N- and C-terminal amino acids of proteolipids from the rat brain and various other organs

N- and C-terminal amino acids of proteolipid proteins from the whole brain and some other organs were investigated. N-terminal amino acids were identified by the dansylation procedure. C-terminal amino acids were determined after the enzymatic hydrolysis with carboxy peptidases A and B with the following dansylation. Phenyl alanine and lysine were identified as C-terminal amino acids of the proteolipids from the whole brain and only lysine--as the C-terminal amino acid of proteolipids from the heart, liver, kidney (cortical and medullary parts) and skeletal muscle. The corresponding N-terminal amino acids of the proteolipids from the whole brain were aspartic acid and glycine and of proteolipids from the heart, liver, kidney (cortical and medullary parts) and skeletal muscle--only aspartic acid. A comparison of the data obtained with the previous ones has shown that in the brain there exist only two types of proteolipids--one characteristic of myelin, another-- of mitochondria, and in other organs--only one characteristic of mitochondria.     

Analysis of amino acids in nectar from pitchers of Sarracenia purpurea (Sarraceniaceae)

Sarracenia purpurea L. (northern pitcher plant) is an insectivorous plant with extrafloral nectar that attracts insects to a water-filled pitfall trap. We identified and quantified the amino acids in extrafloral nectar produced by pitchers of S. purpurea. Nectar samples were collected from 32 pitchers using a wick-sampling technique. Samples were analyzed for amino acids with reverse-phase high-performance liquid chromatography with phenylisothiocyanate derivatization. Detectable amounts of amino acids were found in each of the 32 nectar samples tested. Mean number of amino acids in a nectar sample was 9 (SD = 2.2). No amino acid was detected in all 32 samples. Mean amount of amino acids in a nectar sample (i.e., amount per wick) was 351.4 ng (SD = 113.2). Nine amino acids occurred in 20 of the 32 samples (aspartic acid, cysteine, glutamic acid, glycine, histidine, hydroxyproline, methionine, serine, valine) averaging 263.4 ng (SD = 94.9), and accounting for ~75% of the total amino acid content. Nectar production may constitute a significant cost of carnivory since the nectar contains amino acids. However, some insects prefer nectar with amino acids and presence of amino acids may increase visitation and capture of insect prey.     

E.s.r. of spin-trapped radicals in aqueous solutions of amino acids. Reactions of the hydroxyl radical.

The radicals produced by reactions of hydroxyl radicals with amino acids in aqueous solutions have been investigated. Hydroxyl radicals were formed by U.V.-photolysis of hydrogen peroxide and the short-lived amino acid radicals were spin-trapped by tert-nitrosobutane and identified by electron spin resonance spectroscopy. Nineteen amino acids were studied, and several radicals were identified which have not been observed previously by other methods. Only side-chain radicals were identified for alanine, threonine, aspartic acid, asparagine, lysine, phenylalanine, tyrosine, proline and hydroxyproline; whereas for glycine the C(2) carbon radical was spin-trapped. Both C(2) carbon radicals and side-chain radicals were assigned to valine, leucine, isoleucine, serine, glutamic acid, glutamine, arginine and methionine.     

Amino acid sequence of protein B23 phosphorylation site

A major phosphopeptide labeled in vivo, was identified in nucleolar protein B23 (Mr/pI = 37,000/5.1) after tryptic digestion. This peptide was purified by high performance liquid chromatography using reverse-phase (C8 and C18) columns. The phosphopeptide contains 20 amino acids including 1 phosphoserine, 7 glutamic acids, and 4 aspartic acids. The amino acid sequence is: His-Leu-Val-Ala-Val-Glu-Glu-Asp-Ala-Glu-Ser(P)-Glu-Asp-Glu-Asp- Glu-Glu-Asp-Val-Lys. This amino acid sequence is similar to that of nucleolar phosphoprotein C23 (8 consecutive amino acids were identical), and to the regulatory subunit (RII) of cAMP-dependent protein kinase (7 consecutive amino acids were identical, which is phosphorylated by casein kinase II (Carmichael, D.F., Geahlen, R.L., Allen, S.M., and Krebs, E.G. (1982) J. Biol. Chem 257, 10440-10445). The regions near these phosphorylation sites are enriched with glutamic and aspartic acids, suggesting that this acidic amino acid cluster may be essential for kinase recognition.     

