only slight depression of the immune responses. These diets had previously been shown to produce a proportional decrease in both tumor growth and host body weight. Moderate leucine restriction resulted in a paradoxical depression of cytotoxic cell-mediated immunity with little effect on serum blocking activity. Slight increases had previously been noted in the weight of tumors in mice fed leucine-restricted diets. Deficiency or imbalance of essential amino acids in the diet may produce profound depression of immune responses and apparent, marked changes in the immune resistance of the host animal to tumors.

scholarly journals The sites of hydrolysis of dipeptides containing leucine and glycine by rat jejunum in vitro

1969 ◽  
Vol 114 (4) ◽  
pp. 855-861 ◽  
Author(s):  
E. B. Fern ◽  
R. C. Hider ◽  
D. R. London
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
The Other ◽  
Peptidase Activity ◽  
Rat Jejunum ◽  
Effective Inhibitor ◽  
Inhibitory Effects ◽  
Molar Basis ◽  
Hydrolysis Of

1. The effect of peptides containing leucine and glycine on accumulation of leucine and glycine by everted jejunal rings was studied. 2. It was shown that, on a molar basis, leucyl-leucine is a more effective inhibitor of uptake of [14C]leucine than is either leucylglycine or glycyl-leucine. These latter dipeptides behave alike. 3. The concentration of the dipeptides and their constituent amino acids in both the incubation medium and the tissue has been followed in these experiments by amino acid analysis. No leucine-containing peptides were observed in the tissue. 4. The inhibitory effects of the mixed dipeptides are altered by pH changes in an analogous way to the alterations in peptidase activity. 5. The experimental results indicate that leucine-containing peptides are hydrolysed before the transport step. 6. Glycylglycine, on the other hand, has only a small effect on the accumulation of glycine, although large amounts of the peptide accumulate unchanged in the tissue. This suggests that glycylglycine is taken up by a different mechanism to that for the leucine dipeptides.

Evidence for Active Transport of the Dipeptide Carnosine (β-Alanyl-l-Histidine) by Hamster Jejunum in Vitro

1974 ◽  
Vol 46 (6) ◽  
pp. 693-705 ◽  
Author(s):  
D. M. Matthews ◽  
Jill M. Addison ◽  
D. Burston
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Active Transport ◽  
Amino Acid Mixture ◽  
Intestinal Wall ◽  
Free Form ◽  
Acid Mixture ◽  
Total Uptake ◽  
Hydrolysis Of

1. The characteristics of intestinal transport and hydrolysis of carnosine (β-alanyl-l-histidine) have been studied in rings of everted hamster jejunum in vitro. 2. During incubation with carnosine, large amounts of intact peptide appeared in the intestinal wall, accompanied by small amounts of the constituent amino acids in the free form. Although there was some extracellular hydrolysis, the free amino acids appearing in the intestinal wall were almost entirely derived from intracellular hydrolysis of the peptide. Incubation in l-alanyl-l-histidine resulted in uptake of the constituent amino acids in the free form without appearance of intact peptide in the intestinal wall. 3. Total uptake of β-alanine (both peptide-bound and free) and total uptake of histidine were greater from a low concentration (1 μmol/ml) of carnosine than uptake of these amino acids from the equivalent amino acid mixture. At a high concentration of carnosine (20 μmol/ml), total uptake of β-alanine was greater from the peptide than from the equivalent amino acid mixture but total uptake of histidine was less. At this concentration, total uptake of β-alanine plus total uptake of histidine from the peptide was approximately the same as from the amino acid mixture. 4. Uptake of carnosine by jejunal rings was the result of a saturable process (Kt 9·4 μmol/ml, Vmax. 2·7 μmol g−1 initial wet wt. min−1). Intact carnosine was concentrated in the intestinal wall, the concentration ratio between intracellular fluid and incubation medium being up to 3·4/1. Uptake of carnosine was reduced by anoxia, metabolic inhibitors and replacement of medium Na+. Na+-dependent active transport was shown to be involved in uptake of carnosine by hamster jejunum in vitro.

