Amino acid residues important for substrate specificity of the amino acid permeases Can1p and Gnp1p in Saccharomyces cerevisiae.

Deletion of the general amino acid permease gene GAP1 abolishes uptake of L-citrulline in Saccharomyces cerevisiae, resulting in the inability to grow on L-citrulline as sole nitrogen source. Selection for suppressor mutants that restored growth on L-citrulline led to isolation of 21 mutations in the arginine permease gene CAN1. One similar mutation was found in the glutamine-asparagine permease gene GNP1. L-[(14)C]citrulline uptake measurements confirmed that suppressor mutations in CAN1 conferred uptake of this amino acid, while none of the mutant permeases had lost the ability to transport L-[(14)C]arginine. Substrate specificity seemed to remain narrow in most cases, and broad substrate specificity was only observed in the cases where mutations affect two proline residues (P148 and P313) that are both conserved in the amino acid-polyamine-choline (APC) transporter superfamily. We found mutations affecting six predicted domains (helices III and X, and loops 1, 2, 6 and 7) of the permeases. Helix III and loop 7 are candidates for domains in direct contact with thetransported amino acid. Helix III was affected in both CAN1 (Y173H, Y173D) and GNP1 (W239C) mutants and has previously been found to be important for substrate preference in other members of the family. Furthermore, the mutations affecting loop 7 (residue T354, S355, Y356) are close to a glutamate side chain (E367) potentially interacting with the positively charged substrate, a notion supported by conservation of the side chain in permeases for cationic substrates.     

Inhibitory effect of arginine on proline utilization in Saccharomyces cerevisiae

Proline is a predominant amino acid in grape must, but it is poorly utilized by the yeast Saccharomyces cerevisiae in wine-making processes. This sometimes leads to a nitrogen deficiency during fermentation and proline accumulation in wine. Although the presence of other nitrogen sources under fermentation conditions is likely to interfere with proline utilization, the inhibitory mechanisms of proline utilization remain unclear. In this study, we examined the effect of arginine on proline utilization in S. cerevisiae. We first constructed a proline auxotrophic yeast strain and identified an inhibitory factor by observing the growth of cells when proline was present as a sole nitrogen source. Intriguingly, we found that arginine, and not ammonium ion, clearly inhibited the growth of proline auxotrophic cells. In addition, arginine prevented the proline consumption of wild-type and proline auxotrophic cells, indicating that arginine is an inhibitory factor of proline utilization in yeast. Next, quantitative polymerase chain reaction (PCR) analysis showed that arginine partially repressed the expression of genes involved in proline degradation and uptake. We then observed that arginine induced the endocytosis of the proline transporters Put4 and Gap1, whereas ammonium induced the endocytosis of only Gap1. Hence, our results may involve an important mechanism for arginine-mediated inhibition of proline utilization in yeast. The breeding of yeast that utilizes proline efficiently could be promising for the improvement of wine quality.     

Construction of phosphatidylethanolamine-less strain of Saccharomyces cerevisiae. Effect on amino acid transport

A triple yeast mutant was constructed which lacks BST1, the gene for sphingosine lyase, besides the phosphatidylserine decarboxylases PSD1 and PSD2. In this yeast mutant, which can only be grown in the presence of exogenous ethanolamine, phosphatidylethanolamine can be depleted to very low levels. Under those conditions, respiration as well as glucose and 3-O-methylglucose uptake proceed unaffected. Plasma membrane ATPase is as active in these cells as that of control cells grown in the presence of ethanolamine. Drastically decreased, however, are H+/amino acid symporters. The activities of arginine (Can1p), proline (Put4p) and general amino acid permease (Gap1p) are decreased more than 20-fold. Amino acid transport in yeast is dependent on coupling to the proton motive force. It can be envisaged that phosphatidylethanolamine might play a role in this process or in the early steps of the secretion pathway common for all amino acid permeases or, eventually, it could affect the transport proteins directly at the plasma membrane Transformation of the triple mutant with a CEN plasmid harbouring BST1 wild-type gene totally reversed its phenotype to that observed in the double mutant.     

