Effect of Dipeptides on the Growth of <Emphasis Type="Italic">Oenococcus oeni</Emphasis> in Synthetic Medium Deprived of Amino Acids

Oenococcus oeni has numerous amino acid requirements for growth and dipeptides could be important for its nutrition. In this paper the individual or combined effect of dipeptides on growth of O. oeni X₂L in synthetic media deficient in one or more amino acids with L-malic acid was investigated. Utilization of dipeptides, glucose, and L-malic acid was also analyzed. Dipeptides were constituted by at least one essential amino acid for growth. Dipeptides containing two essential amino acids, except leucine, had a more favorable effect than free amino acids on the growth rate. Gly-Gly was consumed to a greater extent than Leu-Leu and a rapid exodus of glycine to the extracellular medium accompanied it. The microorganism could use glycine in exchange for other essential amino acids outside the cell, favoring growth. In the presence of Leu-Leu, the increase in glucose consumption rate could be related to the additional energy required for dipeptide uptake.     

Some Factors Which Affect Amino Acid Uptake by Saccharomyces carlsbergensis

When fully grown cells of Saccharomyces carlsbergensis were suspended in a solution of glucose and labeled amino acids, there was a lag phase before rapid uptake of certain amino acids. During this lag, significant amounts of sugar were utilized. The lag phase varied in length, depending upon the amino acid under study, but could be shortened by aeration of the cells and eliminated by their preincubation in glucose solution. Divalent metal ions, especially Ca²⁺ added during the early stages of the lag phase, increased the length of the lag, an effect that could be reversed by washing with ethylenediaminetetraacetate, but amino acids which normally showed little or no lag before uptake were insensitive to Ca²⁺. The rate of uptake of amino acids or of sugar was essentially unaffected by Ca²⁺, whereas 2,4-dinitrophenol caused an overall decrease in the rate of uptake of all amino acids tested. The relevance of these observations to commercial brewing practice is shown.     

The Effect of Some Protein and Non-Protein Amino Acids on the Growth of Cladosporium herbarum and Trichothecium roseum

The effect of ten amino acids as the sole nitrogen source for the growth of Cladosporium herbarum (Link.) Fr. and Trichothecium roseum (Bull.) Link. was studied in order to clarify the fungus-host plant relationship. Special attention was paid to some rare non-protein amino acids of legumes. The best nitrogen sources for both fungi were γ-aminobutyric acid, arginine, serine and proline. Cladosporium could use homoarginine and canavanine, but these two amino acids were not used by Trichothecium when each was given as the only nitrogen source. Both fungi utilized ornithine, homoserine and a,γ-diaminobutyric acid to a limited extent. Pipecolic acid was not growth promoting. The growth-retarding effects of rare non-protein amino acids (homoarginine, canavanine, a,γ-diaminobutyric acid and pipecolic acid) were usually reversed by higher concentrations of their normal analogues. It is possible that rare non-protein amino acids may slightly protect the host plant against fungal infections, but there are clear differences between fungi in their reaction to non-protein amino acids.     

The uptake of amino acids from a chemically defined medium byFusobacterium species

The uptake of amino acids from a chemically defined medium was determined for various species ofFusobacterium. Reference strains utilized a wider range and higher levels of amino acids than clinical isolates. Among the acidic and basic amino acids, arginine, histidine, and glutamate were used by most species. In general, nonpolar neutral amino acids such as alanine, valine, leucine, isoleucine, proline, and methionine were poorly utilized. Apart from glycine and tyrosine, the neutral but polar amino acids such as serine, cysteine, and asparagine were incorporated at significantly high levels. ThusFusobacterium species, unlike several Gram-negative anaerobic bacteria, have the capacity to utilize both free amino acids and peptides as energy sources and may partly account for the wide distribution of these species in areas of tissue degradation.     

Amino acids in exudates of healthy and fungus-affected pea roots

Summary Amino acids in exudates of uninoculated pea roots were compared quantitatively and qualitatively with exudates of roots inoculated with Gliocladium catenulatum. This fungus has the potential of causing severe root necrosis. Twenty-one amino acids were found in exudates of healthy roots and apparently some of these were utilized by the fungus. A relatively high concentration of ammonia was detected in exudates of inoculated pea roots, indicating an intense deamination by the fungus. No other imbalance in amino acids was found which could be related to known toxic effects of amino acids on plant tissues.     

