Non-ribosomal biosynthesis of the cyclic octadecapeptide alamethicin.

The biosynthesis of the cyclic octadecapeptide, alamethicin, in a cell-free system of Trichoderma viride has been investigated. It was shown that nucleic acid- and ribo-some-free extracts of Trichoderma viride could catalyze alamethicin biosynthesis. Puromycin, erythromycin and RNAse did not inhibit this synthesis. The Sephadex G 200 filtrate contains a fraction (Kav=0.1) that catalyzes the biosynthesis of alamethicin and shows an ATP-32PPi exchange with 6 of the 8 constituent amino acids of alamethicin. The activated amino acids are bound to the enzyme as aminoacyl adenylates and as thiolesters in a proportion of 1 : 1. About 50% of each bound amino acid could be split off with 7% TCA. The TCA-stable bound amino acid could be split by mercury acetate, hydroxylamine and performic acid. N-ethylmaleimide blocked the binding of 50% of the amino acids to the enzyme, proving that some of the amino acids first bound as aminoacyl adenylates are then transferred into a thiolester bond.     

Research for amino acids in lunar samples

Water extracts of lunar fines were analyzed for amino acids by a gas-liquid chromatographic technique whereby amino acids were converted to the N-trifluoroacetyln-butyl, esters prior to analysis. The lunar material studied included both Apollo 14 (14240 SESC and 14298) and Apollo 12 (12023) samples. The water extract of the special Apollo 14 sample (14240 SESC) was analyzed both for free and bound amino acids (hydrolysis with 6 N hydrochloric acid). In both the hydrolyzed and unhydrolyzed extracts, the amino acids were not observed above background levels.The analysis of Apollo 12 and 14 samples (12023 14298) yielded similar results. Detection limits were established at 300 pg to 1 ng for different amino acids. A large chromatographic peak with a retention temperature of 126°C was observed on analysis of sample, (12023); it was identified as oxalic acid by GC-MS. The concentration of amino acids in the Apollo 14 SESC samples processed and analyzed in the joint experiments at Ames by GLC and IEC were found to be extremely low (glycine at 3 to 4 ng g^–1). As the quantities were so minute, these identifications could not be confirmed by GLC-MS and therefore should still be considered as tentative. Other studies included the analysis of performance standards at the 2 to 6 ng level of each of 17 amino acids, and the analysis of 5 ml of H₂O containing 2 ppb of each amino acid. Recovery of amino acids added to lunar fines were conducted at the 10, 50, and 70 ng level of each amino acid with 50 to 70 mg of lunar material. The recoveries varied from as high as 80% for some of the aliphatics to complete loss of the amino acids ornithine and lysine.Contributed from Missouri Agricultural Experiment Station Journal Series No. 6255. Approved by the Director. Supported in part by grants from the National Aeronautics and Space Administration (NGR 26-004-011) and the Experiment Station Chemical Laboratories.     

鳖甲抗肝纤维化活性物质的氨基酸分析

采用日立L8800全自动氨基酸分析仪,对鳖甲抗肝纤维化有效提取部位中游离和水解氨基酸进行了分析,结果表明,该有效部位中游离氨基酸质量分数为1.32%,水解氨基酸质量分数为58.0%。     

Reactions between amino acid compounds and phenols during oxidation

Summary About 30 per cent of organic soil nitrogen can be hydrolized with HCl to amino acids; about 30 per cent is nonhydrolizable. In contrast to this high content of amino acid nitrogen is the small availability of the nitrogen to micro-organisms. In light of the theory proposing a reaction between the -amino group of amino acids or peptides and quinones formed during oxidation of lignin degradation products or other phenolic compound, different types of phenols were oxidized by phenolases in presence of amino acid compounds.It could be shown that the reaction of binding of nitrogen started at pH values higher than 6.5, and that only such phenols reacted which had no methoxylated hydroxyl groups. The reaction of some phenols during oxidation in presence of amino acids was accompanied by deamination and decarboxylation of the latter.The reaction products of phenols with amino acids were stable against hydrolysis. Using peptides it was found that all amino acids, except the N-terminal which is bound to oxidized phenols, could be hydrolyzed normally.With serum albumin it could be shown that there is a reaction with the amino group of the N-terminal amino acid and also with the -amino group of lysine residues with phenols during oxidation. The reacted protein seemed to be degraded normally with a protease ofBacillus subtilis.Guest Scientist as Fulbright Research Scholar from the Agronomy Department of the Iowa State University, Ames, Iowa, U.S.A.     

