Differential effects of metal ligands on synaptic membrane glutamate binding and uptake systems

The high affinity, Na⁺-independentl-[³H]glutamate binding process in synaptic membranes and in the purified binding protein was shown to be inhibited to an almost equal extent by the metal ligands NaN₃, KCN, ando-phenanthroline, and by 2,4,5-trihydroxyphenylalanine (6-OH DOPA). The high affinity, Na⁺-dependent glutamate transport activity in these membranes was almost totally insensitive to NaN₃,o-phenanthroline, KCN, and 6-OH DOPA. These agents, especially 6-OH DOPA, may be useful tools in achieving a discrimination between putative physiologic receptors and uptake carrier sites forl-glutamate in synaptic membranes. The sensitivity of the glutamate binding sites to the effects of the metal ligands may be correlated to the presence of an iron-sulfur center in the purified glutamate binding protein. Some of the characteristics of this metallic center were explored by optical and paramagnetic resonance spectroscopic techniques and are described in this study.This research was supported by grants DAAG29-79-C-0156 from the Army Research Office and AA 04732 from NIAAA.     

Immune labeling and purification of a 71-kDa glutamate-binding protein from brain synaptic membranes. Possible relationship of this protein to physiologic glutamate receptors

Immunoblot studies of synaptic membranes isolated from rat brain using antibodies raised against a previously purified glutamate-binding protein (GBP) indicated labeling of an approximately 70-kDa protein band. Since the antibodies used were raised against a 14-kDa GBP, the present studies were undertaken to explore the possibility that the 14-kDa protein may have been a proteolytic fragment of a larger Mr protein in synaptic membranes. Protease activity during protein purification was prevented by introducing five protease inhibitors, and a three-step purification procedure was developed that yielded a high degree of purification of glutamate-binding proteins. The major protein enriched in the most highly purified fractions was a 71-kDa glycoprotein, but a 63-kDa protein was co-purified during most steps of the isolation procedure. The glutamate-binding characteristics of these isolated protein fractions were very similar to those previously described for the 14-kDa GBP, including estimated dissociation constants for L-glutamate binding of 0.25 and 1 microM, inhibition of glutamate binding by azide and cyanide, and a selectivity of the ligand binding site for L-glutamate and L-aspartate. The neuroexcitatory analogs of L-glutamate and L-aspartate, ibotenate, quisqualate, and D-glutamate, inhibited L-[3H]glutamate binding to the isolated proteins, as did the antagonist of L-glutamate-induced neuronal excitation, L-glutamate diethylester. On the basis of the lack of any detectable glutamate-related enzyme activity associated with the isolated proteins and the presence of distinguishing sensitivities to analogs that inhibit glutamate transport carriers in synaptic membranes, it is proposed that the 71-kDa protein may be a component of a physiologic glutamate receptor complex in neuronal membranes.     

Activation of rat adipocyte plasma membrane adenylate cyclase by sodium azide

The adenylate cyclase of rat adipocyte plasma membrane is stimulated by sodium azide with a half maximal activation of 100–150% occuring at 50 mM NaN₃. Studies of the effects of azide and fluoride indicate different mechanisms of stimulation of the enzyme by these ions. Comparable stimulation of the activity is obtained by 100 mM NaN₃ or 10 mM NaF but unlike azide, higher concentrations of fluoride cause inhibition of the enzyme. Fluoride activated adenylate cyclase is further stimulated by azide. Epinephrine stimulation of the enzyme is absent in the presence of fluoride but the hormone enhances the activity in the presence of azide. Reversal of the inhibitory action of GTP on adenylate cyclase by epinephrine is demonstrated even in the presence of azide but not in the presence of fluoride.     