Complete nucleotide sequences and comparison of the structural genes of two 2-haloalkanoic acid dehalogenases from Pseudomonas sp. strain CBS3.

The nucleotide sequences of two DNA segments from Pseudomonas sp. strain CBS3 that code for two different haloalkanoic acid halidohydrolases were determined. Two open reading frames with coding capacities of 227 amino acids (corresponding to a molecular mass of 25,401 Da) and 229 amino acids (corresponding to a molecular mass of 25,683 Da) were identified as structural genes of 2-haloalkanoic acid dehalogenases I (dehCI) and II (dehCII) by comparison with the N-terminal amino acid sequences of these enzymes. Comparison of the two sequences revealed 45% homology on the DNA level and 37.5% homology on the amino acid level. No homology with other known protein or nucleotide sequences was found.     

Conversion of amino acid residues in proteins and amino acid homopolymers to carbonyl derivatives by metal-catalyzed oxidation reactions

A number of metal-catalyzed oxidation (MCO) systems mediate the oxidative inactivation of enzymes. This oxidation is accompanied by conversion of the side chains of some amino acid residues to carbonyl derivatives (for review, see Stadtman, E. R. (1986) Trends Biochem. Sci. 11, 11-12). To identify the amino acid residues which are sensitive to MCO oxidation, several enzymes/proteins and amino acid homopolymers were exposed to various MCO systems. The carbonyl groups which were formed were converted to their corresponding 3H-labeled hydroxy derivatives. After acid hydrolysis, the labeled free amino acids were separated by ion exchange chromatography. Each protein or polymer gave rise to several different labeled amino acids. The elution profiles of the labeled amino acids obtained from preparations of Escherichia coli glutamine synthetase which had been oxidized by MCO systems comprised of either Fe(II)/O2 or ascorbate/Fe(II)/O2 both in the presence and absence of EDTA were qualitatively the same. From a comparison of the elution profiles of labeled amino acids from various proteins with those obtained from homopolymers, it is evident that the side chains of histidine, arginine, lysine, and proline are particularly sensitive to oxidation by the MCO systems. This conclusion is supported also by direct amino acid analysis of acid hydrolysates which shows that the oxidation of glutamine synthetase, enolase, and phosphoglycerate kinase is associated with the loss of at least 1 histidine residue per subunit. From the results of studies with homopolymers, it is apparent that glutamic semialdehyde is a major product of both proline and arginine residues. In addition, hydroxyproline and unlabeled glutamic acid were identified among the hydrolysis products of oxidized poly-L-proline, and unlabeled aspartic acid was identified as a product of poly-L-histidine oxidation.     

End group analysis of the cytosolic and mitochondrial fumarases from rat liver

Some molecular properties of the cytosolic and mitochondrial fumarases were compared. The carboxyl(C)-terminal amino acid of both the cytosolic and mitochondrial fumarases of rat liver cell was identified as leucine by using carboxypeptidase (CPase) A. As the amino(N)-terminal amino acid of both the cytosolic and mitochondrial fumarases could not be identified by the dansyl chloride method or by the cyanate method, the N-termini of these two fumarases seems to be masked. Both fumarases, after S-carboxymethylation, were completely digested with pronase E and CPase A and B, and the amino acids with blocked amino group were analyzed by high voltage paper electrophoresis and amino acid analysis after acid hydrolysis of these amino acid derivatives. The N-termini of the mitochondrial and cytosolic fumarases were identified as pyroglutamic acid and N-acetylalanine, respectively. To compare the primary structures of the two fumarases in detail, each fumarase was digested with an arginine-specific protease or cleaved with cyanogen bromide. The electrophoretic profiles of the digests of these fumarases were indistinguishable from each other.     