臭灵丹草氨基酸成分分析

用氨基酸分析仪分析中药臭灵丹草叶片的氨基酸成分,并比较其水解氨基酸和游离氨基酸含量.结果表明:臭灵丹草中鉴定出18种氨基酸成分,水解氨基酸总量为18.68%,游离氨基酸总量为2.75%.γ-氨基丁酸含量可达0.1%以上.     

Some Properties of Three Proteolytic Enzymes Excreted by Bacillus cereus Kp 931

The crude enzyme preparation obtained from culture media of Bacillus cereus Kp 931 was fractionated into three active fractions by Sephadex G-100 gel filtration. These three enzymes had pH optima at between 10.5 and 11.0. One of them, the largest molecular weight species, the enzyme I, was purified extensively. The enzyme catalyzes the release of a number of free amino acids from casein. Large amounts of l-alanine and l-glutamic acid and small amounts of l-leucine, l-serine, glycine, l-cysteic acid and l-arginine were released from oxidized insulin B-chain by the action of the purified enzyme I. It is also suggested that the other two enzymes, II and III, belong to so-called bacterial proteninases.     

Contribution of hydrolyzed nucleic acids and their constituents to the apparent amino acid composition of biological compounds.

Acid hydrolysis of protein-free mixtures of nucleotides, nucleosides, and nucleic acids yields amino acids, free bases, and possibly other unidentified fragments when analyzed by thin-layer chromatography and by standard amino acid analysis. Glycine is the predominant amino acid detected, which may constitute 47–97% of the apparent amino acid composition, depending on the type of material subjected to hydrolysis. Obviously, hydrolyzed nucleic acids or their constituents can therefore contribute to the apparent amino acid composition of a supposedly pure peptide or of other more complex mixtures of compounds mistakenly believed to contain only protein. To circumvent this problem, we suggest that nucleotides or nucleic acid moieties should be removed from any product for which the amino acid composition is desired, and that whenever a large glycine peak is noted in a hydrolyzed sample, the presence of nucleic acids or their constituents should be suspected.     

Amino acid composition of aragonitic concholin in the shell of Arctica islandica

Haugen, J. E. & Sejrup, H. P. 1990 04 15: Amino acid composition of aragonitic conchiolin in the shell of Arctica islandica. Lethaia , Vol. 23, pp. 133–141. Oslo. ISSN 0024–1164.
The distribution of amino acids within the two aragonitic shell layers of modern specimens of the mollusc Arctica islandica (Linné) has been studied in detail. The mean total hydrolyzed amino acid content was 19 nmol*mg in the inner layer and 15 nmol/mg in the outer layer. No significant difference in amino acid composition could be found between the two layers. The layers contained minor amounts of free amino acids which made up 0.3–0.7% of the total hydrolyzed amino acid content. The composition of the free amino acid fraction was very similar in each of the two layers, but differed somewhat from the total hydrolyzed fraction. The total hydrolyzed fraction was dominated by aspartic acid, glycine. alanine and glutamic acid, which together made up 62% of the total amount of amino acids, whereas the free fraction was dominated by Asp. Ser, Gly and Tyr. Amino acid content and composition within a single layer showed little variation from umbo to the ventral margin. The amino acid composition is in accordance with previously reported data on similar mineral structures which support the theory of structure specific rather than species specific amino acid composition. * Arctica islandica. amino acids. shell structure .     

Effect of exogenous amino acids,glucose and citric acid on the patterns of short-term accumulation and loss of amino acids in the root-zone of sand-cultured forage rape (Brassica napus L.)

Amino acid loss from the roots of 25-day-old, sterile and non-sterile sand-grown forage rape plants, was determined over periods of up to 3.5 hours. Amino acid accumulation in the root-zone of sterile plants was concentration-dependent giving a convex accumulation profile. Amino acid levels in the root zone of non-sterile plants rapidly attained steady state values. Microbial assimilation of amino acids within the root zone appeared to lower amino acid concentrations, resulting in an underestimation of rates of amino acid loss from roots. The concentrations of most amino acids were higher after selected amino acids were supplied to the root zone. The response to exogenous acids was dependent on the concentration and composition of the acids added. Addition of a mixture containing ASN, GLN and GABA, each at 0.25 mM resulted in a greater increase in individual and total acid levels compared with a mixture containing ALA, SER, GLY and THR at the same concentration. Apparently, amino acids supplied exogenously competed with acids lost from the plant, by providing an alternative nutrient source for root zone micro-organisms. Addition of glucose and citric acid had a similar effect to addition of ALA, SER, GLY and THR, but were less effective than ASN, GLN and GABA at all concentrations tested. The nitrogen-rich amino acids ASN and GLN, and the -amino acid, GABA, appeared to compete more effectively with plant-derived acids than did ALA, SER, GLY and THR, the most abundent constituents of the plant-derived acids, which had the highest calculated rates of microbial consumption. Therefore, although bacterial consumption showed a dependence on amino acid concentration, a degree of selectivity for nitrogen-rich acids and gaba was also apparent.     