Viability of some metabolic processes in the isolated toad brain adapted to two osmotic environments

The viability of the isolated toad brain in an aerated Ringer-like medium has been evaluated by the following criteria: 1) amino acid content before and after incubation; 2) accumulation of amino acids in the incubation medium; 3) a comparison of glucose utilization and [U-¹⁴C]glucose metabolism with that occurring in vivo; 4) tissue swelling; and 5) tissue lactate content. On the basis of these criteria, the isolated toad brain, from toads adapted to a fresh-water or a salt-water environment, retains considerable metabolic integrity for at least 2 hr of incubation at 25° C. Specifically, there was no swelling of the tissue, no apparent accumulation of lactate in the tissue, glucose appeared to be utilized at a rate not too different from that calculated for the toad brain in vivo, and the distribution of label from [U-¹⁴C]glucose had an overall pattern which resembled that observed in vivo. The tissue levels of amino acids were generally stable in vitro; however, there was a marked decline in the content of aspartate. The accumulation of amino acids in the medium varied considerably from one amino acid to another. Thus, there was very little net efflux of aspartate, GABA, and glutamate from the tissue but considerable net efflux of glutamine. This efflux of amino acids was greater from brains of hyperosmotically adapted toads than from the brains of toads adapted to fresh water by amounts proportional to their initial tissue contents.     

Nutritive effects of d-amino acids on the silkworm, Bombyx mori

Nutritive effects of d-amino acids on the silkworm, Bombyx mori, were investigated by growth experiments using defined diets and also by analysis of free amino acids in the larval haemolymph. None of the d-forms of the usual ten essential amino acids could be utilized effectively, although d-methionine was utilized in lieu of the l-form only to a limited extent and d-histidine gave a positive but smaller effect than d-methionine. d-Proline, its l-form being semi-essential for the silkworm, was not utilized. d-Leucine, and to a lesser extent d-alanine and d-serine, were found to be somewhat toxic. Comparison of free amino acid patterns in the haemolymph of the fifth-instar larvae, which fed on diets either lacking l-forms of histidine, methionine and leucine singly or including the d-forms singly in place of these l-forms, supported the results of the growth experiments.     

Characterization of system L and system y+ amino acid transport activity in cultured vascular smooth muscle cells

The uptake of L-leucine and L-lysine into vascular smooth muscle cells cultured from the aortas of rats has been investigated. Both amino acids are taken up by saturable systems that are independent of the presence of a ^·Na⁺ gradient and can be stimulated in trans by neutral bulky amino acids for leucine and cationic amino acids for lysine. Leucine uptake is inhibited competitively in cis by several neutral amino acids, whereas lysine uptake is inhibited strongly by other cationic amino acids but also significantly by neutral amino acids such as leucine. The leucine inhibition is noncompetitive. Cells preloaded with leucine and lysine could also export these amino acids and the rate of efflux was stimulated by the presence of appropriate amino acids in trans. These data are all consistent with leucine being transported largely if not entirely by System L and lysine by the System y⁺ transporter. © 1993 Wiley-Liss, Inc.     

Growth of Certain Aquatic Oomycetes on Amino Acids II. Apodachlya, Aphanomyces, and Pythium

Four aquatic fungi —Apodachlya brachynema and A. minima (Leptomitales), Aphanomyces laevis (Saprolegniales), and Pythium ultimum (Peronosporales) —were tested for growth in synthetic media containing one of a variety of carbon sources. Apodachlya brachynema readily utilized five amino acids — alanine, glutamate, aspartate, proline and leucine — as well as glucose and acetate. Growth on sucrose as a carbon source was slight. Apodachlya minima differed from A. brachynema in that it could not utilize proline and leucine. Aphanomyces laevis grew well on only three of the substrates tested — glucose, alanine and glutamate. Pythium ultimum utilized glucose, sucrose, maltose, cellobiose, alanine, glutamate, aspartate, proline, asparagine, ornithine, and serine, but not eight other amino acids. All of these fungi hydrolyzed gelatin. Radioactively labeled carbon dioxide was released during incubation of Aphanomyces laevis in media containing labeled leucine, proline, or phenylalanine. These data provide evidence of some catabolism of the three substrates although none of these substrates can support the growth of Aphanomyces laevis as a sole source of carbon and nitrogen.     

