Rate Of Amino Acid Release Research Articles

Enantioselective transport of D,L-phenylalanine and D,L-phenylglycine through a bulk liquid membrane containing cinchona alkaloid derivatives as chiral selectors

Different cinchona derivatives were tested as chiral mobile carriers for enantioselective transport of D,L-phenylglycine and D,L-phenylalanine through a bulk organic liquid membrane (BLM). The effects of several parameters such as carrier nature, membrane solvent, buffer, solute and carrier concentrations on BLM enrichment were evaluated. Only D,L-phenylglycine is subjected to enantioselective transport at a certain degree; the maximum enantioselectivity ratio (the initial transport rates of the L-enantiomer relative to the antipode) was reached by employing O-allyl-N-(9-anthracenylmethyl) cinchonidinium bromide as carrier. In all cases the highest selectivity was observed during the initial stages of the process, indicating the rate of amino acid release from the source phase (SP) to the membrane organic phase (MP) as the driving factor. Chiral enrichment appeared to depend on thermodynamic factors more than on kinetic ones, since the complex formation was observed at (SP)/MP) interface, while complex decomposition was evidenced at (RP)/(MP) interface, where RP is receiving phase. The last phenomenon was promoted by H+ ions present in the RP.

Modulation of the swelling-activated amino acid channel of Leishmania major promastigotes by protein kinases

Leishmania promastigotes respond to hypotonic challenges by a mechanism of regulatory volume decrease (RVD), whereby anionic amino acid channels (HAAC) are hypotonically-activated and intracellular amino acids are released from the cells. Irrespective of the experimental conditions, restoration of isotonicity triggered an immediate blockage of the amino acid release. Both the speed and amplitude of the response depended on the hypotonic stimulus and on the operation of intracellular signaling mechanisms. The initial (5 s) hypotonic-induced release of amino acids ( r i) and the steady state levels of amino acids attained (5 min) or amplitude (A), were markedly affected by modulators of protein kinase C: phorbol 12-myristate 13-acetate, 1-oleoyl-2-acetylglycerol and phorbol 12,13-diacetate whereas staurosporine and the related analog, bis-indolylmaleimide I (GF-109203.X) inhibited the RVD response. Agonists of cAMP-dependent protein kinase A such as forskolin or (8-(4-chlorophenylthio))-adenosine-3′,5′cyclic-monophosphate enhanced the speed of the response but had little effect on its amplitude. Neither 4 α-phorbol 12,13-didecanoate,1,9-dideoxyforskolin nor genistein, tamoxifen or thapsigargin had any apparent effect on either parameter tested. The most striking stimulation of hypotonic-induced amino acid release was exerted by arachidonic acid or by its non-metabolizable analog, 5,8,11,14-eicosatetraynoic acid (ETYA). These agents caused a major increase in the initial rate of amino acid release as well as a higher amplitude of the response, both of which were markedly inhibited by an anion channel blocker. The present studies indicate not only that hypotonicity is an obligatory and dominant component in HAAC activation, but implicate specific second messengers in the modulation of the RVD response. The modes of activation or attenuation of HAAC activity apparently differ for PKC and PKA modulators as well as for arachidonic acid. The involvement of Ca 2+ in HAAC was studied in hypotonic challenged cells which were treated with intracellular Ca 2+-chelators or Ca 2+-free medium. These cells showed a lag in AA release and a modest inhibition of the amplitude. The inhibition of HAAC was markedly increased when cells were treated with the ionophore A23187 in Ca 2+-free media. The HAAC activity was accompanied by a significant increase in internal Ca 2+ when performed in Ca 2+-containing medium (from 88±9 to 179±22 nM) but by no significant change when measured in Ca 2+-free medium. These studies indicate that although Ca 2+ might be involved in the early activation phase of HAAC, it is either not absolutely required or its action might be associated with localized events.

Effects of hypothermia on the rate of excitatory amino acid release after ischemic depolarization.

