Synaptic Membrane Fractions Research Articles

Binding of beta-bungarotoxin to synaptic membrane fractions of chick brain.

beta-Bungarotoxin (beta-BuTx) is a presynaptically active snake venom phospholipase A2 which, in the chick, is cytotoxic for certain subclasses of central and peripheral neurons. In order to elucidate whether the toxin's specificity is due to the existence of specific neuronal binding sites, the binding of 125I-labeled beta-BuTx to synaptic membrane fractions of chick brain was investigated. These experiments defined a single class of saturable high affinity binding sites (KD = 0.47 +/- 0.14 nM, k+1 = 4.3 x 10(6) M-1 s-1, k-1 = 1.08 x 10(-4) s-1) whose pharmacological properties correlated with that of the cytotoxic action of beta-BuTx on cholinergic neurons in chick retina cultures. The density of 125I-beta-BuTx binding sites in synaptic membrane fractions was low (50 fmol/mg of protein). Also specific toxin binding occurred only to membrane fractions of chick organs known to contain beta-BuTx-sensitive cells or nerve endings, i.e. brain, retina, and muscle, but not liver and heart. The binding of 125I-beta-BuTx to synaptic membrane fractions was dependent on Ca2+; Co2+ and Sr2+, but not Mg2+, could replace Ca2+ in the binding reaction. The membrane binding site for 125I-beta-BuTx was sensitive to heat and high concentrations of pronase, and thus most likely is a protein.

Effects of trypsin-treatment on agonist binding affinity to the muscarinic acetylcholine receptor

Trypsin-treatment of the microsome fraction of the ileum and the synaptic membrane fraction of the cerebral cortex of guinea-pig caused selective reduction in the apparent affinity of an agonist (carbachol), but not an antagonist (atropine), to muscarinic acetylcholine receptors (mAChR), measured as inhibition of binding of 3H-quinuclidinyl benzilate ( 3H-QNB). This effect was similar to that of Gpp(NH)p. The effects of trypsin and Gpp(NH)p were not additive. On the other hand, treatment of these fractions with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) increased the apparent affinity of agonist, but not antagonist. The effect of DTNB predominated over those of trypsin and Gpp(NH)p, when the fractions were treated with two reagents simultaneously.

The blocking action of baclofen on excitatory transmission in the rat hippocampal slice

The mode of action of baclofen on the physiology of the rat hippocampus was investigated by studying its effect on electrophysiological responses in the hippocampal slice preparation and by measuring biochemical parameters related to glutamate uptake, binding, and release. Baclofen inhibited, in a dose-dependent fashion, the dendritic field potentials in field CA1 produced by stimulation of the Schaffer commissural fiber system. The drug was inactive in this respect at concentrations of 10 and 100 nM but consistently reduced the amplitude of both the dendritic field potential and the population spike at a concentration of 1 microM. At a concentration of 25 microM, baclofen virtually abolished the dendritic and cell body responses to afferent stimulation. Recovery of field potentials required between 7 and 10 min following the addition of 1 microM baclofen. The levorotatory form of baclofen was much more potent in suppressing synaptic responses than was the dextrorotatory enantiomer. Baclofen, at a concentration of 5 microM, strongly antagonized the excitation of pyramidal neurons evoked by iontophoretically applied glutamate. The antagonism of the glutamate effect was much reduced when the slices were maintained in low calcium, high magnesium perfusion medium. Moreover, under low calcium conditions, baclofen did not interfere with the effects of bath-applied glutamate on antidromically elicited responses. Baclofen did not affect the Na+-dependent or Na+-independent binding of [3H]glutamate to crude synaptic membrane fractions from the hippocampus. However, at a concentration of 1 microM, it markedly inhibited potassium-induced release of [3H]glutamate from hippocampal synaptosomes. Taken together, the present results strongly suggest that baclofen suppresses synaptic responses in the Schaffer commissural fiber system of the hippocampus by blocking the release of an excitatory amino acid transmitter.

