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PubMed1990

Purification and characterization of an acidic amino acid-specific endopeptidase of Bacillus subtilis obtained from a commercial preparation (Protease Type XVI, Sigma).

An acidic amino acid-specific endopeptidase was purified from Protease Type XVI (Sigma), a commercial product from culture filtrate of Bacillus subtilis, by a series of column chromatographies on CM-Toyopearl (Fractogel) and Mono-S, guided by activity assay using Boc-Ala-Ala-Pro-Glu-pNA as a substrate. The final preparation was homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and reversed-phase HPLC. The molecular weight of the protease was estimated to be 18,000 by gel filtration on TSK gel G3000SWXL column using 6 M guanidine hydrochloride as an eluent, and 17,000 by SDS-PAGE in the presence of 2-mercaptoethanol. The isoelectric point of the protease was 7.7. Studies on the substrate specificity with peptide p-nitroanilides and natural peptides revealed that the protease hydrolyzes the peptide bonds on the carboxyl-terminal side of acidic amino acids, especially of glutamic acid. The protease was completely inactivated by DFP, indicating the serine protease nature of the protease. The activity of the protease was also inhibited by EDTA and GEDTA, and reactivated by Ca2+. The protease contained 1.3 +/- 0.2 mol/mol protein of Ca2+. These results suggest that Ca2+ plays a vital role in the protease activity.

Amino Acid Sequence↗
PubMed1999

Energetics and kinetics of the prebiotic synthesis of simple organic acids and amino acids with the FeS-H2S/FeS2 redox couple as reductant.

The thermodynamics of the FeS-H2S/FeS2 redox couple and a select number of reactions critical to the synthesis of simple carboxylic acids and amino acids have been evaluated as a function of temperature. This thermodynamic evaluation shows that the reducing power of the FeS-H2S/FeS2 redox couple decreases drastically with temperature. By contrast the equilibria describing the reduction of CO2 and the formation of simple carboxylic acids and amino acids require an increasingly higher reducing power with temperature. Given these two opposite trends, the thermodynamic driving force for CO2 reduction and amino acid formation with the FeS-H2S/FeS2 redox couple as reductant diminishes with increasing temperature. An evaluation of the mechanism of CO2 reduction by the FeS-H2S/FeS2 couple suggests that the electron transfer from pyrrhotite to CO2 is hindered by a high activation energy, even though the overall reaction is thermodynamically favorable. By comparison the electron transfer from pyrrhotite to either CS2, CO, or HCOOH are far more facile. This theoretical analysis explains the results of experimental work by Keefe et al. (1995), Heinen and Lauwers (1996) and Huber and Wächtershäuser (1997). The implication is that a reaction sequence involving the reduction of CO2 with the FeS-H2S/FeS2 couple as reductant is unlikely to initiate a proposed prebiotic carbon fixation cycle (Wächtershäuser, 1988b; 1990b, 1990a, 1992, 1993).

Amino Acids↗
PubMed1993

Racemization of alpha-amino acid esters by aliphatic ketones in the presence of carboxylic acids.

Amino acid esters are racemized by dissolution in a mixture of aliphatic ketones and carboxylic acids. The racemization rate mainly depends on the structure of the amino acid and on the kind of ketone and carboxylic acid used, the best racemizing medium being acetone containing 15% acetic acid. The mechanism of the racemization and the practical consequences of this study in the optical resolution field are discussed.

Acetates↗
PubMed1982

The intracellular acid-extractable (acid-soluble) amino acid pool in mammalian cells: 2 Displacement.

Amino acid displacement is a separate and easily distinguishable phenomenon from discharge of the acid-extractable pool. It is characterized by its extreme rapidity for certain amino acids, with a resetting of the concentrations of different species in the pool at a new level, from which normal discharge kinetics then continue. It is possible to insert a labelled amino acid into the acid-extractable pool by displacement by the same mechanism as labelled amino acid can be ejected from the pool. The amount of displacement which occurs is dependent on the total number of potential displacing molecules available and not simply on their concentration in the medium. Differences occur between amino acids in their ability to displace the pool or to be displaced from the pool. The major determining factor is the strength with which a resident amino acid in the pool is complexed. Displacement for the more weakly complexed amino acids occurs at 0--2 degrees C, and requires no energy supply. The results are discussed in relation to the nature of the pool itself, and the implications of displacement on the uptake of amino acids into the cell.

Acids↗
PubMed1999

Mutational analysis of RAG1 and RAG2 identifies three catalytic amino acids in RAG1 critical for both cleavage steps of V(D)J recombination.

