Biochemical characterization of rice glutelin

The two major subunits of rice glutelin, the acidic (α) and basic (β) polypeptides were purified by chromatofocusing and cation exchange chromatography, respectively. The molecular weight range of the α polypeptides was 28.5 to 30.8 kilodaltons and the molecular weight range of the β polypeptides was 20.6 to 21.6 kilodaltons. Electrofocusing in polyacrylamide gels showed that the isoelectric points of the α and β polypeptides were 6.5 to 7.5 and 9.4 to 10.3, respectively. At least 12 polypeptides of the α-group and nine polypeptides of the β-group could be separated by electrofocusing. The amino acid compositions of whole glutelin, and the purified α and β subunits were analyzed. The α subunit contained more glutamic acid/glutamine, serine, and glycine, and less alanine, lysine, aspartic acid/asparagine, and isoleucine than the β subunit. A comparison of the amino acid composition of rice glutelin subunits with those of the 11S proteins from eight other plant species indicated that there is more similarity between the β subunits than the α subunits of several diverse plant species.     

Tracer studies on the biosynthesis of amino acids from lactate by Peptostreptococcus elsdenii

Peptostreptococcus elsdenii, a strict anaerobe from the rumen, was grown on a medium containing yeast extract and [1-(14)C]- or [2-(14)C]-lactate. Radioisotope from lactate was found in all cell fractions, but mainly in the protein. The label in the protein fraction was largely confined to a few amino acids: alanine, serine, aspartic acid, glutamic acid and diaminopimelic acid. The alanine, serine, aspartic acid and glutamic acid were separated, purified and degraded to establish the distribution of (14)C from lactate within the amino acid molecules. The labelling patterns in alanine and serine suggested their formation from lactate without cleavage of the carbon chain. The pattern in aspartic acid suggested formation by condensation of a C(3) unit derived directly from lactate with a C(1) unit, probably carbon dioxide. The distribution in glutamic acid was consistent with two possible pathways of formation: (a) by the reactions of the tricarboxylic acid cycle leading from oxaloacetate to 2-oxoglutarate, followed by transamination; (b) by a pathway involving the reaction sequence 2 acetyl-CoA-->crotonyl-CoA-->glutaconate-->glutamate.     

The neurotoxicity of β-N-oxalyl-l-αβ-diaminopropionic acid, the neurotoxin from the pulse Lathyrus sativus

Intraperitoneal administration of β-N-oxalyl-l-αβ-diaminopropionic acid, the neurotoxin from Lathyrus sativus, to 12-day-old rats causes typical convulsions within 10min. There is a striking accumulation of glutamine in the brain, and chronic ammonia toxicity is indicated. There are no changes in the amounts of urea, aspartic acid and glutamic acid in the brain. Adult rats, even when injected with a dose of excess of β-N-oxalyl-l-αβ-diaminopropionic acid, do not develop symptoms, and there are no changes in the amounts of glutamine or ammonia in the brain. A significant concentration of β-N-oxalyl-l-αβ-diaminopropionic acid can be detected in the brain of the young rat but not in that of the adult animal. It is concluded that β-N-oxalyl-l-αβ-diaminopropionic acid interferes with the ammonia-generating or -fixing mechanisms in the brain and leads to chronic ammonia toxicity.     

Gas chromatographic analysis of free amino acids in the hyaloplasm of the hypophysis, pineal gland, thyroid gland, spinal cord, thymus and lymph nodes of the cow

Studies of the qualitative and quantitative composition of free amino acids in the hyaloplasm of the hypophysis, pineal gland, thyroid gland, spinal cord, thymus and lymph nodes of the cow are described. The following findings are reported: the highest levels of alanine, valine, glycine, isoleucine, histidine, leucine, threonine, serine, phenylalanine, tyrosine and lysine are found in the thyroid gland, methionine and aspartic acid in the spinal cord, tryptophan and hydroxyproline in the pineal gland, and proline and glutamic acid in the thymus gland. The highest level by weight is that of glutamic acid in all tissues. The presence of α-aminobutyric acid combined with sarcosine and 4-aminoisobutyric acid with 2-AOA and citrulline with cystine was demonstrated. α-Aminoisobutyric acid and isovaline were found in the spinal cord.     

