Sara Fiszer de Plazas

Isolation of hydrophobic proteins binding amino acids. Stereoselectivity of the binding of L-[14C]glutamic acid in cerebral cortex.

From the total lipid extract of ncrve‐ending membranes or the homogenate of cerebral cortex a hydrophobic protein fraction binding L‐[14C]glutamic acid was separated by chromatography on Sephadex LH20. This protein could only be partially separated from the [14C]GABA‐binding protein and from the lipids that are present in the fraction; however, it was demonstrated that both amino acids bind to different sites. The saturation of the binding showed a high (Kd1= 0.3μM), a medium (Kd, = 5 μM) and a low (Kd, = 55 μM) affinity binding site. The high affinity binding site had a binding capacity of 0.53 nmol/mg of protein and was highly stereoselective for the L‐enantiomer. The binding of L‐[14C]glutamic acid was not inhibited by GABA, was slightly inhibited by glycine and glutamine and was strongly inhibited in a progressive order by DL‐a‐methylglutamic acid, L‐nuciferine, L‐aspartic acid and L‐glutamic acid diethyl ester. These results are compared with those previously obtained with the L‐glutamic acid‐binding protein isolated from crustacean muscle. The stereoselectivity of the binding and the possible role of this protein in synaptic transmission are discussed.

Acetylcholinesterase and acetylcholine proteolipid receptor: Two different components of electroplax membranes

Electroplax membranes from Electrophorus electricus were separated by two methods of subcellular fractionation, and in both cases a parallelism between the content of acetylcholinesterase and the binding of cholinergic drugs was observed. Treatment of the membranes with the -SH reagents, p-hydroxymercuribenzoate and p-chloromercuribenzoate, produced a 16–28% inhibition in the binding of [14C]acetylcholine. With the -S-S- reagent, 1,4-dithiothreitol, followed by N-ethylmaleimide the inhibition was 64–67%. Membranes depleted of acetylcholinesterase with 1 M NaCl showed no quantitative change in the binding of dimethyl (+)-[14C]tubocurarine and methyl[14C]hexamethonium as compared to the controls. Furthermore the same amount of receptor proteolipid, which binds [14C]acetylcholine, was found after the 1 M NaCl treatment. It is concluded that: (a) acetylcholinesterase and the acetylcholine proteolipid receptor are two different macromolecules present in the electroplax membranes; (b) the receptor proteolipid, previously isolated from the intact tissue, is contained in the electroplax membrane.

ISOLATION OF HYDROPHOBIC PROTEINS BINDING AMINO ACIDS: l-ASPARTIC ACID-BINDING PROTEIN FROM THE RAT CEREBRAL CORTEX

Abstract— Lyophilized rat cerebral cortex was treated with chloroform‐methanol (2:1, v/v), and the extracted hydrophobic proteins (i.e. proteolipids) were separated by column chromatography on Sephadex LH‐20. The first peak of protein, eluting with chloroform in the void volume, had high affinity binding for l‐[14C]aspartic acid. The saturation of the binding showed three saturable sites with apparent dissociation constants of 0.2 μm, 10 μm and 50 μm. The binding capacities of the three sites were 2.8, 132 and 617 nmol/mg of protein, respectively. There were 8.0 nmol of high affinity binding sites for l‐aspartic acid and 1.53 nmol for l‐glutamic acid per g of fresh tissue in the cerebral cortex of the rat. Differentiation between binding of l‐aspartic and l‐glutamic acid was clearly established by cross‐binding and competition experiments with agonists and antagonists.

ISOLATION OF HYDROPHOBIC PROTEINS BINDING AMINO ACIDS: γ‐AMINOBUTYRIC ACID BINDING IN THE RAT CEREBRAL CORTEX

—The binding of [14C]GABA to nerve‐ending membranes isolated from rat cerebral cortex follows a hyperbolic curve saturating at 0·4pmol/μg protein. This binding is about 60% inhibited by chloropromazine, and about 40%, inhibited by bicuculline. A hydrophobic protein fraction binding [14C]GABA was separated from the total. lipid extract of nerve‐ending membranes. The binding follows a hyperbolic curve that saturates at 10·5 pmol of [14C]GABA/μg of protein, with an apparent Kd= 30 μm. The binding is competitively inhibited by bicuculline with a Ki= 273 μm. These results are compared with those previously obtained on a GABA binding protein from crustacean muscle.

Isolation of a proteolipid from spleen capsule binding (±)-[3H]norepinephrine

Abstract Bovine spleen capsules were extracted with chloroform-methanol (2:1, v/v) and the proteolipids were separated by column chromatography on Sephadex LH-20. Five proteolipid peaks were eluted with chloroform and one peak with chloroform-methanol (4:1, v/v). Only Peak 1 eluted between 17–22 ml of chloroform showed binding for [ 3 H]norepinephrine. The saturation curve of the binding in Peak 1 suggests that this proteolipid contains two groups of sites with different dissociation constants. For 200 000 g of proteolipid 3 moles of norepinephrine are bound with high affinity and about 27 moles with low affinity. Similar findings were obtained using a partition method for the study of the binding and the competition with some adrenergic antagonists was demonstrated. The possibility that Peak 1 of proteolipid represents the adrenergic receptor of the spleen capsule is discussed.

Subcellular distribution and possible nature of the binding for 14C-dibenamine and 14C-propanolol in the CNS☆

The binding capacity of subcellular fractions, from the cat basal ganglia and brain stem, was studied using respectively α- and β-adrenergic blocking agents 14C-dibenamine and 14C-propanolol. In both cases the highest uptake was obtained in the nerve-ending membrane fractions and was interfered with norepinephrin. In the case of 14C-dibenamine the localization of the binding was in the junctional complex which includes the subsynaptic membrane. Both the 14C-dibenamine and 14C-propanolol in a bound form were extracted with organic solvents. Although the nature of the material binding these drugs could not be ascertained with certainty, several experiments suggest that it may be a special proteolipid present in the nerve-ending membranes.

