Different specific binding sites of [3H]glycine and [3H]strychnine in synaptosomal membranes isolated from frog retina

Synaptosomal fractions were isolated from frog retina: a fraction enriched in photoreceptor terminals (P1) and a second one (P2) containing interneurons terminals. We compared the binding of [³H]glycine and [³H]strychine to membranes of these synaptosomes. The binding of both radioactive ligands was saturable and Na⁺-independent. [³H]Glycine bound to a single site in P1 and P2 synaptosomal fractions, with K_D=12 and 82 nM and B_Max=3.1 and 3.06 pmol/mg protein respectively. [³H]Strychnine bound to two sites in each one of the synaptosomal fractions. For P1 K_D values were 3.9 and 18.7 nM, and B_Max values were 1.1 and 7.1 pmol/mg protein, respecitively. Membranes from the P2 synaptosomal fraction showed K_D's of 0.6 and 48 nM and B_Max's of 0.4 and 4.5 pmol/mg. Specific [³H]glycine binding was displaced by -alanine, l-serine, d-serine and HA966, but not by strychnine 7-chlorokynurenic or 5,7-dichloro-kynurenic acids. Specific [³H]strychnine, binding was partially displaced by glycine and related aminoacids and totally displaced only by 2-NH₂-strychnine. Our results indicate the presence of high affinity binding sites for glycine and strychnine in frog retinal synaptosomal membranes. The pharmacological binding pattern indicates the presence of the strychnine sensitive glycine receptor as well as other sites. These might not include the NMDA receptor-associated glycine site.     

Effect of Glycine and Strychnine on Cl–-Activated Mg2+-ATPase from Bream Brain Microsomes (Abramis bramaL.)

The effect of glycine and strychnine on Mg²⁺-ATPase from the microsomal fraction of the bream (Abramis bramaL.) brain was studied. The glycine in the concentration range 10^–7–10^–4M activates the enzyme. The effect of glycine on Mg²⁺-ATPase is obviated by 100 M strychnine. The strychnine in the concentration range 5–90 M activates the basal Mg²⁺-ATPase but decreases the effect of the enzyme activation by 10^–4M glycine. The effect of Cl^–on Mg²⁺-ATPase depends on the substrate concentration (Mg²⁺-ATP) and is not observed in the presence of 100 M strychnine. A receptor-dependent pathway of glycine and strychnine action on Cl^–-activated Mg²⁺-ATPase from bream brain microsomes is proposed.     

Coordination dynamics of heme-copper oxidases. The ligand shuttle and the control and coupling of electron transfer and proton translocation

Results are presented which, taken with evidence developed by others, suggest a general mechanism for the entry and binding of exogenous ligands (including O₂) at the binuclear site (Cu_B Fe _a3) of the heme-copper oxidases. The mechanism includes a ligand shuttle wherein the obligatory waystation for incoming ligands is Cu_B and the binding of exogenous ligands at this site triggers the exchange and displacement of endogenous ligands at Fe _a3. It is suggested that these ligand shuttle reactions might be functionally important in providing a coupling mechanism for electron transfer and proton translocation. Scenarios as to how this might happen are delineated.     

Synthesis and anti-phlogistic potency of some new non-proteinogenic amino acid conjugates of “Diclofenac”

Summary In search for more potent, particularly less ulcerogenic gastritis that hopefully replace the universal NSAID Diclofenac, (2-[(2,6-di-chlorophenyl)amino]-phenylacetic acid, C.A.S. 15307-86-5), twelve new non-proteinogenic amino acid conjugates of the drug, namely that of sarcosine,-alanine, D-leucine and D-phenylalanine, were synthesized and biologically screened for their anti-inflammatory, analgesic and ulcerogenic activity in rats.Diclofenac amino acid esters (IIa-d), were synthesizedvia the corresponding HOSu or HOBt active esters. Alkaline hydrolysis (NaOH) followed by acidification (KHSO₄) or thioamide formation (Lawsson's Reagent, C.A.S. 19172-47-5), afforded the corresponding free acids IIIa-d or the thioamides IVa-d respectively.Interestingly, in contrary to the parent Diclofenac, the synthesized candidates (except IIId), were entirly nonulcerogenic in rats. Further, they considerably retained a generelized anti-phlogistic activity. The major Diclofenac irritating gastric side effect was thus eliminated.Particularly, the sarcosine conjugate IIa and its thiomimic IVa exhibit promising therapeutic perspectives.Preliminary results were presented and discussed at the 5^th International Congress on Amino Acids, August 25–29, 1997, Chalkidiki, Greece and abstracted in Amino Acids (1997) 15/1: 75.     