A study of the metabolism of l-αγ-diaminobutyric acid in a Xanthomonas species

1. l-αγ-Diaminobutyric acid is metabolized in Xanthomonas sp. to aspartic β-semialdehyde, aspartic acid and oxaloacetic acid. 2. Aspartic β-semialdehyde is formed from diaminobutyric acid by a pyruvate-dependent γ-transamination. 3. The transaminase has a pH optimum of 9 and exhibits a high degree of substrate specificity, as analogues of diaminobutyric acid and pyruvate are inert in the system. The transaminase is inhibited by carbonyl-binding agents such as hydroxylamine. 4. Aspartic acid is formed from aspartic β-semialdehyde by an NAD⁺-dependent dehydrogenation. 5. The dehydrogenase has a pH optimum of 8·5 and is a thiol enzyme. It is specific for aspartic β-semialdehyde but analogues of NAD⁺ such as 3-acetylpyridine–adenine dinucleotide and deamino-NAD are partly active in the system. 6. The significance of these reactions is discussed in relation to diaminobutyric acid metabolism in plants and mammalian systems.     

Amino Acid metabolism of pea leaves: diurnal changes and amino Acid synthesis from N-nitrate

In the young leaves of pea (Pisum sativum L.) plants, there was a diurnal variation in the levels of amino acids. In the light, total amino nitrogen increased for the first few hours, then stabilized; in the dark, there was a transient decrease followed by a gradual recovery. Asparagine, homoserine, alanine, and glutamine accounted for much of these changes. The incorporation of ¹⁵N into various components of the young leaves was followed after supply of ¹⁵N-nitrate. ¹⁵N appeared most rapidly in ammonia, due to reduction in the leaf, and this process took place predominantly in the light. A large proportion of the primary assimilation took place through the amide group of glutamine, which became labeled and turned over rapidly; labeling of glutamic acid and alanine was also rapid. Asparagine (amide group) soon became labeled and showed considerable turnover. Slower incorporation and turnover were found for aspartic acid, γ-aminobutyric acid, and homoserine. Synthesis and turnover of all of the amino acids continued at a low rate in the dark. γ-Aminobutyric acid was the only compound found to label more rapidly in the dark than in the light.     

Metabolic Conversion of Amino Acids Loaded in the Vacuole of Chara australis Internodal Cells

Vacuoles of internodal cells of Chara australis (or Chara corallina) were loaded with a 10 millimolar amount of various amino acids by a perfusion method and incubated under continuous light. After 20 to 24 hours, the cell sap was collected, and free amino acids in it and the rest of the cell (cytoplasm) were analyzed. The only amino acid metabolized completely was alanine. About 40 to 80% of the aspartic acid, glutamine, serine, and glycine were metabolized, whereas less than 30% of the threonine, asparagine, isoasparagine, isoleucine, phenylalanine, γ-aminobutyric acid, lysine, and arginine were metabolized. The figure for glutamic acid fluctuated between 10 and 100%. The main metabolites of alanine were glutamine, glycine and ammonia, which accumulated in the vacuole. Alanine utilization was not affected by l-methionine-d,l-sulfoximine or azaserine, but was strongly inhibited by aminooxyacetate. The cell extract contained enough alanine aminotransferase activity to account for the rate of alanine metabolism.     

