Circadian Rhythm in Amino Acid Uptake by Synechococcus RF-1

In the prokaryote Synechococcus RF-1, circadian changes in the uptake of l-leucine and 2-amino isobutyric acid were observed. Uptake rates in the light period were higher than in the dark period for cultures entrained by 12/12 hour light/dark cycles. The periodic changes in l-leucine uptake persisted for at least 72 hours into continuous light (L/L). The rhythm had a free-running period of about 24 hours in L/L at 29°C. A single dark treatment of 12 hours could initiate rhythmic leucine uptake in an L/L culture. The phase of rhythm could be shifted by a pulse of low temperature (0°C). The free-running periodicity was “temperature-compensated” from 21 to 37°C. A 24 hour depletion of extracellular Ca²⁺ before the free-running L/L condition reduced the variation in uptake rate but had little effect on the periodicity of the rhythm. The periodicity was also not affected by the introduction of 25 mm NaNO₃. The uptake rates for 20 natural amino acids were studied at 12 hour intervals in cultures exposed to 12/12 hour light/dark cycles. For eight of these amino acids (l-Val, l-Leu, l-Ile, l-Pro, l-Phe, l-Trp, l-Met, and l-Tyr), the light/dark uptake rate ratios had values greater than 3 and the rhythm persisted in L/L.     

A Novel Type of Peptidoglycan-binding Domain Highly Specific for Amidated d-Asp Cross-bridge,Identified in Lactobacillus casei Bacteriophage Endolysins

Peptidoglycan hydrolases (PGHs) are responsible for bacterial cell lysis. Most PGHs have a modular structure comprising a catalytic domain and a cell wall-binding domain (CWBD). PGHs of bacteriophage origin, called endolysins, are involved in bacterial lysis at the end of the infection cycle. We have characterized two endolysins, Lc-Lys and Lc-Lys-2, identified in prophages present in the genome of Lactobacillus casei BL23. These two enzymes have different catalytic domains but similar putative C-terminal CWBDs. By analyzing purified peptidoglycan (PG) degradation products, we showed that Lc-Lys is an N-acetylmuramoyl-l-alanine amidase, whereas Lc-Lys-2 is a γ-d-glutamyl-l-lysyl endopeptidase. Remarkably, both lysins were able to lyse only Gram-positive bacterial strains that possess PG with d-Ala⁴→d-Asx-l-Lys³ in their cross-bridge, such as Lactococcus casei, Lactococcus lactis, and Enterococcus faecium. By testing a panel of L. lactis cell wall mutants, we observed that Lc-Lys and Lc-Lys-2 were not able to lyse mutants with a modified PG cross-bridge, constituting d-Ala⁴→l-Ala-(l-Ala/l-Ser)-l-Lys³; moreover, they do not lyse the L. lactis mutant containing only the nonamidated d-Asp cross-bridge, i.e. d-Ala⁴→d-Asp-l-Lys³. In contrast, Lc-Lys could lyse the ampicillin-resistant E. faecium mutant with 3→3 l-Lys³-d-Asn-l-Lys³ bridges replacing the wild-type 4→3 d-Ala⁴-d-Asn-l-Lys³ bridges. We showed that the C-terminal CWBD of Lc-Lys binds PG containing mainly d-Asn but not PG with only the nonamidated d-Asp-containing cross-bridge, indicating that the CWBD confers to Lc-Lys its narrow specificity. In conclusion, the CWBD characterized in this study is a novel type of PG-binding domain targeting specifically the d-Asn interpeptide bridge of PG.     

Evidence for a specific glutamate/h cotransport in isolated mesophyll cells

Mechanically isolated Asparagus sprengeri Regel mesophyll cells were suspended in 1 millimolar CaSO₄. Immediate alkalinization of the medium occured on the addition of 1 millimolar concentrations of l-glutamate (Glu) and its analog l-methionine-d,l-sulfoximine (l-MSO). d-Glu and the l isomers of the protein amino acids did not elicit alkalinization. l-Glu dependent alkalinization was transient and acidification resumed after approximately 30 to 45 minutes. At pH 6.0, 5 millimolar l-Glu stimulated initial rates of alkalinization that varied between 1.3 to 4.1 nmol H⁺/10⁶ cells·minute. l-Glu dependent alkalinization was saturable, increased with decreasing pH, was inhibited by carbonyl cyanide-p-trichloromethoxyphenyl hydrazone (CCCP), and was not stimulated by light. Uptake of l-[U-¹⁴C]glutamate increased as the pH decreased from 6.5 to 5.5, and was inhibited by l-MSO. l-Glu had no influence on K⁺ efflux. Although evidence for multiple amino acid/proton cotransport systems has been found in other tissues, the present report indicates that a highly specific l-Glu/proton uptake process is present in Asparagus mesophyll cells.     