Retinal pigment epithelium contains a distinctive strychnine-binding site

Membranes prepared from the retinal pigment epithelium of several species possess a specific site which binds [³H]strychnine. This binding has a somewhat lower affinity and a much greater density than the corresponding interaction in the hindbrain or neural retina. Binding is not greatly altered in the presence of 10^–3 M glycine,l-alanine, -alanine, taurine, or serine. Thus, the receptor does not resemble the classical glycine receptor of the hindbrain and spinal cord. This new type of binding site appears to be confined to the pigment epithelial layer of the retina.     

Stereospecific assignments of glycine in proteins by stereospecific deuteration and 15N labeling

Summary A method is described for stereospecifically assigning the -protons of glycine residues in proteins. The approach involves the stereospecific deuteration and ¹⁵N labeling of glycine and subsequent selective incorporation of this residue into the protein. The stereospecific assignments of the glycine -protons are obtained from a comparison of a 3D ¹⁵N-resolved TOCSY spectrum of the uniformly ¹⁵N-labeled protein with a 2D/3D ¹⁵N-edited TOCSY spectrum of the protein, containing the stereospecifically deuterated and ¹⁵N-labeled glycine. The approach is demonstrated by stereospecifically assigning the glycine -protons of the FK506 binding protein when bound to the immunosuppressant ascomycin.     

Electrogenic properties of the sodium-alanine cotransporter in pancreatic acinar cells: II. Comparison with transport models

Summary In this paper, the results of the preceding electrophysiological study of sodium-alanine cotransport in pancreatic acinar cells are compared with kinetic models. Two different types of transport mechanisms are considered. In the simultaneous mechanism the cotransporterC forms a ternary complexNCS with Na⁺ and the substrateS; coupled transport of Na⁺ andS involves a conformational transition between statesNCS andNCS with inward- and outward-facing binding sites. In the consecutive (or ping-pong) mechanism, formation of a ternary complex is not required; coupled transport occurs by an alternating sequence of association-dissociation steps and conformational transitions. It is shown that the experimentally observed alanine- and sodium-concentration dependence of transport rates is consistent with the predictions of the simultaneous model, but incompatible with the consecutive mechanism. Assuming that the association-dissociation reactions are not rate-limiting, a number of kinetic parameters of the simultaneous model can be estimated from the experimental results. The equilibrium dissociation constants of Na⁺ and alanine at the extracellular side are determined to beK _N <-64mm andK _S <-18mm. Furthermore, the ratioK _N /K _N ^S of the dissociation constants of Na⁺ from the binary (NC) and the ternary complex (NCS) at the extracellular side is estimated to be <-6. This indicates that the binding sequence of Na⁺ andS to the transporter is not ordered. The current-voltage behavior of the transporter is analyzed in terms of charge translocations associated with the single-reaction steps. The observed voltage-dependence of the half-saturation concentration of sodium is consistent with the assumption that a Na⁺ ion that migrates from the extracellular medium to the binding site has to traverse part of the transmembrane voltage.     

A re-examination of the Na+-independent binding of [3H]β-alanine to rat brain stem-spinal cord

The Na⁺-independent binding of [³H]-alanine to rat brain stem plus spinal cord was reinvestigated, in order to study in more detail the characteristics of previously described -alanine binding processes. Binding was absent when amino acid-free postnuclear supernatants or crude synaptic membranes were used. Experiments performed with several other Na⁺-free preparations showed a sole binding component, irrespective of the preparation used. Biochemical characterization of this Na⁺-independent binding, using frozen/thawed/washed synaptosomal-mitochodrial fractions, showed that binding reached a plateau between 7 min and 13 min, increasing thereafter. Binding was linear with fraction protein over a range of 200–415 g/ml incubation medium. Binding was completely inhibited by glycine, alanine, -aminobutyric acid, -aminoisobutyric acid, hypotaurine and strychnine, and to a lesser extent by 2,2-dimethyl--alanine, brucine and gelsemine. It was insensitive to taurine, -aminobutyric acid (GABA), 2-guanidinoethanesulfonic acid (GES), carnosine, and bicuculline methiodide. Binding was reversible, saturable (K _D 20 M), and heat sensitive.     

Osmotic measurements on stomatal cells ofCommelina communis L.

