Role of lactose on the production of d-arabitol by Kluyveromyces lactis grown on lactose

There are remarkably few reports on d-arabitol production from lactose. Previous studies in our laboratory have shown that the osmophilic yeast Kluyveromyces lactis NBRC 1903 convert lactose to extracellular d-arabitol. The present study was undertaken to determine the participation of osmotic stress caused by lactose on d-arabitol production by K. lactis NBRC 1903 and to provide the information on the kinetics of d-arabitol production from lactose by K. lactis NBRC 1903. It was confirmed that d-arabitol production was triggered when an initial lactose concentration was above 278 mmol L⁻¹. d-Arabitol yield increased with an increase in initial lactose concentration. The highest d-arabitol concentration of 79.5 mmol L⁻¹ was achieved in the cultivation of K. lactis NBRC 1903 in a medium containing 555 mmol L⁻¹ lactose and 40 g L⁻¹ yeast extract. Lactose was found to play two important roles in d-arabitol production by K. lactis NBRC 1903 grown on lactose. First, lactose was assimilated as the substrate both for cell growth and d-arabitol production. Second, a high lactose concentration induced cellular response to high osmotic stress and up-regulated the flow from d-glucose-6-phosphate to d-arabitol. The arrest of cell growth triggered d-arabitol production.     

Characterization of the Na+/H+ Exchanger in the Luminal Membrane of the Distal Nephron

In the rabbit as well as the rat, a Na⁺/H⁺ exchanger is expressed in the apical membrane of both the proximal and distal tubules of the renal cortex. Whereas the isoform derived from the proximal tubule has been extensively studied, little information is available concerning the distal luminal membrane isoform. To better characterize the latter isoform, we purified rabbit proximal and distal tubules, and examined the ethylpropylamiloride (EIPA)-sensitive ²²Na uptake by the luminal membrane vesicles from the two segments. The presence of 100 μm EIPA in the membrane suspension decreased the 15 sec Na⁺ uptake to 75.70 ± 4.70% and 50.30 ± 2.23% of the control values in vesicles from proximal and distal tubules, respectively. The effect of EIPA on 35 mm Na⁺ uptake was concentration dependent, with a IC₅₀ of 700 μm and 75 μm for the proximal and distal luminal membranes. Whereas the proximal tubule membrane isoform was insensitive to cimetidine and clonidine up to a concentration of 2 mm, the 35 mm Na⁺ uptake by the distal membrane was strongly inhibited by cimetidine (IC₅₀ 700 μm) and modestly inhibited by clonidine (IC₅₀ 1.6 mm). The incubation of proximal tubule suspensions with 1 mm (Bu₂) cAMP decreased the 15-sec EIPA-sensitive Na⁺ uptake by the brush border membranes to 24.1 ± 2.38% of the control values. Unexpectedly, the same treatment of distal tubules enhanced this uptake by 46.5 ± 10.3%. Finally, incubation of tubule suspensions with 100 nm phorbol 12-myristate 13-acetate (PMA) decreased the exchanger activity to 58.6 ± 3.04% and 79.7 ± 3.21% of the control values in the proximal and distal luminal membranes, respectively. In conclusion, the high sensitivity of the distal luminal membrane exchanger to various inhibitors, and its stimulation by cAMP-dependent protein kinase A, indicate that this isoform differs from that of the proximal tubule and probably corresponds to isoform 1. Received: 6 March 1998/Revised: 6 July 1998     