Regulation of the amino acid permeases in nitrogen-limited continuous cultures of the yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, there is a general amino acid permease, regulated by nitrogen catabolite repression, and several specific permeases whose nitrogen regulation is not well understood. In this study, we used continuous cultures to analyse the effect of nitrogen limitation and pH on the activity of general and several specific amino acid permeases. General permease activity was maximal in severe nitrogen limitation and diminished 400-fold in cells grown under nitrogen excess. For the specific permeases, the maximal uptake activity was found between mild limitation and nitrogen excess, while very small activity was detected under strict limitation. These results indicate that the nitrogen regulation of the general and the specific amino acid carriers is coordinated in such a way that no redundancy exists in amino acid transport. The regulation of the specific permeases was similar to that found for a system with anabolic function in nitrogen metabolism. All of these permeases are supposed to work through a proton symport mechanism, and thus rely on pH gradients to carry out their function. We studied the effect of pH on the kinetic constants of the general permease. Our results show that the effect of pH on the K_m was different for acidic, neutral and basic amino acids, while the effect on V_max was independent of the electrical charge of the amino acids.     

The response of proline metabolism to nitrogen deficiency in pods and seeds of French bean (Phaseolus vulgaris L cv Strike) plants

The objective of the present work was to determine the impact of nitrogen deficiency on proline metabolism in order to use this amino acid as a bioindicator of the N status of the pods and seeds of French bean (Phaseolus vulgaris L cv Strike) plants. We also identify the pathway of proline synthesis which is favoured under our experimental conditions. N was applied to the nutrient solution in the form of NH₄NO₃ at 1.45 mM (N1), 2.90 mM (N2) and 5.80 mM (N3, optimal level). Our results indicate that N deficiency is characterised by a decline in proline accumulation in both the pod and the seed, fundamentally because proline degradation is encouraged by stimulation of the enzyme proline dehydrogenase under these conditions. However, although the enzymes in charge of proline biosynthesis (ornithine‐δ‐aminotransferase and Δ¹‐pyrroline‐5‐carboxylate synthetase) vary in behaviour depending on the N status, this amino acid appeared to be synthesised mainly by the enzyme ornithine‐δ‐aminotransferase, suggesting predominance of the ornithine pathway over the glutamine pathway. Finally, under our experimental conditions, proline can be regarded as a good indicator of N deficiency, particularly in the seeds of French bean plants. © 2001 Society of Chemical Industry     

Effect of proline on arginine uptake and nitrogen metabolism of lactating goats

The hypothesis that addition of proline may save dietary protein for milk production was tested by administration of proline to two goats through a duodenal cannula. Because proline is synthesized by the mammary gland in vitro from arginine, the effect of proline supplementation on arginine uptake by the gland was tested in vivo. Arginine uptake, calculated from arterio-venous difference, dropped significantly in both animals, especially in the morning 1 h after milking, when a low-protein diet was fed. Milk production and total nitrogen in milk were not affected significantly by proline supplementation. A trend was toward decrease of milk orotic acid and an increase of milk fat due to proline supplementation.     

The use of proline as a nitrogen source causes hypersensitivity to, and allows more economical use of 5FOA in Saccharomyces cerevisiae.

The use of proline as a nitrogen source causes hypersensitivity to 5-fluoro-orotic acid (5FOA) and allows up to 40-fold less of this drug to be used to select for the loss of URA3 function in Saccharomyces cerevisiae. 5FOA hypersensitivity is presumably due to the absence of nitrogen catabolite repression when proline is substituted for (NH4)2SO4 as a nitrogen source. There are two constraints to the use of the proline-5FOA combination: (1) S288c genetic background strains are hypersensitive to 5FOA when grown in proline as a nitrogen source but at least one other genetic background is resistant to low levels of 5FOA under these conditions. (2) The addition of some nutritional supplements confers phenotypic resistance to the 5FOA-proline combination.     