The effect of nitrogen and carbon sources on proteinase production by Pseudomonas fluorescens

Some factors influencing the production of an extracellular proteinase by Pseudomonas fluorescens NCDO 2085 were studied. Proteinase production was optimal at 20C and pH 69 in static culture when calcium was included in the medium. Proteinase was not detectable in basal medium but could be induced by organic nitrogen compounds. The proteinase was produced in the exponential phase of growth on protein substrates but not until early stationary phase during growth on amino acids. The organism did not utilize lactose, the most abundant carbohydrate in milk. Citrate was readily utilized as an energy source but had a strong repressive effect on proteinase production. A medium containing sodium caseinate and pyruvate supported good growth and enzyme production. All the amino acids utilized as a sole carbon source, with the exception of serine, could induce proteinase production. Asparagine was the most effective amino acid inducer. Particular combinations of amino acids could induce or repress proteinase production. The regulation of proteinase production by Ps. fluorescens NCDO 2085 appears to be based on a balance between induction by low concentrations of low molecular weight degradation products and sensitivity to end product catabolite repression. The results suggest that the function of the proteinase is to ensure a supply of carbon rather than amino acids for protein synthesis.     

The effect of nitrogen and carbon sources on proteinase production by Pseudomonas fluorescens

Some factors influencing the production of an extracellular proteinase by Pseudomonas fluorescens NCDO 2085 were studied. Proteinase production was optimal at 20 degrees C and pH 6.9 in static culture when calcium was included in the medium. Proteinase was not detectable in basal medium but could be induced by organic nitrogen compounds. The proteinase was produced in the exponential phase of growth on protein substrates but not until early stationary phase during growth on amino acids. The organism did not utilize lactose, the most abundant carbohydrate in milk. Citrate was readily utilized as an energy source but had a strong repressive effect on proteinase production. A medium containing sodium caseinate and pyruvate supported good growth and enzyme production. All the amino acids utilized as a sole carbon source, with the exception of serine, could induce proteinase production. Asparagine was the most effective amino acid inducer. Particular combinations of amino acids could induce or repress proteinase production. The regulation of proteinase production by Ps. fluorescens NCDO 2085 appears to be based on a balance between induction by low concentrations of low molecular weight degradation products and sensitivity to end product catabolite repression. The results suggest that the function of the proteinase is to ensure a supply of carbon rather than amino acids for protein synthesis.     

Utilization of the mixtures of amino acids by helicostylum piriforme bainier

Summary The utilization of three different mixtures of amino acids was studied. Paper chromatography was employed to detect various amino acids present in the medium. The fungus grew well on all the mixtures of amino acids. The rate of growth and the final amount of mycelium produced on the first two mixtures were better than that of the same amino acids when supplied singly. On the other hand, rate of growth and the final amount of mycelium on mixture No. 3 were not better than that of all the individual amino acids.All the amino acids were completely utilized within the incubation period from mixtures 1 and 2. On the other hand, none of the amino acids could be consumed completely by the fungus from mixture 3.     

Hydrolysis of alpha(s, 1)-Casein B by Streptococcus lactis Membrane Proteinase

The membrane-associated proteinase of Streptococcus lactis strain 3 hydrolyzed α_{s, 1}-casein B into 11 peptide fragments. Eight of the 11 peptides were purified and partially characterized. Each peptide contained several, but not all six, essential amino acids required for growth. The culture was able to utilize one peptide as the sole source for the essential amino acid leucine. Leucine, serine, valine, and glycine were found to be NH₂-terminal residues. Two of the peptides were phosphopeptides. The data support the functional role of the membrane-associated proteinase as being involved in the initial breakdown of proteins to peptides.     