Summary Observations of aperture changes as sucrose is added to the solution bathing epidermal strips ofCommelina communis L. allow calculation of the osmotic changes required to open or close the stomatal pore, for comparison with changes in potassium content. With isolated guard cells, in strips in which all cells other than guard cells have been killed, the internal osmotic changes required are 83 mosmol kg^–1 m^–1 below 10m aperture, 129 mosmol kg^–1 m^–1 in the range 10–15 m, and 180 mosmol kg^–1 m^–1 above 15 m. For opening against subsidiary cell turgor in addition to guard cell turgor, in intact strips with live subsidiary and epidermal cells, these figures should each be increased by about 33 mosmol kg^–1 m^–1. A change in subsidiary cell turgor is magnified in its effects on the water relations of the guard cell by a factor greater than 3.7 for equal changes in the water potential of the two cells, or greater than 4.7 at constant volume of the guard cell.     

Effect of Activators and Blockers of Ligand-Regulated Ion Channels on the Activity of the Cl−-Stimulated Mg2+-ATPase of the Plasma Membrane Fraction from Bream (Abramis brama L.) Brain

Effects of GABA, glycine, acetylcholine, and glutamate (agonists of the GABA_a/benzodiazepine, glycine, choline, and glutamate receptors, respectively) at concentrations in the range 10^–8-10^–4 M on the activity of basal Mg²⁺-ATPase of the plasma membrane fraction from bream brain and on its activation by Cl^– were investigated. GABA and glycine activated basal Mg²⁺-ATPase activity and suppressed its activation by Cl^–. Acetylcholine and glutamate activated basal Mg²⁺-ATPase to a lesser extent and did not suppress the activation of the enzyme by Cl^–.The activation of basal Mg²⁺-ATPase by neuromediators was decreased by blockers of the corresponding receptors (picrotoxin, strychnine, benztropine mesylate, and D-2-amino-5-phosphonovaleric acid). In addition, picrotoxin and strychnine eliminated the inhibiting effect of GABA and glycine, respectively, on the Cl^–-stimulated Mg²⁺-ATPase activity. Agonists of the GABA_a/benzodiazepine receptor–phenazepam (10^–8-10^–4 M) and pentobarbital (10^–6-10^–3 M)–activated the basal Mg²⁺-ATPase activity and decreased the Cl^–-stimulated Mg²⁺-ATPase activity. The dependence of both enzyme activities on ligand concentration is bell-shaped. Moreover, phenazepam and pentobarbital increased the basal Mg²⁺-ATPase activity in the presence of 10^–7 M GABA and did not influence it in the presence of 10^–4 M GABA and 10^–6 M glycine. The data suggest that in the fish brain membranes the Cl^–-stimulated Mg²⁺-ATPase interacts with GABA_a/benzodiazepine and glycine receptors but not with m-choline and glutamate receptors.     

NMDA receptor function is enhanced in the hippocampus of aged rats

The density and functional activity of theN-methyl-D-aspartate (NMDA)-sensitive glutamate receptor was examined in various brain areas of 3-, 18- and 24-month-old rats. The total numbers of binding sites for the NMDA receptor antagonists [³H]CGP 39653 and [³H]MK 801 binding sites were decreased in the hippocampus, cerebral cortex and striatum of 18- and 24-month-old rats, relative to 3-month-old animals. In the hippocampus of 18-month-old rats, the reduced number of NMDA receptors was associated with an increased sensitivity of [³H]MK 801 binding to the stimulatory action of glycine and glutamate. Thus, 10 M glycine and 10 M glutamate increased [³H]MK 801 binding in the hippocampus of 18-month-old rats by 75 and 160%, respectively; in 3-month-old animals, the same concentration of these amino acids increased binding by 37 and 95%, respectively. The sensitivity of [³H]MK 801 binding to glycine and glutamate was not increased in the cerebral cortex and striatum of aged rats. Moreover, an increased efficacy of glycine and glutamate in stimulating the binding of [³H]MK 801 in the hippocampus was no longer apparent in the 24-month-old rats. The increased sensitivity of [³H]MK 801 binding to glycine and glutamate in the hippocampus of 18-month-old rats may reflect an increase in NMDA receptor activity to compensate for the decrease in receptor number.     

Biphasic Modulation of GABAA Receptor Binding by Steroids Suggests Functional Correlates

Neuroactive steroids and other positive modulators of GABA_A receptors showed regional variation in both the efficacy and potency for modulation of [³⁵S]TBPS binding to rat brain membrane homogenates, with biphasic concentration-dependence. GABA present in the binding assays prevented the enhancement phase of the steroid concentration-dependence plot while the antagonists bicuculline and RU5135 prevented the inhibition phase. Using recombinant GABA_A receptors, expressed in insect cell line Sf9 using baculovirus, enhancement by steroids of [³⁵S]TBPS binding was sensitive to the presence of the 2 subunit and the nature of the subunit (122S > 12, 62, 622S, and 62). As in cerebellum, addition of RU5135 reduced the inhibitory phase and revealed a small enhancement of TBPS binding by neuroactive steroids. The subunit-dependent interactions of steroid and GABA site ligands are consistent with a three-state model in which the receptor mono-liganded by GABA or steroid has a different affinity for TBPS than the resting state, and the receptor biliganded by GABA, steroid, or both has little affinity for TBPS.     