Hypothermia slows the increase in extracellular excitatory amino acid (EAA) concentrations during temporary cerebral ischemia. However, it is unclear whether hypothermia slows the rate of EAA release or just delays the time until the first sharp increase (which occurs coincident with terminal depolarization). Pericranial temperatures were adjusted to 38 degrees C, 34 degrees C, 31 degrees C, or 25 degrees C in halothane-anesthetized rats. The cortical DC voltage was recorded from a glass microelectrode while the cortical concentrations of glutamate, aspartate, glycine, and gamma-aminobutyric acid (GABA) were measured by microdialysis. A cardiac arrest was induced with intravenous KCl, and the times until electroencephalograph isoelectricity and terminal depolarization were recorded. Dialysate concentrations of the four compounds were measured at 10, 20, and 30 minutes after depolarization. The times to isoelectricity and depolarization varied inversely with temperature; depolarization time increased from 70 +/- 9 seconds at 38 degrees C (mean +/- SD) to 294 +/- 34 seconds at 25 degrees C. The dialysate concentrations of all four compounds increased during ischemia, and the rate of increase was inhibited by cooling. After 30 minutes of ischemia, glutamate concentration in 38 degrees C animals was 58.4 +/- 31.8 mumol/L; this decreased to 15.9 +/- 8.4 mumol/L at 25 degrees C. The magnitude of the effects of temperature on amino acid release differed with the compound measured. For glutamate, the calculated Q10 was 3.63. Corresponding values for aspartate and glycine were 3.68 and 1.95, respectively. By contrast, Q10 for GABA release was 6.31, indicating greater sensitivity to cooling. These results suggest that effects of hypothermia on EAA concentrations during cerebral ischemia may be the result of both a delay until initial EAA release as well as a direct effect of temperature on the rate of amino acid release. The observed temperature effects are more consistent with carrier-mediated processes controlling EAA release.

Differences in release of endogenous sugars and amino acids from attached and detached seed coats of developing pea seeds

The effect of p‐chloromercuribenzenesulfonic acid (PCMBS), carbonylcyanide‐m‐chlorophenylhydrazone (CCCP) and a high apoplastic pH (pH 7.5 compared with pH 5.5) on the release of sugars (sucrose and glucose) and amino acids from attached and detached seed coats of Pisum sativum L. cv. Marzia into a bathing solution was measured by means of the ‘empty seed coat technique’. PCMBS reduced the release of sugars and amino acids from attached as well as from detached seed coats, suggesting that carrier‐mediated transport might be involved. CCCP reduced sugar release from attached seed coats while amino acid release was hardly affected. In experiments with detached seed coats CCCP had no effect on release of either sugar or amino acids, suggesting that it is not energy‐dependent. Raising the pH of the bathing solution from pH 5.5 to pH 7.5 slightly increased sugar release from both attached and detached seed coats while amino acid release was not affected. This might indicate a role of the apoplastic pH in regulating sugar release from the seed coat via a retrieval mechanism. The presented data indicate that there are important differences between sugars and amino acids with respect to transport processes in the seed coat. This is supported by the observation that the rate of amino acid release from the seed coat was higher than the rate of sugar release. The release data of detached seed coats were subjected to compartmental analysis in order to calculate rate constants for release from cell compartments. In the case of sugars, the half‐times for emptying the cytoplasmic and vacuolar compartment were 0.8 h and 12.5 h. respectively. For amino acids the half‐times were 0.5 h for emptying the cytoplasmic and 3.8 h for emptying the vacuolar compartment.

Patterns of short-term amino acid accumulation and loss in the root-zone of liquid-cultured forage rape (Brassica napus L.)

Amino acid release from roots of sterile and non-sterile, solution-grown, 7-, 21- and 60-days-old forage rape plants (Brassica napus L.), was measured over periods of up to 6 hours. With sterile plants, release of amino acids into a fixed volume of collection medium (6, 12, 70 mL) was concentration-limited, giving rise to similar convex accumulation profiles for individual acids. In contrast, amino acid accumulation in continuously circulating collection medium was not concentration limited, giving a linear accumulation pattern. The compositions of accumulating amino acids, which were similar to those measured in root extracts, did not change significantly. However, the proportions of ALA, GABA, GLU and ILE in both root extracts and root-derived amino acids increased as plants aged. Older plants released more amino acids per plant, while younger plants released more amino acids g-1 root DW. Using non-sterile plants, the patterns of change in amino acid concentration and composition in the collection medium were completely different from those determined with sterile plants. In general, with 7-days-old plants, and 60-days-old plants that had recently become non-sterile, an initial rise in the concentration of all acids was followed by a fall to low levels. The loss of amino acids was apparently due to microbial consumption. Individual amino acids attained maximum concentration at different times during the collection process. This is attributed mainly to concentration-dependent differential assimilation of amino acids, since those with the highest initial concentrations, the major components of the mixtures released from roots, declined the earliest. When calculated rates of amino acid release from roots (Rr) and microbial consumption of amino acids (Rc) were compared (for 7-days-old plants), the highest ratios of Rc/Rr were found for ASN, ARG, GLU, GLN, and LYS. This suggests a degree of selectivity for glutamate and nitrogen-rich acids on the part of the consuming micro-organisms. With 21-days old plants and 60-days old plants grown entirely under non-sterile conditions, fluctuations in amino acid concentration were similar for all acids.

