A combined physiological and proteomic approach to reveal lactic-acid-induced alterations in <Emphasis Type="Italic">Lactobacillus casei</Emphasis> Zhang and its mutant with enhanced lactic acid tolerance

Lactobacillus casei has traditionally been recognized as a probiotic and frequently used as an adjunct culture in fermented dairy products, where acid stress is an environmental condition commonly encountered. In the present study, we carried out a comparative physiological and proteomic study to investigate lactic-acid-induced alterations in Lactobacillus casei Zhang (WT) and its acid-resistant mutant. Analysis of the physiological data showed that the mutant exhibited 33.8% higher glucose phosphoenolpyruvate:sugar phosphotransferase system activity and lower glycolytic pH compared with the WT under acidic conditions. In addition, significant differences were detected in both cells during acid stress between intracellular physiological state, including intracellular pH, H⁺-ATPase activity, and intracellular ATP pool. Comparison of the proteomic data based on 2D-DIGE and i-TRAQ indicated that acid stress invoked a global change in both strains. The mutant protected the cells against acid damage by regulating the expression of key proteins involved in cellular metabolism, DNA replication, RNA synthesis, translation, and some chaperones. Proteome results were validated by Lactobacillus casei displaying higher intracellular aspartate and arginine levels, and the survival at pH 3.3 was improved 1.36- and 2.10-fold by the addition of 50-mM aspartate and arginine, respectively. To our knowledge, this is the first demonstration that aspartate may be involved in acid tolerance in Lactobacillus casei. Results presented here may help us understand acid resistance mechanisms and help formulate new strategies to enhance the industrial applications of this species.     

An aromatic side chain is required at residue 8 of SU for fusion of ecotropic murine leukemia virus

The surface glycoprotein (SU) of most gammaretroviruses contains a conserved histidine at its amino terminus. In ecotropic murine leukemia virus SU, replacement of histidine 8 with arginine (H8R) or deletion of H8 (H8del) abolishes infection and cell-cell fusion but has no effect on binding to the cellular receptor. We report here that an aromatic ring side chain is essential to the function of residue 8. The size of the aromatic ring appears to be important, as does its ability to form a hydrogen bond. In addition, infection by all of the nonaromatic amino acid substitutions could be partially rescued by the addition of two suppressor mutations (glutamine 227 to arginine [Q227R] and aspartate 243 to tyrosine [D243Y]) or by exposure to chlorpromazine, an agent that induces fusion pores in hemifusion intermediates to complete fusion, suggesting that, like the previously described H8R mutant, the mutants reported here also arrest membrane fusion at the hemifusion state. We propose that H8 is a key switch-point residue in the conformation changes that lead to membrane fusion and present a possible mechanism for how its substitution arrests fusion at the hemifusion state.     

Myelin-associated Glycoprotein Interacts with Neurons via a Sialic Acid Binding Site at ARG118 and a Distinct Neurite Inhibition Site

Inhibitory components in myelin are largely responsible for the lack of regeneration in the mammalian CNS. Myelin-associated glycoprotein (MAG), a sialic acid binding protein and a component of myelin, is a potent inhibitor of neurite outgrowth from a variety of neurons both in vitro and in vivo. Here, we show that MAG's sialic acid binding site is distinct from its neurite inhibitory activity. Alone, sialic acid–dependent binding of MAG to neurons is insufficient to effect inhibition of axonal growth. Thus, while soluble MAG-Fc (MAG extracellular domain fused to Fc), a truncated form of MAG-Fc missing Ig-domains 4 and 5, MAG(d1-3)-Fc, and another sialic acid binding protein, sialoadhesin, each bind to neurons in a sialic acid– dependent manner, only full-length MAG-Fc inhibits neurite outgrowth. These results suggest that a second site must exist on MAG which elicits this response. Consistent with this model, mutation of arginine 118 (R118) in MAG to either alanine or aspartate abolishes its sialic acid–dependent binding. However, when expressed at the surface of either CHO or Schwann cells, R118-mutated MAG retains the ability to inhibit axonal outgrowth. Hence, MAG has two recognition sites for neurons, the sialic acid binding site at R118 and a distinct inhibition site which is absent from the first three Ig domains.     

Incorporation of nitrate nitrogen into amino acids during the anaerobic germination of rice

Summary Incorporation of¹⁵NO₃-into amino acids was studied during the anaerobic germination of rice seeds. In treated coleoptiles, the label was incorporated into glutamine, glutamate, alanine,-aminobutyric acid (Gaba), arginine, aspartate and methionine. These findings are consistent with a primary incorporation of nitrate nitrogen into glutamine, glutamate and aspartate, and their further conversion to alanine, Gaba, arginine and methionine.     