Metabolic control of the K+ channel of human red cells

Summary The effects of cAMP, ATP and GTP on the Ca²⁺-dependent K⁺ channel of fresh (1–2 days) or cold-stored (28–36 days) human red cells were studied using atomic absorption flame photometry of Ca²⁺-EGTA loaded ghosts which had been resealed to monovalent cations in dextran solutions. When high-K⁺ ghosts were incubated in an isotonic Na⁺ medium, the rate constant of Ca²⁺-dependent K⁺ efflux was reduced by a half on increasing the theophylline concentration to 40mm. This effect was observed in ghosts from both fresh and stored cells, but only if they were previously loaded with ATP. The inhibition was more marked when Mg²⁺ was added together with ATP, and it was abolished by raising free Ca²⁺ to the micromolar level. Like theophylline, isobutyl methylxanthine (10mm) also affected K⁺ efflux. cAMP (0.2–0.5mm), added both internally and externally (as free salt, dibutyryl or bromide derivatives), had no significant effect on K⁺ loss when the ghost free-Ca²⁺ level was below 1 m, but it was slightly inhibitory at higher concentrations. The combined presence of cAMP (0.2mm) plus either theophylline (10mm), or isobutyl methylxanthine (0.5mm), was more effective than cAMP alone. This inhibition showed a strict requirement for ATP plus Mg²⁺ and it, was not overcome by raising internal Ca²⁺. Ghosts from stored cells seemed more sensitive than those from fresh cells, to the combined action of cAMP and methylxanthines. Loading ATP into ghosts from fresh or stored cells markedly decreased K⁺ loss. Although this effect was observed in the absence of added Mg²⁺ (0.5mm EDTA present), it was potentiated upon adding 2mm Mg²⁺. The K⁺ efflux from ATP-loaded ghosts was not altered by dithio-bis-nitrobenzoic acid (10mm) or acridine orange (100 m), while it was increased two-to fourfold by incubating with MgF₂ (10mm), or MgF₂ (10mm)+theophylline (40mm), respectively. By contrast, a marked efflux reduction was obtained by incorporating 0.5mm GTP into ATP-containing ghosts. The degree of phosphorylation obtained by incubating membranes with (-³²P)ATP under various conditions affecting K⁺ channel activity, was in direct correspondence to their effect on K⁺ efflux. The results suggest that the K⁺ channel of red cells is under complex metabolic control, via cAMP-mediated and nonmediated mechanisms, some which require ATP and presumably, involve phosphorylation of the channel proteins.     

Calcium-dependent Modulation of the Agonist Affinity of the Mammalian Olfactory Cyclic Nucleotide-gated Channel by Calmodulin and a Novel Endogenous Factor

The calcium-dependent modulation of the affinity of the cyclic nucleotide-gated (CNG) channels for adenosine 3′,5′-cyclic monophosphate (cAMP) was studied in enzymatically dissociated rat olfactory receptor neurons, by recording macroscopic cAMP-activated currents from inside-out patches excised from their dendritic knobs. Upon intracellular addition of 0.2 mm Ca²⁺ (0.2 Ca) the concentration of cAMP required for the activation of half-maximal current (EC₅₀) was reversibly increased from 3 μm to about 30 μm. This Ca²⁺-induced affinity shift was insensitive to the calmodulin antagonist, mastoparan, was abolished irreversibly by a 2-min exposure to 3 mm Mg²⁺+ 2 mm EGTA (Mg + EGTA), and was not restored by the application of calmodulin (CAM). Addition of CAM plus 0.2 mm Ca²⁺ (0.2 Ca + CAM), further reversibly shifted the cAMP affinity from 30 μm to about 200 μm. This affinity shift was not affected by Mg + EGTA exposure, but was reversed by mastoparan. Thus, the former Ca²⁺-only effect must be mediated by an unknown endogenous factor, distinct from CAM. Removal of this factor also increased the affinity of the channel for CAM. The affinity shift induced by Ca²⁺-only was maintained in the presence of the nonhydrolyzable cAMP analogue, 8-bromo-cAMP and the phosphatase inhibitor, microcystin-LR, ruling out modulation by phosphodiesterases or phosphatases. Our results indicate that the olfactory CNG channels are modulated by an as yet unidentified factor distinct from CAM. Received: 26 December 1995/Revised: 14 March 1996     

l-Ascorbic-acid biosynthesis in the euryhaline diatom Cyclotella cryptica

l-Ascorbic acid (AA) production in cells of Cyclotella cryptica Reimann, Lewin, Guillard (Bacillariophyceae) is enhanced when darkadapted cells are exposed to light.Heterotrophically grown cells incubated with d-[6-³H,6-¹⁴C]glucose and d-[1-³H,6-¹⁴C]glucose (2 h in dark followed by 15 h light) produced labeled AA with significantly different ratios of ³H and ¹⁴C. Comparisons of labeling patterns in AA and chitin-derived d-glucosamine support a path of conversion in Cyclotella from d-glucose to AA that inverts the carbon chain of the sugar. This process resembles similar conversions found in AA-synthesizing animals and species from two other algal classes.Abbreviations AA l-Ascorbic acid - glc d-glucose - glcN d-glucosamine     

Partial purification of the Na+-dependentd-glucose transport system from renal brush border membranes