RAG1 and RAG2 initiate V(D)J recombination, the process of rearranging the antigen-binding domain of immunoglobulins and T-cell receptors, by introducing site-specific double-strand breaks (DSB) in chromosomal DNA during lymphocyte development. These breaks are generated in two steps, nicking of one strand (hydrolysis), followed by hairpin formation (transesterification). The nature and location of the RAG active site(s) have remained unknown. Because acidic amino acids have a critical role in catalyzing DNA cleavage by nucleases and recombinases that require divalent metal ions as cofactors, we hypothesized that acidic active site residues are likewise essential for RAG-mediated DNA cleavage. We altered each conserved acidic amino acid in RAG1 and RAG2 by site-directed mutagenesis, and examined >100 mutants using a combination of in vivo and in vitro analyses. No conserved acidic amino acids in RAG2 were critical for catalysis; three RAG1 mutants retained normal DNA binding, but were catalytically inactive for both nicking and hairpin formation. These data argue that one active site in RAG1 performs both steps of the cleavage reaction. Amino acid substitution experiments that changed the metal ion specificity suggest that at least one of these three residues contacts the metal ion(s) directly. These data suggest that RAG-mediated DNA cleavage involves coordination of divalent metal ion(s) by RAG1.

Amino Acid Sequence↗
PubMed1997

Cloning, expression, and chromosomal localization of mouse liver bile acid CoA:amino acid N-acyltransferase.

A mouse liver lambda Zap XR cDNA library was screened using the coding region of human bile acid CoA:amino acid N-acyltransferase (BAT) cDNA as a probe. Ten positive clones were isolated and purified, two of which apparently possessed complete open reading frames for BAT based on sequence analysis of the ends of the cDNAs. One clone (mBAT#9) was selected for sequence analysis and characterization. mBAT#9 is 1869 basepairs in length and the full-length cDNA possesses a 189 basepair 5'-nontranslated region, an open-reading frame of 1260 basepairs, and a 404 basepair 3'-nontranslated region followed by a poly(A) tail. The open-reading frame codes for a 420 amino acid protein with a calculated molecular mass of 46,525 daltons. The structural gene for mBAT was mapped to mouse Chromosome 4. The amino acid sequence of mBAT is 69% identical and 84% similar to that of hBAT, and 86% identical and 95% similar to that of kan-1, a putative rat liver BAT. Enzymatically active mBAT was expressed in E. coli using the bacterial expression vector pKK233-2. Immunoblot analysis of expressed mBAT with rabbit anti-human BAT polyclonal antibodies detected a single protein with a molecular mass of approximately 45,000 daltons. Cytosol from cells transformed with mBAT#9/pKK233-2 possessed significant amounts of BAT-catalyzed conjugating activity with taurine as substrate but the expressed enzyme did not use glycine or fluoro-beta-alanine as substrates. The K(m) value for taurine was 1.9 mM +/- 0.1 mM in reactions with cholyl CoA as a cosubstrate. The specificity of mBAT for taurine as a substrate was confirmed by the demonstration, using HPLC-electrospray ionization mass spectrometry, that mouse gallbladder bile contained only taurine conjugates of bile acids. The identification of the types of amino acid conjugates of bile acids present in mouse bile had not been previously reported. These results indicate that a taurine-specific form of BAT has been cloned and expressed from mouse liver.

Acyltransferases↗
PubMed1978

Effect of acylated amino acids and acylated amino acid analogs on microbial antitumor screen.

A series of N-acetyl,-N-propionyl, and N-chloroacetyl derivatives of amino acids and amino acid analogs was tested for growth-inhibitory activity using a Lactobacillus casei system as a prescreen for possible antitumor activity. While none of the acetyl or propionyl derivatives of these amino acids and amino acid analogs caused any remarkable inhibition, certain chloroacetyl derivatives exhibited significant activity. The chloroacetyl derivatives, especially those of essential amino acids and of analogs of essential amino acids, showed modest, but pharmacologically significant, inhibition; those of nonessential amino acids exhibited no activity. When two such inhibitory acyl derivatives were combined in a single assay, the extent of inhibition was neither additive nor synergistic but was that of the more active of the two test components.

Amino Acids↗
PubMed1978

Purification and characterization of bile acid-CoA:amino acid N-acyltransferase from rat liver.

