Dependence of apparent viscosity on mycelial morphology of Streptomyces fradiae culture in various nitrogen sources

To examine what causes increased viscosity in culture broth in Streptomyces fradiae culture, various natural nitrogen sources were investigated. Extracellular protease activity increased with culture time and decomposed the natural nitrogen source into amino acids. In the case of gluten meal, after a culture time of 5 d, concentrations of glutamic acid and aspartic acid had increased to 600 and 200 mg/L, respectively, which were about 3- and 2-fold as high as levels in cultures under similar conditions using Pharmamedia. For various amino acids tested, the addition of glutamic acid or aspartic acid mixture to the culture medium raised the apparent viscosity to its highest demonstrated value, 260 mPa.s after 5 d of culture, which was 3-fold higher than without amino acids. Consumption of the decomposed glutamic acid and aspartic acid was dependent on the activities of glutamate dehydrogenase and aspartate aminotransferase, respectively. When ammonium ion was used as the nitrogen source, cell concentration reached 1.75 g/L measured as an intracellular nucleic acid concentration, which was about 2.3-fold higher than that with any other natural nitrogen source. However, apparent viscosity was only 75 mPa.s, a value one-third that of the amino acid mixture, and 70% of the pellets were bigger than 1.2 x 10(4) microm(2). In the case of gluten meal or the amino acid mixture, pellets bigger than 1.2 x 10(4) microm(2) comprised only 8%. This demonstrates that consumption of some amino acids affected the formation of filamentous morphology, which caused an increase in the apparent viscosity of the culture broth, and the apparent viscosity was not caused by the mycelial concentration but the mycelial morphology.     

Thermal condensation of a mixture of six amino acids

Amino acids can be converted to peptides by the process of heating if the mixture is dry and if the appropriate proportion of glutamic acid as the dominant monomer is present. Three thermal peptide fractions of the diffusible fraction were isolated in this study. Trifluoracetic acid (TFA) hydrolysis was utilized to open the blocked N-terminal group before performing the dansylation reaction. When the molecular weight of the thermal peptide increased, the proportion of neutral amino acids increased slightly, with a decrease in the concentration of glutamic acid. Quantitative analysis of amino acid composition is also reported.     

Influence of the nitrogen source on Saccharomyces cerevisiae anaerobic growth and product formation.

To prevent the loss of raw material in ethanol production by anaerobic yeast cultures, glycerol formation has to be reduced. In theory, this may be done by providing the yeast with amino acids, since the de novo cell synthesis of amino acids from glucose and ammonia gives rise to a surplus of NADH, which has to be reoxidized by the formation of glycerol. An industrial strain of Saccharomyces cerevisiae was cultivated in batch cultures with different nitrogen sources, i.e., ammonium salt, glutamic acid, and a mixture of amino acids, with 20 g of glucose per liter as the carbon and energy source. The effects of the nitrogen source on metabolite formation, growth, and cell composition were measured. The glycerol yields obtained with glutamic acid (0.17 mol/mol of glucose) or with the mixture of amino acids (0.10 mol/mol) as a nitrogen source were clearly lower than those for ammonium-grown cultures (0.21 mol/mol). In addition, the ethanol yield increased for growth on both glutamic acid (by 9%) and the mixture of amino acids (by 14%). Glutamic acid has a large influence on the formation of products; the production of, for example, alpha-ketoglutaric acid, succinic acid, and acetic acid, increased compared with their production with the other nitrogen sources. Cultures grown on amino acids have a higher specific growth rate (0.52 h-1) than cultures of both ammonium-grown (0.45 h-1) and glutamic acid-grown (0.33 h-1) cells. Although the product yields differed, similar compositions of the cells were attained. The NADH produced in the amino acid, RNA, and extracellular metabolite syntheses was calculated together with the corresponding glycerol formation. The lower-range values of the theoretically calculated yields of glycerol were in good agreement with the experimental yields, which may indicate that the regulation of metabolism succeeds in the most efficient balancing of the redox potential.     

