Functional and biochemical characteristics of a putative quisqualate-type receptor in rat striatum: Effect of brain lesions

Excitatory amino acids such asl-glutamate (Glu) and quisqualate (QUIS) markedly potentiated K⁺-evoked release of exogeneous [³H]dopamine (DA) from rat striatal slices. Intranstriatal kainic acid injections resulted in a total disappearance of the stimulatory effects of Glu on evoked-release of [³H]DA as well as in a parallel reduction in the maximal number (B_max) of ad-aspartate-insensitivel-[³H]Glu binding site in striatal particulate fractions. Following cortical ablation, the potentiating effect of Glu on [³H]DA release in decorticated striatal slices lasted longer, compared to normal slices, and occured during the 2nd min following K⁺-depolarization. However, the extent (%) of Glu stimulation on [³H]DA release remained the same in decorticated and normal striatal slices. Cortical ablation produced also a significant decrease in the B_max and in theK _d of thed-aspartateinsensitive binding site towardsl[³H]Glu. These results support the proposal that thed-aspartate-insensitive Glu binding site is somehow related to an amino acid receptor-mediated modulation of dopaminergic transmission in the rat corpus striatum.     

Regulation of [<Superscript>3</Superscript>H] <Emphasis Type="SmallCaps">d</Emphasis>-Aspartate Release from Mammalian Isolated Retinae by Hydrogen Sulfide

Hydrogen sulfide (H₂S), can produce pharmacological effects on neural and non-neural tissues from several mammalian species. The present study investigates the pharmacological action of H₂S, (using sodium hydrosulfide, NaHS, and/or sodium sulfide, Na₂S as donors) on amino acid neurotransmission (using [³H] d-aspartate as a marker for glutamate) from isolated, superfused bovine and porcine retinae. Isolated neural retinae were incubated in Krebs solution containing [³H] d-aspartate at 37°C. Release of [³H] d-aspartate was elicited by high potassium (K⁺ 50 mM) pulse. Both NaHS and Na₂S donors caused an inhibition of K⁺-evoked [³H] d-aspartate release from isolated bovine retinae without affecting basal [³H] d-aspartate efflux yielding IC₅₀ values of 0.006 and 6 μm, respectively. Furthermore, NaHS inhibited depolarization-evoked release of [³H] d-aspartate from isolated porcine retinae with an IC₅₀ value of 8 μM. The inhibitory action of NaHS on [³H] d-aspartate release from porcine retinae was blocked by propargyglycine, a selective inhibitor of cystathionine γ-lyase (CSE). Our results indicate that H₂S donors can inhibit amino acid neurotransmission from both isolated bovine and porcine retinae, an effect that is dependent, at least in part, on intramural biosynthesis of H₂S.     

Characterization of quisqualate recognition sites in rat brain tissue usingDl-[3H]α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and a filtration assay

The binding of [³H]AMPA (Dl--amino-3-hydroxy-5-methylisoxazole-4-propionic acid), a ligand for the putative quisqualate excitatory amino acid receptor subtype, was evaluated using centrifugation and filtration receptor binding techniques in rat brain crude synaptosomal membrane preparations. Maximal specific binding of [³H]AMPA occurred in Triton X-100 treated membranes in the presence of the chaotropic agent potassium thiocyanate (KSCN). The effects of KSCN on binding were reversible and optimal at 100 mM. Supernatant obtained from detergent-treated membranes inhibited specific [³H]AMPA and [³H]kainic acid binding, suggesting the presence of an inhibitory agent which was tentatively identified as glutamate. Using centrifugation, saturation analysis revealed two distinct binding sites in both the absence and presence of KSCN. The chaotrope was most effective in increasing binding at the low affinity binding site, enhancing the affinity (K _d) without a concommitant change in the total number of binding sites. Using filtration, a single binding site was detected in Triton-treated membranes. Like the data obtained by centrifugation, KSCN enhanced the affinity of the receptor (K _d value=10 nM) without altering the number of binding sites (B _max=1.2 pmol/mg protein). The rank order of potency of various glutamate analogs in the [³H]AMPA binding assay was quisqualate > AMPA > l-glutamate > kainate > d-glutamate, consistent with the labeling of a quisqualate-type excitatory amino acid receptor subtype.l-glutamic acid diethylester, and 2-amino-7-phosphonoheptanoic acid (AP₇) were inactive. The present technique provides a rapid, reliable assay for the evaluation of quisqualate-type excitatory amino acid agonists and/or antagonists that may be used to discover more potent and selective agents.     