Effect of proline and arginine metabolism on freezing stress of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the PUT1-encoded proline oxidase and the PUT2-encoded delta1-pyrroline-5-carboxylate dehydrogenase are required to convert proline to glutamate. We recently showed that a put1 disruptant accumulated higher levels of proline intracellularly and conferred higher resistance to freezing stress. Here, we determined the effect of put2 disruption on yeast cell viability under freezing stress. When grown on arginine as the sole nitrogen source, the put2 disruptant showed a significant decrease in cell viability after freezing despite the high proline and arginine contents. This result suggests that delta1-pyrroline-5-carboxylate or glutamate-gamma-semialdehyde, a proline catabolism intermediate, is toxic to yeast cells under freezing stress. In contrast, the survival rate of the wild-type and the put1-disruptant strains was found to increase after freezing in proportion to their arginine contents. This indicates that arginine has a cryoprotective function in yeast. Furthermore, the yeast cells accumulated proline as well as arginine in the vacuole, suggesting that there is a system for the transport of excess proline to the vacuole and that this vacuolar accumulation may be important in the freezing resistance of yeast cells.     

Degradation of N‐(1‐Deoxy‐d‐xylulos‐1‐yl)glycine and N‐(1‐Deoxy‐d‐xylulos‐1‐yl)proline using thermal treatment

The formation and degradation of N‐(1‐Deoxy‐d ‐xylulos‐1‐yl)glycine and N‐(1‐Deoxy‐d ‐xylulos‐1‐yl)proline, derived from the secondary amine Maillard reaction in xylose‐amino acid model solutions, were detailed in this study. The identification and quantitative analysis of N‐(1‐Deoxy‐d‐ xylulos‐1‐yl)glycine and N‐(1‐Deoxy‐d‐ xylulos‐1‐yl)proline were carried out using high‐performance anion‐exchange chromatography and high‐performance liquid chromatography. The formation of intermediate and advanced products derived from N‐(1‐Deoxy‐d‐ xylulos‐1‐yl)glycine and N‐(1‐Deoxy‐d‐ xylulos‐1‐yl)proline was also tested using an UV‐Vis spectrophotometer to gain a better comparing of the degradation process of the two important Maillard reaction products using thermal treatment. Results showed that the degradation of N‐(1‐Deoxy‐d‐ xylulos‐1‐yl)glycine was more significant than N‐(1‐Deoxy‐d‐ xylulos‐1‐yl)proline. Moreover, xylose was tested in the degradation products of both N‐(1‐Deoxy‐d‐ xylulos‐1‐yl)glycine and N‐(1‐Deoxy‐d‐ xylulos‐1‐yl)proline, which indicated that the degradation of N‐substituted 1‐amino‐1‐deoxyketoses was a reversible reaction to form reducing sugar.     

Methods for extracting the taste compounds from water soluble extract of Jinhua ham

The present paper reports the determination of 22 amino acids and ammonium along the acetification process by high-performance liquid chromatography (HPLC), employing 6-aminoquinolyl-N-hydrosysuccinimidyl carbamate (AQC) as precolumn derivatization reagent. The method was successfully validated obtaining adequate values to selectivity, response linearity, precision and accuracy, as well as low detection and quantification limits. Its utility for routine analysis of amino acids along acetification has been proved. Three red wine substrates were fermented employing two kinds of acetification methods (surfaced and submerged culture) and the requirements of amino acids and ammonium as nitrogen source by acetic acid bacteria were evaluated. The identification of the acetic acid bacteria at species level was also done. At the beginning, the principal amino acid, in terms of abundance, was proline followed by arginine. A different behavior between both the acetification methods was observed. First of all, the consumption of amino acids is much lower in submerged than in surface acetifications. For surface culture acetification, the most consumed was proline, arginine being the main nitrogen source for submerged acetification systems. On the other hand, it was also observed that the nitrogen requirement of the bacteria is proportional to the time spent in the acetification process.     

Relative reactivity of amino acids with chlorine in mixtures

The relative reactivity of chlorine with amino acids is an important determinant of the resulting chlorination products in systems where chlorine is the limiting reagent, for example, in the human gastrointestinal tract after consumption of chlorine-containing water, or during food preparation with chlorinated water. Since few direct determinations of the initial reactivity of chlorine with amino acids have been made, 17 amino acids were compared in this study using competitive kinetic principles. The experimental results showed that (1) most amino acids have similar initial reactivities at neutral pH; (2) amino acids with thiol groups such as methionine and cysteine are exceptionally reactive and produce sulfoxides; (3) amino acids without thiol groups primarily undergo monochlorination of the amino nitrogen; and (4) glycine and proline are the least reactive. Dichlorination was estimated to occur with approximately 26% of the amino acid groups when the total amino acid: chlorine concentrations were equal.     