Formation of specific amino acid sequences during carbodiimide-mediated condensation of amino acids in aqueous solution

Carbodiimide-mediated peptide synthesis in aqueous solution at room temperature has been studied with respect to self-ordering of amino acids. Inasmuch as glutamic acid is readily converted into pyroglutamic acid, the peptides formed by copolymerization of glutamic acid with other amino acids are preferentially pyroglutamyl-peptides. Competition experiments were carried out to determine the influence of the amino acid side chains on the reactivity of amino acids and peptides during the dehydration condensation. The results show that the self-ordering process is controlled by both the activated carboxyl component (amino acid or growing peptide) and the incoming amino acid. A condensation of pyroglutamic acid, alanine and another amino acid component Xxx (Xxx = Gly, Val, Leu or Gly-Tyr) preferentially yielded the dipeptide pyroGlu-Ala, but the formation of the tripeptide pyroGlu-Ala-Ala became strongly reduced because of competing reactions. A simple explanation for the observed selectivities is not at hand. Polypeptides were so far only obtained when they were allowed to precipitate in the reaction system. Evidence for the non-random copolymerization of larger peptides is presented as well.     

TRANSPORT OF AMINO ACIDS BY THE SOIL ALGA STICHOCOCCUS BACILLARIS1

The green alga Stichococcus bacillaris Naeg. is able to take up at least eleven amino acids. All of these except glutamic and aspartic acids are transported by carrier systems that obey saturation kinetics. The acidic amino acids enter the cell by passive diffusion. Michaelis-Menten parameters (K_s and V_max) were calculated for several amino acids. All obey simple Michaelis-Menten behavior except for 2-methylalanine and leucine which may have double carrier systems of different affinities. Interactions between pairs of amino acids suggest that there is at least one carrier system specific for basic amino acids and probably several systems specific for neutral amino acids. Further analysis of neutral amino acid interactions reveal that the uptake of several amino acids is incompletely inhibited by competitor uptake at infinite concentration. The simplest interpretation of the data is the operation of three carrier systems for neutral amino acids, one of which has higher affinity and broader specificity than the other two. The amino acid carrier systems appear to operate by an active mechanism. The metabolic poison DCCD inhibits uptake up to 99%. The capacities of the neutral amino acid carrier systems are increased when cells are grown in medium containing suboptimal concentrations of nitrogen.     

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.     

Identification and fine mapping of IgG and IgE epitopes in ovomucoid

Ovomucoid is a major allergen in hen egg white which causes a serious IgE-mediated food allergy reaction. This study determined eight IgG epitopes, 5-11 amino acids in length, and nine IgE epitopes, 5-16 amino acids in length, within the primary sequence in ovomucoid using arrays of overlapping peptides synthesized on cellulose membranes. Pooled sera from eight egg-allergic patients were used to probe the membrane. We also analyzed the amino acids that are critical for antibody binding by substituting a single amino acid within each epitope. Mutational analysis of the epitopes indicated that charged amino acids (aspartic acid, glutamic acid, and lysine) and some hydrophobic (leucine, phenylalanine, and glycine) and polar (serine, threonine, tyrosine, and cystein) amino acids were important for antibody binding. These results provide useful information for the molecular design necessary to reduce the allergenicity of ovomucoid, and a better understanding of structure-function relationships of allergic epitopes in food proteins.     

Ammonium and amino acids as regulators of nitrate reductase in corn roots

When amino acids or ammonia are added to plant systems, the effects on the development of nitrate-dependent nitrate reductase activity are variable. In addition, amino acids added singly or as casein hydrolysate may not support a normal growth. A physiologically correct mixture of amino acids, one similar in composition to amino acids released by the endosperm, has been shown to support normal growth and protein synthesis in corn (Zea mays) embryos. In this investigation, we have used the mixture of corn amino acids to determine whether amino acids have an effect on the appearance or disappearance of nitrate reductase activity. The results show that these amino acids partially inhibit the induction of nitrate reductase in corn roots. The effect is more pronounced in mature root than in root tip sections. When glutamine and asparagine are included along with the "corn amino acid mixture," the inhibition is more severe. Amino acids or amino acid analogues added singly to the induction medium have a similar effect: i.e. when the induction of nitrate reductase is inhibited in the root tips (lysine, canavanine, azaserine, azetidine-2-carboxylic acid, dl-4-azaleucine, asparagine, and glutamine), that inhibition is more severe in mature root sections. Arginine enhanced the recovery of nitrate reductase in root tips but inhibited it in mature root sections. The effect of the amino acids is apparently on some phase of the induction processes (i.e. the uptake or distribution of nitrate or a direct effect on the synthesis of the enzyme) and not on the turnover of the enzyme.     