Effects of amphipathic drugs onl-[3H]glutamate binding to synaptic membranes and the purified binding protein

Four amphipathic molecules with known local anesthetic activity, dibucaine, tetracaine, chlorpomazine, and quinacrine, inhibited the binding ofl-[³H]glutamic acid to rat brain synaptic plasma membranes and to the purified glutamate binding protein. Neither haloperidol nor diphenylhydantoin had significant inhibitory effects on the glutamate binding activity of the membranes or of the purified protein. The amphipathic drugs apparently inhibitedl-[³H]glutamate binding to synaptic membranes by a mixed type of inhibition. The inhibitory activity of quinacrine on glutamate binding to the synaptic membranes was greater in a low ionic strength, Ca²⁺-free buffer medium, than in a physiologic medium (Krebs-Henseleit buffer). Removal of Ca²⁺ from the Krebs solution enhanced quinacrine's inhibition of glutamate binding. Quinacrine up to 1 mM concentration did not inhibit the high affinity Na⁺-dependentl-glutamate transport in these membrane preparations. The importance of Ca²⁺ in the expression of quinacrine's effects on the glutamate binding activity of synaptic membranes and the observed tetracaine and chlorpromazine-induced increases in the transition temperature for the glutamate binding process of these membranes, were indicative of an interaction of the local anesthetics with the lipid environment of the glutamate binding sites.     

Molecular characteristics of glutamate receptors in the mammalian brain

Summary The strong excitatory activity of L-glutamic acid on central nervous system neurons is thought to be produced by interaction of this amino acid with specific neuronal plasma membrane receptors. The binding of L-glutamate to these surface receptors brings about an increase in membrane permeability to Na⁺ and Ca²⁺ ions presumably through direct activation of ion channels linked to the membrane receptors. The studies described in this paper represent attempts to define the subcellular distribution and pharmacological properties of the recognition site for L-glutamic acid in brain neuronal preparations, to isolate and explore the molecular characteristics of the receptor recognition site, and, finally, to demonstrate the activation of Na⁺ channels in synaptic membranes following the interaction of glutamate with its receptors.Radioligand binding assays with L-[³H] glutamic acid have been used to demonstrate a relative enrichment of these glutamate recognition sites in isolated synaptic plasma membranes. The specific binding of L-[³H] glutamate to these membrane sites exhibits rapid association and dissociation kinetics and rather complex equilibrium binding kinetics. The glutamate binding macromolecule from synaptic membranes has been solubilized and purified and was shown to be a small molecular weight glycoprotein (M_T 13 000). This protein tends to form aggregates which have higher specific activity at low concentrations of glutamate than the M_T 13 000 protein has. The overall affinity of the purified protein is lower than that of the high affinity sites in the membrane. Nevertheless, the purified protein exhibits pharmacological characteristics very similar to those of the membrane binding sites. On the basis of its pharmacological properties this protein belongs in the category of the physiologic glutamate preferring receptors.By means of differential solubilization of membrane proteins with Na-cholate, it was shown that this recognition site is an intrinsic synaptic membrane protein whose binding activity is enhanced rather than diminished by cholate extraction of the synaptic membranes. The role of membrane constituents in regulating the binding activity of this protein has been explored and a possible modulation of glutamate binding by membrane gangliosides has been demonstrated. Finally, this glutamate binding glycoprotein is a metalloprotein whose activity is dependent on the integrity of its metallic (Fe) center. This is a clear distinguishing characteristic of this protein vis-à-vis the glutamate transport carriers.The presence of functional glutamate receptors in synaptosomes and resealed synaptic plasma membranes has also been documented by the demonstration of glutamate-activated Na⁺ flux across the membrane of these preparations. The bidirectionality, temperature independence, and apparent desensitization of this stimulated flux following exposure to high concentrations of glutamate are properties indicative of a receptor-initiated ion channel activation. It would appear, then, that the synaptic membrane preparations provide a very useful system for the study of both recognition and effector function of the glutamate receptor complex.     

Binding of l-[3H]glutamate to repeatedly frozen-thawed rat brain membranes

l-[³H]Glutamate binding to synaptic plasma membranes from rat cerebral cortices was carried out at 2–4°C in 50 mM Tris-acetate buffer (pH 7.4) using a microfuge centrifugation method. Binding was increased by repeated freezing-thawing and washing in either crude or partially purified synaptic membranes. Scatchard analysis showed a single binding site (dissociation constant, K_D = 697 nM; maximal binding capacity, B_max = 7.5 pmol/mg protein) in four times distilled water washed crude synaptic membrane. After six times freezing-thawing and washing, a new high affinity site (K_D1 = 26 nM, B_max1 = 1.8 pmol/mg protein) appeared and the number of low affinity site was increased with no apparent change in affinity (K_D2 = 662 nM, B_max2 = 10.5 pmol/mg protein). l-[³H]Glutamate binding was inhibited by acidic amino acid analogues that interact with N-methyl-d-aspartate- and quisqualate-sensitive sites of glutamate receptors. Binding was marginally inhibited by kainate and l-2-amino-4-phosphonobutyrate. These results indicate that repeatedly frozen-thawed and washed synaptic plasma membrane is suitable for studying the subtypes and regulation of glutamate receptors.     