Intermediates of the gamma-glutamyl cycle in mouse tissues. Influence of administration of amino acids on pyrrolidone carboxylate and gamma-glutamyl amino acids.

GAMMA-Glutamyl transpeptidase, gamma-glutamyl cyclotransferase, L-pyrrolidone carboxylate hydrolase, gamma-glutamylcysteine synthetase and glutathione synthetase, the enzymes of the gamma-glutamyl cycle, were found in mouse brain, liver and kidney. The activity of L-pyrrolidone carboxylate hydrolase was many times lower than the activities of the other enzymes, and thus the conversion of L-pyrrolidone carboxylate to L-glutamate is likely to be the rate-limiting step of the cycle. The specificity of gamma-glutamyl cyclotransferase from mouse tissues was similar to that from rat tissues. The concentration of pyrrolidone carboxylate and gamma-glutamyl amino acids, intermediates of the gamma-glutamyl cycle, was determined by a gas chromatographic procedure coupled with electron capture detection. Administration of L-2-aminobutyrate, an amino acid that is utilized as substrate in the reaction catalyzed by gamma-glutamylcysteine synthetase, led to a large accumulation of gamma-glutamyl-2-aminobutyrate and pyrrolidone carboxylate in mouse tissues. L-Methionine-RS-sulfoximine, an inhibitor of gamma-glutamylcysteine synthetase, abolished the increase in concentration of pyrrolidone carboxylate. No accumulation of pyrrolidone carboxylate was observed after L-cysteine. The separate administration of several protein amino acids had little effect on the concentration of pyrrolidone carboxylate; however formation of small amounts of the corresponding gamma-glutamyl derivatives (e.g. gamma-glutamylmethionine and gamma-glutamylphenylalanine) was detected. These intermediates are probably formed by transpeptidation between glutathione and the corresponding amino acid, catalyzed by gamma-glutamyl transpeptidase. The concentration of pyrrolidone carboxylate increased significantly after administration of a mixture containing all protein amino acids, the highest increase occurring in the kidney. The results suggest that two separate pathways for the formation of gamma-glutamyl amino acids and pyrrolidone carboxylate exist in vivo. One of these results from the function of gamma-glutamylcysteine synthetase in glutathione synthesis. The other pathway involves the amino-acid-dependent degradation of glutathione, mediatedby gamma-glutamyl transpeptidase. Only very small amounts of free intermediates are apparently derived from the latter pathway, suggesting that the gamma-glutamyl amino acids formed in this pathway are either enzyme-bound or are directly hydrolyzed to glutamate and free amino acid.     

Taurine Levels in Some Tissues of Yellowtail (Seriola quinqueradiata) and Mackerel (Scomber japonicus)

The mechanism of stereospecific production of l-amino acids from the corresponding 5-substituted hydantoins by Bacillus brevis AJ-12299 was studied. The enzymes involved in the reaction were partially purified by DEAE-Toyopearl 650M column chromatography and their properties were investigated. The conversion of dl-5-substituted hydantoins to the corresponding l-amino acids consisted of the following two successive reactions. The first step was the ring-opening hydrolysis to N-carbamoyl amino acids catalyzed by an ATP dependent l-5-substituted hydantoin hydrolase. This reaction was stereospecific and the N-carbamoyl amino acid produced was exclusively the l-form. N-Carbamoyl-l-amino acid was also produced from the d-form of 5-substituted hydantoin, which suggests that spontaneous racemization occurred in the reaction mixture. In the second step, N-carbamoyl-l-amino acid was hydrolyzed to l-amino acid by an N-carbamoyl-l-amino acid hydrolase, which was also an l-specific enzyme. The ATP dependency of the l-5-substituted hydantoin hydrolase was supposed to be the limiting factor in the production of l-amino acids from the corresponding 5-substituted hydantoins by this bacterium.     