Variable homeoviscous responses of different brain membranes of thermally-acclimated goldfish

The effects of thermal acclimation of goldfish upon the bulk fluidity of synaptic, mitochondrial and myelin membrane fractions of brain was determined using steady-state and differential polarised phase fluorimetry. Membrane fluidity decreased in the order, mitochondria>synaptic membranes>myelin. In each case membranes from cold-acclimated goldfish were more fluid than the corresponding membranes of warm-acclimated goldfish, though the adjustment of fluidity in each case was insufficient to compensate for the direct effects of the temperature difference. The extent of fluidity compensation was greatest in the mitochondrial fraction and least in the myelin fraction, indicating heterogeneous responses in different membrane-types. Steady-state and dynamic fluorimetric techniques provided identical estimates of the homeoviscous responses, indicating that despite its short-comings, the steady-state technique provided as good a measure of adaptive responses as the more complex and sophisticated technique.

Cysteine sulfinic acid in the central nervous system: specific binding of [35S]cysteic acid to cortical synaptic membranes--an investigation of possible binding sites for cysteine sulfinic acid.

Specific binding sites for cysteine sulfinic acid, an excitatory amino acid, in crude synaptic membrane fractions of rat cerebral cortex were examined, using L-[35S]cysteic acid as a ligand. Two specific binding systems of [35S]cysteic acid were found, one Na+-dependent and the other Na+-independent. The Na+-independent specific binding of [35S]cysteic acid was saturable, with a Kd of 474 nM and Bmax of 3.29 pmol/mg protein. The binding was optimal at pH 7.4 and at 37 degrees C. Treatment of the membranes with proteases, concanavalin A, or Triton X-100 markedly reduced the binding. Of various compounds related to cysteic acid, L-cysteine sulfinic acid was the most effective competitor of this binding. These results indicate the existence of an Na+-independent specific binding site for cysteic acid in the synaptic membrane of rat cerebral cortex, which may be different from that for glutamate. Possible involvement of cysteine sulfinic acid as an endogenous ligand for this binding site is discussed.

Binding of toxic bicyclic phosphates to rat brain synaptic membrane fractions.

A 3H-labeled bicyclic phosphate (BP), 4-[2,3-3H]propyl-2,6,7-trioxa-1-phosphabicyclo-[2.2.2]octane 1-oxide, bound specifically to rat brain synaptic membrane preparations. The apparent dissociation constant and the quantity of the BP bound at saturation were estimated to be about 30 μm and about 2 pmol per mg of protein, respectively. BP analogs except 4-amino BP, which is extremely low in toxicity, inhibited the binding but no obvious correlation was observed between the toxicity and the inhibitory potency of the BP analogs. The binding was also inhibited by high concentrations of GABA and picrotoxinin, whereas it was enhanced by bicuculline methiodide.

Enzymic inactivation of substance P in the central nervous system.

A membrane-bound 'substance P degrading enzyme' (EC 3.4.24.-) from human brain has been purified to apparent homogeneity. The enzyme was extracted from a membrane fraction of human diencephalon with a non-ionic detergent, Brij 35, and activity was monitored by measuring the rate of disappearance of added substance P using radioimmunoassay, bioassay or radiochemical assay. The enzyme is a thermolabile, neutral metallo-endopeptidase with a relative molecular mass of about 50000. It cleaves substance P between Gln6-Phe7, Phe7-Phe8 and Phe8-Gly9, with a ratio of 0.7:1:1. The breakdown products have been identified by a combination of reverse-phase high-performance liquid chromatography and amino acid analysis. A similar cleavage pattern of substance P has also been demonstrated in a synaptic membrane fraction prepared from rat brain, indicating that a 'substance P-degrading enzyme' is the major peptidase responsible for inactivating the peptide in rat brain membranes. The properties of this enzyme distinguished it from previously described peptidases for which substance P is a substrate. Its high selectivity and its affinity for substance P, among many other neuropeptides, suggest that it may be involved in the physiological inactivation of the peptide by neural tissues.

Affinity-purified anti-protein I antibody. Specific inhibitor of phosphorylation of protein I, a synaptic protein.

