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 μ m , 10 μ m and 50 μ m . 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.
It is suggested that the isolated protein fraction may correspond to a synaptic receptor and not to the transport system. It is concluded that in the cerebral cortex there is a separate receptor for l -aspartic acid. This is further support to the possible role of this amino acid as a central excitatory transmitter.     

Benzodiazepine Receptor and Thyroid Hormones: In Vivo and In Vitro Modulation

In rats rendered hyperthyroid by chronic treatment with L-triiodothyronine (T3) hormone there was a 21 and 27% decrease, respectively, in the number of binding sites for [3H]flunitrazepam ([3H]FNZ) and [3H]ethyl-beta-carboline-3-carboxylate ([3H]beta-CCE) without changes in affinity for the two ligands. Two weeks after thyroidectomy there was a 44% increase in [3H]FNZ sites and a 17% increase in [3H]beta-CCE binding sites. In vitro we found that T3 produces a decrease in Bmax and an increase in KD, both changes being characteristic of a mixed type of inhibition. Thyroid status dramatically affected the Ki of T3 in displacing [3H]FNZ from sites on isolated membranes of the cerebral cortex: in hypothyroid rats the Ki value was 0.9 microM, whereas in hyperthyroid rats, it was 83 microM, a 92-fold difference. In control rats, the Ki was 11 microM. These findings are discussed in relation to a possible modulation of benzodiazepine receptors by thyroid hormones.     

Differences in the cholinergic proteolipid isolated by affinity chromatography from normal and denervated rat leg muscle

The cholinergic proteolipid from rat leg muscle, isolated by Sephadex LH-20 column chromatography, was purified about 5 fold further by affinity chromatography in organic solvents. The affinity column consisted of p-phenyltrimethylammonium as the active group and a pulse of acetylcholine or acid was used to desorb the specific fraction. From normal controls 36 μg of specific protein per g fresh tissue were obtained. In the denervated muscle there was no increase in this protein after three days, but it increased by 120% after six days. Binding studies carried out with ¹⁴C-acetylcholine, ³H-α-bungarotoxin and ¹⁴C-d-tubocurarine showed that only the specific fraction was able to bind the ligands. Three days after denervation the specific activity (nmoles/mg protein) for ¹⁴C-acetylcholine increased 400% and for ³H-α-bungarotoxin 100% over the control; on the contrary, there was no change in the binding of ¹⁴C-d-tubocurarine. These results are discussed in relation to the different pharmacological properties of the functional and extrajunctional receptors in skeletal muscle.     

ALTERATION OF GABA SYSTEM AND PURKINJE CELLS IN RAT CEREBELLUM BY THE CONVULSANT 3-MERCAPTOPROPIONIC ACID

Abstract— The effect of the convulsant, 3-mercaptopropionic acid (MP) on the content of free amino acids and on the activity of some enzymes related to their metabolism was studied in the rat cerebellum. A decrease in the activity of glutamate decarboxylase (EC 4.1.1.15) and in the level of GABA was found; at the same time, the activity of GABA-aminotransferase (EC 2.6.1.19) was increased. These changes coincided with a profound alteration of the morphology of the Purkinje cells which was related to the dose of MP. These findings, plus some changes in the content of other free amino acids and the activities of related enzymes, suggest that 3-mercaptopropionic acid induces in the cerebellum an imbalance among the amino acids involved in the excitation-inhibition mechanisms.     

The Xenopus laevis Hox 2.1 homeodomain protein is expressed in a narrow band of the hindbrain

The expression pattern of the Xenopus homeodomain protein Hox 2.1 during development was determined using an affinity-purified antibody directed against a carboxyterminal peptide. Nuclear staining was detected in a very narrow band of the hindbrain. This pattern was compared to that of the previously described Xenopus gene XIHbox 1 in serial sections and found to be more anterior than the XIHbox 1 long protein expression but overlapping with that of the short protein. Xenopus Hox 2.1 protein expression is restricted to a much narrower antero-posterior band than that reported for mouse Hox 2.1 RNA expression by in situ hybridization.     

32P incorporation into different membranous structures separated from rat cerebral cortex

Abstract— The time course of incorporation, between 3 hr and 16 days, of ortho[³²P]phosphate into different membranous structures isolated from the rat cerebral cortex was studied. After subarachnoideal administration into the CSF it was found that myelin, mitochondria, microsomes and purified nerve-ending membranes and synaptic vesicles incorporate ³²P at the same rate. Most of the individual phospholipids of the synaptic vesicles and nerve-ending membranes also have similar rates of incorporation. Only phosphoinositides and/or phosphatidylserine may have a more rapid metabolism. The incorporation of ³²P into phosphoproteins follows a different pattern from that of the phospholipids. The intraperitoneal route is less effective in the ³²P incorporation and differences among the fractions may be found. These results are discussed in relation to the problem of the blood-brain barrier to phosphate and to the labelling of individual phospholipids in the different membranes.