A microbiological assay for sodium azide

A reproducible and sensitive method is presented for quantitating sodium azide (NaN₃) that exploits the fact that NaN₃ inhibits Escherichia coli RNA synthesis. A linear correlation is observed between incorporation of [³H]uridine into a trichloroacetic acid-precipitable form and NaN₃ concentration over a 31- to 2000-μg range of azide. This technique was used to determine the azide content of a complex enzyme solution where established colorimetric azide determinations proved to be unworkable. This technique when properly controlled should be applicable to a variety of similar solutions.     

L-Glutamate Receptor Heterogeneity: Labeling of Distinct Receptor Sub-Types Using Radioligand Binding Techniques

Abstract

This study demonstrates (1) that L-[³H]glutamate labels 3 distinct binding sites (types A1, A2 and A4) in isolated rat brain membranes and (2) that only the N-methyl-aspartate (A1) and quisqualate (A2) receptor classes are associated with the postsynaptic density (PSD). L-[³H]glutamate bound to PSDs with K_d 339 nM and B_max 6.1 pmol/mg protein. These sites were resolved into 2 distinct sub-types on the basis of inhibition studies. N-Methyl-aspartate maximally inhibited 57% of PSD-located L-glutamate binding sites (the A1 site) and quisqualate 43% (the A2 site); the effects of both substances were additive. The ligand selectivities of these 2 sites indicated their identity with the N-methyl-D-aspartate and quisqualate receptor classes defined electrophysiologically. The Cl^?-dependent population of L-glutamate binding sites (the A4 site) which predominates in synaptic membranes was absent from PSDs.     

Purification, Biochemical Characterization, Binding Activity, and Selectivity of a Glutamate Binding Protein from Bovine Brain

A glutamate binding protein was purified from bovine brain to apparent homogeneity. The procedure used for the purification of this protein involved extraction of a crude synaptic membrane fraction with Na-cholate, followed by solubilization of the binding protein from the membranes by Triton X-100, and, finally, affinity batch separation of the protein on L-glutamate-loaded glass fiber. The molecular characteristics of the purified protein were similar to those previously described for the glutamate binding protein from rat brain synaptic membranes and included the following: small Mr (14,000), acidic (pI = 4.7) protein with a single NH2-terminal amino acid (tyrosine), and significant absorption at wave-lengths greater than 300 nm. Complete amino acid analysis of the protein was not achieved, either because of destruction of some amino acids or of incomplete hydrolysis of the protein. The protein bound L-glutamate with high affinity (KD = 0.87 microM), exhibited one class of L-glutamate binding sites, and bound glutamate with a stoichiometry of 0.7 mol ligand/mol protein. The displacement of protein-bound L-glutamic acid by other neuroactive amino acids had characteristics similar to those observed for the displacement of L-glutamate from rat brain synaptic membrane or purified protein binding sites. Finally, the metal ligand formers KCN and NaN3 inhibited the activity of this protein just as they have been shown to do in rat brain synaptic membranes or the purified protein.     