Chemical Composition of Cell-Wall Preparations from Strains of Various Form-Genera of Aerobic Actinomycetes

Cell-wall preparations were made from more than 140 strains of aerobic actinomycetes representing most of the form-genera that have been proposed. All cell-wall preparations contained as major constituents glucosamine, muramic acid, alanine, and glutamic acid. In addition, cell-wall preparations from various types of streptomycetes and strains of Microëllobosporia contained glycine and ll-α,ε-diaminopimelic acid; those from strains of most Actinoplanaceae and micromonosporae contained glycine and meso-α-ε-diaminopimelic acid; those from strains of Thermoactinomyces, Microbispora, Dermatophilus, and nocardiae of the madurae-pelletieri group contained meso-α,ε-diaminopimelic acid; and those from strains of Thermomonospora, Micropolyspora, and most nocardiae contained meso-α,ε-diaminopimelic acid, arabinose, and galactose. All the strains used were also studied morphologically.     

Amino Acid Substitutions in Cold-Adapted Proteins from Halorubrum lacusprofundi,an Extremely Halophilic Microbe from Antarctica

The halophilic Archaeon Halorubrum lacusprofundi, isolated from the perennially cold and hypersaline Deep Lake in Antarctica, was recently sequenced and compared to 12 Haloarchaea from temperate climates by comparative genomics. Amino acid substitutions for 604 H. lacusprofundi proteins belonging to conserved haloarchaeal orthologous groups (cHOGs) were determined and found to occur at 7.85% of positions invariant in proteins from mesophilic Haloarchaea. The following substitutions were observed most frequently: (a) glutamic acid with aspartic acid or alanine; (b) small polar residues with other small polar or non-polar amino acids; (c) small non-polar residues with other small non-polar residues; (d) aromatic residues, especially tryptophan, with other aromatic residues; and (e) some larger polar residues with other similar residues. Amino acid substitutions for a cold-active H. lacusprofundi β-galactosidase were then examined in the context of a homology modeled structure at residues invariant in homologous enzymes from mesophilic Haloarchaea. Similar substitutions were observed as in the genome-wide approach, with the surface accessible regions of β-galactosidase displaying reduced acidity and increased hydrophobicity, and internal regions displaying mainly subtle changes among smaller non-polar and polar residues. These findings are consistent with H. lacusprofundi proteins displaying amino acid substitutions that increase structural flexibility and protein function at low temperature. We discuss the likely mechanisms of protein adaptation to a cold, hypersaline environment on Earth, with possible relevance to life elsewhere.     

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.     

An electrophoretic investigation of groundnut proteins: the structure of arachins A and B

1. The proteins of the groundnut cotyledon have been fractionated and analysed by DEAE-Sephadex chromatography and acrylamide-gel electrophoresis. Seventeen components were detected. 2. A new method is described for the preparation of arachin, using calcium precipitation. The product contains at least 99% of arachin. 3. The theory of acrylamide-gel electrophoresis is developed and applied to the arachin system to predict the molecular weight of one sub-unit of arachin. 4. A variant form of arachin, arachin B, has been discovered. Of 81 nuts, 27 contained only arachin B, 53 contained both arachin A and B, and one contained arachin A only. This is almost certainly a polymorphism of arachin; this is the first example of polymorphism to be reported in plant proteins. 5. A combination of controlled denaturation, electrophoretic analysis, ultracentrifuge and Sephadex filtration data has shown that arachin A contains four different kinds of peptide chains (α, β, γ and δ). Arachin B contains only β, γ and δ chains. 6. The most probable structure for arachin B, mol.wt. 330000 form, is 8 β, 2 γ and 2 δ chains, and for arachin A, 4 α, 4 β, 2 γ and 2 δ chains. Arachin without β chains was not found. 7. The α and β chains have mol.wts. of about 35000 and the γ and δ chains of about 10000. 8. Three N-terminal groups were found: the α and β chains both terminate in glycine; the γ and δ chains terminate in isoleucine and glutamic acid. 9. Arachin contains no carbohydrate. 10. Disulphide bonds are not important in arachin: there are none between the α, β, γ and δ chains. 11. The amino acid compositions of arachins A and B are very similar. Glutamic acid and aspartic acid residues are exceptionally frequent.     