Protein I is a synaptic protein which serves as an endogenous substrate for cyclic AMP- and calcium-dependent protein kinases. Antibodies raised against purified Protein I have been isolated from rabbit antiserum by affinity chromatography on Protein I-conjugated agarose column. The purified antibodies were identified as immunoglobulin G by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Ouchterlony double immunodiffusion precipitation test. The purified antibodies not only inhibited the phosphorylation of purified Protein I by exogenous cyclic AMP-dependent protein kinase, but also inhibited specifically the phosphorylation of Protein I by endogenous cyclic AMP-dependent protein kinase in a synaptic vesicle fraction, synaptic junctional complex fraction, synaptic membrane fraction, and crude homogenate of cerebrum. The purified anti-Protein I antibodies also inhibited with a similar potency calcium-dependent phosphorylation of Protein I without affecting the phosphorylation of other proteins. The Fab(t) fragment of anti-Protein I immunoglobulin G, which was produced by tryptic digestion, retained the ability to inhibit specifically the phosphorylation of Protein I. This substrate-directed, specific inhibitor of the phosphorylation of Protein I may provide a unique probe for investigating the function of Protein I phosphorylation.

Characterization of gamma-aminobutyric acid binding sites on crude synaptic membranes.

[3H]GABA binding to crude synaptic membranes of rat brain was studied in an attempt to identify GABA binding to its synaptic receptor in the presence of Na+. Membrane vesicles prepared from crude synaptic membrane fractions were useful as a tool to differentiate synaptic GABA receptors from GABA uptake sites. The crude synaptic membranes treated with Triton X-100 [membranes (TX)] involved two classes of GABA binding sites (KD = 38.7 and 78.0 nM) in the absence of Na+, but the high-affinity sites disappeared in the presence of Na+ and a single class of GABA binding sites (KD = 75.0 nM) was detected. The failure to detect an active uptake of [3H]GABA into the vesicles prepared from membranes (TX) suggests that the [3H]GABA binding in the presence of Na+ was related to synaptic GABA receptors. It is probable that Na+ could mask the presence of the high-affinity class of GABA receptor.

Postnatal development of synaptic glycine receptors in normal and hyperglycinemic rats.

The postnatal development of glycine synaptic receptors has been studied. Strychnine binding to the synaptic membrane fraction is very low at birth, increases thereafter, and reaches adult values at the 15th day in the brain, and at the 30th day in the spinal cord. Throughout postnatal development, there are more glycine receptors in the spinal cord than in the brain. The development of receptors in the spinal cord displays a pattern similar to that reported previously for the glycine reuptake system in spinal cord slices and in the activity of spinal cord glycine synthase. In rats with experimental hyperglycinemia strychnine binding to spinal cord glycine receptors increases much more rapidly, reaching a level 1.5 times the control value by day 10. When the hyperglycinemia was induced after the 10th postnatal day, however, no effect on the glycine receptors was observed. This increased number of receptors could be explained by an effect of glycine on the synaptic stabilisation process. No changes in the KD for strychnine were observed either during postnatal development or in hyperglycinemic rats. The KD remained approximately 10 nM in the spinal cord and 50 nM in the brain. Results are discussed with respect to the ontogeny of glycinergic synapses and the pathogenesis of nonketotic hyperglycinemia.

Influence of prenatal hypoxia on brain development: effects on body weight, brain weight, DNA, protein, acetylcholinesterase, 3-quinuclidinyl benzilate binding, and in vivo incorporation of [14C]lysine into subcellular fractions.

The influence of prenatal hypoxia on subsequent brain development in the young rat was investigated by examining body and brain weight, cerebral cortex wet weight, protein and DNA concentrations, acetylcholinesterase (AChE) activity, 3-quinuclidinyl benzilate (QNB)-binding levels, the relative amounts of protein in various subcellular fractions, and the in vivo incorporation of [14C]lysine into the protein of homogenate and subcellular fractions. Exposure of pregnant females to a mild hypoxia (9.1% O2, 10 h per day for the 9--11 days preceding birth) resulted in a reduced body weight in the pups and days 1 and 5 after birth; total cortical DNA was reduced but brain weight and protein content were unaffected, leading to a higher protein/DNA ratio in prenatally hypoxic pups. By 10 days of age these differences between prenatally hypoxic and control animals were no longer apparent. There were no differences between prenatally hypoxic and control animals in AChE and QNB binding per milligram cortex protein. The relative amount of synaptic membrane protein from the cerebral cortex was reduced at day 1 in prenatally hypoxic animals and the synaptic membrane fraction showed a higher level of incorporation of [14C]lysine on days 1, 5, and 10. The developmental profile of [14C]lysine incorporation showed a peak on day 10 which was higher in prenatally hypoxic rats. By 46 days after birth little difference could be found between prenatally hypoxic and control animals.