Isolation and partial characterization of rat brain synaptic plasma membranes

Abstract— Synaptic plasma membranes from the cortices of adult rat brain were isolated from synaptosomes prepared by flotation of a washed mitochondrial pellet (P2) in a discontinuous Ficoll-sucrose gradient. Contamination of the synaptosome fraction by microsomes was estimated by enzymic and chemical analysis to be less than 15 per cent. (2) The purified synaptosome fraction was subjected to osmotic shock, subfractionated on a discontinuous sucrose gradient and the distribution of enzymic and chemical markers for synaptic plasma membranes, microsomal membranes and mitochondria was determined. (3) Comparison of synaptosome subfractions prepared in the presence and absence of 1 mM NaH₂ PO₄/0.1 mM EDTA buffer pH 7.5, indicated that the ionic composition of the isolation medium markedly affected the distribution and enzymic composition of the subfractions. (4) Synaptic plasma membranes prepared in the presence of PO₄/EDTA exhibited a 10-fold enrichment in [Na⁺+ K⁺] ATPase and were characterized by less than 15 and 10 per cent contamination by microsomes and mitochondria respectively. (5) The polypeptide composition of the purified synaptic plasma membranes was compared with the microsomes and mitochondria by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. No differences between the protein and glycoprotein composition of the synaptic plasma membranes and microsomes were detected. The mitochondria, in contrast, possessed a unique protein composition.     

Carbohydrate-binding profile of a pregnancy-associated rat uterine glycoprotein

Sugar-binding proteins obtained from the peri-implantation uterine tissue have been thought in recent years to have significant roles in embryo implantation, where carbohydrate moieties of the protein are actively involved. Based on this rationale a mannose-containing glycoprotein/lectin (named uterine agglutinin or UA) was purified by Concanavalin A (Con A) affinity chromatography in a previous study. A modification of the original purification procedure to include a 33% ammonium sulfate fractionation improves the yield of the protein significantly. An alternative purification procedure by Mannan affinity matrix, indicates that apart from containing mannose, UA possesses mannose-binding properties as well.In this paper, we report some of the biochemical and more specifically, the carbohydrate-binding characteristics of UA. The protein is seen to contain mannose-6-phosphate (M-6-P)-binding sites, which is of importance since M-6-P receptors have a large number of biologically significant roles, including that of binding to growth factors.SDS-PAGE, gel filtration chromatography and alkaline PAGE indicate the homogenous nature of the protein with subunit molecular weights of 36 kDa and 19 kDa, and a native size of 64kDa. Amino acid analysis shows glycine, glutamic acid and aspartic acid to be the major constituents.UA is a glycoprotein and shows presence of N-acetyl glucosamine and galactose, apart from mannose.De nove synthesis studies in the presence of tunicamycin show that the carbohydrate moiety of the glycoprotein is attached by N-linkage to the protein. Binding characteristics of the protein is studied quantitatively in which (¹²⁵I)-labelled lectin is bound to Mannan-Sepharose affinity matrix. The sugar inhibition pattern of this binding shows -methyl mannopyranoside and M-6-P to be equally effective as inhibitors. Scatchard analysis of the binding of UA to (¹⁴C)-mannose shows a Ka of 6.43×10⁵ (M^–1) and that 1 mole of UA can bind to 8 moles of mannose. The possible role of the protein in implantation has also been discussed.Abbreviations b.w. body weight - BSA Bovine Serum Albumin - Con A Concanavalin A - cpm counts per minute - Endo H endoglycosidase H - GlcNAc N-acetyl glucosamine - Man mannose - M-6-P mannose-6-phosphate - MEM-deficient Minimum Essential Medium Eagle-deficient modification - NaBH₄ sodium borohydride - NaN₃ sodium azide - (NH₄)₂SO₄ ammonium sulphate - p.c. post coitum - PMSF phenyl methyl sulphonyl fluoride - PTA phosphotungstic acid - RCA Ricinus communis Agglutinin - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid - UA Uterine Agglutinin - WGA Wheat-germ Agglutinin     

Correlation of glutamate binding activity with glutamate-binding protein immunoreactivity in the brain of control and alcohol-treated rats

Specific antibodies raised against a glutamate binding protein purified from bovine brain were used to trace the immunoreactivity of this protein in rat brain subcellular fractions. In the subcellular fractions obtained from whole brain homogenates, the synaptic membranes had the highest immunochemical reactivity towards the anti-glutamate-binding protein antibodies. The combination of measurements of glutamate binding activity and glutamate-binding protein immunoreactivity indicated that in brain synaptic membranes from control animals the highest activity in these two measures was associated with a synaptic plasma membrane subfraction that was enriched with synaptic junctions. In animals treated with ethanol for 14 days, there was a significant increase in the density of synaptic membrane glutamate binding sites. This increase in glutamate binding capacity was correlated with a greater than two-fold increase in the glutamate binding activity and binding protein immunoreactivity of the light synaptic membrane subfraction, a subfraction which does not contain many recognizable synaptic junctions. Acute administration of ethanol to rats produced a moderate but non-significant decrease in glutamate binding capacity of synaptic membranes. The increase in the number of glutamate binding protein subunits in brain plasma membranes may be an adaptive response of central nervous system neurons to the acute effects of ethanol on glutamate synaptic transmission.     