Effect of ammonium and other ions on the glucose-dependent active transport of l-histidine in slices of rat-brain cortex

1. The influence of cations on the active transport into cells of rat-brain-cortex slices of l-histidine, an amino acid that is not metabolized by this tissue, has been studied. 2. Like other amino acids, l-histidine accumulated in the cells in the presence of glucose in concentrations up to over double that in the incubation medium. 3. The active transport of l-histidine was highest in a medium containing Ca²⁺ (3mm). The addition of K⁺ (27mm) led to a marked decrease in the intracellular concentration of l-histidine, though the oxygen uptake of the slices was higher. 4. The active l-histidine transport was inhibited by NH₄⁺. The inhibitory effect increased with the NH₄⁺ concentration, being about 25% at 8mm, 65% at 20mm, and 90% at 27 and 50mm. The oxygen uptake of the brain slices was depressed by only 25% by the highest NH₄⁺ concentration used, and less by lower concentrations.     

The use of potential-sensitive cyanine dye for studying ion-dependent electrogenic renal transport of organic solutes. Spectrophotometric measurements

Renal transport of four different categories of organic solutes, namely sugars, neutral amino acids, monocarboxylic acids and dicarboxylic acids, was studied by using the potential-sensitive dye 3,3′-diethyloxadicarbocyanine iodide in purified luminal-membrane and basolateral-membrane vesicles isolated from rabbit kidney cortex. Valinomycin-induced K⁺ diffusion potentials resulted in concomitant changes in dye–membrane-vesicle absorption spectra. Linear relationships were obtained between these changes and depolarization and hyperpolarization of the vesicles. Addition of d-glucose, l-phenylalanine, succinate or l-lactate to luminal-membrane vesicles, in the presence of an extravesicular>intravesicular Na⁺ gradient, resulted in rapid transient depolarization. With basolateral-membrane vesicles no electrogenic transport of d-glucose or l-phenylalanine was observed. Spectrophotometric competition studies revealed that d-galactose is electrogenically taken up by the same transport system as that for d-glucose, whereas l-phenylalanine, succinate and l-lactate are transported by different systems in luminal-membrane vesicles. The absorbance changes associated with simultaneous addition of d-glucose and l-phenylalanine were additive. The uptake of these solutes was influenced by the presence of Na⁺-salt anions of different permeabilities in the order: Cl⁻>SO₄²⁻>gluconate. Addition of valinomycin to K⁺-loaded vesicles enhanced uptake of d-glucose and l-phenylalanine in the presence of an extravesicular>intravesicular Na⁺ gradient. Gramicidin or valinomycin plus nigericin diminished/abolished electrogenic solute uptake by Na⁺- or Na⁺+K⁺-loaded vesicles respectively. These results strongly support the presence of Na⁺-dependent renal electrogenic transport of d-glucose, l-phenylalanine, succinate and l-lactate in luminal-membrane vesicles.     

Emission of Hydrogen Sulfide by Leaf Tissue in Response to l-Cysteine

Leaf discs and detached leaves exposed to l-cysteine emitted a volatile sulfur compound which was proven by gas chromatography to be H₂S. This phenomenon was demonstrated in all nine species tested (Cucumis sativus, Cucurbita pepo, Nicotiana tabacum, Coleus blumei, Beta vulgaris, Phaseolus vulgaris, Medicago sativa, Hordeum vulgare, and Gossypium hirsutum). The emission of volatile sulfur by cucumber leaves occurred in the dark at a similar rate to that in the light. The emission of leaf discs reached the maximal rate, more than 40 picomoles per minute per square centimeter, 2 to 4 hours after starting exposure to l-cysteine; then it decreased. In the case of detached leaves, the maximum occurred 5 to 10 h after starting exposure. The average emission rate of H₂S during the first 4 hours from leaf discs of cucurbits in response to 10 millimolar l-cysteine, was usually more than 40 picomoles per minute per square centimeter, i.e. 0.24 micromoles per hour per square decimeter. Leaf discs exposed to 1 millimolar l-cysteine emitted only 2% as much as did the discs exposed to 10 millimolar l-cysteine. The emission from leaf discs and from detached leaves lasted for at least 5 and 15 hours, respectively. However, several hours after the maximal emission, injury of the leaves, manifested as chlorosis, was evident. H₂S emission was a specific consequence of exposure to l-cysteine; neither d-cysteine nor l-cystine elicited H₂S emission. Aminooxyacetic acid, an inhibitor of pyridoxal phosphate dependent enzymes, inhibited the emission. In a cell free system from cucumber leaves, H₂S formation and its release occurred in response to l-cysteine. Feeding experiments with [³⁵S]l-cysteine showed that most of the sulfur in H₂S was derived from sulfur in the l-cysteine supplied and that the H₂S emitted for 9 hours accounted for 7 to 10% of l-cysteine taken up. ³⁵S-labeled SO₃²⁻ and SO₄²⁻ were found in the tissue extract in addition to internal soluble S²⁻. These findings suggest the existence of a sulfur cycle which converts l-cysteine to SO₄²⁻ through cysteine desulfhydration.     

