Isolation and characterization of glycosaminoglycans in peripheral nerve and spinal cord of monkey.

The isolation of uronic acid-containing glycosaminoglycans from peripheral nerve and spinal cord of monkey was done by combining the cetyl pyridinium procedure and DEAE-Sephadex column chromatography. The constituent analyses of the isolated GAG-fractions indicated that hyaluronic acid, chondroitin-4-sulphate, chondroi tin-6sulphate, heparan sulphate and a testicular hyaluronidase-resistant galactosamine-containing GAG were present in both tissues. Hyaluronic acid was the predominant GAG (63 per cent) in both tissues and its level was much higher than in brain. Chondroitin-4-sulphate constituted 16 per cent in both tissues. The levels of heparan sulphate and hyaluronidaseresistant galactosamine-containing GAG in these tissues were much lower than in brain. The results indicate that the patterns of GAGs in peripheral nerve and spinal cord of monkey are similar but differ from that of brain. SEVERAL laboratories have reported the identification of glycosaminoglycans (GAGs) in different nervous tissues. ABOOD and ABUL-HAJ (1956) showed histochemically the presence of hyaluronic acid in peripheral nerve of bullfrogs. The work of SZABO and ROBOZ-EINSTEIN (1962) indicated the occurrence of sulphated and non-sulphated GAG in bovine spinal cord and brain. Further they noticed that the concentration of sulphated GAG in spinal cord was much less than in brain. This finding prompted us to extend our investigation on the nature of GAG in brain to spinal cord as well as to peripheral nerve. The present paper reports on the isolation and characterization of uronic acid-containing GAGs in spinal cord and peripheral nerve of monkey. MATERIALS A N D METHODS Cetyl pyridinium bromide was obtained from Mann Research Laboratories, U.S.A. and chondroitin-6-sulphate from Miles Research Laboratories. Glucosamine-IiCI, galactosamine-HCI, hyaluronic acid and testicular hyaluronidase type I were supplied by Sigma Chemicals, U.S.A. Sephadex G-75 and DEAE-Sephadex A-25 were obtained from Pharmacia Uppsala, Sweden. Papain (crude) and pronase B were received respectively from Central Food Technological Research Institute, Mysore, and California Biochemical Corporation, U.S.A. Other reagents used were of analytical grade. The processing of the tissues for the isolation of GAGs was carried out as described earlier (SINGH and BACHHAWAT, 1968), and 12.2 g of lipid free dry tissue were obtained from 77.3 g spinal cord and 11.1 g from 52.7 g peripheral nerves (median and lateral popliteal). Amdyes. The modification of the carbazole reaction (DISCHE, 1947) by BITTER and MUIR (1962) was used to estimate uronic acid. Dermatan sulphate was identified by making use of its very low colour yield in carbazole reaction without borate. The total hexosamine, galactosamine, sulphate, non-acetylated hexosamine (N-sulphate), chondroitin-6-sulphate and hyaluronidase resistant GAG were estimated as described earlier (CHANDRASEKARAN and BACHHAWAT, 1969). RESULTS Table 1 presents the concentration of the three GAG fractions isolated from the tissues by the cetyl pyridinium procedure. Peripheral nerve contains twice the amount of GAG present in spinal cord. In both tissues the ratios of fractions I to IT are Abbreviation used: GAG, glycosaminoglycans. 1529 1530 E. V. CHANDRASEKARAN and B. K. BACHHAWAT TABLE 1 .-CONCENTRATION OF GAG-FRACTIONS IN NERVOUS TISSUES f ig uronic acid/g dry defatted tissue Tissue Fraction I Fraction 11 Fraction 111 Total Ratio 1/11 .. Spinal cord 486 209 14 709 2.33 Peripheral nerve 1010 41 5 31 1456 2.44 Brain* 155