Preparation and reactivity studies of synthetic microperoxidases containing<Emphasis Type="Italic"> b</Emphasis>-type heme

In order to create a heme environment that permits biomimicry of heme-containing peroxidases, a number of new hemin–peptide complexes—hemin-2(18)-glycyl-l-histidine methyl ester (HGH), hemin-2(18)-glycyl-glycyl-l-histidine methyl ester (HGGH), and hemin-2,18-bis(glycyl-glycyl-l-histidine methyl ester) (H2GGH)—have been prepared by condensation of glycyl-l-histidine methyl ester or glycyl-glycyl-l-histidine methyl ester with the propionic side chains of hemin. Characterization by means of UV/vis- and ¹H NMR spectroscopy as well as cyclic- and differential pulse voltammetry indicates the formation of five-coordinate complexes in the case of HGH and HGGH, with histidine as an axial ligand. In the case of H2GGH, a six-coordinate complex with both imidazoles coordinated to the iron center appears to be formed. However, ¹H NMR of H2GGH reveals the existence of an equilibrium between low-spin six-coordinate and high-spin five-coordinate species in solution. The catalytic activity of the hemin–peptide complexes towards several organic substrates, such as p-cresol, l-tyrosine methyl ester, and ABTS, has been investigated. It was found that not only the five-coordinate HGH and HGGH complexes, but also the six-coordinate H2GGH, catalyze the oxidation of substrates by H₂O₂. The longer and less strained peptide arm provides the HGGH complex with a slightly higher catalytic efficiency, as compared with HGH, due to formation of more stable intermediate complexes.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0532-5.Abbreviations ABTS 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) - DCC dicyclohexylcarbodiimide - HGH hemin-2(18)-glycyl-l-histidine methyl ester - HGGH hemin-2(18)-glycyl-glycyl-l-histidine methyl ester - H2GGH hemin-2,18-bis(glycyl-glycyl-l-histidine methyl ester) - HOBt N-hydroxybenzotriazole     

On the activity of γ-aminobutyric acid and glutamate transporters in chick embryonic neurons and rat synaptosomes

The uptake of radioactive -aminobutyric acid (GABA) andd-aspartate and the effect of SKF 89976-A, a non-substrate inhibitor of the GABA transporter, on this uptake have been investigated. Neuronal cultures from eight-day-old chick embryos grown for three or six days in vitro, were used as a model. For comparison, we also used the P₂-fraction from rat. Neuronal cultures grown for three and six days expressed high-affinity uptake systems for [³H]GABA and ford-[³H]aspartate with an increasing V_max during this period. The lipophilic non-substrate GABA uptake inhibitor, SKF 89976-A, inhibited transporter mediated uptake of GABA both in cell cultures from chicken, and in P₂-fractions from rat. The results also showed that SKF 89976-A was a poor inhibitor of the uptake ofd-aspartate. We found no non-saturable uptake ofd-aspartate.     

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.     

Inhibition of endothelial cell morphogenetic interactions in vitro by alpha- and beta-xylosides

Summary Bovine aortic endothelial cells retain the ability to undergo histotypic morphogenetic interactions in vitro as evidenced by a) the reversible expression of a sprouting cell phenotype and b) the patterned self-association of these sprouting cells into three-dimensional meshworks and tubule-like structures. These morphogenetic events are inhibited by xylosides in a dose-dependent manner. Two types of beta-xylosides (p-nitrophenyl-beta-d-xylopyranoside and 4-methylumbelliferyl-beta-d-xylopyranoside) and one alpha-xyloside (p-nitrophenyl-alpha-d-xylopyranoside) were tested. Beta-xylosides are well characterized acceptors of glycosaminoglycan chains, whereas alpha-xylosides do not function in this capacity and have been extensively used as negative controls when studying the effects of beta-xylosides. Both alpha-and beta-xylosides inhibited endothelial morphogenetic interactions. This inhibition was slowly reversed during the 6- to 7-d period following removal of the xyloside. Inhibition of morphogenetic interactions by xylosides occurred at concentrations (0.5 to 2.0 mM) that had no demonstrable effects on cell proliferation, migration, or adhesion to 2-D plastic or collagen substrata. The xylosides seemed to inhibit cell spreading on a 3-D environment, they also inhibited the incorporation of [³H]-proline and Na₂ ³⁵SO₄ into the extracellular matrix deposited by the cells, suggesting that the inhibition of morphogenesis may be related to the inhibition of matrix deposition. Endothelial morphogenetic interactions were not inhibited by the extracellular matrix or by the conditioned medium produced by cells cultured in the presence of xylosides.     