Interaction of Synthetic Peptides from Fasciculin with Acetylcholinesterase

Fasciculins (Fas) are three-looped polypeptides isolated from mamba venom which exert their toxic action by inhibiting noncompetitively acetylcholinesterase (AChE). A peptide (Fas-D) encompassing the first loop sequence was synthesized and characterized chemically, structurally, and functionally. Fas-D possesses an intramolecular disulfide bridge, present in the native toxin. Circular dichroism (CD) indicated the existence of 21.8% -sheet content and 24.2% -turn in this peptide, compatible with crystallographic data of the native toxin. The peptide showed only low partial AChE inhibition at submillimolar concentrations, much lower than that observed with Fas and a peptide (Fas-B) encompassing the second loop sequence. The simultaneous presence of Fas-D and Fas-B produced an additive inhibitory effect on AChE activity; calculated K _i and K _i values (7.3 ± 2.4 M and 10.0 ± 1.8 M, respectively) were not significantly different, thus indicating noncompetitive inhibition. These results are consistent with site-directed mutagenesis studies and analysis of the crystal structure of the Fas–AChE complex, which indicate that residues from loops I and II contribute to Fas binding to the enzyme.     

A K+-selective,three-state channel from fragmented sarcoplasmic reticulum of frog leg muscle

Summary Sarcoplasmic reticulum (SR) vesicles from frog leg muscle were fused with a planar phospholipid bilayer by a method described previously for rabbit SR. As a result of the fusion, K⁺-selective conduction channels are inserted into the bilayer. Unlike the two-state rabbit channel, the frog channel displays three states: a nonconducting (closed) state and two conducting states and . In 0.1m K⁺ the single-channel conductances are 50 and 150 pS for and , respectively. The probabilities of appearearance of the three states are voltage-dependent, and transitions between the closed and states proceed through the state. Both open states follow a quantitatively identical selectivity sequence in channel conductance: K⁺>NH ₄ ⁺ >Rb⁺>Na⁺>Li⁺>Cs⁺. Both open states are blocked by Cs⁺ asymmetrically in a voltage-dependent manner. The zero-voltage dissociation constant for blocking is the same for both open states, but the voltage-dependences of the Cs⁺ block for the two states differ in a way suggesting that the Cs⁺ blocking site is located more deeply inside the membrane in the than in the state.     

Mode of uptake of sterol by Arthrobacter simplex

The mechanism of uptake of water-insoluble -sitosterol by a newly isolated strain of Arthrobacter simplex SS-7 was studied. The production of an extracellular sterol-pseudosolubilizing protein during growth of A. simplex on -sitosterol was demonstrated by isolating the factor from the cell-free supernatant and its subsequent purification by Sephadex G-150 column chromatography. The M _r of the purified sterol-pseudosolubilizing protein determined by SDS–PAGE was 19kDa. The rate of sterol pseudosolubilization (5.2×10^–3g l^–1h^–1) could not adequately account for the rate of sterol uptake (72×10^–3g l^–1h^–1) and the specific growth rate (56×10^–3 h^–1). However in the unfavourable growth condition, when the cells were treated with sodium azide at the level of 30–60% of MIC, the sterol pseudosolubilization accounted for nearly 74% of the total growth containing 96% free cells. Cellular adherence to substrate particles was found to play an active role in the normal growth of the strain on -sitosterol. Unlike sodium acetate-grown cells, whose surface activity was negligible (60mNm^–1), the sterol-grown cells had strong surface activity (40mNm^–1). The high lipid content and long chain fatty acids in the cell-wall of -sitosterol-grown cells probably contribute to the high sterol adherence activity of the cells.     

New expanded bed adsorbents for the recovery of DNA

A 20–40 m pellicular high density (3.7 g cm^–3) expanded bed material has been designed for the capture of DNA and other large macromolecules. Anion exchangers fashioned out of these supports exhibited dramatically enhanced DNA binding capacities over commercial anion exchange adsorbents (6 mg ml^–1, c.f. 50 g ml^–1 at 10% breakthrough), due to a combination of small particle and fuzzy surface architecture created through the coupling of polyethylene imine chains.     