Function of a calmodulin in postsynaptic densities. III. Calmodulin-binding proteins of the postsynaptic density.

A method has been developed for binding calmodulin, radioiodinated by the lactoperoxidase method, to denaturing gels and has been used to attempt to identify the calmodulin-binding proteins of cerebral cortex postsynaptic densities (PSDs). Calmodulin primarily bound to the major 51,000 Mr protein in a saturatable manner; secondarily bound to the 60,000 Mr region, 140,000 Mr region, and 230,000 Mr protein; and bound in lesser amounts to a number of other proteins. The major 51,000 Mr calmodulin-binding protein is one of unknown identity. Binding of iodinated calmodulin to these proteins was blocked by EDTA, EGTA, chlorpromazine, and preincubation with unlabeled calmodulin. Calmodulin iodinated by the chloramine-T method, which inactivates calmodulin did not bind to the PSD but bound nonspecifically to histone. Calmodulin did not bind to proteins from a variety of sources for which calmodulin interactions have not been found. Except for three proteins, all of the proteins of synaptic membranes that bind calmodulin could be accounted for by proteins of the PSD which are a part of the synaptic membrane fraction. The major 51,000 M, protein and the corresponding iodinated calmodulin binding were greatly reduced in cerebellar PSDs and this difference between cerebral cortex and cerebellar PSDs is discussed in light of the possible function of calmodulin in synaptic excitatory responses.

Increased incorporation of [3H]lysine into synaptic membranes of the visual cortex following first exposure of dark-reared rats to light is confined to particular polypeptides.

The incorporation of [3H]lysine into separated polypeptides of synaptic-membrane fractions prepared from the visual cortices of dark-reared rats and littermates exposed to light for 1 h was examined. Increased incorporation of [3H]lysine was found in synaptic membranes from light-exposed compared to dark-reared rats in polypeptides of four molecular weights: 100,000, 71,000, 44,000, and 38,000. The 44,000-molecular-weight peak has been suggested to be actin on the basis of its comigration with pure brain actin. These results indicate that increased incorporation of [3H]lysine into synaptic membranes of the visual cortex, following first exposure of dark-reared rats to light, is confined to particular polypeptides.

An endogenous substance from porcine brain antagonizes the anticonvulsant effect of diazepam

An endogenous substance /ES/ obtained from porcine brain /MW~3000/ partially inhibited the specific 3H-diazepam binding in the P 2-fraction but not in the synaptic membrane fraction of rat brain. ES inhibited specific 3H-GABA binding to synaptic membranes in a dose-related manner. Intraamygdaloid injection of diazepam fully inhibited the convulsions evoked by systemic administration of pentylenetetrazol in the rat. This anticonvulsant action of diazepam was antagonized by a previous intraamygdaloid injection of ES but not of myoglobin. ES does not seem to be an endogenous ligand of benzodiazepine receptors; it probably exerts its effect via GABA receptors.

Activation of adenylate cyclase of rat brain by lipid peroxidation.

The relationship between adenylate cyclase activity in the synaptic membrane fraction (M1) of rat brain and lipid peroxidation of these membranes was examined. In the presence of 5 mM dithiothreitol (DTT), 1 to 10 microM Fe/+ activated adenylate cyclase 2- to 4-fold. Of several metal ions, Fe2+ was the most effective. Other enzymes in M1, such as Mg2+-ATPase, (Na+-K+)-ATPase, 5'-nucleotidase, acetylcholinesterase, and phosphodiesterase, were not activated by Fe2+ plus DTT. Activation of adenylate cyclase by Fe2+ plus DTT was accompanied by production of malondialdehyde, a product of lipid peroxidation. Formation of malondialdehyde was completely parallel with enzyme activation. Ascorbic acid or a NADPH system also stimulated enzyme activity and caused lipid peroxidation. Activation of the enzyme and lipid peroxidation induced by Fe2+ plus DTT, ascorbic acid, or NADPH was completely prevented by simultaneous addition of N,N'-diphenyl-p-phenylenediamine, an inhibitor of lipid peroxidation. This inhibitor also prevented the decrease in turbidity of the enzyme preparation induced by Fe2+ plus DTT. The stimulatory effects of NaF, guanylyl-5'-imidodiphosphate and calmodulin, respectively, and that of Fe2+ plus DTT on the enzyme activity were additive. Activation of adenylate cyclase by Fe2+ plus DTT was only observed in brain synaptic membranes, not in erythrocyte ghosts, liver plasma membranes, or cardiac sarcolemma. These results indicate that lipid peroxidation of synaptic membranes was accompanied by specific stimulation of adenylate cyclase activity.

