GLUTAMYL, ASPARTYL AND AMIDE MOIETIES OF CEREBRAL PROTEINS: METABOLIC ASPECTS <i>IN VITRO</i>

Abstract

Abstract— The total mixed proteins (excluding proteolipids) were isolated from cat cerebral cortex and subjected to acid and enzymic hydrolyses. Analyses on the hydrolysates were carried out by specific enzymic procedures to determine the glutamyl, glutaminyl, aspartyl and asparaginyl composition. The content of total glutamyl and total aspartyl residues was the same in all types of protein samples, with average values of 78 and 58 /miol/100 mg of protein, respectively. In biopsy samples approximately 45 per cent of each total was in the amide form. Preparation of slices of cerebral cortex for incubation was associated with deamidation in situ of 16 per cent of the protein‐bound glutaminyl residues. The extent of deamidation was not increased by incubation or by prolonged hypoxia and was unaffected by prior anaesthesia or by incubation of slices with 10 mM‐NH 4 Cl or 40 mM‐malonate. Slices prepared from animals intoxicated with methionine sulphoximine exhibited no deamidation. No deamidation was observed for slices of subcortical white matter, liver, kidney, testis or diaphragm of the cat. Cortical proteins from other species appeared to behave similarly to those of the cat. The 5‐4 μmol of NH 3 released/g of fresh cortex could account for about 85 per cent of the endogenous free ammonia regularly encountered in such slices. Hence the labile fraction of protein‐bound glutaminyl amide groups represents, as previously suspected, a major source of endogenous cerebral NH 3 . Proteins isolated from cerebral cortical slices incubated with L‐[U‐ 14 C]glutamic acid or L‐[U‐ 14 C]glutamine contained 105 (±0.095) per cent of the total 14 C metabolized. The ratios (x 100) of protein to free pool specific radioactivities (c.p.m.μmol) of glutamic acid and of glutamine were in the range 0‐22 to 0‐42, or of the same order as previously reported for other amino acids. Comparable results were obtained with proteins isolated from cerebral cortical slices incubated with 10 mM‐ 15 NH 4 Cl or L‐[amide‐ 15 N]glutamine or both. In the amide N of protein‐bound glutaminyl residues the atoms per cent excess 15 N ranged from 007 to 0‐42. This degree of labelling could be accounted for completely by the turnover of the entire glutaminyl moiety, as indicated by the 14 C studies. Simultaneous analyses of free pool NH 3 and glutamine suggested that transfer of glutamine from medium to slice involves deamidation as it is taken up and reamidation after entry.