Cyclic AMP-dependent stimulation of Na,K-ATPase in shark rectal gland

Summary Scatchard analysis of³H ouabain bound to isolated rectal gland cells as a function of increasing ouabain concentrations produced a concave curvilinear plot that was resolved into two specific sites with either a high (I) or low (II) affinity for ouabain. Cyclic cAMP/theophylline (±furosemide, 10^–4 m) increased the amount of³H ouabain bound to the high-affinity site I. Vanadate, a phosphate congener which promotes formation of the ouabain-binding state of the enzyme, mimicked the effects of cAMP/theophylline at low concentrations of ouabain, suggesting that cAMP/theophylline increases binding to site I by enhancing the rate of turnover of resident enzyme. Enhanced⁸⁶Rb uptake seen following cAMP/theophylline administration was primarily associated with increased flux through the high-affinity ouabain site, and this stimulation was not obliterated by the co-administration of furosemide. A model was presented which suggested the presence of two noninteracting pools of enzyme or isozymes which exhibit either a high or low affinity for ouabain. Cyclic AMP both stimulated turnover via site I, and modified the kinetics of binding of³H ouabain to site II. The (ave)K _d of³H ouabain for site II was increased from 3.6 m (controls) to 0.5 m (cAMP/theophylline) and the Hill coefficient was modified from 0.45 (controls) to 1.12 (caMP/theophylline), suggesting a transition from a negative- to a noncooperative binding state. While furosemide reversed the effects of cAMP/theophylline on site II kinetics, it did not obliterate cAMP/theophylline effects on site I. This suggests that cAMP may alter the intrinsic turnover rate of this particular pool of Na,K-ATPase in shark rectal gland.     

Effects of spinal transection on presynaptic markers for glutamatergic neurons in the rat

To evaluate the hypothesis that glutamic acid may be the neurotransmitter of descending, excitatory supraspinal pathways, the uptake and release ofl-[³H] glutamate and the levels of endogenous glutamate were measured in preparations from rat lumbar spinal cord following complete mid-thoracic transection. Following transection, the activity of the synaptosomal high-affinty glutamate uptake process was increased in both dorsal and ventral halves of lumbar cord between 1 and 14 days after transection and returned to control levels by 21 days posttransection. At 7 days, the increased activity of the uptake process forl-[³H] glutamate resulted in elevation ofV _max with no significant alteration inK _t as compared to age-matched controls. Depolarization-induced release ofl-[³H]glutamate from prelabeled slices did not differ significantly from control in the lesioned rat except at 21 days after lesion when the amount of tritium release was significantly greater in the transected preparations than in control. Amino acid analysis of the lumbar cord from control and transected rats indicated only a 10% decrease in the level of endogenous glutamate and no alterations in the concentration of GABA and glycine 7 days after lesion. These findings do not support the hypothesis that glutamate serves as a major excitatory neurotransmitter in supraspinal pathways innervating the lumbar cord of the rat.     

Effects of ammonia onl-glutamate uptake in cultured astrocytes

The effect of ammonia onl-glutamate (L-GLU) uptake was examined in cultured astrocytes. Acute ammonia treatment (5–10 mM) enhanced L-[³H]GLU uptake by 20–42% by increasing the V_max; this persisted for 2 days and then started to decline. Ammonia, however, did not affect the uptake ofd-[³H]aspartate (D-ASP), a non-metabolizable analog of L-GLU, that uses the same transport carrier as L-GLU. Also, L-GLU uptake was not affected during the first 2 min of the assay. Thus, ammonia did not have an acute effect on L-GLU transport (translocation); rather, ammonia enhanced the accumulation or “trapping” of L-GLU or its by-products. Chronic ammonia treatment, on the other hand, inhibited L-GLU transport in astrocytes by ∼30–45% and this was due to a decrease in V_max, suggesting that the number of L-GLU transporters was decreased. This inhibitory effect was observed after 1 day of treatment and persisted for at least 7 days. The inhibition of L-GLU transport was partially reversible following removal of ammonia. The effects of ammonia on L-GLU transport and uptake may explain the abnormal L-GLU neurotransmission observed in hyperammonemia/hepatic encephalopathy, and the brain swelling associated with fulminant hepatic failure.     