Uptake of Dipeptides Containing Basic and Acidic Amino Acids by Rat Small Intestine in Vitro

1972 ◽  
Vol 43 (6) ◽  
pp. 823-837 ◽  
Author(s):  
D. Burston ◽  
Jill M. Addison ◽  
D. M. Matthews
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Glutamic Acid ◽  
Aspartic Acid ◽  
Free Amino Acids ◽  
Free Amino ◽  
Tissue Uptake ◽  
Acidic Amino Acids ◽  
Hydrolysis Of

1. The characteristics of transport and hydrolysis of twenty-two dipeptides containing basic and acidic amino acids by rat ileal rings were investigated in vitro. The peptides included combinations of basic and neutral, basic and basic, basic and acidic, acidic and acidic, and acidic and neutral amino acids. 2. All peptides studied were removed intact from the bulk phase of the incubation medium, though, in general, only free amino acids appeared in the tissue. Uptake of one or both constituent amino acids was greater from the peptide than from the equivalent amino acid or amino acid mixture in the case of at least one peptide from each group and in eighteen of the twenty-two peptides studied. In general, there was no relationship between the extent of uptake of amino acids from peptides and the extent of their hydrolysis by the system. The results support the hypothesis that there is more than one mode of uptake of amino acids from peptides. 3. Hydrolysis of γ-glutamyl-l-glutamic acid by intact intestine or intestinal homogenate was slight, and intact peptide was taken up by the tissue. Uptake of free glutamic acid from this peptide was poor. Comparison of γ-glutamyl-l-glutamic acid with three other slowly hydrolysed dipeptides, glycyl-d-valine, sarcosylglycine and glycylsarcosine, suggested that all four were transported into the mucosal cells and hydrolysed intracellularly. The results indicate that the presence of a γ-linkage or a d-amino acid, or methylation of the free amino group as in sarcosylglycine, impair both transport and hydrolysis of peptide, but that attachment of a methyl group to the N of the peptide bond, as in glycylsarcosine, impairs hydrolysis but has no effect on peptide transport. 4. l-Aspartic acid and l-glutamic acid were extensively transaminated by the intestine, whether presented as free amino acids or in peptides. Evidence was obtained suggesting that production of alanine from aspartic acid resulted from direct transamination of aspartic acid with pyruvic acid, rather than from a sequence of two reactions involving aspartate and alanine aminotransferases. 5. The results show that more rapid uptake of amino acids from peptides than from free amino acids is not confined to peptides made up of neutral amino acids, and probably occurs with many small peptides. Uptake of lysine and the dicarboxylic amino acids, which are particularly slowly absorbed from free solution, was much greater from several dipeptides than from the free amino acids. The results suggest the importance of mucosal peptide uptake in protein absorption.

Amino acid digestibility of meat and bone meals for broiler chickens

2002 ◽  
Vol 53 (11) ◽  
pp. 1257 ◽  
Author(s):  
V. Ravindran ◽  
W. H. Hendriks ◽  
B. J. Camden ◽  
D. V. Thomas ◽  
P. C. H. Morel ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Crude Protein ◽  
Ash Content ◽  
Meat And Bone Meal ◽  
Bone Meal ◽  
Vitro Assay ◽  
Ileal Digestibility ◽  
Gross Energy

Variation in the apparent ileal digestibility of amino acids in 19 meat and bone meal samples, obtained from commercial rendering plants in New Zealand, was determined using 5-week-old broilers. Assay diets contained meat and bone meal as the only source of protein, and chromic oxide as an indigestible marker for the calculation of amino acid digestibility values. Correlations of chemical composition (crude protein, ash, crude fat, and gross energy) and in vitro assays (protein solubility in 0.2% potassium hydroxide and nitrogen digestibility by 0.2% pepsin hydrolysis) with ileal amino acid digestibility were also examined. Considerable variation was observed in the contents of crude protein (38.5–67.2 g/100 g), ash (13.0– 56.5 g/100 g), crude fat (4.3–15.3 g/ 100�g), and gross energy (9.4–22.3 MJ/kg) of meat and bone meal samples. The amino acid concentrations and ileal digestibility of amino acids also varied substantially. Cystine, the first limiting amino acid in meat and bone meal, had the lowest digestibility estimates. Correlation analyses showed that the ash content was the only chemical parameter that was consistently correlated with amino acid digestibility. Digestibility of amino acids, with the exception of aspartic acid, threonine, serine, tyrosine, histidine, and cystine, was negatively correlated with ash content, with samples with high ash levels having lower digestibility. Both in vitro assay measurements were found to be insensitive indicators of variations in amino acid digestibility.