Quantitative analysis of free amino acids and carbohydrates in spring barley anthers at different stages of maturity

The content of the carbohydrates glucose, fructose and sucrose was determined in spring barley anthers at different stages of maturity. During maturation the sucrose content of the anthers increased markedly. The following 17 free amino acids were detected in anthers of different stages of maturity: aspartic acid, glutamic acid, serine, alanine, arginine, leucine, isoleucine, lysine, α-aminobutyric acid, glutamine, proline, tyrosine, phenylalanine, valine, threonine, cystine and glycine. Quantitative analysis was only carried out in amino acids present in higher concentrations in the analysed samples. These were: aspartic acid, glutamic acid, α-aminobutyric acid, proline, serine, valine and glutamine, and a mixture of amino acids (leucine, isoleucine, valine and phenylalanine). The total content of free amino acids increased with increasing maturity of the anthers. However, not all amino acids followed contributed to this increase, but only proline, glutamic acid, aspartic acid and glutamine. A small difference was found in the variety Gopal in which the aspartic acid content did not increase significantly, but the content of the mixture of amino acids and serine did. With the exception of green anthers of the variety Firlbecks Union, proline was present in the highest concentration in all samples analysed.     

Amino acid distribution in some fungal melanins and of soil humic acids from Brazil

Summary Humic acids from four Brazilian topsoils of different origins and four soil fungal melanins, synthesized under two cultural conditions, were subjected to 6N HCl hydrolysis and their amino acid distribution patterns qualitatively and quantitatively determined. Both soil and fungal polymers showed similar patterns with aspartic acid, glutamic acid, glycine and alanine as the dominant amino acids. Some variations noted were more quantitative than qualitative, the similarities were more pronounced than differences, indicating that the fungal melanins may play a significant role in the formation of soil humic acid polymers. The humic acids of Brazilian soils had amino acid distribution patterns similar to those reported for humic acids of other tropical and temperate soils.     

Oxidation of lipids and membranes I: In vitro formation of peroxidative lipid polymers

Fluorescent polymers were obtained by oxidizing partly emulsified linolenic acid with different oxidants. The speed of formation of polymers differed for the various oxidants, and the difference was not a simple function of the oxidation potential. The speed of polymerization also depended on the nature of the emulsion. The presence of egg albumen in the emulsion enhanced polymer formation with all oxidants. When the oxidants used are arranged in the order of decreasing speed of polymer formation, the order is different in the presence of albumen from what it is in the absence of albumen. With different oxidation catalysts most antioxidants and amino acids tested enhanced polymerization. In oxidation with ferric ions, with K-dichromate, and without added oxidants the only antioxidants which delayed polymerization were “inhibitors”. “Retarders” enhanced polymerization. With KMnO₄ slight delay was caused by some retarders. The findings indicate that not only oxidation catalysts, but also proteins, amino acids, and antioxidants enhance polymerization. The possibility is suggested that in animal cells lipid pigment formation might represent a mechanism for neutralizing free radicals.     

The importance of dietary amino acids on the reproduction and longevity of adult Dacus oleae (Gmelin) (Diptera Tephritidae)

When the amino-acid mixture of an effective chemically defined diet was replaced by single amino acids, keeping the total nitrogen at the same level, the egg production of Dacus oleae was minimal with all the 19 amino acids tested. Male survival was adversely affected by the amino acids : alanine, aspartic acid, glutamic acid, glycine, hydroxyproline, lysine, serine and tyrosine, while female survival was shortened when the amino acids : glycine, hydroxyproline and lysine were added. The creation of amino-acid imbalances, by deleting the 19 amino acids individually, from the complete amino-acid mixture, showed that the amino acids : arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophane and valine were indispensable to the adult Dacus oleae flies, as far as egg production is concerned. Survival of the male flies was significantly shortened when the amino acids : alanine, hydroxyproline and tryptophane were omitted. Significant differences in longevity between males and females were scored, when the amino acids : alanine, aspartic acid, cystine, glycine and tryptophane, were omitted.     

Purification, characterization and gene cloning of a novel glutamic acid-specific endopeptidase from Staphylococcus aureus ATCC 12600.