Biosynthesis of (+)-Tartaric Acid from l-[4-C]Ascorbic Acid in Grape and Geranium

The metabolic fate of l-[4-¹⁴C]ascorbic acid has been examined in the grape (Vitis labrusca L.) and lemon geranium (Pelargonium crispum L. L'Hér. cv. Prince Rupert) under conditions comparable to data from l-[1-¹⁴C]ascorbic acid and l-[6-¹⁴C]ascorbic acid experiments. In detached grape leaves and immature berries, l-[4-¹⁴C]ascorbic acid and l-[1-¹⁴C]ascorbic acid were equivalent precursors to carboxyl labeled (+)-tartaric acid. In geranium apices, l-[4-¹⁴C]ascorbic acid yielded internal labeled (+)-tartaric acid while l-[6-¹⁴C]ascorbic acid gave an equivalent conversion to carboxyl labeled (+)-tartaric acid. These findings clearly show that two distinct processes for the synthesis of (+)-tartaric acid from l-ascorbic acid exist in plants identified as (+)-tartaric acid accumulators. In grape leaves and immature berries, (+)-tartaric acid synthesis proceeds via preservation of a four-carbon fragment derived from carbons 1 through 4 of l-ascorbic acid while carbons 3 through 6 yield (+)-tartaric acid in geranium apices.     

l-Glutamate-Dependent Medium Alkalinization by Asparagus Mesophyll Cells : Cotransport or Metabolism?

Mechanically isolated Asparagus sprengeri Regel mesophyll cells cause alkalinization of the suspension medium on the addition of l-glutamate or its analog l-methionine-d,l-sulfoximine. Using a radiolabeled pH probe, it was found that both compounds caused internal acidification whereas l-aspartate did not. Fusicoccin stimulated H⁺ efflux from the cells by 111% and the uptake of l-[U-¹⁴C]glutamate by 55%. Manometric experiments demonstrated that, unlike l-methionine-d,l-sulfoximine, l-glutamate stimulated CO₂ evolution from nonilluminated cells. Simultaneous measurements of medium alkalinization and ¹⁴CO₂ evolution upon the addition of labeled l-glutamate showed that alkalinization was immediate and reached a maximum value after 45 minutes whereas ¹⁴CO₂ evolution exhibited a lag before its appearance and continued in a linear manner for at least 100 minutes. Rates of alkalinization and uptake of l-[U-¹⁴C]glutamate were higher in the light while rates of ¹⁴CO₂ evolution were higher in the dark. The major labeled product of glutamate decarboxylation, γ-aminobutyric acid, was found in the cells and the suspension medium. Its addition to the cell suspension did not result in medium alkalinization and evidence indicates that it is lost from the cell to the medium. The data suggest that the origin of medium alkalinization is co-transport not metabolism, and that the loss of labeled CO₂ and γ-aminobutyric acid from the cell result in an overestimation of the stoichiometry of the H⁺/l-glutamate uptake process.     

Substrate Specificity of a Mannose-6-Phosphate Isomerase from Bacillus subtilis and Its Application in the Production of l-Ribose

The uncharacterized gene previously proposed as a mannose-6-phosphate isomerase from Bacillus subtilis was cloned and expressed in Escherichia coli. The maximal activity of the recombinant enzyme was observed at pH 7.5 and 40°C in the presence of 0.5 mM Co²⁺. The isomerization activity was specific for aldose substrates possessing hydroxyl groups oriented in the same direction at the C-2 and C-3 positions, such as the d and l forms of ribose, lyxose, talose, mannose, and allose. The enzyme exhibited the highest activity for l-ribulose among all pentoses and hexoses. Thus, l-ribose, as a potential starting material for many l-nucleoside-based pharmaceutical compounds, was produced at 213 g/liter from 300-g/liter l-ribulose by mannose-6-phosphate isomerase at 40°C for 3 h, with a conversion yield of 71% and a volumetric productivity of 71 g liter⁻¹ h⁻¹.l-Ribose is a potential starting material for the synthesis of many l-nucleoside-based pharmaceutical compounds, and it is not abundant in nature (5, 19). l-Ribose has been produced mainly by chemical synthesis from l-arabinose, l-xylose, d-glucose, d-galactose, d-ribose, or d-mannono-1,4-lactone (2, 17, 23). Biological l-ribose manufacture has been investigated using ribitol or l-ribulose. Recently, l-ribose was produced from ribitol by a recombinant Escherichia coli containing an NAD-dependent mannitol-1-dehydrogenase (MDH) with a 55% conversion yield when 100 g/liter ribitol was used in a 72-h fermentation (18). However, the volumetric productivity of l-ribose in the fermentation is 28-fold lower than that of the chemical method synthesized from l-arabinose (8). l-Ribulose has been biochemically converted from l-ribose using an l-ribose isomerase from an Acinetobacter sp. (9), an l-arabinose isomerase mutant from Escherichia coli (4), a d-xylose isomerase mutant from Actinoplanes missouriensis (14), and a d-lyxose isomerase from Cohnella laeviribosi (3), indicating that l-ribose can be produced from l-ribulose by these enzymes. However, the enzymatic production of l-ribulose is slow, and the enzymatic production of l-ribose from l-ribulose has been not reported.Sugar phosphate isomerases, such as ribose-5-phosphate isomerase, glucose-6-phosphate isomerase, and galactose-6-phosphate isomerase, work as general aldose-ketose isomerases and are useful tools for producing rare sugars, because they convert the substrate sugar phosphates and the substrate sugars without phosphate to have a similar configuration (11, 12, 21, 22). l-Ribose isomerase from an Acinetobacter sp. (9) and d-lyxose isomerase from C. laeviribosi (3) had activity with l-ribose, d-lyxose, and d-mannose. Thus, we can apply mannose-6-phosphate (EC 5.3.1.8) isomerase to the production of l-ribose, because there are no sugar phosphate isomerases relating to l-ribose and d-lyxose. The production of the expensive sugar l-ribose (bulk price, $1,000/kg) from the rare sugar l-ribulose by mannose-6-phosphate isomerase may prove to be a valuable industrial process, because we have produced l-ribulose from the cheap sugar l-arabinose (bulk price, $50/kg) using the l-arabinose isomerase from Geobacillus thermodenitrificans (20) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Schematic representation for the production of l-ribulose from l-arabinose by G. thermodenitrificans l-arabinose isomerase and the production of l-ribose from l-ribulose by B. subtilis mannose-6-phosphate isomerase.In this study, the gene encoding mannose-6-phosphate isomerase from Bacillus subtilis was cloned and expressed in E. coli. The substrate specificity of the recombinant enzyme for various aldoses and ketoses was investigated, and l-ribulose exhibited the highest activity among all pentoses and hexoses. Therefore, mannose-6-phosphate isomerase was applied to the production of l-ribose from l-ribulose.     