Lipoic acid-dependent oxidative catabolism of alpha-keto acids in mitochondria provides evidence for branched-chain amino acid catabolism in Arabidopsis

Lipoic acid-dependent pathways of alpha-keto acid oxidation by mitochondria were investigated in pea (Pisum sativum), rice (Oryza sativa), and Arabidopsis. Proteins containing covalently bound lipoic acid were identified on isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis separations of mitochondrial proteins by the use of antibodies raised to this cofactor. All these proteins were identified by tandem mass spectrometry. Lipoic acid-containing acyltransferases from pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase complex were identified from all three species. In addition, acyltransferases from the branched-chain dehydrogenase complex were identified in both Arabidopsis and rice mitochondria. The substrate-dependent reduction of NAD(+) was analyzed by spectrophotometry using specific alpha-keto acids. Pyruvate- and alpha-ketoglutarate-dependent reactions were measured in all three species. Activity of the branched-chain dehydrogenase complex was only measurable in Arabidopsis mitochondria using substrates that represented the alpha-keto acids derived by deamination of branched-chain amino acids (Val [valine], leucine, and isoleucine). The rate of branched-chain amino acid- and alpha-keto acid-dependent oxygen consumption by intact Arabidopsis mitochondria was highest with Val and the Val-derived alpha-keto acid, alpha-ketoisovaleric acid. Sequencing of peptides derived from trypsination of Arabidopsis mitochondrial proteins revealed the presence of many of the enzymes required for the oxidation of all three branched-chain amino acids. The potential role of branched-chain amino acid catabolism as an oxidative phosphorylation energy source or as a detoxification pathway during plant stress is discussed.     

珠子参地上部分氨基酸测定及营养评价

对珠子参茎、叶、花的氨基酸组成与含量进行测定和营养评价分析.结果表明,珠子参茎、叶、花中均含有17种常见氨基酸,氨基酸总量分别为3.73％,14.80％和8.72％;珠子参的茎中必需氨基酸与总氨基酸的比值(E/T)为0.39,必需氨基酸与非必需氨基酸的比值(E/N)为0.64,叶中E/T为0.41,E/N为o.71,花中E/T为0.39,E/N为0.63,氨基酸配比较为合理;珠子参茎、叶、花中蛋氨酸+胱氨酸均为第一限制氨基酸;珠子参茎、叶、花中均含有γ-氨基丁酸,含量分别为0.12％,0.26％和0.16％,叶片中γ-氨基丁酸的含量最高.     

Amino Acids from Heterodera glycines

Amino acids emitted and extracted from surface-sterilized larvae and adults of Heterodera glycines were identified by paper chromatography and quantitatively analyzed by column chromatography. Five amino acids (alanine, aspartic acid, glutamic acid, glycine and serine) were emitted by H. glycines larvae and eight others (asparagine, glutamine, leucine/isoleucine, lysine, methionine sulfoxide, threonine, tyrosine, valine/methionine) were found in extracts from crushed larvae.In addition to the amino acids emitted or extracted from larvae, four others were emitted by adults (γ-aminobutyric acid, histidine, phenylalanine, and proline). Four different amino acids (arginine, cystathionine, hydroxyproline, and ornithine) were found only in the extract from crushed adults. Greater quantities of alanine, aspartic acid and glycine were emitted than could be detected in nematode extracts suggesting selective emission.Subsamples of nematode populations were taken from growing plants 19, 26, 33, and 40 days after inoculation and extracted to determine whether changes in specific amino acid content correlated with aging. Proline content shifted most, increasing from 4.1% to 21.5% of the total amino acid complement from the 19th to the 40th days.     

Amino acid sequence of the b subunit of human factor XIII, a protein composed of ten repetitive segments