Peptidoglycan of a Chemolithotrophic Bacterium, Ferrobacillus ferrooxidans

The rigid layer (peptidoglycan) of the wall of the chemolithotroph Ferrobacillus ferrooxidans was isolated after various chemical treatments. The removal of specific components was followed by noting in an electron microscope changes in the appearance of the cell surface. The final peptidoglycan was virtually free from proteins and was sensitive to the action of lysozyme. Results of chemical analyses of acidhydrolyzed peptidoglycan revealed three major amino acids and two amino sugars: glutamic acid, α,ε-diaminopimelic acid, alanine, glucosamine, and muramic acid in a ratio of 1:1:2.33:062:088.     

Comparative Genomics of DNA Fragments from Six Antarctic Marine Planktonic Bacteria

Six environmental fosmid clones from Antarctic coastal water bacterioplankton were completely sequenced. The genome fragments harbored small-subunit rRNA genes that were between 85 and 91% similar to those of their nearest cultivated relatives. The six fragments span four phyla, including the Gemmatimonadetes, Proteobacteria (α and γ), Bacteroidetes, and high-G+C gram-positive bacteria. Gene-finding and annotation analyses identified 244 total open reading frames. Amino acid comparisons of 123 and 113 Antarctic bacterial amino acid sequences to mesophilic homologs from G+C-specific and SwissProt/UniProt databases, respectively, revealed widespread adaptation to the cold. The most significant changes in these Antarctic bacterial protein sequences included a reduction in salt-bridge-forming residues such as arginine, glutamic acid, and aspartic acid, reduced proline contents, and a reduction in stabilizing hydrophobic clusters. Stretches of disordered amino acids were significantly longer in the Antarctic sequences than in the mesophilic sequences. These characteristics were not specific to any one phylum, COG role category, or G+C content and imply that underlying genotypic and biochemical adaptations to the cold are inherent to life in the permanently subzero Antarctic waters.     

Changes in the Free Amino Compounds in Young Tomato Plants in Light and Darkness with particular Reference to {gamma}-Aminobutyric Acid

Tomato plants were grown to the five-leaf stage under uniformconditions in a growth room with a daily light period of 15h. Plants were sampled at intervals through 24 h periods andthe free ninhydrin-positive compounds determined in roots, bleedingsap, stems and shoots (mainly leaves), using ion-exchange columnchromatography and a lithium-buffer separation system. The compoundspresent and their range of concentrations are given for twooccasions: after illumination for 8 hand after 5 h of darkness. Data for -aminobutyric acid (GAB), glutamic acid, glutamine,alanine, aspartic acid and ammonia are summarized graphicallyfor all occasions and for all parts of the plant; asparaginefor sap only. The data were examined for correlations betweenthese substances for both light and dark conditions. Relative amounts of free acids were: root glutamine> glutamicand GAB > aspartic > alanine; bleeding sap glutamine >asparagine > GAB > aspartic> alanine; stem glutamine> glutamic > GAB and aspartic > alanine; shoot (leaf)GAB and glutamine > aspartic > alanine and glutamic. Patternsof change were as follows: in the root GAB and glutamic weresimilar and unlike glutamine; alanine did not change;sap ammonia,GAB and alanine were parallel, glutamine was similar to theseonly in light; in the stem glutamine and glutamic tended toaccumulate in parallel in light, but GAB did not; in the shoot(leaf) GAB and glutamine were similar except that the formeraccumulated more rapidly in the initial light period; glutamicacid and alanine were similar to each other but distinct fromGAB and glutamine. The relatively large amounts of GAB in tomato plants and themagnitude of the changes occurring in light and darkness seemindicative of its importance as a temporary storage productfor protein amino acids, but the factors controlling accumulationand utilization in different parts of the plant are unknown.     