An in vitro study of bile acid-CoA:amino acid N-acyltransferase activity of rat liver was undertaken in order to determine whether separate amino acid-specific enzymes catalyzed the formation of glycine and taurine conjugates of bile acids as postulated by others. Polyacrylamide gel electrophoresis of 200-fold purified enzyme localized the glycine- and taurine-dependent activities to a single band. Both activities were optimal at pH 7.8 and showed similar loss of activity at pH 6.0, pH 9.0, in the presence of 5,5'-dithiobis(2-nitrobenzoic acid), and at temperatures exceeding 50 degrees. With the purified fraction, Km for glycine was 31 mM and Km for taurine was 0.8 mM. Km for several bile acid-CoA substrates was approximately 20 micron and independent of the amino acid acceptor. Only amino acids with terminal alpha- or beta-amino groups were active as acyl acceptors. Acyl donors were limited to bile acid-CoA derivatives. The data support the conclusion that the rat has a single bile acid-CoA:amino acid N-acyltransferase. The substrate kinetics are consistent with previous observations that taurine conjugates predominate in rat bile at normal hepatocellular concentrations of glycine and taurine.

Acyltransferases↗
PubMed1982

Plasma amino acid levels and amino acid losses during continuous ambulatory peritoneal dialysis.

Free amino acid losses into dialysate during a 24-h collection period and postabsorptive plasma amino acid concentrations were measured in 14 studies in nine clinically stable men undergoing continuous ambulatory peritoneal dialysis. Patients ingested diets containing 97 +/- 18 (SD) g/day of protein in a metabolic research unit. Total amino acid losses were 3.4 +/- 1.2 g/24 h and represented 3.9 +/- 1.9% ot total nitrogen output (in dialysate, urine and feces). The sum of plasma essential, nonessential, and total amino acid concentrations was normal, although plasma valine, leucine, and isoleucine were decreased and several amino acids were elevated. Neither plasma nor dialysate amino acids, varied with protein intake. Dialysate amino acid concentrations averaged 72 +/- 18% of plasma levels, and 29.0 +/- 3.6% of dialysate amino acids were essential. Dialysate concentrations and 24 h losses of individual amino acids each correlated with plasma levels (r = 0.83 and 0.78, respectively). Thus during continuous ambulatory peritoneal dialysis, postabsorptive plasma amino acids are, in general, well maintained. Daily losses of free amino acids during continuous ambulatory peritoneal dialysis are rather small and are easily replaced by food intake.

Adult↗
PubMed2001

Effects of branched-chain amino acids on placental amino acid transfer and insulin and glucagon release in the ovine fetus.

OBJECTIVE: Competition for placental amino acid transporters can affect the fetal supply of amino acids. Specifically, the branched-chain amino acids-isoleucine, leucine, and valine-may inhibit the transfer of other amino acids. This study was undertaken to determine the effect of branched-chain amino acids on the umbilical uptake of amino acids. STUDY DESIGN: Six late-gestation ewes were infused sequentially for 2 hours with 3 different mixtures of amino acids: (1) one that was comparable to commercial parenteral nutrition preparations, (2) the same solution without branched-chain amino acids, and (3) branched-chain amino acids alone. Maternal and fetal blood samples were collected simultaneously for the determination of uterine and umbilical uptake values of amino acids, and for concentrations of arterial insulin, glucagon, glucose, and lactate before (control) and during (experimental) infusion. RESULTS: Umbilical uptake of branched-chain amino acids increased significantly when they were present in the infusates. The fetal uptake of several other amino acids could be increased by increasing their maternal concentrations. Inhibition of umbilical uptake by branched-chain amino acids could be shown for threonine and methionine. The infusion of branched-chain amino acids alone did not affect maternal and fetal insulin or glucagon concentrations. CONCLUSIONS: In late-gestation sheep, an increase in maternal plasma concentration of branched-chain amino acids led to increased branched-chain amino acid umbilical uptake, but branched-chain amino acids can also inhibit the transport of some amino acids to the fetus. Changes in fetal plasma concentration and uptake of branched-chain amino acid appear to have no significant effect on fetal insulin or glucagon.

Amino Acids↗
PubMed1994

Glycine and taurine conjugation of bile acids by a single enzyme. Molecular cloning and expression of human liver bile acid CoA:amino acid N-acyltransferase.