Summary A membrane extract enriched with the Na⁺-dependentd-glucose transport system was obtained by differential cholate solubilization of rat renal brush border membranes in the presence of 120mm Na⁺ ions. Sodium ions were essential in stabilizing the transport system during cholate treatment. This membrane extract was further purified with respect to its Na⁺-coupledd-glucose transport activity and protein content by the use of asolectin-equilibrated hydroxylapatite. The reconstituted proteoliposomes prepared from this purified fraction showed a transient accumulation ofd-glucose in response to a Na⁺ gradient. The observed rate of Na⁺-coupledd-glucose uptake by the proteoliposomes represented about a sevenfold increase as compared to that of the reconstituted system derived from an initial 1.2% cholate extract of the membranes. Other Na⁺-coupled transport systems such asl-alanine, -ketoglutarate and phosphate were not detected in these reconstituted proteoliposomes.     

Differences in the release ofl-glutamate andd-aspartate from primary neuronal chick cultures

Primary neuronal cultures were made from eight-day-old embryonic chick telencephalon. Ten-day-old cultures were used to study the release ofd-[³H]aspartate andl-[³H]glutamate. Thed-[³H]aspartate release was stimulated by increasing potassium concentrations, but it was not calcium dependent. In contrast, the potassium dependentl-[³H]glutamate release was calcium dependent, and furthermorel-[³H]glutamate release was optimal at potassium concentrations<30 mM. The inhibitors of glutamate uptake, dihydrokainate and 1-aminocyclobutane-trans-1,3-dicarboxylic acid (CACB), also referred to as cis-1-aminocyclobutane-1,3-dicarboxylate, were used in the release experiments. Dihydrokainate had no effect on aspartate release, whereas CACB increased both the basal efflux ofd-[³H]aspartate and the potassium evoked release. CACB had no effect on the potassium stimulatedl-glutamate release. We believe thatl-glutamate is released mainly by a vesicular mechanism from the presumably glutamatergic neurons present in our culture.d-aspartate release observed by us, could be mediated by a transporter protein. The cellular origin of this release remains to be assessed.     

Intracellular potassium activity and the role of potassium in transepithelial salt transport in the human reabsorptive sweat duct

Summary We have measured the intracellular potassium activity, [K⁺]_i and the mechanisms of transcellular K⁺ transport in reabsorptive sweat duct (RSD) using intracellular ion-sensitive microelectrodes (ISMEs). The mean value of [K⁺]_i in RSD is 79.8±4.1mm (n=39). Under conditions of microperfusion, the [K⁺]_i is above equilibrium across both the basolateral membrane, BLM (5.5 times) and the apical membrane, APM (7.8 times). The Na⁺/K⁺ pump inhibitor ouabain reduced [K⁺]_i towards passive distribution across the BLM. However, the [K⁺]_i is insensitive to the Na⁺/K⁺/2 Cl^– cotransport inhibitor bumetanide in the bath. Cl^– substitution in the lumen had no effect on [K⁺]_i. In contrast, Cl^– substitution in the bath (basolateral side) depolarized BLM from –26.0±2.6 mV to –4.7^*±2.4 mV (n=3;^* indicates significant difference) and decreased [K⁺]_i from 76.0±15.2mm to 57.7^* ±12.7mm (n=3). Removal of K⁺ in the bath decreased [K⁺]_i from 76.3±15.0mm to 32.3^*±7.6mm (n=4) while depolarizing the BLM from –32.5±4.1 mV to –28.3^*±3.0 mV (n=4). Raising the [K⁺] in the bath by 10-fold increased [K⁺]_i from 81.7±9.0mm to 95.0^*±13.5mm and depolarized the BLM from –25.7±2.4 mV to –21.3^*±2.9 mV (n=4). The K⁺ conductance inhibitor, Ba²⁺, in the bath also increased [K⁺]_i from 85.8±6.7mm to 107.0^*±11.5mm (n=4) and depolarized BLM from –25.8±2.2 mV to –17.0^*±3.1 mV (n=4). Amiloride at 10^–6 m increased [K⁺]_i from 77.5±18.8mm to 98.8^*±21.6mm (n=4) and hyperpolarized both the BLM (from –35.5±2.6 mV to –47.8^*±4.3 mV) and the APM (from –27.5±1.4 mV to –46.0^* ±3.5 mV,n=4). However, amiloride at 10^–4 m decreased [K⁺]_i from 64.5±0.9mm to 36.0^*±9.9mm and hyperpolarized both the BLM (from –24.7±1.4 mV to –43.5^*±4.2 mV) and APM (from –18.3±0.9 mV to –43.5^*±4.2 mV,n=6). In contrast to the observations at the BLM, substitution of K⁺ or application of Ba²⁺ in the lumen had no effect on the [K⁺]_i or the electrical properties of RSD, indicating the absence of a K⁺ conductance in the APM. Our results indicate that (i) [K⁺]_i is above equilibrium due to the Na⁺/K⁺ pump; (ii) only the BLM has a K⁺ conductance; (iii) [K⁺]_i is subject to modulation by transport status; (iv) K⁺ is probably not involved in carrier-mediated ion transport across the cell membranes; and (v) the RSD does not secrete K⁺ into the lumen.     