Developmental changes in morphology and molecular composition of isolated synaptic junctional structures

Synaptic junctional fractions which display subcellular purity that compares favorably to similar fractions prepared from adult have been isolated from immature rat brains. Electron microscopic analysis of immature fractions has revealed age-dependent changes in the morphology of isolated synaptic structures. The recovery of total synaptic junctional protein increased in a linear fashion and was temporally correlated with the appearance of asymmetric synapses in brain. Systematic age-dependent changes were observed in the protein and glycoprotein composition of synaptic membrane and synaptic junction fractions during postnatal development. In isolated synaptic junctions, the major postsynaptic density protein increased approximately 20-fold during postnatal development. Immature synaptic junction fractions contained tubulin and actin in larger relative quantities than are present in synaptic junction fractions isolated from adult brain tissues. Immature synaptic junctions also contained appreciable amounts of postsynaptic membrane glycoproteins that bind concanavalin A (con A).

Attachment of the synapse-specific phosphoprotein protein I to the synaptic membrane: a possible role of the collagenase-sensitive region of protein I.

The purified synapse-specific phosphoprotein Protein I was previously shown to be degraded by a bacterial collagenase, through a series of intermediates, to a collagenase-resistant fragment of molecular weight about 48,000 containing a phosphorylated serine residue. In this study, a purified synaptic membrane fraction containing Protein I was treated with Cl. histolyticum collagenase; membrane-bound and membrane-free proteins were then phosphorylated using [gamma-32P]ATP and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. It was observed that Protein I bound to the synaptic membrane was susceptible to the collagenase and degraded to fragments of molecular weights about 68,000, 62,000, and 48,000; the 68,000 fragment remained bound to the membrane whereas the 62,000 and 48,000 fragments were dissociated from the membrane. These observations suggest that the peptide moiety of mol. wt. 6000, present in the 68,000 fragment but absent from the 62,000 fragment, may play a crucial role in anchoring Protein I to the synaptic membrane.

O-68) - Specific binding of cysteine sulfinic acid to synaptic membrane fractions

O-68) - Specific binding of cysteine sulfinic acid to synaptic membrane fractions

Influence of Sodium-Independent ?-Aminobutyric Acid Binding on the Assay of Sodium-Dependent ?-Aminobutyric Acid Binding in a Membrane Preparation of Rat Brain

A large amount of [3H]GABA was bound to crude synaptic membrane fractions of rat, by sodium-independent process in a medium that contained 100 microM [3H]GABA used for assaying GABA uptake site. This [3H]-GABA binding was different from receptor binding of GABA. It was confirmed that this sodium-independent [3H]GABA binding scarcely occurred in the presence of a physiological concentration of sodium chloride, and that sodium-independent GABA binding had a negligible influence on sodium-dependent GABA binding.

Subcellular locaization of increased incorporation of [ 3H]fucose following passive avoidance learning in the chick

The effect of training on a passive avoidance test on incorporation of [ 3H]fucose into subcellular fractions of the anterior forebrain roof of 1-day-old chicks was determined. Isotope was injected intraperitoneally, and birds tested and killed after 3 h. There was an increase in incorporation in trained compared with untrained control birds in the synaptic membrane (19% elevation, P < 0.02) and the mitochondrial (14% elevation, P < 0.05) fractions. Following intracranial injection, increased incorporation in trained birds was seen only in the synaptic membrane fraction (39% elevation, P < 0.01); this increase in incorporation was not limited to any particular electrophoretically separated glycopeptides, but occurred in all 9 detected peaks.