In order to establish whether a single enzyme in human liver was capable of conjugating bile acids with both glycine and taurine, a cDNA encoding human liver bile acid-CoA:amino acid N-acyltransferase (hBAT) has been isolated and characterized. A specific immunoaffinity-purified rabbit anti-hBAT polyclonal antibody was used to screen a lambda Zap XR human liver cDNA library resulting in the isolation of two unique clones. hBAT8 and hBAT9 (1669 and 1491 base pairs in length, respectively) were isolated following screening of 4 x 10(5) clones of the cDNA library. Restriction mapping and sequence analysis demonstrated that the cDNAs were identical except hBAT8 contained an additional 178 bases of 5' sequence; hBAT8 was completely sequenced, characterized, and used for all subsequent studies. hBAT8 consisted of a 184-nucleotide 5'-nontranslated region, an open reading frame of 1,254 bases predicting a protein of 418 amino acids with a molecular mass of 46,296 Da, and a 3'-nontranslated region of 209 nucleotides followed by a poly(A)+ tail. The identity of the cDNA was confirmed by the following findings: 1) the open reading frame began with an ATG codon and was followed by a nucleotide sequence which, when translated, corresponded exactly to the first 17 NH2-terminal amino acids of purified human liver BAT; 2) cytosol of Escherichia coli XL1-Blue cells transfected with hBAT8 subcloned into an expression vector, pKK233-2, demonstrated significant enzymatic activity for the conjugation of both taurine and glycine with cholic acid; 3) bacterial expression of hBAT8 generated a protein that comigrated with hBAT from human liver during SDS-polyacrylamide gel electrophoresis and cross-reacted with a specific polyclonal rabbit anti-hBAT antibody during immunoblot analysis; 4) kinetic characteristics of the expressed enzyme were very similar to those reported for purified liver BAT. These data demonstrate that a single cDNA is present in human liver which codes for a protein capable of catalyzing the conjugation of cholic acid with both glycine and taurine.

Acyltransferases↗
PubMed1996

Effects of truncation of the COOH-terminal region of a Na+-independent neutral and basic amino acid transporter on amino acid transport in Xenopus oocytes.

To determine the role of a neutral and basic amino acid transporter (NBAT) in amino acid transport, we microinjected several COOH-terminal deletion mutants of NBAT cRNA into Xenopus oocytes and measured transport activity for arginine, leucine, and cystine in the presence and absence of sodium. Wild-type NBAT significantly stimulated the uptake of all three amino acids 10-20-fold compared with controls. On the other hand, no mutant, except a Delta511-685 mutant, stimulated the uptake of these amino acids. The Delta511-685 mutant significantly increased the uptake of arginine. In the presence of sodium, the Delta511-685 mutant also increased the uptake of leucine. The Delta511-685 mutant did not stimulate cystine uptake in the presence or absence of sodium. The stimulation of arginine uptake by the Delta511-685 mutant was inhibited by a 100-fold excess of unlabeled leucine in the presence of sodium. Inhibition of L-arginine uptake by L-homoserine was seen only in the presence of sodium, and an increase in the inhibition of L-arginine uptake by L-histidine was seen when the extracellular pH was decreased. Furthermore, an inward current in oocytes injected with the Delta511-685 mutant was recorded electrophysiologically when basic amino acids were applied. Homoserine was also taken up, but sodium was necessary for their transport. These properties of the Delta511-685 mutant correspond to those of the y+ amino acid transporter. If NBAT is a component of the b0,+-like amino acid transport system, it is unlikely that a mutant protein (Delta511-685) is able to stimulate an endogenous y+-like transport system. These results suggest that NBAT functions as a activator of the amino acid transport system in Xenopus oocytes.

Amino Acid Sequence↗
PubMed1994

Permeability of membranes to amino acids and modified amino acids: mechanisms involved in translocation.

The amino acid permeability of membranes is of interest because they are one of the key solutes involved in cell function. Membrane permeability coefficients (P) for amino acid classes, including neutral, polar, hydrophobic, and charged species, have been measured and compared using a variety of techniques. Decreasing lipid chain length increased permeability slightly (5-fold), while variations in pH had only minor effects on the permeability coefficients of the amino acids tested in liposomes. Increasing the membrane surface charge increased the permeability of amino acids of the opposite charge, while increasing the cholesterol content decreased membrane permeability. The permeability coefficients for most amino acids tested were surprisingly similar to those previously measured for monovalent cations such as sodium and potassium (approximately 10(-12)-10(-13) cm s-1). This observation suggests that the permeation rates for the neutral, polar and charged amino acids are controlled by bilayer fluctuations and transient defects, rather than partition coefficients and Born energy barriers. Hydrophobic amino acids were 10(2) more permeable than the hydrophilic forms, reflecting their increased partition coefficient values. External pH had dramatic effects on the permeation rates for the modified amino acid lysine methyl ester in response to transmembrane pH gradients. It was established that lysine methyl ester and other modified short peptides permeate rapidly (P = 10(-2) cm s-1) as neutral (deprotonated) molecules. It was also shown that charge distributions dramatically alter permeation rates for modified di-peptides. These results may relate to the movement of peptides through membranes during protein translocation and to the origin of cellular membrane transport on the early Earth.