Specific role of endopeptidases in modulating the nature of protein digestion products

The impact of various endopeptidases on the nature of protein digestion products was measured with the digestion cell technique. After a 30 min pepsin pre-digestion, casein and rapeseed concentrates were hydrolyzed with various amounts of pancreatin, trypsin and/or chymotrypsin. This hydrolysis was performed in a dialysis tube (molecular weight cut-off 1000) with continuous collection of the digested material. The addition of pure trypsin or chymotrypsin to pancreatin (Enzyme:Substrate 1:25) did not change the digestibility of casein. Only a higher pancreatin level (Enzyme:Substrate 1:12.5) increased the total protein digestibility without affecting the amino acid spectra. Rapeseed digestibility was markedly increased by the addition of pure trypsin to pancreatin. Lysine and arginine, target amino acids of trypsin, were favored at the expense of chymotrypsin and elastase target amino acids. Supplementation of pancreatin with chymotrypsin enhanced rapeseed digestibility without affecting the relative amino acid digestibility. The impact of a higher pancreatin ratio (Enzyme:Substrate 1:12.5) was similar to that of enriched pancreatin but the rate of amino acid release was modified. The differences between protein sources were mainly attributed to protein structure.

Effects of adrenaline and prior exercise on the release of alanine, glutamine and glutamate from incubated rat skeletal muscle

Catecholamines have been proposed as important regulators of the rate of amino acid release from skeletal muscle. In the present study, we have investigated the influence of adrenergic action and its possible interaction with exercise on muscle release and tissue content of alanine, glutamine and glutamate. For this purpose epitrochlearis muscles were dissected from resting and exercised (1 and 2 h) rats and incubated for 1 h in the presence or absence of adrenaline. In addition, muscles from water-immersed resting rats were included to separate the influence of the stress involved in the swimming exercise from that of muscle contractile activity per se. In muscles from untreated resting rats, the release, tissue content and total amount (released amount + tissue content) of the three amino acids were not influenced by 10(-7) M adrenaline; when the adrenaline concentration was raised to 10(-5) M only the tissue content of glutamate was significantly changed (-50%, P less than 0.001). However, in muscles of rats subjected to 2 h prior exercise or water immersion, 10(-7) M adrenaline significantly increased the release of glutamine (+ 48% and +34%, P less than 0.05) and glutamate (+38% and +27%, P less than 0.05). Moreover, 1 h of water immersion resulted in a significant increase in muscle glutamine and glutamate compared to values from the exercised and control rats. The data suggest that adrenergic action is involved in the regulation of muscle amino acid transport during exercise and that the stress involved in exercise may mask the influence of contractile activity per se on formation of amino acids in skeletal muscle.

15] Deblocking in peptide synthesis with immobilized carboxypeptidase Y

This chapter describes carboxypeptidase Y (CPY) that involves the concept of a method for the synthesis of peptides in aqueous systems using very mild conditions for deblocking. Small protected peptides can be deblocked in alcohol/water mixtures. However, as the size of the peptide increases, the solubility decreases. To use the immobilized CPY in the presence of high levels of organic solvent is not possible. CPY is an exopeptidase found in yeast. The enzyme is unusual, because it has a broad specificity. It catalyzes the release of L-amino acids, including proline, from the C terminus of a polypeptide chain. Rates of amino acid release vary but not to the extent exhibited by other exopeptidases such as pancreatic enzymes. CPY also acts as an esterase. It rapidly cleaves the C-terminal alkyl ester group of large and small peptides. In conclusion, immobilized CPY is a versatile tool for the protein chemistry lab. It can be used for deblocking in peptide synthesis, the resolution of racemic mixtures of blocked amino acid esters or peptides, the sequencing of peptides, the total hydrolysis of peptides for analysis, and condensation reactions in peptide synthesis. The attachment of C-terminal amino acid amides may be accomplished using CPY to catalyze the peptide bond formation starting with the peptide ester and the amide. Also, the enzyme should be of interest in peptide modification and fragment condensation of peptides.