Diazepam fails to potentiate GABA-induced chloride uptake and to produce anxiolytic-like action in aged rats.

The pharmacological response to benzodiazepines has been demonstrated to be different in aged individuals in comparison to adults. We studied the age-dependent changes in some of the in vitro and behavioral effects of diazepam in aged (24 months old) rats, comparing them to adults (3 months old). We evaluated the in vitro gamma-aminobutyric acid (GABA)-induced 36Cl- uptake and the diazepam potentiation of GABA-stimulated 36Cl- uptake in microsacs from cerebral cortex of both groups of animals. We found no differences in the GABA-stimulated 36Cl- uptake between adult and aged animals, and diazepam failed to potentiate GABA-induced 36Cl- flux in the aged cortical microsacs. We also examined the effect of 0.03-10 mg of diazepam on locomotor activity in an open-field test and the anxiolytic-like action of diazepam in doses ranging from 0.03 to 1 in a dark-light transition test. We observed no anxiolytic-like action of the drug in the dark-light transition test in the aged rats, while there was a shift to the left in the diminution of locomotor activity evaluated by the open-field test. We conclude that the pharmacodynamic changes observed in cortical GABA(A) receptors in aged rats could partially explain the lack of anxiolytic-like action but not the oversedation evidenced in this group of animals.

Differentiation of L-aspartate and L-glutamate high-affinity binding sites in a protein fraction isolated from rat cerebral cortex

THE possibility that L-glutamate and L-aspartate are excitatory transmitters in the CNS is supported by neurochemical and neurophysiological evidence1. The problems of identifying particular synapses that are excited by one or the other of these dicarboxylic amino acids, and distinguishing between the two receptors are, however, more difficult. At the recent International Meeting of Neurochemistry (September 1975) Graham summarised some of the evidence favouring distinct functions for the two amino acids, concentrating on the possible transmitter role of L-aspartate. For example, a higher content of this amino acid is found in the ventral than in the dorsal grey matter of the spinal cord2, aortic occlusion leads to loss of aspartate content and degeneration of small neurones3, reduction in aspartate content is seen in the spinal cord after section of dorsal roots4 and in the olfactory cortex after olfactory bulbectomy5, which also decreases glutamate. Renshaw cells are slightly more sensitive to the ionotophoretic administration of L-aspartate while the reverse is true of interneurones of the dorsal spinal horn6. It is possible that L-glutamate and L-aspartate, being flexible molecules, fold or unfold and could interact with either receptor and McCulloch et al.7 accordingly used kainate, a conformational restricted analogue of glutamic acid, which would be unlikely to fold to occupy the aspartate receptor, and N-methyl-D-aspartate, which would be too small to interact with the glutamate receptor (Fig. 1). The difference in sensitivity between the two groups of neurones to these agonists was much greater: the Renshaw cells were much more sensitive to N-methyl-D-aspartate and the dorsal interneurones to kainate7.

Binding of α-bungarotoxin to the cholinergic receptor proteolipid from Electrophorus electroplax

Abstract 1. 1. This investigation was initiated to study the similarities or differences between the cholinergic proteolipid first isolated in our laboratory from Electrophorus electroplax and the protein-detergent-α-bungarotoxin complex isolated by others. 2. 2. The binding of α-[ 131 I]bungarotoxin to the proteolipid extracted from electric tissue or electroplax membranes was studied using column chromatography and a partition method. 3. 3. The saturation curve obtained revealed a single site of binding per molecule of proteolipid of molecular weight 37000. 4. 4. The binding of α-bungarotoxin could not be displaced with acetylcholine or decamethonium. 5. 5. It is concluded that the cholinergic proteolipid extracted with organic solvents is similar to the protein-α-bungarotoxin complex separated by the use of strong detergents.

Prolonged exposure to hypobaric hypoxia transiently reduces GABAA receptor number in mice cerebral cortex

The central nervous system is severely affected by hypoxic conditions, which produce alterations in neural cytoarchitecture and neurotransmission, resulting in a variety of neuropathological conditions such as convulsive states, neurobehavioral impairment and motor CNS alterations. Some of the neuropathologies observed in hypobaric hypoxia, corresponding to high altitude conditions, have been correlated with a loss of balance between excitatory and inhibitory neurotransmission, produced by alterations in glutamatergic and GABAergic receptors. In the present work, we have studied the effect of chronic hypobaric hypoxia (506 hPa, 18 h/day x 21 days) applied to adult male mice on GABA(A) receptors from cerebral cortex, to determine whether hypoxic exposure may irreversibly affect central inhibitory neurotransmission. Saturation curves for [3H]GABA specifically bound to GABA(A) receptors in isolated synaptic membranes showed a 30% decrease in maximal binding capacity after hypoxic exposure (Bmax control, 4.70+/-0.19, hypoxic, 3.33+/-0.10 pmol/mg protein), with no effect on GABA binding sites affinity (Kd control: 159.3+/-13.3 nM, hypoxic: 164.2+/-15.1 nM). Decreased B(max) values were observed up to the 10th post-hypoxic day, returning to control values by the 15th post-hypoxic day. Pharmacological properties of GABA(A) receptor were also affected by hypoxic exposure, with a 45 to 51% increase in the maximal effect by positive allosteric modulators (pentobarbital and 5alpha-pregnan-3alpha-ol-20-one). We conclude that long-term hypoxia produces a significant but reversible reduction on GABA binding to GABA(A) receptor sites in cerebral cortex, which may reflect an adaptive response to this sustained pathophysiological state.