Dickenson J M, Huckerby T N, Nieduszynski I A
Division of Biological Sciences, Lancaster University, Bailrigg, U.K.
Biochem J. 1992 Feb 15;282 ( Pt 1)(Pt 1):267-71. doi: 10.1042/bj2820267.
Peptido-keratan sulphate fragments were isolated from the nucleus pulposus of bovine intervertebral discs (2-year-old animals) after digestion with chondroitin ABC lyase followed by digestion with diphenylcarbamoyl chloride-treated trypsin of A1D1 proteoglycans and gel-permeation chromatography on Sepharose CL-6B. The peptido-keratan sulphate fragments were subjected to alkaline borohydride reduction. The reduced chains were treated with keratanase in the presence of the sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, and the digest was subjected to alkaline borohydride reduction. This produced oligosaccharides with galactitol at their reducing ends. This reduced digest was chromatographed on a Nucleosil 5 SB anion-exchange column and individual oligosaccharides were isolated. One of these was shown by 600 MHz 1H-n.m.r. spectroscopy to have the following structure: NeuAc alpha 2-6Gal beta 1-4GlcNAc(6-SO4)beta 1-3Gal-ol The structure of this oligosaccharide shows that keratan sulphate chains from bovine intervertebral disc have non-reducing termini with N-acetylneuraminic acid linked alpha(2----6) as well as alpha(2----3) to an unsulphated galactose.
用软骨素ABC裂解酶消化牛椎间盘（2岁动物）的髓核，然后用二苯基甲酰氯处理的A1D1蛋白聚糖胰蛋白酶消化，并在Sepharose CL - 6B上进行凝胶渗透色谱，从其中分离出肽角蛋白硫酸盐片段。对肽角蛋白硫酸盐片段进行碱性硼氢化还原。还原后的链在唾液酸酶抑制剂2,3 - 脱氢 - 2 - 脱氧 - N - 乙酰神经氨酸存在下用角蛋白酶处理，消化产物再进行碱性硼氢化还原。这产生了在其还原端带有半乳糖醇的寡糖。将这种还原后的消化产物在Nucleosil 5 SB阴离子交换柱上进行色谱分离，分离出各个寡糖。其中一个通过600 MHz 1H - 核磁共振光谱显示具有以下结构：NeuAcα2 - 6Galβ1 - 4GlcNAc(6 - SO4)β1 - 3Gal - ol。这种寡糖的结构表明，来自牛椎间盘的硫酸角质素链具有非还原末端，其中N - 乙酰神经氨酸以α(2→6)以及α(2→3)连接到一个未硫酸化的半乳糖上。