Studies on the mucin derived from human colloid breast carcinoma

1. A non-diffusible mucoid, showing a single peak in the ultracentrifuge, was isolated from human colloid breast carcinoma by treatment with trypsin and pepsin. The material contained threonine, leucine (isoleucine), valine, proline, glycine and glutamic acid in the approximate molar proportions 5:1:1:2:1:1. Smaller amounts of aspartic acid and serine were also found. For each 5 threonine residues, 6 N-acetylgalactosamine and 3–4 galactose residues were present. 2. The mucoid possessed reducing properties by the Park & Johnson (1949) procedure; these were attributable to the action of mild alkali, as employed in this procedure. Mild alkaline treatment by the Aminoff, Morgan & Watkins (1952) procedure gave rise to a diffusible N-acetylgalactosamine chromophore that gave an enhanced colour with Ehrlich's reagent. That galactosyl-(1→3)-N-acetylgalactosamine residues were liberated was supported by periodate studies. 3. Alkaline liberation of hexosamine residues was accompanied by a specific destruction of threonine. After 40 min. at 100° in 0·18 n-lithium hydroxide, both moieties had almost completely disappeared from the ninhydrin-positive components formed on subsequent acid hydrolysis. Glycine and α-oxobutyric acid were present in the acid hydrolysate, showing that both possible pathways of a β-elimination reaction were involved. Formation of diffusible peptide on very mild alkaline treatment was attributable to the rupture of the original peptide core, necessitated by the second of these two pathways. 4. Hydroxamate formation on treatment with hydroxylamine showed the presence of carbohydrate linkage to glutamic acid or aspartic acid residues or both. This could account for the single N-acetylgalactosamine residue not linked to threonine. 5. The native mucin contained sialic acid, which was cleaved by the acid environment used in the treatment with pepsin. A statistical model of the mucin would require each prosthetic group to be linked, via N-acetylgalactosamine, to threonine, which would occupy every alternate position among the amino acids in the peptide core.     

Florida citrus molasses as a fermentation substrate: I. Free amino acids in molasses from early and midseason varieties of citrus fruits during 2 years of production

Samples of midseason Florida citrus molasses from 10 different manufacturing plants were studied during 2 years of production for suitability as fermentation substrates. They were analyzed for degrees Brix, pH, percentage of protein, and free amino acid content. The following ranges of values were obtained: degrees Brix, 65.4 to 73.0; pH, 4.2 to 6.4; and protein, 3.3 to 5.1%. Although the general characteristics of all samples were quite similar, the total free amino acid content and the number of amino acids differed from year to year and from plant to plant. The free amino acids consistently present were asparagine, aspartic acid, alanine, γ-amino-butyric, glycine, glutamic acid, proline, serine, and threonine. Occasionally found were arginine, histidine, isoleucine, leucine, lysine, phenylalanine, tyrosine, and valine. An extremely high level of proline was detected in all samples.     

Biosynthesis of amino acids from sucrose and Krebs cycle metabolites by Rhizobium lupini bacteroids

Summary The possibility of amino acids biosynthesis from sucrose, metabolites of Krebs cycle or glyoxylate and ammonium by intact bacteroids has been studied. The suspension of intact Rhizobium lupini bacteroids in phosphate buffer solution pH 7.8 was shown to catalyse the biosynthesis from sucrose and ammonium of some amino acids, such as alanine, aspartic and glutamic acids, glycine and serine. The yield of alanine and aspartic acid was 2.5–3 times higher than that of other amino acids, which were formed in almost equal quantities. Intact bacteroids were also found to catalyse the biosynthesis of aspartic and glutamic acids, alanine and glycine from ammonium and Krebs cycle metabolites such as fumaric acid (FA), oxaloacetic acid (OAA), pyruvic acid (PA), a-ketoglutaric acid (a-KGA), malic acid (MA), as well as from glyoxylic acid (GOA). The biosynthesis of aspartic acid from fumaric acid was dominant. Besides that, the suspension of intact bacteroids catalysed transamination of aspartic and glutamic acids, the transamination of aspartic acid being especially intense with -KGA and GOA. Aspartic acid was synthesized most efficiently through the amination of fumaric acid, while glutamic acid was better synthesized through the transamination of aspartic acid with -KGA than through reductive amination of -KGA.The experimental data proved that intact bacteroids posess Krebs cycle enzymes and primary ammonia assimilation enzymes. This enzyme complex permits bacteroids to detoxify ammonia, which they produce using sucrose and metabolites of Krebs cycle as the sources of carbon.The data obtained are of great interest as they prove the importance of bacteroids in the synthesis of amino acids from ammonium which is formed in the course of N₂-fixation, and sucrose available from leaves.     