l-Ribose Production from l-Arabinose by Using Purified l-Arabinose Isomerase and Mannose-6-Phosphate Isomerase from Geobacillus thermodenitrificans

Two enzymes, l-arabinose isomerase and mannose-6-phosphate isomerase, from Geobacillus thermodenitrificans produced 118 g/liter l-ribose from 500 g/liter l-arabinose at pH 7.0, 70°C, and 1 mM Co²⁺ for 3 h, with a conversion yield of 23.6% and a volumetric productivity of 39.3 g liter⁻¹ h⁻¹.l-Ribose, a potential starting material for the synthesis of many l-nucleoside-based pharmaceutical compounds, is not abundant in nature (4, 15, 20). l-Ribose has been synthesized primarily from l-arabinose, l-xylose, d-glucose, d-galactose, d-ribose, and d-mannono-1,4-lactone (1, 13, 20). Recombinant cells containing a NAD-dependent mannitol-1-dehydrogenase produced 52 g/liter l-ribose from 100 g/liter ribitol after fermentation for 72 h (14). However, the volumetric productivity of l-ribose was 26-fold lower than that of the chemical synthetic method starting from l-arabinose (6). l-Ribose isomerase from an Acinetobacter sp., which is most active with l-ribose, showed poor efficiency in the conversion of l-ribulose to l-ribose (9). Recently, l-ribulose was produced with a conversion yield of 19% from the inexpensive sugar l-arabinose using l-arabinose isomerase (AI) from Geobacillus thermodenitrificans (18). l-Ribose has been produced from l-ribulose using mannose-6-phosphate isomerase (MPI) from Bacillus subtilis with a conversion yield of 70% (17). In this study, the production of l-ribose from l-arabinose was demonstrated via a two-enzyme system from G. thermodenitrificans, in which l-ribulose was first produced from l-arabinose by AI and subsequently converted to l-ribose by MPI.The analysis of monosaccharides and the purification and thermostability of AI and MPI from G. thermodenitrificans (2) isolated from compost were performed as described previously (7, 18, 19). The cross-linked enzymes were obtained from the treatment of 0.5% glutaraldehyde (10, 16). The reaction was performed by replacing the reaction solution with 100 g/liter l-arabinose and 1 mM Co²⁺ every 6 h at 70°C and pH 7.0. The reaction volume of 10 ml contained 5 g of the cross-linked enzymes with 8 U/ml AI and 20 U/ml MPI. One unit of AI or MPI activity, which corresponded to 0.0625 or 2.5 mg protein, respectively, was defined as the amount of enzyme required to produce 1 μmol of l-ribulose or l-ribose, respectively, per min at 70°C, pH 7.0, and 1 mM Co²⁺. Unless otherwise stated, the reaction was carried out in 50 mM piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) buffer (pH 7.0) in the presence of 1 mM Co²⁺ at 70°C for 4 h. All experiments were performed in triplicate.The recombinant Escherichia coli ER2566 (New England Biolabs, Ipswich, MA) containing pTrc99A plasmid (Pharmacia Biotech, Piscataway, NJ) and the AI or MPI gene was cultivated in a 7-liter fermentor containing 3 liters of chemically defined medium (11). When the cell mass reached 2 g/liter, 10 g/liter lactose was added for enzyme induction. After 14 h, 40 g/liter cells with 13,400 U/liter of AI or 34 g/liter cells with 630 U/liter of MPI was obtained. The enzyme was purified by heat treatment and Hi-Trap anion-exchange chromatography. The purification yields of AI and MPI were 21 and 78%, respectively, and the levels of purity for the concentrated AI and MPI by gene scanning were 48 and 92%, respectively. Maximum l-ribose production from l-arabinose by AI and by MPI in 10 ml of total volume was observed at pH 7.0, 70°C, and 1 mM Co²⁺ (data not shown). Half-lives for the two-enzyme system containing 10 mM l-arabinose, 0.2 U/ml AI, and 0.5 U/ml MPI at 60, 65, 70, 75, and 80°C were 1,216, 235, 48, 26, and 12 h, respectively. The use of Co²⁺ may be disadvantageous, as it is fairly toxic. This problem can be solved by using Mn²⁺ instead of Co²⁺. When Mn²⁺ was used in the reaction with the same amounts of enzymes, the conversion yield was the same as that obtained with Co²⁺, even though the volumetric productivity was lower than that with Co²⁺ (data not shown).The effect of the ratio of AI to MPI in the two-step enzymatic production of l-ribose from l-arabinose was investigated by mixing the enzyme solutions (8 U/ml AI and 20 U/ml MPI) to obtain AI/MPI ratios ranging from 10:90 to 90:10 (vol/vol) (Fig. ​(Fig.1).1). The reactions were run with 300 g/liter l-arabinose. Maximum l-ribose production was observed at a volume ratio of 50:50 of the enzyme solutions. The effects of enzyme concentration on l-ribose production were investigated at the