Thornton D J, Morris H G, Cockin G H, Huckerby T N, Nieduszynski I A, Carlstedt I, Hardingham T E, Ratcliffe A
Department of Biological Sciences, University of Lancaster, U.K.
Biochem J. 1989 May 15;260(1):277-82. doi: 10.1042/bj2600277.
Two populations of alkaline-borohydride-reduced keratan sulphate (KS) chains were prepared from the two peptido-keratan sulphate trypsin fragments of proteoglycan aggregates isolated from bovine femoral head cartilage (6-year-old animals). Each population was separated by high-performance ion-exchange chromatography on a Pharmacia Mono-Q column into eight pools, Q1-Q8. These were analysed by gel permeation chromatography, radioimmunoassay with the monoclonal antibody MZ15, and 500 MHz 1H n.m.r. spectroscopy. Upon chromatography on Sephadex G-75 the Mono-Q fractions were shown to increase in hydrodynamic size progressively from Q1 to Q8 for both KS populations. For each population the strongest antigenic response with the anti-KS monoclonal antibody MZ15 was expressed by the two fractions of greatest size and charge density, Q7 and Q8. Proton n.m.r. spectroscopic studies on the two series of fractions demonstrated: (i) a progressive increase in the level of galactose sulphation from Q1 to Q8, (ii) the presence of approximately one alpha(1-3)-linked fucose residue per chain in every sample, and (iii) the presence of N-acetylneuraminic acids in three discrete environments, two alpha(2-3)- and one alpha(2-6)-linked in every sample. The results are discussed in terms of a possible heterogeneity in the carbohydrate-protein linkage region of keratan sulphates from bovine articular cartilage.
从6岁牛股骨头软骨分离的蛋白聚糖聚集体的两个肽段硫酸角质素胰蛋白酶片段制备了两群碱硼氢化物还原的硫酸角质素（KS）链。每一群都通过在Pharmacia Mono-Q柱上的高效离子交换色谱法分离成八个组分，Q1-Q8。通过凝胶渗透色谱法、用单克隆抗体MZ15进行放射免疫测定以及500 MHz 1H核磁共振光谱对这些组分进行分析。在Sephadex G-75上进行色谱分析时，对于两个KS群体，Mono-Q组分的流体力学尺寸从Q1到Q8逐渐增加。对于每个群体，最大尺寸和电荷密度的两个组分Q7和Q8与抗KS单克隆抗体MZ15表现出最强的抗原反应。对这两个系列组分的质子核磁共振光谱研究表明：（i）从Q1到Q8半乳糖硫酸化水平逐渐增加，（ii）每个样品中每条链大约存在一个α(1-3)连接的岩藻糖残基，以及（iii）在三个不同环境中存在N-乙酰神经氨酸，每个样品中有两个α(2-3)连接的和一个α(2-6)连接的。根据来自牛关节软骨的硫酸角质素的碳水化合物-蛋白质连接区域中可能存在的异质性对结果进行了讨论。