Factor XIII is a plasma protein that participates in the final stages of blood coagulation. The complete amino acid sequence of the b subunit of human factor XIII was determined by a combination of cDNA cloning and amino acid sequence analysis. A lambda gt11 cDNA library prepared from human liver mRNA was screened with an affinity-purified antibody against the b subunit of human factor XIII. Nine positive clones were isolated from 2 X 10(6) phage and plaque-purified. The largest cDNA insert was sequenced and shown to contain 2180 base pairs coding for a portion of the leader sequence (19 amino acids), the mature protein (641 amino acids), a stop codon (TGA), a 3' noncoding region (187 nucleotides), and a poly(A) tail. When the b subunit of human factor XIII was digested with cyanogen bromide, nine peptides were isolated by gel filtration and reverse-phase high-performance liquid chromatography. Amino acid sequence analyses of these peptides were performed with an automated sequenator, and 299 amino acid residues were identified. These amino acid sequences were in complete agreement with the amino acid sequence predicted from the cDNA. The b subunit of factor XIII contained 10 repetitive homologous segments, each composed of about 60 amino acids and 4 half-cystine residues. Each of these repeated segments is a member of a family of repeats present in human beta 2-glycoprotein I, complement factor B, and haptoglobin alpha 1 chain. Three potential Asn-linked carbohydrate attachment sites were also identified in the b subunit of factor XIII.     

Piperidinyl-3-phosphinic acids as novel uptake inhibitors of the neurotransmitter gamma-aminobutyric acid (GABA)

Piperidinyl-3-phosphinic acid 2, piperidinyl-3-methylphosphinic acid 3 and N-(4,4-diphenyl-3-butenyl)piperidinyl-3-phosphinic acid 4 have been synthesized as bioisosteres of the corresponding amino carboxylic acids, which are potent and specific GABA-uptake inhibitors. The novel amino phosphinic acids were tested for their GABA-uptake inhibitory activity and 2 and 4 were identified as the first phosphinic acid based GABA-uptake inhibitors. The methylphosphinic acid 3 was found to be inactive.     

镉胁迫对丹参生理特性和代谢特征的影响

采用盆栽实验以及液相色谱-质谱联用技术,对镉(Cd)胁迫下丹参(Salvia miltiorrhiza Bunge)的生理和代谢特征进行研究。结果显示：Cd胁迫下丹参根中的Cd、脯氨酸和可溶性蛋白含量均显著增加;丙二醛(Malondialdehyde, MDA)含量显著下降;还原型谷胱甘肽(Glutathione, GSH)含量和超氧化物歧化酶(Superoxide dismutase, SOD)活性变化不显著;过氧化物酶(Peroxidase, POD)活性显著下降,但过氧化氢酶(Catalase, CAT)活性显著增加。同时也发现丹参代谢物含量发生了变化,共筛选出51个差异标志代谢物(主要是有机酸和氨基酸);L-脯氨酸和L-组氨酸的差异倍数(Fold change, FC)均大于2.5,是上调较多的氨基酸;3,4,5-三甲氧基苯甲酸和迷迭香酸FC值均大于5,是上调较多的有机酸;Cd与差异标志代谢物(尤其是氨基酸和有机酸)呈显著正相关。研究结果说明丹参可积累一定量的Cd,高浓度的Cd胁迫可引起丹参膜脂过氧化,限制其抗氧化酶的活性,影响其代谢过程。丹参主要通过调节氨基酸和有机酸代谢,上...     

Amino acids in seeds and seedlings of the genus Lens

The amino acid content of seeds and 4-day-old seedlings were studied in five species of lentil: Lens culinaris, L. orientalis, L. ervoides, L. nigricans and L. odemensis. Free amino acid and also total protein amino acid content after HCl hydrolysis were determined by HPLC. The nonprotein UV-absorbing amino acids were determined by capillary zone electrophoresis (CZE). The content of free protein amino acids in seeds varied among species and increased dramatically after germination. Asparagine is quantitatively most important in both seed and seedling. The content of free nonprotein amino acids is variable in seeds and seedlings. gamma-Hydroxyarginine, gamma-hydroxyornithine, alpha-aminobutyric acid and taurine were found in both seeds and seedlings. Homoarginine was found in four species but not in L. orientalis while gamma-aminobutyric acid (GABA), alpha-aminoadipic acid (alpha-aaa) and three isoxazolinone derivatives: beta-(isoxazolin-5-on-2-yl)-alanine (BIA), gamma-glutamyl-BIA (gamma-glu-BIA) and 2-carboxymethyl-isoxazolin-5-one (CMI) were found exclusively in the seedlings. CMI was identified for the first time in lentil species. Lathyrine, beta-(2-amino-pyrimidine-4-yl)-alanine, which was reported to be in the seeds of some Lathyrus species was confirmed to be present also in the seedling of L. culinaris (trace amount), L. nigricans and L. odemensis. Trigonelline (N-methyl-nicotinic acid), a plant hormone, is present both in seeds and seedlings in different concentrations except in L. ervoides. The different combination of nonprotein amino acids among the species gives indication of their genetic relationship and might partly explain the varying compatibility for interspecies crossing.     