Structural characteristics of the saxitoxin receptor on nerve

Summary The effects of uranyl ion (UO ₂ ²⁺ ; at low concentrations binds specifically to phosphate groups) and the cationic dye methylene blue (MB⁺; binds strongly to carboxyl groups) on saxitoxin (STX) potency in crayfish axon has been studied by means of intracellular microelectrodes. At pH 6.00±0.05 and 13.5mm Ca²⁺, addition of 10.0 m UO ₂ ²⁺ +5.0nm STX had only slightly, if any, less effect on the spike's maximum rate of rise [0.79±0.04 (viz., mean±sem) of control value] than did addition of 5.0nm STX alone (0.72±0.05). Under the same conditions of pH and Ca²⁺ concentration, 1.0mm MB⁺ had approximately the same effect: 1.0mm MB⁺+5.0nm STX, 0.76±0.03; 5.0nm STX alone, 0.70±0.04. However, at pH 7.00±0.05 and lower Ca²⁺ concentrations, 1.0mm MB⁺ significantly reduced STX potency. Using 6.0mm Ca²⁺: 1.0mm MB⁺+5.0nm STX, 0.92±0.01; 5.0nm STX alone, 0.68±0.08. Using 3.0mm Ca²⁺, the corresponding values were 0.94±0.03 and 0.67±0.04. It is concluded that: (1) In accord with previous suggestions, the ionized acidic group known to exist in the Na channel (and to which a guanidinium group of STX appears to bind) is very likely a carboxyl group and not a phosphate group. (2) The accessible part of the Na channel mouth serving as the saxitoxin receptor probably does not include phospholipid in its structure proper.     

The localized incorporation of 3H-L-fucose into cell-wall polysaccharides of the cap and epidermis of corn roots

Summary When ³H-L-fucose is provided to corn roots, a large proportion of the radioactivity is recovered in the polysaccharides extracted from the cell wall. Hydrolysis of this material yields ³H-L-fucose as the sole radioactive product. Two metabolites, identified tentatively as L-fucose-1-phosphate and a nucleoside diphosphate derivative of L-fucose have been isolated from the ethanol soluble fractions of the roots and are possibly precursors of the polysaccharide. Autoradiographs of tissue sections indicate that the synthesis of polysaccharides containing L-fucose is confined largely to the root-cap and epidermis. The outer epidermal wall and root-cap slime are particularly radioactive and, therefore, likely to be relatively rich in fucose. By contrast the cell walls from more deeply lying tissues incorporate negligible amounts of the sugar.     

The cytosolic pathway of l-malic acid synthesis in Saccharomyces cerevisiae: the role of fumarase

Saccharomyces cerevisiae accumulates l-malic acid but only minute amounts of fumaric acid. A ¹³C-nuclear magnetic resonance study following the label from glucose to l-malic acid indicates that the l-malic acid is synthesized from pyruvic acid via oxaloacetic acid. From this, and from previously published studies, we conclude that a cytosolic reductive pathway leading from pyruvic acid via oxaloacetic acid to l-malic acid is responsible for the l-malic acid production in yeast. The non-production of fumaric acid can be explained by the conclusion that, in the cell, cytosolic fumarase catalyzes the conversion of fumaric acid to l-malic acid but not the reverse. This conclusion is based on the following findings. (a) The cytosolic enzyme exhibits a 17-fold higher affinity towards fumaric acid than towards l-malic acid; the K _m for l-malic acid is very high indicating that l-malic acid is not an in vivo substrate of the enzyme. (b) Overexpression of cytosolic fumarase does not cause accumulation of fumaric acid (but rather more l-malic acid). (c) According to ¹³C NMR studies there is no interconversion of cytosolic l-malic and fumaric acids.     