[3H]2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalen (ADTN)

The regional distribution and in vivo binding of the dopamine analog 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalen (ADTN) was studied in the brain. The highest density of binding sites was in the striatum, with virtually no binding in the cerebellum. The binding of [³H]ADTN reflects an occupation of specific dopamine sites because the binding was diminished by the simultaneous administration of the dopamine antagonist haloperidol or the dopamine precursorl-3,4-dihydroxyphenylalanine (l-dopa). Chronic administration of haloperidol orl-dopa prior to assaying for in vivo binding resulted in an increase in the number of sites for [³H]ADTN which correlates to the increase observed in in vitro assays following long-term treatment with these agents. The subcellular distribution of in vivo labeled ADTN sites in the caudate nucleus indicate a high density of specific binding sites in the microsomal fraction, P₃. Overall, these data demonstrate that the aminotetralins, such as ADTN, which bind with high affinity to the dopamine receptor in the caudate nucleus in vitro and in vivo, can provide precise information on the topography of this receptor.     

Baclofen-induced,calcium-dependent stimulation of in vivo release ofd-[3H]aspartate from rat hippocampus monitored by intracerebral microdialysis

The release ofd-[³H]aspartate (used as a tracer for endogenous glutamate and aspartate) was studied at high K⁺ (100 mM) and under ischemia in rats implanted with 0.3 mm diameter dialysis tubing through the hippocampus. The effect on thed-[³H]aspartate release of the two -aminobutyric acid (GABA) agonists 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol (THIP) and (±)--(p-chlorophenyl)GABA (baclofen), which specifically activate GABA_A and GABA_B receptors, respectively, was studied. Initial experiments employing HPLC analysis showed a coincident increase in the amounts of glutamate, aspartate and the amount of radioactivity following introduction of K⁺ (100 mM) or a period of ischemia suggesting that thed-[³H]aspartate labels the transmitter pools of the two amino acids under the present experimental conditions. The presence of 10 mM baclofen or 10 mM THIP in the perfusion medium did not inhibit ischemia inducedd-[³H]aspartate release. On the contrary, 10 mM baclofen alone (but not 0.1 or 1 mM) in the perfusion medium induced release ofd-[³H]aspartate in a calcium dependent manner, whereas 10 mM THIP had no significant releasing effect.Special issue dedicated to Dr. Elling Kvamme     

Differential effect of ethanol on muscarinic cholinergic binding to rat and locust neural membranes

We have compared the effect of ethanol, a membrane perturbant, on the muscarinic binding sites in neural membranes from a vertebrate (rat) and an insect (locust). The binding of the muscarinic antagonist [³H]quinuclidinyl benzilate ([³H]QNB) to both rat and locust neural membranes was inhibited by ethanol at 10–500 mM concentrations; but this inhibition was greater in the locust. Ethanol (500 mM) increased the apparent dissociation constant (K d) of [³H]QNB binding to rat membranes from 0.13±0.01 nM in control to 0.20±0.02 nM; there was also an small but significant reduction in the number of binding sitesB _max. In locust, 500 mM ethanol reduced theB _max of [³H]QNB binding from 590±30 in control to 320±40 pmol/g protein; no significant alteration in theK _D was detected. The dissociation rate constant (k _off) of [³H]QNB increased from 0.020±0.003 in controls to 0.031±0.004 (min^–1) in the presence of 500mM ethanol, the association rate constant (k _on) did not change significantly. In locust, 500 mM ethanol did not affect eitherk _on ork _off. Competition experiments revealed that the binding affinities of both the agonist carbamylcholine and the antagonist atropine to the rat membranes were reduced in the presence of ethanol. In contrast, ethanol caused no alteration in the binding affinities of these ligands to the locust membranes. This differential effect of ethanol on rat and locust muscarinic binding suggests a difference in the hydrophobic domains and/or the membrane interactions of the muscarinic receptors in the two species.     