Total Amino Acid Release Rates of Soluble and Insoluble Protein Fractions of Concentrate Feedstuffs by Streptomyces griseus

Streptomyces griseus (5mg) protease was incubated with increasing protein concentrations of casein, soybean meal, or corn gluten meal. Rates of amino acid release were 1.04, .72, and .36 μmoles of amino acid per hour with enzyme saturation at 7.5 and 8.5mg of casein and soybean meal protein. A ratio of protein to enzyme of 1.25 gave rates of release within saturation limits for protein sources differing in degradability. Partitioning of nitrogen fractions for 11 protein and 6 energy concentrate ingredients showed dehydrated alfalfa and corn gluten feed had largest proportions of soluble nitrogen and amino acids and least amount of potentially available insoluble protein compared to other protein ingredients. Incubation of Streptomyces griseus protease with soluble and insoluble protein fractions indicated that most feedstuff soluble protein fractions have amino acid release rates two to three times faster than insoluble protein fractions. Ratio of soluble to insoluble protein amino acid release were 1.2 for corn gluten meal and peanut meal and .5 for dehydrated alfalfa and corn gluten feed. Soluble protein fractions of certain protein feedstuffs may not be as available in the rumen as insoluble protein fractions.

Muscle proteolysis induced by a circulating peptide in patients with sepsis or trauma.

Accelerated proteolysis of muscle is characteristic in patients with trauma or sepsis, but its cause is not well understood. Using rat muscle in vitro, we developed a bioassay to compare the proteolytic activity of plasma from 50 patients with trauma or sepsis with that of plasma from 14 normal volunteers and from 15 patients who had undergone "clean" elective surgical procedures. The mean proteolytic activity in the plasma of patients with trauma or sepsis was found to be 190 +/- 8.0 per cent of the control value (rat muscle incubated in medium alone), whereas the activity in normal plasma was 124 +/- 4.5 per cent (P less than 0.001). The activity in the plasma of patients who had undergone elective surgery was slightly elevated at 142 +/- 2.5 per cent (P less than 0.005). In 25 of the patients with trauma or sepsis the rate of amino acid release from one leg was measured by subtracting the concentration of tyrosine plus phenylalanine in the femoral artery plasma from that in the femoral vein; this rate correlated well with the bioactivity of the plasma in the bioassay system (r = 0.67, P less than 0.001). By means of ultrafiltration and chromatography, the plasma factor inducing proteolysis was isolated and found to be a peptide, probably containing sialic acid, with a chain of 33 amino acids and a molecular weight of approximately 4274 daltons.

Rates of peptide bond hydrolysis by cobalt(III) complexes: observation by rate of amino acid release

The rates of hydrolysis of eleven dipeptides (Gly-Gly; Gly- l-Leu; Gly- d,l-Val; Gly- l-Phe: d,l-Leu-Gly; l-Leu- l-Tyr; l-Pro- l-Tyr; l-Ala- l-Phe; l-Val-Gly; l-Val- l-Ile; l-Ile- l-Val) by cis-β-Co(trien)(OH)(H 2O) 2+ under pseudo first order conditions (excess Co(III)) are reported. The rates are determined by the rate of amino acid release by liquid chromatography. The range of rates observed for the eleven dipeptides is 10.1. Two of the dipeptides are hydrolyzed more rapidly than Gly-Gly, the remaining six more slowly. Phosphate does not have any significant effect on the rate of peptide hydrolysis. Aquohydroxy-1,8-diamino-3,6-dithiaoctanecobalt(III) 2+ did not hydrolyze Gly-Gly at 25°, pH8, and aquohydroxy-1,6-bis-(α-pyridyl)-2,5-diazahexanecobalt(III) 2+ did not hydrolyze Gly-Gly at 65°, pH 8, to any significant extent.