optimal unit ratio (AI/MPI ratio, 1:2.5) with 500 g/liter l-arabinose and AI and MPI concentrations from 0.4 and 1.0 U/ml, respectively, to 9.2 and 23.0 U/ml, respectively (Fig. ​(Fig.2A).2A). l-Ribose production increased with increasing amounts of enzymes until reaching a plateau at 8 U/ml AI and 20 U/ml MPI. The effect of substrate concentration on l-ribose production was evaluated at l-arabinose concentrations ranging from 15 to 500 g/liter with 8 U/ml AI and 20 U/ml MPI (Fig. ​(Fig.2B).2B). The production of both l-ribose and l-ribulose, an intermediate, increased with increasing substrate level. The results suggest that concentrations of substrate above 500 g/liter l-arabinose might cause the increased production. The conversion yields of l-ribose and l-ribulose from l-arabinose were constant at 32% and 14%, respectively, within an initial concentration of 100 g/liter l-arabinose, indicating that the reactions reached equilibrium at an l-arabinose/l-ribulose/l-ribose ratio of 54:14:32, which was in agreement with the calculated equilibrium (17). However, at l-arabinose concentrations above 100 g/liter, the conversion yields of l-ribose and l-ribulose from l-arabinose decreased with increasing l-arabinose concentration. The l-arabinose/l-ribulose/l-ribose ratio, with an initial l-arabinose concentration of 300 g/liter, was 71:6:23 after 4 h of reaction. To obtain near-equilibrium (54:14:32) at this high concentration of l-arabinose, more effective enzymes are required.Open in a separate windowFIG. 1.Effect of the ratio of AI to MPI on l-ribose production from l-arabinose by the purified AI and MPI from G. thermodenitrificans. Data are the means for three separate experiments, and error bars represent standard deviations. Symbols: •, l-ribose; ▪, l-ribulose.Open in a separate windowFIG. 2.(A) Effect of enzyme concentration on l-ribose production from l-arabinose at the optimal unit ratio (AI/MPI ratio, 1:2.5). Symbols: •, l-ribose; ▪, l-ribulose; ○, l-arabinose. (B) Effect of l-arabinose concentration on l-ribose production. Symbols: •, l-ribose; ▪, l-ribulose. Data are the means for three separate experiments, and error bars represent standard deviations.A time course reaction of l-ribose production from l-arabinose was monitored for 3 h with 8 U/ml AI and 20 U/ml MPI (Fig. ​(Fig.3).3). As a result, 118 g/liter l-ribose was obtained from an initial l-arabinose concentration of 500 g/liter after 3 h, with a conversion yield of 23.6% and a productivity of 39.3 g liter⁻¹ h⁻¹. Recombinant E. coli containing MDH yielded 52 g/liter l-ribose from an initial ribitol concentration of 100 g/liter after 72 h, with a productivity of 0.72 g liter⁻¹ h⁻¹ (14). The production and productivity obtained in the current study using AI and MPI from G. thermodenitrificans were 2.3- and 55-fold higher, respectively, than those obtained from ribitol and 17- and 21-fold higher than those obtained with the production of l-ribose from l-arabinose using resting cells of recombinant Lactobacillus plantarum (5). The chemical synthetic method is capable of producing 56.5 g/liter l-ribose from 250 g/liter l-arabinose after 3 h, corresponding to a productivity of 18.8 g liter⁻¹ h⁻¹ (6). Still, both the production and productivity of l-ribose using the method described herein were 2.1-fold higher. Thus, the method of production of l-ribose in the present study exhibited the highest productivity and production, compared to other fermentation methods and chemical syntheses.Open in a separate windowFIG. 3.Time course of l-ribose production from l-arabinose by purified AI and MPI from G. thermodenitrificans. Data are the means for three separate experiments, and error bars represent standard deviations. Symbols: •, l-ribose; ▪, l-ribulose; ○, l-arabinose.Several rounds of conversion reusing the cross-linked enzymes were performed (Fig. ​(Fig.4).4). The immobilized enzymes showed more than 20% conversion of l-ribose from l-arabinose for the 9th batch, and the concentration of l-ribose was reduced to 43% after the 20th batch. These results suggest that the immobilization of enzyme facilitates separation of product and enzyme, and it enables the enzyme to function continuously, as reported previously (3, 8, 12). Thus, the reuse of enzyme by immobilization improves the economic viability of this enzymatic process.Open in a separate windowFIG. 4.Reuse of immobilized AI and MPI from G. thermodenitrificans for l-ribose production from 100 g/liter l-arabinose. Data are the means for three separate experiments, and error bars represent standard deviations.     