Selective modification of tyrosine and cysteine residues in aspartate aminotransferase from pig heart cytosol

In the region of the active site of aspartate amino-transferase two amino acid residues — one Tyr and one Cys — are accessible to selective modification by appropriate reagents. Modification of each of the two residues singly results in certain changes of the enzyme's physico-chemical properties, but does not abolish its ability to catalyse the transamination reaction. Complete inactivation, associated with irreversible amination of the protein-bound pyridoxal-P to pyridoxamine-P, is observed only on modification of both residues.     

Distribution and metabolism of xylem-borne ureido and amino compounds in developing soybean shoots

Pulse-chase feeding (30-120 minutes) of ¹⁴C-labeled nitrogenous compounds to cut transpiring shoots was used to investigate the early fate of the major xylem-borne solutes in N₂-fixing soybean (Glycine max) plants at the V₄ growth stage. By comparison with the foliar distribution of [¹⁴C]inulin (a xylem marker), it was determined that the phloem supply of allantoin, allantoic acid, asparagine, glutamine, aspartate, and arginine, respectively, provided about 20, 10, three, two, five, and 20 times the ¹⁴C delivered to the developing trifoliolate in the xylem stream. Recovery of unmetabolized asparagine, aspartate, and arginine in this indicator trifoliolate, and significant declines in the percentage of ¹⁴C from allantoic acid and allantoin recovered in the first trifoliolate, provided some support for the direct xylem-to-phloem transfer of these compounds, but did not preclude the involvement of indirect transfer. Data on stem retention and foliar distribution, expressed as a function of the relative xylem sap composition, indicated that ureides provide the major sources of nitrogen to all plant parts. There was no consistent distinction in distribution patterns between pairs of similar anionic and neutral compounds. The extent of xylem-to-phloem transfer among the ureido or the amino compounds was inversely related to its prominence in xylem sap.     

Enhanced acid tolerance in <Emphasis Type="Italic">Lactobacillus casei</Emphasis> by adaptive evolution and compared stress response during acid stress

This study aimed to improve the acid tolerance of Lactobacillus casei Zhang and compare the stress response of the parental strain and the acid-resistant mutant during acidic conditions. Adaptive evolution was conducted for 70 days to generate acid-tolerant L. casei. The evolved mutant lb-2 exhibited more than a 60% increase in biomass as well as a 13.6 and 65.6% increase in concentrations of lactate and acetate, respectively, when cultured at pH 4.3 for 64 h. Lactic acid tolerances of the parental strain and the evolved mutant were determined. As a result, the evolved mutant showed a 318-fold higher survival rate than that of the parental strain. Physiological analysis showed that the evolved mutant exhibited higher intracellular pH (pH_i), NH₄ ⁺ concentration and lower inner membrane permeability than that of the parental strain during acid stress. Moreover, higher amounts of intracellular arginine and aspartate were also detected in lb-2 under acid stress. Validation of the relationship between the acid tolerance and the intracellular arginine and aspartate accumulation was conducted by experiments that showed the survival of L. casei at pH 3.3 was improved 1.36-, 2.10-, or 3.42-fold by the addition of 50 mM aspartate, arginine or both of them, respectively. Taken together, results presented here not only supply an effective way to select acid-resistant strains for the food industry, but also contribute to reveal the mechanisms of acid tolerance and provide new strategies to enhance the industrial utility and health-promoting properties of this species.     

Modification of ferredoxin-NADP+ reductase from the alga Bumilleriopsis with butanedione and dansyl chloride

Modification of ferredoxin-NADP⁺ reductase from the alga Bumilleriopsis with butanedione (diacetyl) and dansyl chloride results in loss of enzymatic activity. Under pseudo-first order conditions the rate of inactivation by butanedione is directly proportional to the concentration of the modifying reagent with a slope of unity. The protective effect of pyridine nucleotides, as well as their analogs against inactivation by butanedione indicates involvement of arginine in the binding of pyridine nucleotides at the active site. Inactivation by dansyl chloride suggests that a further amino acid is involved, possibly lysine. Amino acid analyses of the butanedione-treated reductase show that the degree of inactivation correlates well with the decrease in arginine. Furthermore, two arginine residues are modified concomitant with complete inactivation of the enzyme, although this does not imply that both residues participate necessarily in the binding of pyridine nucleotides. Fingerprint analysis of the carboxymethylated, trypsin-digested enzyme indicates loss of one arginine-containing peptide when the protein had been modified by butanedione. There was no change in cysteine-containing peptides.     