Nitrogen starvation induces expression of Lg‐FLO1 and flocculation in bottom‐fermenting yeast

When exponentially growing cells of bottom‐fermenting yeast were starved for nitrogen or were grown on proline (a non‐preferred nitrogen source), flocculation was induced. This flocculation was not induced by starvation for either carbon or amino acids. Expression of Lg‐FLO1, which is required for flocculation of bottom‐fermenting yeast, was also found to be induced by starvation for nitrogen. This suggests that the flocculation of bottom‐fermenting yeast is under the control of a nitrogen catabolite repression (NCR)‐like mechanism. Copyright © 2012 John Wiley & Sons, Ltd.     

Characterization of the sec1-1 and sec1-11 mutations.

Sec1 proteins are implicated in positive and negative regulation of SNARE complex formation. To better understand the function of Sec1 proteins we have identified the nature of the temperature-sensitive mutations in sec1-1 and sec1-11. The sec1-1 mutation changes a conserved glycine(443) to glutamic acid. The sec1-11 mutation changes a highly conserved arginine(432) to proline. Based on homology and the crystal structure of the mammalian nSec1p, the corresponding amino acids localize to the 3b domain of nSec1p. Compared to the wild-type Sec1p the mutant proteins are less abundant even at the permissive temperature. Thus, the R432P and G443E mutations may cause structural alterations that affect folding and make the mutant proteins more susceptible to degradation. The remaining part is sufficient for growth and protein secretion at 24 degrees C and thus is likely to be properly folded. At 37 degrees C the mutant proteins become non-functional. In pulse-chase-type experiments the newly synthesized Sec1-1 and Sec1-11 proteins decayed similarly with the wild-type protein. Thus, the non-functionality of the mutant proteins cannot be explained by denaturation-induced degradation only. It is possible that the newly synthesized mutant proteins fold slowly and are susceptible to degradation before they have managed to fold and associate with other proteins. The mutant proteins were unable to interact with the Sec1p-interacting proteins Mso1p and Sso2p in the two-hybrid assay, even at the permissive temperature. These results localize sec1-1 and sec1-11 mutations to a domain of Sec1p and suggest a mechanism by which sec1-1 and sec1-11 cells become temperature-sensitive.     

High-performance liquid chromatography determination of proline isomers in Italian wines

This paper deals with the quantitative reverse-phase high-performance liquid chromatography (RP-HPLC) analysis of the isomers of proline, the most abundant amino acid in wine, and with the relative isomeric presence in samples of different vintage and geographical origin. The sample preparation was carried out by using Marfey's reagent ((-fluoro-2,4-dinitrophenyl)-5-L-alanine amide (FDAA)) for the derivatisation reaction in order to obtain dl and ll diastereomers of the corresponding optically active amino acids. The separation was performed with an analytical column for RP-HPLC Nucleosil 100-5 C18 (25 × 0.4 cm id), adopting both isocratic and gradient procedures and making use of water/acetonitrile buffers as the mobile phase. It has been observed that the best resolution is achieved under gradient conditions, although the analysis takes a long time. L-Norleucine was used as the internal standard for quantitative determination and the detection of components was made at the fixed wavelength of 340 nm. Peak identifications were performed by comparing retention times and observing on-line UV spectra. Under these conditions the detection limit for proline isomers reached the value of 0–5 mg liter^?1. Wine samples of different ages were analysed and it is possible to see that L-proline is present only in ‘young’ wines, while the D-isomer appears in wines that are at least 5 years old. The most abundant D-proline content was observed in a 30 years vintage red wine of Puglie (southern Italy) with about 15% of the ratio D to D + L.     