Effects of various metabolites on two phosphoglucomutase allozyme activities from Drosophila melanogaster

The effects of various metabolites on the two most common phosphoglucomutase allozymes (PGM^A and PGM^B) in Drosophila melanogaster have been investigated in vitro. 2,3-Diphosphoglycerate (2,3DPG) inhibited PGM^A and PGM^B to the same degree in the presence of 25 µM glucose-1,6-diphosphate (G1, 6P₂). However a higher concentration of G1,6P₂ partially reversed the inhibition of PGM^A exerted by 2,3DPG, so that in the presence of 150 µM G1,6P₂ the inhibition of PGM^A was half that of PGM^B at pH 6.0. Glycerol-3-phosphate (G3P) had no significant effect at pH 7.4 but exerted an activating effect at pH 6.0 which was more pronounced in the case of PGM^B. ATP, citrate, and fructose-1, 6-diphosphate (F1,6P₂) inhibited both PGM^A and PGM^B. The differences found in vitro between these two allozymes can have a significant impact on in vivo function and, therefore, on the maintenance of PGM polymorphism in experimental populations of D. melanogaster studied in the laboratory.     

Specific binding sites for corticosterone in isolated cells and plasma membrane from rat liver

Summary The specific binding of [³H]corticosterone to hepatocytes is a nonsaturable, reversible and temperature-dependent process. The binding to liver purified plasma membrane fraction is also specific, reversible and temperature dependent but it is saturable. Two types of independent and equivalent binding sites have been determined from hepatocytes. One of them has high affinity and low binding capacity (K _D=8.8nm andB _max=1477 fmol/mg protein) and the other one has low affinity and high binding capacity (K _D=91nm andB _max=9015 fmol/mg). In plasma membrane only one type of binding site has been characterized (K _D=11.2nm andB _max=1982 fmol/mg). As it can be deduced from displacement data obtained in hepatocytes and plasma membrane the high affinity binding sites are different from the glucocorticoid, progesterone nuclear receptors and the Na⁺,K⁺-ATPase digitalis receptor. Probably it is of the same nature that the one determinate for [³H]cortisol and [³H]corticosterone in mouse liver plasma membrane. Beta-and alpha-adrenergic antagonists as propranolol and phentolamine did not affect [³H]corticosterone binding to hepatocytes and plasma membranes; therefore, these binding sites are independent of adrenergic receptors. The binding sites in hepatocytes and plasma membranes are not exclusive for corticosterone but other steroids are also bound with very different affinities.     

l-[3H]lysine binding to rat retinal membrane: I. Quantitative determination and characterization of the binding sites

A saturable reversible binding to membranes from rat retina has been found forl-[³H]lysine. Specific binding is time, temperature and protein concentration-dependent, and shows stereospecificity. The best computer fits of the experimental data are obtalned with a receptor medel based on two independent binding sites, of which only one site with a K_d value of 229.4±14.23 nM and a B_max of 2.04 ±0.11 pmol/mg protein could be characterized satisfactorily. Several compounds included putative neurotransmitters have moderate or no affinity forl-lysine binding sites. A different pattern of distribution ofl-[³H]lysine binding sites is observed among various regions of the brain, with the highest density in the occipital cortex, and the lowest density in ponsmedulla.The existence of binding sites in rat retinal membranes forl-lysine, as well as in the areas involved in the visual pathway, suggests a role for this amino acid in the physiological mechanism of the visual function.     

Vergleichende untersuchung des peptonabbaus und der damit verknüpften ciliatenbesiedlung in strömenden und stagnierenden modellgewässern

Summary The decomposition of peptone and the associated species numbers and individual counts of ciliates has been investigated in flowing (current speed 40 cm/sec) and stagnant model ecosystems. In order to imitate the ecological conditions in natural waters receiving sewage small quantities of peptone were added at regular intervals.The most important environmental factors such as pH, dissolved oxygen, NH₄ ⁺-, NO₃ ^--levels, and the organisms occurring in the free water as well as in the periphyton community on microscopic slides, were investigated for three weeks. Both the population dynamics of organisms and the environmental conditions within the ecosystems are figured (Figs. 1–6).The ecosystem with flowing water, and the aerated stagnant model showed high contents of O₂ and fast mineralisation of peptone up to the NO₃ ^--level. The unaerated model showed a retarded decomposition of peptone and accumulation of ammonia.The unaerated stagnant ecosystem showed the highest individual counts of bacteria and ciliates, while in the flowing model a limited number of organisms only was found.Most of the differences between flowing and stagnant waters are due to the varying O₂-level. Only two species were directly inhibited by the current speed tested in these experiments.

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Zoologisches Institut der Universität Bonn Hydrobiologische Arbeitsgruppe (Leiter: Prof. Dr. H. Bick)

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Mit Untersützung der Weltgesundheitsorganisation.

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Herrn Prof. Dr. Rolf Danneel zum 70. Geburtstag gewidmet.