Effect of antagonists of ethylene action on binding of ethylene in cut carnations

Five hours after cut carnations had been treated with a pulse of 1 or 4 mM silver thiosulfate (STS), in vivo ethylene binding in petals was inhibited by 22 and 29%, respectively. When binding was measured 4 days after the 4-mM STS treatment, binding was inhibited by 81%. 2,5-Norbornadiene, which substantially delays carnation senescence, inhibited ethylene binding by 41% at a concentration of 1000 l/l. The Kd for ethylene binding in carnations was estimated to be 0.1 l/l in petals and 0.09 l/l in leaves. The concentration of binding sites was estimated to be 6.0×10^–9 mol/kg of petals and 2.0×10^–9 mol/kg of leaves     

Comparison of the carbohydrate moieties of recombinant soluble Fcε receptor (sFcε RII/sCD23) expressed inSaccharomyces cerevisiae and Chinese hamster ovary cells. Different O-glycosylation sites are used by yeast and mammalian cells

Recombinant human soluble low affinity receptor for the Fc portion of IgE (sFcRII/sCD23) was produced inSaccharomyces cerevisiae or Chinese hamster ovary cells and subjected to carbohydrate analysis. Applied methods included analytical SDS-PAGE, reversed phase HPLC, methylation analysis and sequential degradation with exoglycosidases. The results revealed that sFcRII derived from Chinese hamster ovary cells is glycosylated exclusively at Ser-147, containing mainly the trisaccharide Sia(2–3)Gal(1–3)GalNAc, whereas the yeast derived glycoprotein was glycosylated at Ser-167 and contained only -mannosyl residues. It is shown here for the first time that different amino acids of a given protein can be O-glycosylated when expressed in yeast or Chinese hamster ovary cells.     

A quantitative method for separating reaction components of CMP-sialic acid: Lactosylceramide sialyltransferase using Sep Pak C18 cartridges

A rapid procedure is described for the separation of CMP-sialic acid:lactosylceramide sialyltransferase reaction components using Sep Pak C₁₈ cartridges. The quantitative separation of the more polar nucleotide sugar, CMP-sialic acid, and its free acid from the less polar G_M3-ganglioside is simple and rapid relative to previously described methods. Recovery of G_M3 is optimized by the addition of phosphatidylcholine to the reaction mixture prior to the chromatographic step. Using rat liver Golgi membranes as a source of CMP-sialic acid: lactosylceramide sialyltransferase activity (G_M3 synthase; ST-1), the transfer of [¹⁴C] sialic acid from CMP-[¹⁴C] sialic acid to lactosylceramide can be quantified by this assay. The procedure is reliable and may be applicable to the isolation of ganglioside products in otherin vitro glycosyltransferase assays.Abbreviations G_M3 G_M3-ganglioside - II³NeuAc-LacCer NeuAc2-3Gal1-4Glc1-1Cer - G_D1a G_D1a-ganglioside, IV³NeuAc, II³NeuAc-GgOse₄Cer, NeuAc2-3Gal1-3GalNac1-4(NeuAc2-3)Gal1-4Glc1-1Cer - G_D3 G_D3-ganglioside, II³(NeuAc)₂LacCer, NeuAc2-8NeuAc2-3Gal1-4Glc1-1Cer - GgOse₄Cer asialo-G_M1 Gal1-3GalNAc1-4Gal1-4Glc1-1Cer - FucG_MI fucosyl-G_MI-ganglioside, Fuc1-2Gal1-3GalNAc1-4Gal1-4 Glc1-1Cer - ST-1 G_M3 synthase, CMP-sialic acid:lactosylceramide sialyltransferase - LacCer lactosylceramide, Gal1-4Glc1-1Cer - CMP-NeuAc cytidine 5-monophospho-N-acetylneuraminic acid - PC phosphatidylcholine - PMSF phenylmethylsulfonyl fluoride     

13Cα and 13Cβ chemical shifts as a tool to delineate β-hairpin structures in peptides

Unravelling the factors that contribute to the formation and the stability of -sheet structure in peptides is a subject of great current interest. A -hairpin, the smallest -sheet motif, consists of two antiparallel hydrogen-bonded -strands linked by a loop region. We have performed a statistical analysis on protein -hairpins showing that the most abundant types of -hairpins, 2:2, 3:5 and 4:4, have characteristic patterns of ¹³C and ¹³C conformational shifts, as expected on the basis of their and angles. This fact strongly supports the potential value of ¹³C and ¹³C conformational shifts as a means to identify -hairpin motifs in peptides. Their usefulness was confirmed by analysing the patterns of ¹³C and ¹³C conformational shifts in 13 short peptides, 10–15 residues long, that adopt -hairpin structures in aqueous solution. Furthermore, we have investigated their potential as a method to quantify -hairpin populations in peptides.