Accumulation,elimination, release and metabolism of pipecolic acid in the mouse brain following intraventricular injection

Following i.c.v. (intracerebral ventricular) injections ofd,l-[³H]pipecolic acid (PA), it is reabsorbed from the ventricles and redistributed to various brain regions. The highest accumulation is found in three brain regions ipsilateral to the injection site, hippocampus, neocortex, striatum, and in the diencephalon. Following preloading in vivo, the radioactivity is released from hippocampus slices in the perfusion medium after depolarization induced by high K⁺. During perfusion with a Ca⁺⁺ free medium containing EGTA, a significant reduction of release is observed.The radioactivity ofd,l-[³H]PA in the brain shows a more rapid phase of decrease from 0 to 2 hours and a slower phase from 2 to 5 hours. At 5 hours, only 28% radioactivity, represented mainly by PA, is left in the brain. Kidney secretion represents the major route of elimination of the injected PA. The presence of -aminoadipic acid both in brain and urine was observed. Probenecid (200 mg/kg) significantly increases the accumulation of i.c.v. injectedd,l-[³H]PA in brain and kidney. The presence of a regional accumulation of PA in certain brain regions, its metabolism in brain, its enhanced retention following probenecid administration and its Ca⁺⁺ dependent release following high K⁺ stimulation, all constitute indirect evidence for a neuronal localization of this brain endogenous iminoacid.     

Ion and water balance in isolated epithelial cells of the abdominal skin of the frogLeptodactylus ocellatus

Summary Isolated epithelial cells were obtained from abdominal skin of the frogLeptodactylus ocellatus by a trypsination-dissection method. As estimated by nigrosin staining, the amount of damaged cells is only 6.6±0.7 per cent. When washed briefly after incubation the ionic concentrations in these cells were (mm): K⁺ 14.20±4.0; Na⁺ 15.8±1.8; and Cl^– 57.2±5.3. If they are not washed, the concentration of K⁺ remains essentially the same (131.2±1.4mm) but the Na⁺ concentration is much higher (38.5±0.9mm). It is shown that a large fraction of Na⁺ is contained in a compartment that is freely connected with the bathing solution. Ouabain (10^–4 m) elicits a marked decrease of K⁺, a slight decrease of Cl^–, and an increase of Na⁺ content. In an equal period, low temperature (3°C) produces a similar effect, although less marked than ouabain.     

ATP-sensitive potassium channels in adult mouse skeletal muscle: Characterization of the ATP-binding site

Summary Single K⁺-selective channels were studied in excised inside-out membrane patches from dissociated mouse toe muscle fibers. Channels of 74 pS conductance in symmetrical 160mm KCl solutions were blocked reversibly by 10 m internal ATP and thus identified as ATP-sensitive K⁺ channels. The channels were also blocked reversibly bymm concentrations of internal adenosine, adenine and thymine, but not by cytosine and uracil. The efficacy of the reversible channel blockers was higher when they were present in internal NaCl instead of KCl solutions. An irreversible inhibition of ATP-sensitive K⁺ channels was observed after application of several sulphydryl-modifying substances in the internal solution: 0.5mm chloramine-T, 50mm hydrogen peroxide or 2mm n-ethylmaleimide (NEM). Largeconductance Ca-activated K⁺ channels were not affected by these reagents. The presence of 1mm internal ATP prevents the irreversible inhibition of ATP-sensitive K⁺ channels by NEM. The results suggest that internal Na⁺ ions increase the affinity of the ATP-sensitive K⁺ channel to ATP and to other reversible channel blockers and that a functionally important SH-group is located at or near the ATP-binding site.     

Calcium-Sensitive Nonselective Cation Channel Identified in the Epithelial Cells Isolated from the Endolymphatic Sac of Guinea Pigs