Phosphorylation site analysis of Semliki forest virus nonstructural protein 3

Nonstructural protein 3 (Nsp3) is an essential subunit of the alphavirus RNA replication complex, although its specific function(s) has yet to be well defined. Previously, it has been shown that Semliki Forest virus Nsp3 (482 amino acids) is a phosphoprotein, and, in the present study, we have mapped its major phosphorylation sites. Mass spectrometric methods utilized included precursor ion scanning, matrix-assisted laser desorption/ionization mass spectrometry used in conjunction with on-target alkaline phosphatase digestions, and tandem mass spectrometry. Two-dimensional peptide mapping was applied to separate tryptic (32)P-labeled phosphopeptides of Nsp3. Radiolabeled peptides were then subjected to Edman sequencing, and phosphoamino acid analysis. In addition, radiolabeled Nsp3 was cleaved successively with cyanogen bromide and trypsin, and microscale iron-chelate affinity chromatography was used to enrich phosphopeptides. By combining these methods, we showed that Nsp3 is phosphorylated on serine residues 320, 327, 332, 335, 356, 359, 362, and 367, and is heavily phosphorylated on peptide Gly(338)-Lys(415), which carries 7-12 phosphates distributed over its 13 potential phosphorylation sites. These analytical findings were corroborated by constructing a Nsp3 derivative devoid of phosphorylation. The results represent the first determination of phosphorylation sites of an alphavirus nonstructural protein, but the approach can be utilized in phosphoprotein analysis in general.     

Variation in amino acid content and its relationship to nitrogen content and growth rate in Ulva ohnoi (Chlorophyta)

To evaluate the quantitative and qualitative changes in amino acids related to internal nitrogen content and growth rate of Ulva ohnoi, the supply of nitrogen to outdoor cultures of the seaweed was manipulated by simultaneously varying water nitrogen concentrations and renewal rate. Both internal nitrogen content and growth rate varied substantially, and the quantitative and qualitative changes in amino acids were described in the context of three internal nitrogen states: nitrogen‐limited, metabolic, and luxury. The nitrogen limited state was defined by increases in all amino acids with increasing nitrogen content and growth up until 1.2% internal nitrogen. The metabolic nitrogen state was defined by increases in all amino acids with increasing internal nitrogen content up to 2.6%, with no increases in growth rate. Luxury state was defined by internal nitrogen content above 2.6%, which occurred only when nitrogen availability was high but growth rates were reduced. In this luxury circumstance, excess nitrogen was accumulated as free amino acids, in two phases. The first phase was distinguished by a small increase in the majority of amino acids up to ≈3.3% internal nitrogen, and the second by a large increase in glutamic acid, glutamine, and arginine up to 4.2% internal nitrogen. These results demonstrate that the relationship between internal nitrogen content and amino acid quality is dynamic but predictable, and could be used for the selective culture of seaweeds.     

Resistance of proline-containing peptides to ruminal degradation in vitro.

Mixed ruminal bacteria utilized an enzymatic digest of casein at a rate faster than that for an enzymatic digest of gelatin, but neither amino acid source was completely utilized even when the incubation period was as long as 96 h. Since the reaction of ninhydrin with the residual nonammonia, nonprotein nitrogen was more than twofold stronger when the samples were hydrolyzed with 6 N HCl, it appeared that much of the residual nitrogen was from peptides. Approximately 66% of the nonammonia, nonprotein, ninhydrin-reactive material could not be recovered as amino acids, but there was a significant decrease in total amino acid nitrogen when the samples were pretreated with a C18 Sep-Pak column to remove peptides. The resistant peptides had an abundance of proline, and subsequent incubations showed that synthetic dipeptides which contained proline were hydrolyzed slowly. Lysine appears to be the amino acid which is most apt to limit ruminant production. Dipeptides containing proline and lysine were hydrolyzed at least fivefold slower than lysine-alanine. Methionine, another potentially limiting amino acid, was also degraded at a slower (2.5-fold) rate when it was present as part of a proline dipeptide.     

Resistance of proline-containing peptides to ruminal degradation in vitro.

Mixed ruminal bacteria utilized an enzymatic digest of casein at a rate faster than that for an enzymatic digest of gelatin, but neither amino acid source was completely utilized even when the incubation period was as long as 96 h. Since the reaction of ninhydrin with the residual nonammonia, nonprotein nitrogen was more than twofold stronger when the samples were hydrolyzed with 6 N HCl, it appeared that much of the residual nitrogen was from peptides. Approximately 66% of the nonammonia, nonprotein, ninhydrin-reactive material could not be recovered as amino acids, but there was a significant decrease in total amino acid nitrogen when the samples were pretreated with a C18 Sep-Pak column to remove peptides. The resistant peptides had an abundance of proline, and subsequent incubations showed that synthetic dipeptides which contained proline were hydrolyzed slowly. Lysine appears to be the amino acid which is most apt to limit ruminant production. Dipeptides containing proline and lysine were hydrolyzed at least fivefold slower than lysine-alanine. Methionine, another potentially limiting amino acid, was also degraded at a slower (2.5-fold) rate when it was present as part of a proline dipeptide.     