Amino Acids↗
PubMed2005

Evidence of selection for low cognate amino acid bias in amino acid biosynthetic enzymes.

If the enzymes responsible for biosynthesis of a given amino acid are repressed and the cognate amino acid pool suddenly depleted, then derepression of these enzymes and replenishment of the pool would be problematic, if the enzymes were largely composed of the cognate amino acid. In the proverbial "Catch 22", cells would lack the necessary enzymes to make the amino acid, and they would lack the necessary amino acid to make the needed enzymes. Based on this scenario, we hypothesize that evolution would lead to the selection of amino acid biosynthetic enzymes that have a relatively low content of their cognate amino acid. We call this the "cognate bias hypothesis". Here we test several implications of this hypothesis directly using data from the proteome of Escherichia coli. Several lines of evidence show that low cognate bias is evident in 15 of the 20 amino acid biosynthetic pathways. Comparison with closely related Salmonella typhimurium shows similar results. Comparison with more distantly related Bacillus subtilis shows general similarities as well as significant differences in the detailed profiles of cognate bias. Thus, selection for low cognate bias plays a significant role in shaping the amino acid composition for a large class of cellular proteins.

Amino Acids↗
PubMedDate not supplied

Amino acid uptake by amino acid analog resistant tobacco cell lines.

Two tobacco cell lines resistant to p-fluorophenylalanine (PFP) and one resistant to 5-methyltryptophan (5-MT) are compared with wild type cells in their ability to absorb amino acids from the medium. One p-fluorophenylalanine-resistant cell line shows greatly reduced uptake of all amino acids so is resistant to growth inhibition by other amino acid analogs. The impaired absorption is noted with amino acids, amino acid analogs and shikimate, but not with cinnamate, salicylate, nicotine, glucose, 3-O-methylglucose and palmitate. The phenylalanine transport system of the PFP-resistant cell line and the wild type both have Km values of 90 micrograms, but have different Vmax values. Several analogs of phenylalanine and several neutral L-amino acids inihibt the phenylalanine transport system, while L-aspartic acid, L-arginine, D-phenylalanine or chlorogenic acid do not interfere with the L-phenylalanine uptake. The results indicate the presence of more than one transport system for amino acid uptake. The lessened uptake of all amino acids, the specificity of the uptake systems and the unchanged binding let us conclude that a pleiotropic mutation or that some inhibitor causes the reduced uptake of all amino acids by the PFP-resistant cell line.

Amino Acids↗
PubMed1991

Purification and characterization of bile acid-CoA:amino acid N-acyltransferase from human liver.

The bile acid-conjugating enzyme, bile acid-CoA: amino acid N-acyltransferase, was purified 480-fold from the soluble fraction of homogenized frozen human liver. Purification was accomplished by a combination of anion exchange chromatography, chromatofocusing, glycocholate-AH-Sepharose affinity chromatography, and high performance liquid chromatography (HPLC) gel filtration. Following purification, the reduced, denatured enzyme migrated as a single 50-kDa protein band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A similar molecular mass was obtained for the native enzyme by HPLC gel filtration. Elution from the chromatofocusing column suggested an apparent isoelectric point of 6.0 (+/- 0.2). Using a rabbit polyclonal antibody raised against the purified enzyme, Western blot analysis using 100,000 x g human liver supernatant confirmed that the affinity-purified polyclonal antibody was specific for human liver bile acid-CoA:amino acid N-acyltransferase. The purified enzyme utilized glycine, taurine, and 2-fluoro-beta-alanine (a 5-fluorouracil catabolite), but not beta-alanine, as substrates. Kinetic studies revealed apparent Km values for taurine, 2-fluoro-beta-alanine, and glycine of 1.1, 2.2, and 5.8 mM, respectively, with corresponding Vmax values of 0.33, 0.19, and 0.77 mumol/min/mg protein. These data demonstrate that a single monomeric enzyme is responsible for the conjugation of bile acids with glycine or taurine in human liver.

Acyltransferases↗