Na+-independent binding of GABA to the triton X-100 treated synaptic membranes from cerebellum of rat brain

The treatment of the membranes from cerebellum of rat brain with 0.5% Triton X-100 increases both the affinity and the density of the Na⁺-independent binding sites for ³H-GABA (γ-aminobutyric acid) from the values obtained from membranes of rat brain after an extensive freezing and thawing treatment (Young $\text{et al.}$, 1976). Upon repeated washings of the Triton-treated membranes, the binding of ³H-GABA is further increased and follows biphasic kinetics which indicates two binding components having dissociation constants of 5.9 and 27 nM and densities of 1.35 and 3.9 pmole/mg protein, respectively. GABA agonist, imidazoleacetic acid, and the GABA antagonists, bicuculline and d-tubocurarine, inhibit 50% of ³H-GABA binding at 1, 47 and 85 μM concentrations (IC₅₀ values), respectively. The IC₅₀ values for these compounds are unchanged by Na⁺. Thus, the Na⁺-independent binding of ³H-GABA to the Triton-treated membranes may represent binding to the synaptic GABA receptors.     

Involvement of Singlet Oxygen in 5-Aminolevulinic Acid-Induced Photodynamic Damage of Cucumber (Cucumis sativus L.) Chloroplasts

Cucumber (Cucumis sativus L., cv Poinsette) plants were sprayed with 20 millimolar 5-aminolevulinic acid and then incubated in the dark for 14 hours. The intact chloroplasts were isolated from the above plants in the dark and were exposed to weak light (250 micromoles per square meter per second). Within 30 minutes, photosystem II activity was reduced by 50%. The singlet oxygen (¹O₂) scavengers, histidine and sodium azide (NaN₃) significantly protected against the damage caused to photosystem II. The hydroxyl radical scavenger formate failed to protect the thylakoid membranes. The production of ¹O₂ monitored as N,N-dimethyl p-nitrosoaniline bleaching increased as a function of light exposure time of treated chloroplasts and was abolished by the ¹O₂ quencher, NaN₃. Membrane lipid peroxidation monitored as malondialdehyde production was also significantly reduced when chloroplasts were illuminated in the presence of NaN₃ and histidine. Protochlorophyllide was the most abundant pigment accumulated in intact chloroplasts isolated from 5-aminolevulinic acid-treated plants and was probably acting as type II photosensitizer.     

Specific Binding of L-[3h]-Glutamic Acid to Rat Substantia Nigra Synaptic Membranes

Abstract

The specific binding of L-[³H] -glutamic acid (GLU) was investigated in synaptic membranes from rat substantia nigra. L-[³H]-GLU binding to the membrane preparations occurred in a reversible and saturable way. The specific binding was stimulated by the presence of CaCl₂ and was reduced by freezing and thawing the membranes. Scatchard analysis of the saturation isotherms yielded a non-linear plot suggesting that the binding reaction does not occur through a simpla bimolecular association. Assuming non-interacting binding sites, a high (K_D1, 139 nM; Bmax1, 3.5 pmoles/mg protein) and a low (K_D2, 667 nM; Bmax2, 15.1 pmoles/mg protein) affinity L-[³H]-GLU binding site were obtained. The kinetics of dissociation of bound L-[³H]-GLU was biphasic; the respective dissociation rate constant (k-1) being 0.20 min^?1 and 0.013 min^?1. A series of amino acid receptor agonists and antagonists were tested as inhibitors of L-[³H]-GLU specific binding. Quisqualic acid, L-GLU and D-α-aminoadipate (D-α-AA) were the most potent inhibitors. DL-2-amino-4-phosphonobutyrate (APB), N-Methy1-D-aspartate (NMDA) and D-GLU were moderate inhibitors, whereas diamino-pimelic acid (DAPA) and glutamate diethyl ester (GDEE) exhibited the lowest relative potency. Kainic acid (KA), γ-aminobutyric acid (GABA) and bicuculline were not able to modify at any concentration used the specific binding of L-[³H]-GLU. These data demonstrate the presence of specific GLU binding sites in synaptic structures at substantia nigra level and support the idea that excitatory amino acids may play a role in synaptic transmission in this brain region.     