Assessment of egg quality in atlantic salmon,Salmo salar,treated with testosterone—II. Amino acids

• 1.1. Atlantic salmon (Satmo salar) were treated with Silastic pellet implants containing testosterone (200 μg/g body weight) four times in a year. Eggs stripped from control (sham implantation) and testosterone-treated fish were fertilized and comparisons of free and total amino acid compositions made until first feeding.
• 2.2. Despite having eggs which were smaller in diameter, lighter in weight and lower in total amino acid contents, alevins from testosterone-treated fish were heavier in wet weight and larger in body length, and exhibited enhanced free amino acid contents at first feeding.
• 3.3. The qualitative composition of total amino acids in eggs from treated and control fish did not differ.
• 4.4. Total amino acid pool of eggs and alevins declined during development, but an increase in the free amino acid pool was noticed through development. The increase in free amino acid pool was higher in eggs and alevins from treated fish than controls, perhaps due to enhanced mobilization of the free amino acid pool.

     

Location and State of Methionine Residues in a Papain-synthesized Plastein from a Mixture of Soybean Protein Hydrolysate and l-Methionine Ethyl Ester

With the aid of papain, a plastein was synthesized from a 1 : 10 mixture of l-methionine ethyl ester and a peptic hydrolysate of soybean protein. Dialysis of the whole reaction-product yielded a methionine-incorporated plastein (Met-plastein) as the nondialyzable fraction, its yield being 78.2% on a dry-matter basis, of the whole reaction-product. Methionine content in this Met-plastein was 7.22% on a weight basis, while its content in the material hydrolysate was only 1.25%. Carboxypeptidase A treatment of Met-plastein liberated methionine at an outstandingly rapid rate. A similar, but not so outstanding, rate was observed for the methionine liberation from Met-plastein by treatment with leucine aminopeptidase. Methyl isothiocyanate treatment and subsequent cyclization yielded a mixture of methylthiohydantoins from Met-plastein. Gas chromatographic analysis of this mixture after trimethylsilylation showed a result that methionine occupied 33.2%, on a molar basis, of the total N-terminal amino acids. Lithium borohydride reduction and 6 n hydrochloric, acid hydrolysis of Met-plastein produced monomeric aminois, and their 2,4-dinitrophenylation followed by thin-layer chromatography gave a result that methionine occupied 84.9%, on a molar basis, of the total C-terminal amino acids; the residues amounting to 14.4% of the C-terminal methionine residues remained as an ethyl eater form. The selective degradation probe employing cyanogen bromide to generate free homoserine disclosed that the occurrence of the polymeric methionine-methionine sequence was little if any in Met-plastein. Based on the above experiments as well as an evaluation of the esterase activity against l-methionine ethyl ester, a possible mechanism was discussed of the papain-catalyzed synthesis of plastein in a system containing such an ester.     

Polymerization of amino acids under primitive earth conditions

Summary Chemical evolution on the primitive earth must have involved the condensation of-amino acids to peptides under a variety of conditions. Subjecting a mixture of methane, ammonia, and water to an electric discharge in the presence of free amino acids yields small peptides. The dehydration-condensation may have taken place via ammonium cyanide, the hydrogen cyanide tetramer, or aminonitriles. The experiments may be considered genuinely prebiotic and significant in the context of chemical evolution.     

Tissue specificity of metabolism in the bivalve mollusc <Emphasis Type="Italic">Anadara inaequivalvis</Emphasis> Br. under conditions of experimental anoxia

Peculiarities of the course of metabolic processes in tissues of the bivalve mollusc Anadara inaequivalvis Br. were studied under conditions of experimental anoxia. In the absence of oxygen, in gill and foot the protein catabolism processes were found to be enhanced; this led to a decrease of the protein content and to an increase of the free amino acid and urea levels. Predominantly hydrolyzed were low molecular peptides, which was indicated by a decrease of the cathepsin D activity on the background of a rise of the γ-glutamyltranspeptidase activity. Anoxia was accompanied by enhancement of the succinate thiokinase and fumarate reductase reactions controlled by alanine and aspartate aminotransferases. This prevented accumulation of toxic lactate in tissues and allowed obtaining an additional macroerg resource. Metabolic processes in the mollusc hepatopancreas were oriented to production of amino acids.     