scholarly journals Interaction of the Sliding Clamp β-Subunit and Hda, a DnaA-Related Protein

2004 ◽  
Vol 186 (11) ◽  
pp. 3508-3515 ◽  
Author(s):  
Mareike Kurz ◽  
Brian Dalrymple ◽  
Gene Wijffels ◽  
Kritaya Kongsuwan
Keyword(s):  
Amino Acid ◽  
Consensus Sequence ◽  
Nitrilotriacetic Acid ◽  
Replication Initiation ◽  
Binding Motif ◽  
Β Subunit ◽  
Related Protein ◽  
Sliding Clamp ◽  
Hydrolysis Of

ABSTRACT In Escherichia coli, interactions between the replication initiation protein DnaA, the β subunit of DNA polymerase III (the sliding clamp protein), and Hda, the recently identified DnaA-related protein, are required to convert the active ATP-bound form of DnaA to an inactive ADP-bound form through the accelerated hydrolysis of ATP. This rapid hydrolysis of ATP is proposed to be the main mechanism that blocks multiple initiations during cell cycle and acts as a molecular switch from initiation to replication. However, the biochemical mechanism for this crucial step in DNA synthesis has not been resolved. Using purified Hda and β proteins in a plate binding assay and Ni-nitrilotriacetic acid pulldown analysis, we show for the first time that Hda directly interacts with β in vitro. A new β-binding motif, a hexapeptide with the consensus sequence QL[SP]LPL, related to the previously identified β-binding pentapeptide motif (QL[SD]LF) was found in the amino terminus of the Hda protein. Mutants of Hda with amino acid changes in the hexapeptide motif are severely defective in their ability to bind β. A 10-amino-acid peptide containing the E. coli Hda β-binding motif was shown to compete with Hda for binding to β in an Hda-β interaction assay. These results establish that the interaction of Hda with β is mediated through the hexapeptide sequence. We propose that this interaction may be crucial to the events that lead to the inactivation of DnaA and the prevention of excess initiation of rounds of replication.

Mechanisms of Dipeptide Uptake by Rat Small Intestine in Vitro

1971 ◽  
Vol 40 (3) ◽  
pp. 247-259 ◽  
Author(s):  
B. Cheng ◽  
F. Navab ◽  
M. T. Lis ◽  
T. N. Miller ◽  
D. M. Matthews
Keyword(s):  
Amino Acids ◽  
Small Intestine ◽  
Amino Acid ◽  
Acid Uptake ◽  
Rat Small Intestine ◽  
Amino Acid Oxidase ◽  
Wide Range ◽  
Mucosal Cells ◽  
Hydrolysis Of

1. The uptake of l-methionine and glycine as free amino acids, and from their dipeptides by everted rings of rat small intestine in vitro has been investigated. The concentrations used covered a wide range, including values likely to be near those found in the lumen of the intestine. 2. Though no intact peptides were found in the mucosal cells, evidence was obtained which showed that hydrolysis of the peptides was cellular at all concentrations. Total hydrolysis of peptides by the intestine was very great in relation to amino acid uptake over very short incubation times, suggesting that much hydrolysis took place superficially. 3. Except at the lowest concentrations, the rates of uptake of amino acids from the peptides were more rapid than from the equivalent amino acid mixtures. Competition for uptake between glycine and methionine was avoided when they were presented in the form of l-methionylglycine. 4. Anoxia inhibited uptake of methionine from free l-methionine and from l-methionyl-l-methionine. It also inhibited hydrolysis of l-methionyl-l-methionine by intact intestine, but not by intestinal homogenates, suggesting that peptide uptake may be energy-dependent. The l-amino acid oxidase of snake venom, which destroys l-methionine but has no effect on glycine or on the peptides studied, inhibited methionine uptake from peptides when present at high concentrations, suggesting that a major site of hydrolysis is enzyme-accessible. 5. It is suggested that there may be two modes of uptake of amino acids from oligopeptides: (1) surface hydrolysis by mechanisms closely linked to the amino acid entry mechanisms, and (2) peptide entry into the mucosal cells by a special mechanism, followed by intracellular hydrolysis.