HIGH AFFINITY l-[3H]GLUTAMATE BINDING TO POSTSYNAPTIC RECEPTOR SITES ON RAT CEREBELLAR MEMBRANES

l [³H]glutamate binding was investigated in membrane preparations derived from rat cerebellum, an area of the brain where it is likely that a high density of postsynaptic glutamate receptors occurs. Glutamate was hound specifically and, in freshly prepared membranes, was optimal under physiological conditions of pH and temperature and was associated with the synaptic membrane fraction of the cell. Specific binding occurred through a single, high-affinity process with a K_D, of 744 nM and a capacity of 73 pmol/mg protein. Unlike the findings reported for GABA, the specific binding of glutamate to fresh membranes did not involve an uptake site. Comparison of the potencies of a wide range of compounds with known pharmacological activities, demonstrated that their ability to displace specific glutamate binding was consistent with specific interactions with glutamate receptors.     

Transformation inBacillus subtilis. Initial stages of the transformation process and their energy dependence

NaN₃ was found to inhibit transformation but not the irreversible binding of donor³H-DNA in competent cells of the original low-transformable strainBacillus subtilis 168trp ₂ . Addition of NaN₃ to cells of two mutantsBacillus subtilis HT39 and HT46 with an increased transformability decreased substantially the irreversible binding of the donor DNA to the competent cells. The decreased irreversible binding of DNA is caused by an increased osmotic sensitivity of competent cells of the mutants HT39 and HT46 in the presence of NaN₃, leading preferentially to lysis of the competent cells.     

Levorphanol inhibition of Ca++ binding to synaptic membranes in vitro

Levorphanol inhibits Ca⁺⁺ binding to synaptic membranes in a dose dependent fashion (0.5–10nM) and induces release of calcium previously bound to membranes. The inhibition is noncompetitive and exhibits a K_i of 9 × 10^?9M. Naloxone (10 nM) blocks this inhibition while dextrophan (100nM) is without effects. The Ca⁺⁺ binding exhibits a high affinity (K_D=0.84μM) and a saturable capacity of 350 picomles/mg protein. The binding curve is sigmoid with a Hill coefficient of 2.2, while in the presence of levorphanol (0.5 to 50nM), the curve becomes hyperbolic with a Hill coefficient of 1.1. The data suggest the calcium and opiate receptor binding sites are in close proximity and may be functionally linked through sub-unit interactions.     

Microphotometric determination of enzymes in brain sections

Summary An incubation medium was established for the microphotometric demonstration of glutamate dehydrogenase (Gldh) in cryostat sections of the rat hippocampus which served as an exemplary brain region. The final incubation medium consisted of 100 mM l-glutamic acid monosodium salt, 5 mM NAD, 10 mM sodium azide (NaN₃), 5 mM ADP, 20 mM sodium chloride, 0.15 mM phenazine methosulfate (PMS), 5 mM nitroblue tetrazolium chloride and 22% polyvinyl alcohol (PVA) in 0.05 M Hepes buffer; the final pH was 7.5. — The study showed that in the histochemical demonstration of Gldh the use of relatively high PVA concentrations were necessary to avoid diffusion artefacts because Gldh seems to be only loosely bound to the mitochondrial matrix. The use of NaN₃ as a blocker of the respiratory chain was indispensible, because without NaN₃ most reduction equivalents were lost through the respiratory chain. With PMS as an exogenous electron carrier, the demonstrable Gldh activities increased significantly indicating that, in the case of Gldh, the endogenous NADH tetrazolium reductase was not sufficiently effective. Furthermore, it was shown that Gldh was affected by many small molecules (e.g. activation by sodium ions, inhibition by magnesium and calcium ions) so that minor variations of the incubation conditions may cause major differences in demonstrable activities. Supported by the Deutsche Forschungsgemeinschaft (Ku 541/2-2)