Twenty strains of Staphylococcus aureus from ATCC type cultures and strains found in clinical studies were cultivated, and their endopeptidase activity specific for glutamic acid was surveyed using benzyloxycarbonyl-Phe-Leu-Glu-p-nitroanilide (Z-Phe-Leu-Glu-pNA) as a substrate. The activity was found in two of the strains, ATCC 12600 and ATCC 25923. A glutamic acid-specific proteinase, which we propose to call SPase, was purified from the culture filtrate of S. aureus strain ATCC 12600 by a series of column chromatographies on DEAE-Sepharose twice and on Sephacryl S-200. A single band was observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified SPase. The molecular weight of the proteinase was estimated to be 34000 by SDS-PAGE. When synthetic peptides and oxidized insulin B-chain were used as substrates, SPase showed the same substrate specificity as V8 proteinase, EC 3.4.21.9, which specifically cleaves peptide bonds on the C-terminal side of glutamic acid and aspartic acid. Examination with p-nitroanilides of glutamic acid and aspartic acid as substrates, however, revealed that both proteinases are highly specific for a glutamyl bond in comparison with an aspartyl bond. To elucidate the complete primary structure of SPase, its gene was cloned from genomic DNA of S. aureus ATCC 12600, and the nucleotide sequence was determined. Taking the amino acid sequence of SPase from the NH2-terminus to the 27th residue into consideration, the clones encode a mature peptide of 289 amino acids, which follows a prepropeptide of 68 residues. SPase was confirmed to be a novel endopeptidase specific for glutamic acid, being different from V8 proteinase which consists of 268 amino acids.     

Independent and Additive Effects of Glutamic Acid and Methionine on Yeast Longevity

It is established that glucose restriction extends yeast chronological and replicative lifespan, but little is known about the influence of amino acids on yeast lifespan, although some amino acids were reported to delay aging in rodents. Here we show that amino acid composition greatly alters yeast chronological lifespan. We found that non-essential amino acids (to yeast) methionine and glutamic acid had the most significant impact on yeast chronological lifespan extension, restriction of methionine and/or increase of glutamic acid led to longevity that was not the result of low acetic acid production and acidification in aging media. Remarkably, low methionine, high glutamic acid and glucose restriction additively and independently extended yeast lifespan, which could not be further extended by buffering the medium (pH 6.0). Our preliminary findings using yeasts with gene deletion demonstrate that glutamic acid addition, methionine and glucose restriction prompt yeast longevity through distinct mechanisms. This study may help to fill a gap in yeast model for the fast developing view that nutrient balance is a critical factor to extend lifespan.     

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.     

Pyroglutamyl N-termini of thermal polyamino acids

It has been established indirectly that the N-termini of the thermal polyamino acids are pyroglutamic acid. This was determined by trifluoroacetic acid hydrolysis of the lactam ring followed by Dansyl labelling. The polyamino acids contained Ala, Gly, Glu, Leu, Phe, and Pro. In the experiments described here, the presence of pyroglutamic acid at the N-terminus of a polyamino acid was determined directly by the use of pyrrolidone carboxylyl peptidase. The enzyme catalyzes the removal of pyroglutamyl residues at the N-terminus of polypeptide chains. The polyamino acids used in these studies contained glutamic acid, aspartic acid, alanine, glycine, isoleucine, proline and valine. Alkaline hydrolysis was also used to determine indirectly that the N-termini of these polyamino acids are pyroglutamic acid. Another interesting finding was that many of the amino acids in the polymerization mixture were found to occur penultimate to the N-terminal amino acid. This is interpreted to mean that the diffusible fraction contains many polyamino acids.     

Stimulation of Lysine Decarboxylase Production in Escherichia coli by Amino Acids and Peptides

A commercial hydrolysate of casein stimulated production of lysine decarboxylase (EC 4.1.1.18) by Escherichia coli B. Cellulose and gel chromatography of this hydrolysate yielded peptides which were variably effective in this stimulation. Replacement of individual, stimulatory peptides by equivalent amino acids duplicated the enzyme levels attained with those peptides. There was no indication of specific stimulation by any peptide. The peptides were probably taken up by the oligopeptide transport system of E. coli and hydrolyzed intracellularly by peptidases to their constituent amino acids for use in enzyme synthesis. Single omission of amino acids from mixtures was used to screen them for their relative lysine decarboxylase stimulating abilities. Over 100 different mixtures were evaluated in establishing the total amino acid requirements for maximal synthesis of lysine decarboxylase by E. coli B. A mixture containing all of the common amino acids except glutamic acid, aspartic acid, and alanine increased lysine decarboxylase threefold over an equivalent weight of casein hydrolysate. The nine most stimulatory amino acids were methionine, arginine, cystine, leucine, isoleucine, glutamine, threonine, tyrosine, and asparagine. Methionine and arginine quantitatively were the most important. A mixture of these nine was 87% as effective as the complete mixture. Several amino acids were inhibitory at moderate concentrations, and alanine (2.53 mM) was the most effective. Added pyridoxine increased lysine decarboxylase activity 30%, whereas other B vitamins and cyclic adenosine 5′-monophosphate had no effect.     