A yeast arginine specific tRNA is a remnant aspartate acceptor

High specificity in aminoacylation of transfer RNAs (tRNAs) with the help of their cognate aminoacyl-tRNA synthetases (aaRSs) is a guarantee for accurate genetic translation. Structural and mechanistic peculiarities between the different tRNA/aaRS couples, suggest that aminoacylation systems are unrelated. However, occurrence of tRNA mischarging by non-cognate aaRSs reflects the relationship between such systems. In Saccharomyces cerevisiae, functional links between arginylation and aspartylation systems have been reported. In particular, it was found that an in vitro transcribed tRNA^Asp is a very efficient substrate for ArgRS. In this study, the relationship of arginine and aspartate systems is further explored, based on the discovery of a fourth isoacceptor in the yeast genome, tRNA₄^Arg. This tRNA has a sequence strikingly similar to that of tRNA^Asp but distinct from those of the other three arginine isoacceptors. After transplantation of the full set of aspartate identity elements into the four arginine isoacceptors, tRNA₄^Arg gains the highest aspartylation efficiency. Moreover, it is possible to convert tRNA₄^Arg into an aspartate acceptor, as efficient as tRNA^Asp, by only two point mutations, C38 and G73, despite the absence of the major anticodon aspartate identity elements. Thus, cryptic aspartate identity elements are embedded within tRNA₄^Arg. The latent aspartate acceptor capacity in a contemporary tRNA^Arg leads to the proposal of an evolutionary link between tRNA₄^Arg and tRNA^Asp genes.     

Cyanophycin granule polypeptide protease in a unicellular cyanobacterium

An assay was developed to measure the proteolysis of cyanophycin granule polypeptide in crude extracts of a unicellular cyanobacterium. The substrate was radioactively labeled cyanophycin granule polypeptide formed by an unicellular cyanobacterium grown in the presence of chloramphenicol. Substrate polypeptide displayed identical chemical properties with polypeptide isolated from non-chloramphenicol-treated cells. Solubilization of radioactivity as arginine indicated hydrolysis of polypeptide. Radioactively labeled aspartate and arginine from hydrolyzed polypeptide was related to nmol amino acid using a combination of paper chromatography, liquid scintillation analysis, and ninhydrin quantitation. Protease activity was found in extracts of nitrogen-limited cells harvested 16–24 h after a nitrogen source was added back. Optimal pH for protease activity was 8.0 and optimum temperature was 35°C. Protease activity in crude extracts followed Michaelis-Menten kinetics with a V _max of 92 nmol arginine per 15 min/mg protein and a K _m of 2.1×10³ nmol arginine. Protease activity was inhibited by arginine and by high concentrations of aspartate.     

Solubility of hemoglobin S by sedminetation, equilibrium, and antisickling compounds.

The effect of the compounds guanidine, arginine, lysine, and aspartic acid and the salt arginine aspartate on the solubility of deoxyhemoglobin S (Hb S) was studied by sedimentation equilibrium at 20–22 °C. Guanidine and arginine were found to be most effective, whereas aspartic acid and lysine had only a small effect. The effectiveness of these compounds in solubilizing Hb S is relatively pH independent. It is unlikely that the small effect of lysine and aspartic acid on the solubility of Hb S can account for the antisickling properties of lysine and aspartic acid previously reported (Sophianopoulos, A. J., et al. (1974) Clin. Biochem.7, 112–118). The effect of guanidine and arginine is large enough to account for a large part of such antisickling properties (Sophianopoulos et al. (1974). The nonideality of concentrated hemoglobin solutions (up to 0.3 g cm^?3) has been studied in detail. By using the liganded as well as the unliganded forms of both Hb S and Hb A, it was found that the magnitude of the virial (nonideality) coefficients can change with varying solution conditions. A comparison of pure Hb S with hemolysates is made using viscosity and sedimentation velocity.     