Desiccation-induced Changes in Antioxidant Enzymes,Fatty Acids,and Amino Acids in the Cyanobacterium <Emphasis Type="Italic">Tolypothrix scytonemoides</Emphasis>

Tolypothrix scytonemoides subjected to desiccation exhibited increased antioxidant enzyme activities when compared to fresh cells. The activities of catalase (EC 1.11.1.6) and superoxide dismutase (EC 1.15.1.1) were enhanced in desiccated cells by 42.8% and 8.1%, respectively. The isoforms of catalase and superoxide dismutase were detected by activity staining of crude samples separated on native gels. The isoforms of superoxide dismutase were identified based on their sensitivity to hydrogen peroxide and cyanide. The changes in fatty acids and amino acids in fresh and desiccated cells were also investigated and it was found that the quantity of certain fatty acids and amino acids was greater in desiccated cells. Palmitic acid, palmitoleic acid, heptadecanoic acid, linoleic acid, and myristic acid were more in desiccated cells when compared to fresh cells. Desiccated cells synthesized myristoleic acid, eicosenoic acid and behenic acid, acids which were not synthesized by the fresh cells, whereas tricosanoic acid was synthesized by the fresh cells and not by desiccated cells. The levels of lysine, serine, glycine, proline and cysteine were also comparatively greater in the desiccated cells.     

Electrophysiological evidence for acidic, basic, and neutral amino acid olfactory receptor sites in the catfish

Electrophysiological experiments indicate that olfactory receptors of the channel catfish, Ictalurus punctatus, contain different receptor sites for the acidic (A), basic (B), and neutral amino acids; further, at least two partially interacting neutral sites exist, one for the hydrophilic neutral amino acids containing short side chains (SCN), and the second for the hydrophobic amino acids containing long side chains (LCN). The extent of cross-adaptation was determined by comparing the electro-olfactogram (EOG) responses to 20 "test" amino acids during continuous bathing of the olfactory mucosa with water only (control) to those during each of the eight "adapting" amino acid regimes. Both the adapting and test amino acids were adjusted in concentrations to provide approximately equal response magnitudes in the unadapted state. Under all eight adapting regimes, the test EOG responses were reduced from those obtained in the unadapted state, but substantial quantitative differences resulted, depending upon the molecular structure of the adapting stimulus. Analyses of the patterns of EOG responses to the test stimuli identified and characterized the respective "transduction processes," a term used to describe membrane events initiated by a particular subset of amino acid stimuli that are intricately linked to the origin of the olfactory receptor potential. Only when the stimulus compounds interact with different transduction processes are the stimuli assumed to bind to different membrane "sites." Four relatively independent L-alpha-amino acid transduction processes (and thus at least four binding sites) identified in this report include: (a) the A process for aspartic and glutamic acids; (b) the B process for arginine and lysine; (c) the SCN process for glycine, alanine, serine, glutamine, and possibly cysteine; (d) the LCN process for methionine, ethionine, valine, norvaline, leucine, norleucine, glutamic acid-gamma-methyl ester, histidine, phenylalanine, and also possibly cysteine. The specificities of these olfactory transduction processes in the catfish are similar to those for the biochemically determined receptor sites for amino acids in other species of fishes and to amino acid transport specificities in tissues of a variety of organisms.     