Uptake of Phenylalanine into Isolated Barley Vacuoles Is Driven by Both Tonoplast Adenosine Triphosphatase and Pyrophosphatase : Evidence for a Hydrophobic l-Amino Acid Carrier System

The uptake of phenylalanine was studied with vacuole isolated from barley mesophyll protoplasts. The phenylalanine transport exhibited saturation kinetics with apparent K_m-values of 1.2 to 1.4 millimolar for ATP- or PPi-driven uptake; V_{max app} was 120 to 140 nanomoles Phe per milligram of chlorophyll per hour (1 milligram of chlorophyll corresponds to 5 × 10⁶ vacuoles). Half-maximal transport rates driven with ATP or PPi were reached at 0.5 millimolar ATP or 0.25 millimolar PPi. ATP-driven transport showed a distinct pH optimum at 7.3 while PPi-driven transport reached maximum rates at pH 7.8. Direct measurement of the H⁺-translocating enzyme activities revealed K_{m app} values of 0.45 millimolar for ATPase (EC 3.6.1.3) and 23 micromolar for pyrophosphatase (PPase) (EC 3.6.1.1). In contrast to the coupled amino acid transport, ATPase and PPase activities had relative broad pH optima between 7 to 8 for ATPase and 8 to 9 for PPase. ATPase as well as ATP-driven transport was markedly inhibited by nitrate while PPase and PPi-coupled transport was not affected. The addition of ionophores inhibited phenylalanine transport suggesting the destruction of the electrochemical proton potential difference Δ μH⁺ while the rate of ATP and PPi hydrolysis was stimulated. The uptake of other lipophilic amino acids like l-Trp, l-Leu, and l-Tyr was also stimulated by ATP. They seem to compete for the same carrier system. l-Ala, l-Val, d-Phe, and d-Leu did not influence phenylalanine transport suggesting a stereospecificity of the carrier system for l-amino acids having a relatively high hydrophobicity.     

d-Glucosone and l-Sorbosone, Putative Intermediates of l-Ascorbic Acid Biosynthesis in Detached Bean and Spinach Leaves

d-[6-¹⁴C]Glucosone that had been prepared enzymically from d-[6-¹⁴C]glucose was used to compare relative efficiencies of these two sugars for l-ascorbic acid (AA) biosynthesis in detached bean (Phaseolus vulgaris L., cv California small white) apices and 4-week-old spinach (Spinacia oleracea L., cv Giant Noble) leaves. At tracer concentration, ¹⁴C from glucosone was utilized by spinach leaves for AA biosynthesis much more effectively than glucose. Carbon-14 from [6-¹⁴C]glucose underwent considerable redistribution during AA formation, whereas ¹⁴C from [6-¹⁴C]glucosone remained almost totally in carbon 6 of AA. In other experiments with spinach leaves, l-[U-¹⁴C]sorbosone was found to be equivalent to [6-¹⁴C]glucose as a source of ¹⁴C for AA. In the presence of 0.1% d-glucosone, conversion of [6-¹⁴C] glucose into labeled AA was greatly repressed. In a comparable experiment with l-sorbosone replacing d-glucosone, the effect was much less. The experiments described here give substance to the proposal that d-glucosone and l-sorbosone are putative intermediates in the conversion of d-glucose to AA in higher plants.     

A new unextracted-sample radioimmunoassay method for hepatic endogenous nuclear l-tri-iodothyronine content. Validity of its use in determining nuclear receptor binding characteristics