HeLa ribosomal protein S6. Insulin and dibutyryl cyclic AMP affect different phosphopeptides

Tryptic phosphopeptides from HeLa ribosomal protein S6 have been separated by chromatography, followed by electrophoresis. Three phosphopeptides were found in S6 from cells treated in vivo with dibutyryl cAMP and at least four different phosphopeptides were found from cells treated with insulin plus the essential amino acids. Only phosphoserine was found in S6 isolated from insulin-treated cells.     

Rational Molecular Design of Potent PLK1 PBD Domain‐binding Phosphopeptides Using Preferential Amino Acid Building Blocks

Polo‐like kinase 1 (PLK1) is an important regulator in diverse aspects of the cell cycle and proliferation. The protein has a highly conserved polo‐box domain (PBD) present in C‐terminal noncatalytic region, which exhibits a relatively broad sequence specificity in recognizing and binding phosphorylated substrates to control substrate phosphorylation by the kinase. In order to elucidate the structural basis, thermodynamic property, and biological implication underlying PBD‐substrate recognition and association, a systematic amino acid preference profile of phosphopeptide interaction with PLK1 PBD domain was established via virtual mutagenesis analysis and mutation energy calculation, from which the contribution of different amino acids at each residue position of two reference phosphopeptides to domain–peptide binding was characterized comprehensively and quantitatively. With the profile, we are able to determine the favorable, neutral, and unfavorable amino acid types for each position of PBD‐binding phosphopeptides, and we also explored the molecular origin of the broad sequence specificity in PBD‐substrate recognition. To practice computational findings, the profile was further employed to guide rational design of potent PBD binders; three 6‐mer phosphopeptides (i.e., IQSpSPC, LQSpTPF, and LNSpTPT) were successfully developed, which can efficiently target PBD domain with high affinity (K_d = 5.7 ± 1.1, 0.75 ± 0.18, and 7.2 ± 2.6 μm , resp.) as measured by a fluorescence anisotropy assay. The complex structure of PLK1 PBD domain with a newly designed, potent phosphopeptide LQSpTPF as well as diverse noncovalent chemical forces, such as H‐bonds and hydrophobic interactions at the complex interface, were examined in detail to reveal the molecular mechanism of high affinity and stability of the complex system.     

Changes in Amino Acid Content and the Metabolism of γ-Aminobutyrate in Cucurbita moschata Seedlings

Ungerminated pumpkin (Cucurbita moschata Poir.) cotyledons contained 30 % of their dry weight as lipid and 26 % as protein, of which 93 % was globulin. There was a rapid degradation of these reserves 4 to 8 days after planting when the cotyledons had their maximum metabolic activity. About half of the mole percent of amino acids found in the globulin reserve was in arginine, glutamate, aspartate, and their amides. The cotyledons had a large soluble pool of arginine, glutamine, glutamate, and leucine. Most amino acids increased steadily in amount in the cotyledons during germination, except glutamine, ornithine, alanine, serine, glycine, and γ-aminobutyrate and these appeared in large amounts in the translocation stream to the axis tissue. Little arginine or proline was translocated. By 10 days, when translocation had decreased, amino acids accumulated. Ornithine, γ-aminobutyrate, and aspartate were rapidly utilized in the hypocotyl, while glutamine, glycine, and alanine accumulated there. Cysteine and methionine levels were low in the reserve, trans-location stream and soluble fractions. γ-Aminobutyrate-U^?14C injected into cotyledons or incubated with hypocotyls was utilized in a similar fashion. The label appeared in citric acid cycle acids and in the amino acids closely related to this cycle, but the bulk of the label appeared in CO₂. The labeling pattern suggests that γ-aminobutyrate was utilized via succinate, and thus entered the citric acid cycle. A close relationship between arginine, ornithine, glutamate, and γ-aminobutyrate exists in the cotyledon with all but arginine being translocated rapidly to the axis tissue where these amino acids are rapidly metabolized.