We identified a Ca²⁺-sensitive cation channel in acutely dissociated epithelial cells from the endolymphatic sac (ES) of guinea pigs using the patch-clamp technique. Single-channel recordings showed that the cation channel had a conductance of 24.0 ± 1.3 pS (n= 8) in our standard solution. The relative ionic permeability of the channel was in the order K⁺= Na⁺ > Ca²⁺≫ Cl⁻. This channel was weakly voltage-dependent but was strongly activated by Ca²⁺ on the cytosolic side at a concentration of around 1 mm in inside-out excised patches. With cell-attached patches, however, the channel was activated by much lower Ca²⁺ concentrations. Treatment of the cells, under cell-attached configuration, with ionomycin (10 μm), carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 20 μm), or ATP (1 mm), which increased intracellular Ca²⁺ concentration ([Ca²⁺]_i), activated the channel at an estimated [Ca²⁺]_i from 0.6 μm to 10 μm. It is suggested that some activators of the channel were deteriorated or washed out during the formation of excised patches. Based on this Ca²⁺ sensitivity, we speculated that the channel contributes to the regulation of ionic balance and volume of the ES by absorbing Na⁺ under certain pathological conditions that will increase [Ca²⁺]_i. This is the first report of single-channel recordings in endolymphatic sac epithelial cells. Received: 24 October 2000/Revised: 10 April 2001     

A Metal Ion as a Cofactor Attenuates Substrate Inhibition in the Enzymatic Production of a High Concentration of <Emphasis Type="SmallCaps">D</Emphasis>-glutamate Using <Emphasis Type="Italic">N</Emphasis>-acyl-<Emphasis Type="SmallCaps">D</Emphasis>-glutamate Amidohydrolase

N-Acyl-D-glutamate amidohydrolase (D-AGase) was inhibited by 94 % when 1 mol/l N-acetyl-DL- glutamate was used as a substrate. The addition of 1 mM Co²⁺ stabilized D-AGase. Moreover, the substrate inhibition was weakened to 88% with the addition of 0.4 mM Co²⁺ to the reaction mixture. Although D-AGase is a zinc-metalloenzyme, the addition of Zn²⁺ from 0.01 to 10 mM did not increase the D-glutamic acid production in the saturated substrate. Under optimal conditions, 0.38 M D-glutamic acid was obtained from N-acyl-DL-glutamate with 100% of the theoretical yield after 48 h.     

In vitro study of the antioxidative properties of the glucose derivatives against oxidation of plasma components

Oxidative stress has been implicated in the pathogenesis of variety of diseases. Since the endogenous antioxidant defense may be not adequate to counteract the enhanced generation of oxidants, a growing interest in research for exogenous nutrients has been observed. The present study was designed to assess in vitro the antioxidative properties of the glucose derivatives: calcium d-glucarate, d-gluconic acid lactone, and sodium d-gluconate (0.5–3 mM) in the protection of plasma proteins and lipids, against the damage caused by 0.1 mM peroxynitrite (ONOO⁻). Exposure of plasma to ONOO⁻ resulted in carbonyl groups increase, 3-nitrotyrosine (3-NT) formation, reduction in thiol groups, and enhanced lipid peroxidation. d-Gluconic acid lactone and sodium d-gluconate effectively decreased 3-NT formation; the antinitrative action of calcium d-glucarate was less effective. In plasma samples incubated with ONOO⁻ and tested compounds, the level of carbonyl groups was decreased in comparison to plasma samples treated only with ONOO⁻. The level of protein −SH groups and glutathione was significantly higher in the presence of glucose derivatives than in plasma samples treated with ONOO⁻ only. All the tested compounds had the inhibitory effect on the peroxynitrite-induced plasma lipids peroxidation. The results obtained from our work indicate that calcium d-glucarate, d-gluconic acid lactone, and sodium d-gluconate may partly protect plasma proteins and lipids against peroxynitrite-induced damages.     

Substrate specificity of a recombinant ribose-5-phosphate isomerase from <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> and its application in the production of <Emphasis Type="SmallCaps">l</Emphasis>-lyxose and <Emphasis Type="SmallCaps">l</Emphasis>-tagatose

A putative ribose-5-phosphate isomerase (RpiB) from Streptococcus pneumoniae was purified with a specific activity of 26.7 U mg⁻¹ by Hi-Trap Q HP anion exchange and Sephacryl S-300 HR 16/60 gel filtration chromatographies. The native enzyme existed as a 96-kDa tetramer with activity maxima at pH 7.5 and 35°C. The RpiB exhibited isomerization activity with l-lyxose, l-talose, d-gulose, d-ribose, l-mannose, d-allose, l-xylulose, l-tagatose, d-sorbose, d-ribulose, l-fructose, and d-psicose and exhibited particularly high activity with l-form monosaccharides such as l-lyxose, l-xylulose, l-talose, and l-tagatose. With l-xylulose (500 g l⁻¹) and l-talose (500 g l⁻¹) substrates, the optimum concentrations of RpiB were 300 and 600 U ml⁻¹, respectively. The enzyme converted 500 g l⁻¹ l-xylulose to 350 g l⁻¹ l-lyxose after 3 h, and yielded 450 g l⁻¹ l-tagatose from 500 g l⁻¹ l-talose after 5 h. These results suggest that RpiB from S. pneumoniae can be employed as a potential producer of l-form monosaccharides.     