Endogenous l-tri-iodothyronine content in an hepatic nuclear extract was measured by a new unextracted-sample radioimmunoassay method using 8-anilinonaphthalene-1-sulphonic acid to inhibit the l-[¹²⁵I]tri-iodothyronine binding to the nuclear l-tri-iodothyronine receptor within the extract. For this method, the lower sensitivity limit was 3.125 pg/tube, the recovery of added l-tri-iodothyronine was 90–120%, and the between-assay coefficient of variation was 10%. The amount of endogenous l-tri-iodothyronine was 10–40 pg/0.2 ml of hepatic nuclear extract from euthyroid rats, compared with less than 3.125 pg/0.2 ml from thyroidectomized rats. The results obtained by this new method were compared with a Sephadex G-25 column extracted-sample radioimmunoassay method and showed a good agreement. The values for the endogenous l-tri-iodothyronine content were utilized to correct for the l-tri-iodothyronine concentration within the binding assay mixture in order to accurately determine by Scatchard analysis the binding characteristics of the nuclear l-tri-iodothyronine receptor. The validity of the correction for endogeneous l-tri-iodothyronine was demonstrated by using a nuclear extract from a thyroidectomized rat which was preincubated with a small known amount of l-tri-iodothyronine before determining the nuclear l-tri-iodothyronine receptor binding characteristics. When the Scatchard plots were corrected for the preincubated dose, the results obtained were similar to true values, but they were falsely lower when not corrected. It is concluded that the necessity and validity of using endogenous l-tri-iodothyronine corrections in the Scatchard analytical computations of the nuclear l-tri-iodothyronine receptor binding characteristics has been demonstrated, being particularly more important for affinity constant than maximum binding capacity.     

Site-directed Mutagenesis Switching a Dimethylallyl Tryptophan Synthase to a Specific Tyrosine C3-Prenylating Enzyme

The tryptophan prenyltransferases FgaPT2 and 7-DMATS (7-dimethylallyl tryptophan synthase) from Aspergillus fumigatus catalyze C⁴- and C⁷-prenylation of the indole ring, respectively. 7-DMATS was found to accept l-tyrosine as substrate as well and converted it to an O-prenylated derivative. An acceptance of l-tyrosine by FgaPT2 was also observed in this study. Interestingly, isolation and structure elucidation revealed the identification of a C³-prenylated l-tyrosine as enzyme product. Molecular modeling and site-directed mutagenesis led to creation of a mutant FgaPT2_K174F, which showed much higher specificity toward l-tyrosine than l-tryptophan. Its catalytic efficiency toward l-tyrosine was found to be 4.9-fold in comparison with that of non-mutated FgaPT2, whereas the activity toward l-tryptophan was less than 0.4% of that of the wild-type. To the best of our knowledge, this is the first report on an enzymatic C-prenylation of l-tyrosine as free amino acid and altering the substrate preference of a prenyltransferase by mutagenesis.     

The biological sulphation of l-tyrosyl peptides

1. A rat-liver supernatant preparation can achieve the biological O-sulphation of l-tyrosylglycine and l-tyrosyl-l-alanine at pH7·0. 2. The optimum concentrations of l-tyrosylglycine and l-tyrosyl-l-alanine in this system are 50mm and 60mm respectively. 3. l-Tyrosylglycine yields two sulphated products, whereas l-tyrosyl-l-alanine yields three sulphated products, when used as acceptor for sulphate in the rat-liver system. 4. With both substrates, one of the sulphated products has been identified as the O-sulphate ester of the corresponding parent peptide.     

Regulation of sulfate uptake by amino acids in cultured tobacco cells

The sulfur requirements of tobacco (Nicotiana tabacum L. var. Xanthi) XD cells grown in chemically defined liquid media can be satisfied by sulfate, thiosulfate, l-cyst(e)ine, l-methionine or glutathione, and somewhat less effectively by d-cyst (e) ine, d-methionine or dl-homocyst (e)ine. Sulfate uptake is inhibited after a 2 hr lag by l-cyst (e)ine, l-methionine, l-homocyst(e)ine or l-isoleucine, but not by any of the other protein amino acids, nor by d-cyst(e)ine. l-cyst(e)ine is neither a competitive nor a non-competitive inhibitor of sulfate uptake. Its action most closely resembles apparent uncompetitive inhibition. Inhibition of sulfate uptake by l-cyst(e)ine can be partially prevented by equimolar l-arginine, l-lysine, l-leucine, l-phenylalanine, l-tyrosine or l-tryptophan, but is little affected by any of the other protein amino acids. The effective amino acids are apparent competitive inhibitors of l-cyst(e)ine uptake after a 2 hr lag. Inhibition of sulfate uptake by l-methionine cannot be prevented, nor can uptake of l-methionine be inhibited by any single protein amino acid. The results suggest the occurrence of negative feedback control of sulfate assimilation by the end products, the sulfur amino acids, in cultured tobacco cells.     

Engineering Filamentous Fungi for Conversion of d-Galacturonic Acid to l-Galactonic Acid

d-Galacturonic acid, the main monomer of pectin, is an attractive substrate for bioconversions, since pectin-rich biomass is abundantly available and pectin is easily hydrolyzed. l-Galactonic acid is an intermediate in the eukaryotic pathway for d-galacturonic acid catabolism, but extracellular accumulation of l-galactonic acid has not been reported. By deleting the gene encoding l-galactonic acid dehydratase (lgd1 or gaaB) in two filamentous fungi, strains were obtained that converted d-galacturonic acid to l-galactonic acid. Both Trichoderma reesei Δlgd1 and Aspergillus niger ΔgaaB strains produced l-galactonate at yields of 0.6 to 0.9 g per g of substrate consumed. Although T. reesei Δlgd1 could produce l-galactonate at pH 5.5, a lower pH was necessary for A. niger ΔgaaB. Provision of a cosubstrate improved the production rate and titer in both strains. Intracellular accumulation of l-galactonate (40 to 70 mg g biomass⁻¹) suggested that export may be limiting. Deletion of the l-galactonate dehydratase from A. niger was found to delay induction of d-galacturonate reductase and overexpression of the reductase improved initial production rates. Deletion of the l-galactonate dehydratase from A. niger also delayed or prevented induction of the putative d-galacturonate transporter An14g04280. In addition, A. niger ΔgaaB produced l-galactonate from polygalacturonate as efficiently as from the monomer.     