Non-Decarboxylating transformation of indol-3-ylacetic acid in apple seeds

An enzyme extract from apple(Pyrus malus Borb.) seeds which causes the disappearance of free indol-3-ylacetic acid (IAA) requires the presence of oxygen, but is not inhibited by cyanide. Using 1-¹⁴C-IAA it has been demonstrated that the IAA transformation is not accompanied by its decarboxylation. Decarboxylating IAA oxidase is absent during the whole period of apple seed cold stratification. Free IAA has not been detected in dormant apple seeds and in seeds stratified at low temperature. It appears during stratification at 25 °C. Ethyl ester of IAA and indol-3-ylacetyl aspartate have been identified in dormant and after-ripened seeds. Exogenous 1-¹⁴C IAA taken up by apple embryos is converted into conjugates with aspartate and short peptides containing an aspartate moiety.     

The incorporation of acetate by the chemoautotroph Thiobacillus neapolitanus strain C

Summary Cultures of Thiobacillus neapolitanus strain C assimilate ¹⁴C-labelled acetate and aspartate. Both carbon atoms of acetate are incorporated, and 25% of the cell carbon can arise from acetate. Aspartate-¹⁴C contributes 4–5% of the cell carbon, and is found in pyrimidines and in protein as aspartate and its related amino acids. Acetate-¹⁴C contributes to lipid, glutamate, arginine, proline and leucine, but not to aspartate. Acetate assimilation by washed organisms requires carbon dioxide and energy from thiosulphate oxidation. Degradation of ¹⁴C-glutamic acid from acetate-¹⁴C-labelled bacteria; the accumulation of ¹⁴C-citrate in the presence of fluoroacetate and [¹⁴C] acetate; short-term kinetic experiments on acetate-¹⁴C turnover; and the demonstration of citrate synthesis by cell-free extracts all indicate glutamate synthesis from -ketoglutarate formed by reactions of the tricarboxylic acid cycle. The cycle is believed to be incomplete, probably not proceeding further than -ketoglutarate, and functions as a glutamate-synthesising system, using oxaloacetate derived solely from carbon dioxide fixation. Malate synthase (and the glyoxylate cycle) appear to be insignificant in the metabolism, but extracts did form citramalate from acetate and pyruvate.     

Transport of arginine and aspartic Acid into isolated barley mesophyll vacuoles

The transport of arginine into isolated barley (Hordeum vulgare L.) mesophyll vacuoles was investigated. In the absence of ATP, arginine uptake was saturable with a K_m of 0.3 to 0.4 millimolar. Positively charged amino acids inhibited arginine uptake, lysine being most potent with a K_i of 1.2 millimolar. In the presence of free ATP, but not of its Mg-complex, uptake of arginine was drastically enhanced and a linear function of its concentration up to 16 millimolar. The nonhydrolyzable adenylyl imidodiphosphate, but no other nucleotide tested, could substitute for ATP. Therefore, it is suggested that this process does not require energy and does not involve the tonoplast ATPase. The ATP-dependent arginine uptake was strongly inhibited by p-chloromercuriphenylsulfonic acid. Furthermore, hydrophobic amino acids were inhibitory (I₅₀ phenylalanine 1 millimolar). Similar characteristics were observed for the uptake of aspartic acid. However, rates of ATP-stimulated aspartic acid transport were 10-fold lower as compared to arginine transport. Uptake of aspartate in the absence of ATP was negligible.     

A Conserved Asparagine in a P-type Proton Pump Is Required for Efficient Gating of Protons

The minimal proton pumping machinery of the Arabidopsis thaliana P-type plasma membrane H⁺-ATPase isoform 2 (AHA2) consists of an aspartate residue serving as key proton donor/acceptor (Asp-684) and an arginine residue controlling the pK_a of the aspartate. However, other important aspects of the proton transport mechanism such as gating, and the ability to occlude protons, are still unclear. An asparagine residue (Asn-106) in transmembrane segment 2 of AHA2 is conserved in all P-type plasma membrane H⁺-ATPases. In the crystal structure of the plant plasma membrane H⁺-ATPase, this residue is located in the putative ligand entrance pathway, in close proximity to the central proton donor/acceptor Asp-684. Substitution of Asn-106 resulted in mutant enzymes with significantly reduced ability to transport protons against a membrane potential. Sensitivity toward orthovanadate was increased when Asn-106 was substituted with an aspartate residue, but decreased in mutants with alanine, lysine, glutamine, or threonine replacement of Asn-106. The apparent proton affinity was decreased for all mutants, most likely due to a perturbation of the local environment of Asp-684. Altogether, our results demonstrate that Asn-106 is important for closure of the proton entrance pathway prior to proton translocation across the membrane.     