氨基酸组成对胶原与整合素α2β1结合能力的影响

以不同物种来源的胶原作为研究对象，测定各胶原的氨基酸组成，利用酶联免疫吸附测定实验和细胞黏附实验研究各胶原样本与整合素α2β1及HT1080细胞的结合能力，探讨氨基酸组成对胶原配体-细胞受体α2β1结合能力的影响。结果表明，不同来源的胶原样本在与整合素α2β1和HT1080细胞结合能力方面均存在显著差异，其中，哺乳动物胶原的结合能力明显大于鱼类胶原。氨基酸组成对胶原-整合素α2β1及HT1080细胞结合能力的影响具有相似性，其结合能力均与羟脯氨酸、不带电荷极性氨基酸、亚氨基酸含量和羟基化率呈正相关，而与谷氨酸、甘氨酸、带电荷极性氨基酸和酸性氨基酸含量呈负相关（P<0.01）。同时对胶原与整合素α2β1高亲和力结合位点的信息氨基酸含量进行多元逐步回归分析，建立了羟脯氨酸和精氨酸含量与胶原-整合素α2β1结合能力之间的数学模型，经验证，该数学模型具有较好的预测准确性。     

Determination of endogenous amino acid flow at the terminal ileum of the rat

In a comparative study endogenous amino acid flow was determined in twenty-four 190-g male rats (protein-replete) given diets containing synthetic amino acids as the sole nitrogen source but devoid of specific amino acids, and six rats fed a protein-free diet. Endogenous flows were not significantly different (P > 0.05) for aspartic acid, glutamic acid, serine and lysine but were significantly higher (P < 0.05) under protein-free alimentation than amino acid alimentation for proline, glycine and alanine. This indicates that the protein-free method did not lead to lowered endogenous amino acid excretions owing to an altered amino acid metabolism in the protein-free state. The loss of some amino acids may be enhanced under protein-free alimentation. The high apparent digestibility of the synthetic amino acids not excluded from the diets (histidine, arginine, threonine, valine, isoleucine, leucine, tyrosine, phenylalanine) indicated that their ileal excretions were mainly of endogenous origin. There were no significant differences (P>0.05) between the latter excretions and those obtained by feeding the rats a protein-free diet.     

Evaluation of the potentials of millet,sorghum and barley with similar nitrogen contents malted at their optimum germination temperatures for use in brewing

Studies on the malting physiology of barley have led to similar studies on millet and sorghum. This study compares the outcomes of the malting physiology of millet, sorghum and barley. Results show that optimal development of diastatic power, soluble nitrogen, hot water extract and the wide range of amino acids of these three cereals is related to optimal malting conditions and appropriate mashing procedures. Transfer of the nitrogen/extract/soluble nitrogen/diastatic concepts of barley malt do not apply to millet and sorghum. However, all the cereals studied produced the range of amino acids required by yeast for fermentation. Sorghum malt released the highest amounts of group 1 amino acids, usually taken up faster by yeast. It also produced and released the highest amounts of amino acids, classified as group 2, which are assimilated more slowly than group 1 amino acids. It also produced and released more of the amino acids that are slowest to be assimilated during fermentation, as well as very high levels of proline. Optic barley malt produced and released the least amount of proline. The fate of proline during yeast fermentation is not clear, but it is believed that proline is not utilized during fermentation. Copyright © 2013 The Institute of Brewing & Distilling     

Contribution of Lactococcus lactis subsp lactis IFPL 359 and Lactobacillus casei subsp casei IFPL 731 to the Proteolysis of Caprine Curd Slurries

ABSTRACT: Proteolysis was studied in low-fat caprine curd slurries pre-incubated (24 h at 30 °C) with an extra dose of rennet, 10.0 or 22.0 μg g^-1 (LR and HR slurries, respectively), and treated at 70 °C for 15 min prior to incubation with sonicated cells of Lactococcus lactis subsp lactis IFPL 359 alone or combined with Lactobacillus casei subsp casei IFPL 731. The addition of lactobacilli to slurries containing lactococci increased the conversion of water-soluble nitrogen to nonprotein nitrogen and amine nitrogen. Major differences were found for some free amino acids, such as glutamic acid and proline, and hydrolysis of most hydrophobic peptides produced by lactococci, which are associated with bitterness in cheese.