A metabolic study to decipher amino acid catabolism-directed biofuel synthesis in Acetoanaerobium sticklandii DSM 519

Acetoanaerobium sticklandii DSM 519 is a hyper-ammonia-producing anaerobe. It has the ability to produce organic solvents and acids from protein catabolism through Stickland reactions and specialized pathways. Nevertheless, its protein catabolism-directed biofuel production has not yet been understood. The present study aimed to decipher such growth-associated metabolic potential of this organism at different growth phases using metabolic profiling. A seed culture of this organism was grown separately in metabolic assay media supplemented with gelatin and or a mixture of amino acids. The extracellular metabolites produced by this organism were qualitatively analyzed by gas chromatography–mass spectrometry platform. The residual amino acids after protein degradation and amino acids assimilation were identified and quantitatively measured by high-performance liquid chromatography (HPLC). Organic solvents and acids produced by this organism were detected and the quantity of them determined with HPLC. Metabolic profiling data confirmed the presence of amino acid catabolic products including tyramine, cadaverine, methylamine, and putrescine in fermented broth. It also found products including short-chain fatty acids and organic solvents of the Stickland reactions. It reported that amino acids were more appropriate for its growth yield compared to gelatin. Results of quantitative analysis of amino acids indicated that many amino acids either from gelatin or amino acid mixture were catabolised at a log-growth phase. Glycine and proline were poorly consumed in all growth phases. This study revealed that apart from Stickland reactions, a specialized system was established in A. sticklandii for protein catabolism-directed biofuel production. Acetone–butanol–ethanol (ABE), acetic acid, and butyric acid were the most important biofuel components produced by this organism. The production of these components was achieved much more on gelatin than amino acids. Thus, A. sticklandii is suggested herein as a potential organism to produce butyric acid along with ABE from protein-based wastes (gelatin) in bio-energy sectors.

     

Rhizobium leguminosarum has a second general amino acid permease with unusually broad substrate specificity and high similarity to branched-chain amino acid transporters (Bra/LIV) of the ABC family

Amino acid uptake by Rhizobium leguminosarum is dominated by two ABC transporters, the general amino acid permease (Aap) and the branched-chain amino acid permease (Bra(Rl)). Characterization of the solute specificity of Bra(Rl) shows it to be the second general amino acid permease of R. leguminosarum. Although Bra(Rl) has high sequence identity to members of the family of hydrophobic amino acid transporters (HAAT), it transports a broad range of solutes, including acidic and basic polar amino acids (L-glutamate, L-arginine, and L-histidine), in addition to neutral amino acids (L-alanine and L-leucine). While amino and carboxyl groups are required for transport, solutes do not have to be alpha-amino acids. Consistent with this, Bra(Rl) is the first ABC transporter to be shown to transport gamma-aminobutyric acid (GABA). All previously identified bacterial GABA transporters are secondary carriers of the amino acid-polyamine-organocation (APC) superfamily. Also, transport by Bra(Rl) does not appear to be stereospecific as D amino acids cause significant inhibition of uptake of L-glutamate and L-leucine. Unlike all other solutes tested, L-alanine uptake is not dependent on solute binding protein BraC(Rl). Therefore, a second, unidentified solute binding protein may interact with the BraDEFG(Rl) membrane complex during L-alanine uptake. Overall, the data indicate that Bra(Rl) is a general amino acid permease of the HAAT family. Furthermore, Bra(Rl) has the broadest solute specificity of any characterized bacterial amino acid transporter.     

The identification of N-acetylglycine as a contaminant of glacial acetic acid.

This paper provides evidence by gas chromatography-mass spectrometry (GC-MS) that N-acetylglycine is present, in varying amounts, as a contaminant of all samples of analytical grade glacial acetic acid that have been examined in our laboratory. Supportive evidence for the GC-MS data was obtained by amino acid analyses of evaporated samples of acetic acid which were subjected to acid hydrolysis and then analyzed by ion-exchange chromatography.Although the identification of N-acetylglycine has been established with certainty, small quantities of other amino acid derivatives which have not yet been identified are also present in glacial acetic acid. These additional amino acids have been identified after acid hydrolysis. It should be pointed out that although amino acids are of chief interest here, they comprise approximately 1% or less of the total organic contamination.A very marked reduction of the concentration of N-acetylglycine and all other contaminants was accomplished by slow distillation of the glacial acetic acid through a column of packed Raschig rings.