Chemical modification and amino terminal sequence of calotropin DI from Calotropis gigantea

The effect of chemical modification of histidine, lysine, arginine, tryptophan and methionine residues on the enzymatic activity of calotropin DI has been studied. 1,3-Dibromoacetone inhibited the enzyme completely, indicating that a single histidine residue and a cysteine residue are involved in its catalytic activity. Its second bistidine residue was modified with diethyl pyrocarbonate without loss of activity. Modification of seven of its 13 lysine residues with 2,4,6-trinitrobenzene sulphonic acid led to 90% loss of its activity, but no single lysine residue appears to be essential for its activity. Four of the 12 arginine residues by 1,2-cyclohexanedione can be modified with little loss of activity. Modification of a single tryptophan residue and two methionine residues did not inhibit enzymatic activity. The blocked amino-terminal amino acid residue of calotropin DI has been identified as pyroglutamic acid. Its amino-terminal amino acid sequence to residue 14 has been determined and compared with that of papain. They show an extensive homology in their amino-terminal amino acid sequences.     

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.     

Aspartate Carbamyltransferase : Site of End-Product Inhibition of the Orotate Pathway in Intact Cells of Cucurbita pepo

Lovatt et al. (1979 Plant Physiol 64: 562-569) have previously demonstrated that end-product inhibition functions as a mechanism regulating the activity of the orotic acid pathway in intact cells of roots excised from 2-day-old squash plants (Cucurbita pepo L. cv Early Prolific Straightneck). Uridine (0.5 millimolar final concentration) or one of its metabolites inhibited the incorporation of NaH¹⁴CO₃, but not [¹⁴C]carbamylaspartate or [¹⁴C]orotic acid, into uridine nucleotides (ΣUMP). Thus, regulation of de novo pyrimidine biosynthesis was demonstrated to occur at one or both of the first two reactions of the orotic acid pathway, those catalyzed by carbamylphosphate synthetase (CPSase) and aspartate carbamyltransferase (ACTase). The results of the present study provide evidence that ACTase alone is the site of feedback control by added uridine or one of its metabolites. Evidence demonstrating regulation of the orotic acid pathway by end-product inhibition at ACTase, but not at CPSase, includes the following observations: (a) addition of uridine (0.5 millimolar final concentration) inhibited the incorporation of NaH¹⁴CO₃ into ΣUMP by 80% but did not inhibit the incorporation of NaH¹⁴CO₃ into arginine; (b) inhibition of the orotate pathway by added uridine was not reversed by supplying exogenous ornithine (5 millimolar final concentration), while the incorporation of NaH¹⁴CO₃ into arginine was stimulated more than 15-fold when both uridine and ornithine were added; (c) incorporation of NaH¹⁴CO₃ into arginine increased, with or without added ornithine when the de novo pyrimidine pathway was inhibited by added uridine; and (d) in assays employing cell-free extracts prepared from 2-day-old squash roots, the activity of ACTase, but not CPSase, was inhibited by added pyrimidine nucleotides.     

Analysis of energetic amino acid metabolism in Acyrthosiphon pisum: A multidimensional approach to amino acid metabolism in aphids

Aphids are highly specialized insects that feed on the phloem-sap of plants, the amino acid composition of which is very unbalanced. Amino acid metabolism is thus crucial in aphids, and we describe a novel investigation method based on the use of ¹⁴C-labeled amino acids added in an artificial diet. A metabolism cage for aphids was constructed, allowing for the collection and analysis of the radioactivity incorporated into the aphid body, expired as CO₂, and rejected in the honeydew and exuviae. This method was applied to the study of the metabolism of eight energetic amino acids (aspartate, glutamate, glutamine, glycine, serine, alanine, proline, and threonine) in the pea aphid, Acyrthosiphon pisum. All these amino acids except threonine were subject to substantial catabolism as measured by high ¹⁴CO₂ production. The highest turnover was displayed by aspartate, with 60% of its carbons expired as CO₂. For the first time in an aphid, we directly demonstrated the synthesis of three essential amino acids (threonine, isoleucine, and lysine) from carbons of common amino acids. The synthesis of these three compounds was only observed from amino acids that were previously converted into glutamate. This conversion was important for aspartate, and lower for alanine and proline. To explain the quantitative results of interconversion between amino acids, we propose a compartmentation model with the intervention of bacterial endosymbiotes for the synthesis of essential amino acids and with glutamate as the only amino acid supplied by the insect to the symbiotes. Moreover, proline exhibited partial conversion into arginine, and it is suggested that proline is probably indirectly involved in excretory nitrogen metabolism. © 1995 Wiley-Liss, Inc.