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Anschrift der Verfasser: PProf. Dr. H. Bick und Diol. Biol. W. Schmerenbeck, Hydrobiologische Arbeitsgruppe am Zoologischen Institut der Universität, D05300 Bonn 1, Poppelsdorfer Schlo\.     

Electrophysiological investigation of the amino acid carrier selectivity in epithelial cells fromXenopus embryo

Summary The electrical responses induced by external applications of neutral amino acids were used to determine whether different carriers are expressed in the membrane of embryonic epithelial cells ofXenopus laevis. Competition experiments were performed under voltage-clamp conditions at constant membrane potential.Gly,l-Ala,l-Pro,l-Ser,l-Asn andl-Gln generate electrical responses with similar apparent kinetic constants and compete for the same carrier. They are [Na] _o and voltage-dependent, insensitive to variations in [Cl] _o and [HCO₃] _o , inhibited by pH _o changes, by amiloride and, for a large fraction of the current, by MeAIB. The increase in [K] _o at constant and negative membrane potential reduces the response, whereas lowering [K] _o augments it. l-Leu,l-Phe andl-Pro appear to compete for another carrier. They generate electrogenic responses insensitive to amiloride and MeAIB, as well as to alterations of membrane potential, [Na] _o and [K] _o . Lowering [Cl] _o decreases their size, whereas increasing [HCO₃] _o at neutral pH _o increases it.It is concluded that at least two and possibly three transport systems (A, ASC and L) are expressed in the membrane of the embryonic cells studied. An unexpected electrogenic character of the L system is revealed by the present study and seems to be indirectly linked to the transport function. l-Pro seems to be transported by system A or ASC in the presence of Na and by system L in the absence of Na. MeAIB induces an inward current.     

l-[3H]lysine binding to rat retinal membrane: II. effect of kainic acid,d,l-α-aminoadipic acid,iodoacetic acid,and modification by dark-exposure

The rat retina and the different brain regions contain membranes sites that bindl-lysine in the nanomolar range. These binding sites undergo changes in different experimental conditions, thus: I) intraocular injection of kainic acid induces a reduction of the density ofl-lysine binding sites, II)d,l--aminoadipic acid injected into the eye enhances both kinetic parameters (B _max andK _d) ofl-[³H]lysine binding sites, III) the intraperitoneal injection of iodoacetic acid decreases the sensitivity for its ligand binding sites, and IV) the exposure to darkness of the rats reducesl-[³H]lysine binding in the retina, thalamus, hypothalamus and superior colliculus, but not in the occipital cortex; such a decrease appears to be characterized, at least in the retina, by a lower sensitivity of the binding sites forl-lysine after the exposure to darkness. The results show thatl-lysine binding sites are located on kainic acid-sensitive cells and can be involved in the physiological mechanism of vision.     

Engineering of pyranose 2-oxidase from Peniophora gigantea towards improved thermostability and catalytic efficiency

To improve the stability and catalytic efficiency of pyranose 2-oxidase (P2Ox) by molecular enzyme evolution, we cloned P2Ox cDNA by RACE-PCR from a cDNA library derived from the basidiomycete Peniophora gigantea. The P2Ox gene was expressed in Escherichia coli BL21(DE3), yielding an intracellular and enzymatically active P2OxB with a volumetric yield of 500 units/l. Site-directed mutagenesis was employed to construct the P2Ox variant E540K (termed P2OxB1), which exhibited increased thermo- and pH-stability compared with the wild type, concomitantly with increased catalytic efficiencies (k_cat/K_m) for d-xylose and l-sorbose. P2OxB1 was provided with a C-terminal His₆-tag (termed P2OxB1H) and subjected to directed evolution using error-prone PCR. Screening based on a chromogenic assay yielded the new P2Ox variant K312E (termed P2OxB2H) that showed significant improvements with respect to k_cat/K_m for d-glucose (5.3-fold), methyl--d-glucoside (2.0-fold), d-galactose (4.8-fold), d-xylose (59.9-fold), and l-sorbose (69.0-fold), compared with wild-type P2Ox. The improved catalytic performance of P2OxB2H was demonstrated by bioconversions of l-sorbose that initially was a poor substrate for wild-type P2Ox. This is the first report on the improvement of a pyranose 2-oxidase by a dual approach of site-directed mutagenesis and directed evolution, and the application of the engineered P2Ox in bioconversions.This revised version was published online in February 2005 with corrections to Table 2.