Polymerization on the Rocks: Theoretical Introduction

It is difficult if not impossible to synthesize long polymers of amino acids, nucleotides, etc., in homogeneous aqueous solution. We suggest that long polymers were synthesized on the surface of minerals in a prebiotic process analogous to solid-phase synthesis. Provided that the affinity of a mineral for an oligomer increases with the length of the oligomer, adsorption must become essentially irreversible for sufficiently long oligomers. Irreversibly adsorbed oligomers may be elongated indefinitely by repeated cycles in which the mineral with its adsorbed oligomers is first incubated with activated monomers and then washed free of deactivated monomer and side-products. We discuss in some detail the formation of oligomers of negatively-charged amino acids such as glutamic acid on anion-exchange minerals such as hydroxylapatite or illite. We show that the average length of adsorbed oligomers at steady state, n, depends on the balance between the rate of chain elongation and the rate of hydrolysis, and we derive a very approximate formula for n.     

A putative signal peptidase recognition site and sequence in eukaryotic and prokaryotic signal peptides

Presecretory signal peptides of 39 proteins from diverse prokaryotic and eukaryotic sources have been compared. Although varying in length and amino acid composition, the labile peptides share a hydrophobic core of approximately 12 amino acids. A positively charged residue (Lys or Arg) usually precedes the hydrophobic core. Core termination is defined by the occurrence of a charged residue, a sequence of residues which may induce a beta-turn in a polypeptide, or an interruption in potential alpha-helix or beta-extended strand structure. The hydrophobic cores contain, by weight average, 37% Leu: 15% Ala: 10% Val: 10% Phe: 7% Ile plus 21% other hydrophobic amino acids arranged in a non-random sequence. Following the hydrophobic cores (aligned by their last residue) a highly non-random and localized distribution of Ala is apparent within the initial eight positions following the core: (formula; see text) Coincident with this observation, Ala-X-Ala is the most frequent sequence preceding signal peptidase cleavage. We propose the existence of a signal peptidase recognition sequence A-X-B with the preferred cleavage site located after the sixth amino acid following the core sequence. Twenty-two of the above 27 underlined Ala residues would participate as A or B in peptidase cleavage. Position A includes the larger aliphatic amino acids, Leu, Val and Ile, as well as the residues already found at B (principally Ala, Gly and Ser). Since a preferred cleavage site can be discerned from carboxyl and not amino terminal alignment of the hydrophobic cores it is proposed that the carboxyl ends are oriented inward toward the lumen of the endoplasmic reticulum where cleavage is thought to occur. This orientation coupled with the predicted beta-turn typically found between the core and the cleavage site implies reverse hairpin insertion of the signal sequence. The structural features which we describe should help identify signal peptides and cleavage sites in presumptive amino acid sequences derived from DNA sequences.     

Chiral separation using molecularly imprinted heteroaromatic polymers

Novel molecularly imprinted polymer systems utilizing 4-vinylpyridine and 1-vinylimidazole as functional monomers have been developed for enantioselective recognition of carboxylic and N-protected amino acids. Non-covalent interactions between the functional monomers and the template molecules were the source of the subsequent recognition sites in the resultant polymers. The capacity of the polymers for molecular recognition was investigated by using them as stationary phases in the HPLC mode. Polymers prepared with 4-vinylpyridine were found to be more efficient in racemic resolution than those prepared with 1-vinylimidazole. When applying a racemic mixture of the template molecule, the polymers showed highest affinity for the enantiomer used as template. Imprints of a racemic template molecule, as expected, did not exhibit enantioselectivity. The optimal molar ratio of 4-vinylpyridine to the template Cbz-L -Asp-OH in the polymerization mixture was determined to be 12:1. In addition to enantioselectivity, the investigated polymers demonstrated ‘ligand selectivity’ e.g., a Cbz-L -Asp-OH-imprinted polymer was able to separate Cbz-D ,L -Asp-OH, but was unable to separate Cbz-D ,L -Glu-OH.