Absence of Vitamin K-Dependent γ-Carboxylation in Human Periostin Extracted from Fibrotic Lung or Secreted from a Cell Line Engineered to Optimize γ-Carboxylation

Periostin (PN, gene name POSTN) is an extracellular matrix protein that is up-regulated in bronchial epithelial cells and lung fibroblasts by TH-2 cytokines. Its paralog, TGF-β-induced protein (βig-h3, gene name TGFBI), is also expressed in the lung and up-regulated in bronchial myofibroblasts by TGF-β. PN and βig-h3 contain fasciclin 1 modules that harbor putative recognition sequences for γ-glutamyl carboxylase and are annotated in UniProt as undergoing vitamin K-dependent γ-carboxylation of multiple glutamic acid residues. γ-carboxylation profoundly alters activities of other proteins subject to the modification, e.g., blood coagulation factors, and would be expected to alter the structure and function of PN and βig-h3. To analyze for the presence of γ-carboxylation, proteins extracted from fibrotic lung were reacted with monoclonal antibodies specific for PN, βig-h3, or modification with γ-carboxyglutamic acid (Gla). In Western blots of 1-dimensional gels, bands stained with anti-PN or -βig-h3 did not match those stained with anti-Gla. In 2-dimensional gels, anti-PN-positive spots had pIs of 7.0 to >8, as expected for the unmodified protein, and there was no overlap between anti-PN-positive and anti-Gla-positive spots. Recombinant PN and blood coagulation factor VII were produced in HEK293 cells that had been transfected with vitamin K 2, 3-epoxide reductase C1 to optimize γ-carboxylation. Recombinant PN secreted from these cells did not react with anti-Gla antibody and had pIs similar to that found in extracts of fibrotic lung whereas secreted factor VII reacted strongly with anti-Gla antibody. Over 67% coverage of recombinant PN was achieved by mass spectrometry, including peptides with 19 of the 24 glutamates considered targets of γ-carboxylation, but analysis revealed no modification. Over 86% sequence coverage and three modified glutamic acid residues were identified in recombinant fVII. These data indicate that PN and βig-h3 are not subject to vitamin K-dependent γ-carboxylation.     

Structure-Function Analysis of the Glioma Targeting NFL-TBS.40-63 Peptide Corresponding to the Tubulin-Binding Site on the Light Neurofilament Subunit

We previously reported that a 24 amino acid peptide (NFL-TBS.40-63) corresponding to the tubulin-binding site located on the light neurofilament subunit, selectively enters in glioblastoma cells where it disrupts their microtubule network and inhibits their proliferation. Here, we analyzed the structure-function relationships using an alanine-scanning strategy, in order to identify residues essential for these biological activities. We showed that the majority of modified peptides present a decreased or total loss to penetrate in these cells, or to alter microtubules. Correspondingly, circular dichroism measurements showed that this peptide forms either β-sheet or α-helix structures according to the solvent and that alanine substitution modified or destabilized the structure, in relation with changes in the biological activities. Moreover, substitution of serine residues by phosphoserine or aspartic acid concomitantly decreased the cell penetrating activity and the structure stability. These results indicate the importance of structure for the activities, including selectivity to glioblastoma cells of this peptide, and its regulation by phosphorylation.