Neuropharmacological analysis of synaptic transmission in the Lorenzinian ampulla of the skate Raja clavata

Summary Dissected ampullae of Lorenzini of the skate (Raja clavata) were studied with the aim of determining the synaptic transmitter between electroreceptor cell and afferent fibre. Resting activity and stimulus-evoked activity in response to electrical pulses were recorded in single afferent units at constant perfusion with normal and test solutions containing different putative neurotransmitters. Presynaptic transmitter release was blocked by Mg²⁺ (up to 50 mM) to investigate the effects of the test substances upon the postsynaptic membrane. l-Glutamate (l-GLU) and l-aspartate (l-ASP), both at concentrations between 10^-7 and 10^-3 M, enlarged strongly resting and stimulus-evoked discharge frequency in the afferent fibre. If transmission was blocked by high Mg²⁺, resting discharge frequency could be restored by l-GLU or l-ASP. The glutamate agonists quisqualate (10^-8–10⁵ M) and N-methyl-D-aspartate (10^-5–10^-3 M) enlarged spontaneous activity in the afferent fiber. The same was found for kainic acid (10^-9–10^-5 M). Taurine at concentrations between 10^-5 and 10^-3 M caused a concentration-dependent decrease in afferent activity. The same was found for gammaaminobutyric acid (GABA; 10^-5–10^-4 M), and for the catecholamines adrenaline and noradrenaline, both in concentrations between 10^-5 and 10^-3 M. Serotonine (10^-5–10^-3 M) and dopamine (10^-5-10^-3 M) had no effect on resting or evoked activity in the Lorenzinian ampulla afferents. Acetylcholine (ACh; 10^-4 M) enlarged discharge frequency in those units with initial rates lower than 22–25 Hz, but diminished discharge frequency in fibres with initial activity higher than 25 Hz. When synaptic transmission was blocked by high Mg²⁺ solution, perfusion with additional ACh did not restore resting activity in the afferent fibre. The results suggest that the most probable transmitter in the afferent synapse of the ampullae of Lorenzini is l-GLU or l-ASP, or a substance of similar nature.Abbreviations ACh acetylcholine - GABA gamma aminobutyric acid - KA kainic acid - l-ASP l-aspartate - l-GLU l-glutamate - NMDA N-methyl-D-aspartate - Q quisqualate - n.s. normal solution     

Amino acid transport system y+L of human erythrocytes: Specificity and cation dependence of the translocation step

The transport specificity of system y⁺L of human erythrocytes was investigated and the carrier was found to accept a wide range of amino acids as substrates. Relative rates of entry for various amino acids were estimated from their trans-effects on the unidirectional efflux of l-[¹⁴C]-lysine. Some neutral amino acids, l-lysine and l-glutamic acid induced marked trans-acceleration of labeled lysine efflux; saturating concentrations of external l-leucine and l-lysine increased the rate by 5.3±0.63 and 6.2±0.54, respectively. The rate of translocation of the carrier-substrate complex is less dependent on the structure of the amino acid than binding. Translocation is slower for the bulkier analogues (l-tryptophan, l-phenylalanine); smaller amino acids, although weakly bound, are rapidly transported (l-alanine, l-serine). Half-saturation constants (±sem) calculated from this effect (l-lysine, 10.32±0.49 m and l-leucine, 11.50±0.50 m) agreed with those previously measured in cis-inhibition experiments. The degree of trans-acceleration caused by neutral amino acids did not differ significantly in Na⁺, Li⁺ or K⁺ medium, whereas the affinity for neutral amino acids was dramatically decreased if Na⁺ or Li⁺ were replaced by K⁺. The observation that specificity is principally expressed in substrate binding indicates that the carrier reorientation step is largely independent of the forces of interaction between the carrier and the transport site.We wish to thank Dr C.A.R. Boyd for helpful discussions and Prof. H.N. Christensen for sharing with us very relevant bibliographic material. We are grateful to FONDECYT (1282/91) and DTI (B 2674) (Chile) for financial assistance.