N-ACETYL-l-ASPARTIC ACID IN BRAIN

Abstract

In recent studies on the amino acids and related compounds present in protein-free extracts of cat tissues (l), a relatively large amount of bound aspartic acid was found in the extract from brain. Acid hydrolysis of the extract liberated about 80 mg. of aspartic acid per 100 gm. of tissue, less than 2 per cent of which could have arisen from asparagine. The bound aspartic acid in brain represented the largest quantity of conjugated amino acid of unknown nature found in any of the tissue extracts examined. The isolation of this conjugate and its identification as N-acetyl-n-aspartic acid form the subject of this communication. Since there were no major unidentified ninhydrin-positive peaks on the effluent curve obtained from the extract of brain, it could be concluded that the conjugated amino acid was present in an N-substituted and ninhydrin-negative form. In order to locate compounds of this type in the effluent from the ion exchange columns, aliquots were removed from the fractions for direct ninhydrin analysis and for analysis after acid hydrolysis. In this manner, a relatively large amount of material, which was ninhydrin-positive after hydrolysis, was found in a peak emerging at about 60 effluent ml., just preceding phosphoethanolamine (cf. Fig. 1 (1)). Amino acid analysis of a hydrolyzed sample from this peak showed that it contained nearly all of the conjugated aspartic acid of the brain; 87 per cent of the ninhydrin color of the hydrolysate arose from aspartic acid, and the amount of this amino acid corresponded to 74 mg. per 100 gm. of tissue. The presence of small amounts of glutamic acid, serine, cystine, and tyrosine in the hydrolysate indicated that the compound was not present in a completely pure form. Since the derivative was eluted rapidly from the Dowex 50 column, it appeared likely that the conjugate was an acidic compound and could probably be chromatographed more effectively by using a column of a basic resin, such as Dowex 2-X4, as had been done with phenylacetylglutamine (2) and tyrosine-O-sulfate (3). When an extract of cat brain was chromatographed on a 0.9 X 15 cm. column of Dowex 2-X4, the effluent curve shown in Fig. 1 was obtained. The peak at about 90 ml. was completely ninhydrin-negative before hydrolysis, and the amino acid content of a hydrolysate showed aspartic acid to be the only amino acid present. Rechromatography of a sample from the Dowex 2 experiment on a column