Nutritional value of protein hydrolysis products (Oligopeptides and free amino acids) as a consequence of absorption and metabolism kinetics

When pigs were submitted to duodenal infusion of solutions containing a large percentage of small peptides (PEP) or free amino acids with the same pattern (AAL) amino acids appear in the portal blood more rapidly and more uniformly after infusion of PEP then after infusion of AAL, with the notable exception of methionine for which the opposite was true. These differences were lowered when a carbohydrate (maltose dextrin) was present in the solution, but nevertheless remained significant for the first hour after the infusion. The long‐term (8‐hour) uptake of free amino acids into the liver and the peripheral tissues differed in profile according to the nature of the duodenal infusion. Peripheral uptake was appreciably less well balanced after infusion of free amino acids (deficiency of threonine and phenylalanine) than after infusion of small peptides (deficiency of methionine). Accordingly, in the rat, under conditions of discontinuous enterai nutrition the mixture of small peptides was of greater nutritive value than the mixture of free amino acids. It thus appears that the absorption kinetics which results in important variations in the temporal distribution of free amino acids in the tissues may be at the origin of transitory imbalances in tissue amino acid uptake, and as a result of a lower nutritive value.     

Measurement of total protein in plant samples in the presence of tannins

A method for measuring total protein in situ in plant samples has been developed using the determination of amino acids released by acid hydrolysis of dried plant material. Standard proteins and plant samples were hydrolyzed with 3% sulfuric acid at 100 degrees C for 24 h and the amino acids released were measured with ninhydrin. Unhydrolyzed plant extracts were also analyzed for free amino acids with ninhydrin. Total amino acid equivalents (protein plus free amino acids) of a diverse set of plant samples was significantly correlated with total protein as estimated by elemental analysis (N X 6.25). The Lowry method as modified by precipitation of proteins with trichloroacetic acid was found to be unsatisfactory for dried plant samples due to the incomplete extractability of proteins. Although some alkaloids caused increased absorbance with ninhydrin, interference with quantification of protein is likely to be minimal. Tannins interfered with the Lowry and Bradford methods but not the ninhydrin method.     

Changes in the Levels of Free Amino Acids in Various Regions of Cuscuta during Growth

The angiospermous plant parasite Cuscuta derives reduced carbonand nitrogen compounds primarily from its host. Free amino acidsalong Cuscuta vines in three zones, viz., 0 to 5 cm, 5 to 15cm, and 15 to 30 cm, which in a broad sense represent the regionof cell division, cell elongation and differentiation and vasculartissue differentiation respectively, were quantitatively estimated.The free amino acid content was the highest in the 0 to 5 cmregion and progressively decreased along the posterior regionsof the vine. The haustorial region showed the lowest contentof free amino acids. In general, the free amino acid contentin samples collected at 7 p.m. was found to be higher than thatin the samples collected at 7 a.m. Three basic amino acids,histidine, the uncommon amino acid -hydroxyarginine, and arginineconstituted more than 50% of the total free amino acids in allthe zones studied except the haustorial region. Aspartic acidand glutamic acid constituted the major portion in the acidicand neutral fraction of amino acids. Glutamine, asparagine,threonine, and serine were eluted together and occurred in substantialamounts. -Hydroxyarginine constituted the largest fraction inthe cut end exudate of Cuscuta and presumably appeared to bethe major form of transport amino acid. -Hydroxyarginine wasalso a major constituent of the basic amino acids in Cuscutavines parasitizing host plants from widely separated families,suggesting that this amino acid is a biosynthetic product ofthe parasite rather than that of the hosts. Also, U-¹⁴C argininewas converted to -hydroxyarginine by cut Cuscuta vines, suggestingthat -hydroxyarginine is synthesized de novo from arginine byCuscuta. (Received March 30, 1987; Accepted June 7, 1988)     

Characterization and gene cloning of a novel β-mannanase from alkaliphilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. N16-5

An alkaline -mannanase was purified to homogeneity from a culture broth of alkaliphilic Bacillus sp. N16-5. The enzyme had optimum activity at pH 9.5 and 70°C. It was composed of a single polypeptide chain with a molecular weight of 55 kDa deduced from SDS-PAGE, and its isoelectric point was around pH 4.3. The enzyme efficiently hydrolyzed galactomannan and glucomannan, producing a series of oligosaccharides and monosaccharides. The -mannanase gene (manA) contained an open reading frame (ORF) of 1,479 bp, encoding a 32-amino acids signal peptide, and a mature protein of 461 amino acids, with a calculated molecular mass of 50,743 Da. Strain N16-5 ManA, deduced from the manA ORF, exhibited relatively high amino acid similarity to the members of the glycosyl hydrolase family 5. The eight conserved active-site amino acids in family 5 glycosyl hydrolase were found in the deduced amino acid sequence of strain N16-5 ManA.