An l-glucose Catabolic Pathway in Paracoccus Species 43P

An l-glucose-utilizing bacterium, Paracoccus sp. 43P, was isolated from soil by enrichment cultivation in a minimal medium containing l-glucose as the sole carbon source. In cell-free extracts from this bacterium, NAD⁺-dependent l-glucose dehydrogenase was detected as having sole activity toward l-glucose. This enzyme, LgdA, was purified, and the lgdA gene was found to be located in a cluster of putative inositol catabolic genes. LgdA showed similar dehydrogenase activity toward scyllo- and myo-inositols. l-Gluconate dehydrogenase activity was also detected in cell-free extracts, which represents the reaction product of LgdA activity toward l-glucose. Enzyme purification and gene cloning revealed that the corresponding gene resides in a nine-gene cluster, the lgn cluster, which may participate in aldonate incorporation and assimilation. Kinetic and reaction product analysis of each gene product in the cluster indicated that they sequentially metabolize l-gluconate to glycolytic intermediates, d-glyceraldehyde-3-phosphate, and pyruvate through reactions of C-5 epimerization by dehydrogenase/reductase, dehydration, phosphorylation, and aldolase reaction, using a pathway similar to l-galactonate catabolism in Escherichia coli. Gene disruption studies indicated that the identified genes are responsible for l-glucose catabolism.     

Effects of Hydrogen Sulfide-releasing l-DOPA Derivatives on Glial Activation: POTENTIAL FOR TREATING PARKINSON DISEASE*

The main lesion in Parkinson disease (PD) is loss of substantia nigra dopaminergic neurons. Levodopa (l-DOPA) is the most widely used therapy, but it does not arrest disease progression. Some possible contributing factors to the continuing neuronal loss are oxidative stress, including oxidation of l-DOPA, and neurotoxins generated by locally activated microglia and astrocytes. A possible method of reducing these factors is to produce l-DOPA hybrid compounds that have antioxidant and antiinflammatory properties. Here we demonstrate the properties of four such l-DOPA hybrids based on coupling l-DOPA to four different hydrogen sulfide-donating compounds. The donors themselves were shown to be capable of conversion by isolated mitochondria to H₂S or equivalent SH⁻ ions. This capability was confirmed by in vivo results, showing a large increase in intracerebral dopamine and glutathione after iv administration in rats. When human microglia, astrocytes, and SH-SY5Y neuroblastoma cells were treated with these donating agents, they all accumulated H₂S intracellularly as did their derivatives coupled to l-DOPA. The donating agents and the l-DOPA hybrids reduced the release of tumor necrosis factor-α, interleukin-6, and nitric oxide from stimulated microglia, astrocytes as well as the THP-1 and U373 cell lines. They also demonstrated a neuroprotective effect by reducing the toxicity of supernatants from these stimulated cells to SH-SY5Y cells. l-DOPA itself was without effect in any of these assays. The H₂S-releasing l-DOPA hybrid molecules also inhibited MAO B activity. They may be useful for the treatment of PD because of their significant antiinflammatory, antioxidant, and neuroprotective properties.     

Amino Acid Transport in Suspension-Cultured Plant Cells : VI. Influence of pH Buffers, Calcium, and Preincubation Media on l-Leucine Uptake

The rate at which l-leucine was transported into suspension-cultured Nicotiana tabacum cv Wisconsin 38 cells increased more than 2-fold over a period of hours when the cells were preincubated in a 1% sucrose solution. This increase in uptake rate was eliminated if certain tris buffers were included in the preincubation solution while other buffers had little effect. Calcium could reverse the effect of the inhibitory buffers only if the buffer and calcium were present together from the beginning of the preincubation period. It was the amine group of the inhibitory buffers which was responsible for the inhibition. Preincubation in a complete culture medium (EM Linsmaier, F Skoog 1965 Physiol Plant 18: 100-127) led to minimal changes in l-leucine uptake rate over a 10 hour preincubation period indicating that the uptake rate was stabilized by this medium. The complete medium stabilized the l-leucine uptake rate as a result of its ionic composition and not because of its osmolarity. Most of the increased uptake rate observed after preincubation in a 1% sucrose solution could be inhibited by 2,4-dinitrophenol or carbonyl cyanide m-chlorophenyl hydrazone, or high concentrations of l-phenyl-alanine or l-leucine. Therefore much of the increase could be accounted for by an increase in active transport of l-leucine.