scholarly journals Specificity of the HIV-1 Protease on Substrates Representing the Cleavage Site in the Proximal Zinc-Finger of HIV-1 Nucleocapsid Protein

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1092
Author(s):  
János András Mótyán ◽  
Márió Miczi ◽  
Stephen Oroszlan ◽  
József Tőzsér
Keyword(s):  
Amino Acid ◽  
Zinc Finger ◽  
Cleavage Site ◽  
Potential Role ◽  
Binding Mode ◽  
Interaction Energies ◽  
Vitro Assay ◽  
Hiv 1

To explore the sequence context-dependent nature of the human immunodeficiency virus type 1 (HIV-1) protease’s specificity and to provide a rationale for viral mutagenesis to study the potential role of the nucleocapsid (NC) processing in HIV-1 replication, synthetic oligopeptide substrates representing the wild-type and modified versions of the proximal cleavage site of HIV-1 NC were assayed as substrates of the HIV-1 protease (PR). The S1′ substrate binding site of HIV-1 PR was studied by an in vitro assay using KIVKCF↓NCGK decapeptides having amino acid substitutions of N17 residue of the cleavage site of the first zinc-finger domain, and in silico calculations were also performed to investigate amino acid preferences of S1′ site. Second site substitutions have also been designed to produce “revertant” substrates and convert a non-hydrolysable sequence (having glycine in place of N17) to a substrate. The specificity constants obtained for peptides containing non-charged P1′ substitutions correlated well with the residue volume, while the correlation with the calculated interaction energies showed the importance of hydrophobicity: interaction energies with polar residues were related to substantially lower specificity constants. Cleavable “revertants” showed one residue shift of cleavage position due to an alternative productive binding mode, and surprisingly, a double cleavage of a substrate was also observed. The results revealed the importance of alternative binding possibilities of substrates into the HIV-1 PR. The introduction of the “revertant” mutations into infectious virus clones may provide further insights into the potential role of NC processing in the early phase of the viral life-cycle.

scholarly journals FRUCTOSE-AMINO ACIDS IN LIVER: STIMULI OF AMINO ACID INCORPORATION IN VITRO

1955 ◽  
Vol 215 (1) ◽  
pp. 111-124 ◽  
Author(s):  
Henry Borsook ◽  
Adolph Abrams ◽  
Peter H. Lowy
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Incorporation ◽  
Acid Incorporation

scholarly journals Determination of amino acids in human biological fluids by high-performance liquid chromatography: critical review

Amino Acids ◽  
2021 ◽  
Author(s):  
Grażyna Gałęzowska ◽  
Joanna Ratajczyk ◽  
Lidia Wolska
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
High Performance ◽  
Hplc Analysis ◽  
Limit Of Detection ◽  
Biological Fluids ◽  
Analytical Techniques ◽  
Epidemiological Studies ◽  
Preparation Conditions

AbstractThe quantitation and qualification of amino acids are most commonly used in clinical and epidemiological studies, and provide an excellent way of monitoring compounds in human fluids which have not been monitored previously, to prevent some diseases. Because of this, it is not surprising that scientific interest in evaluating these compounds has resurfaced in recent years and has precipitated the development of a multitude of new analytical techniques. This review considers recent developments in HPLC analytics on the basis of publications from the last few years. It helps to update and systematize knowledge in this area. Particular attention is paid to the progress of analytical methods, pointing out the advantages and drawbacks of the various techniques used for the preparation, separation and determination of amino acids. Depending on the type of sample, the preparation conditions for HPLC analysis change. For this reason, the review has focused on three types of samples, namely urine, blood and cerebrospinal fluid. Despite time-consuming sample preparation before HPLC analysis, an additional derivatization technique should be used, depending on the detection technique used. There are proposals for columns that are specially modified for amino acid separation without derivatization, but the limit of detection of the substance is less beneficial. In view of the fact that amino acid analyses have been performed for years and new solutions may generate increased costs, it may turn out that older proposals are much more advantageous.

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