Sundaram L, Deloria L B, Oegema T R
Department of Orthopaedic Surgery, University of Minnesota, Minneapolis, Minnesota, 55455, USA.
Arch Biochem Biophys. 1997 Feb 15;338(2):213-9. doi: 10.1006/abbi.1996.9827.
Aggrecan (PG) was isolated from Swarm rat chondrosarcoma and the chondroitin 4-sulfate removed with chondroitinase ABC (ABC) or ACII (AC), leaving a 4-deoxy-beta-d-gluc-4-enuronosyl (DeltaGlcA) residue on the nonreducing terminus of the attached chondroitin sulfate chains. Mercuric acetate (as low as 5 mm) removed the DeltaGlcA from the PG-ABC within 10 min at 25 degrees C at pH 5.0, and the rate was pH independent between pH 3.0 and 5.0. The reaction was readily monitored by following the loss of reactivity to the monoclonal antibodies specific for 4-sulfated and nonsulfated unsaturated disaccharides in ELISA. After mercury treatment, there was a loss of carbazole-positive material and a decrease in the size of the linkage region oligosaccharides consistent with DeltaGlcA being removed. Aside from the loss of DeltaGlcA, neutral sugar composition and sialic acid content remained unchanged. After electrophoresis in a 4% polyacrylamide gel, Hg-treated PG-ABC and PG-AC migrated as single major bands, but with reduced mobilities, which is consistent with a loss of charge. There was a loss of reactivity to specific monoclonal antibodies. Treated aggrecan did not bind hyaluronic acid. This loss was not completely prevented by being present in a complex with link protein and hyaluronic acid. However, link protein could partially restore the hyaluronic acid interaction, so the effect of mercuric acetate on biological function will have to be assessed on an individual basis. Treatment with mercuric acetate is an effective, rapid, reproducible way of removing DeltaGlcA from both chondroitinase ABC and ACII-digested proteoglycan.
从群大鼠软骨肉瘤中分离出聚集蛋白聚糖（PG），并用软骨素酶ABC（ABC）或ACII（AC）去除硫酸软骨素4-硫酸酯，在连接的硫酸软骨素链的非还原末端留下4-脱氧-β-D-葡糖-4-烯糖醛酸（ΔGlcA）残基。在25℃、pH 5.0条件下，乙酸汞（低至5 mM）在10分钟内即可从PG-ABC中去除ΔGlcA，且在pH 3.0至5.0之间反应速率与pH无关。通过在ELISA中跟踪对4-硫酸化和非硫酸化不饱和二糖特异性单克隆抗体的反应性丧失，可轻松监测该反应。汞处理后，咔唑阳性物质减少，连接区寡糖大小减小，这与ΔGlcA被去除一致。除了ΔGlcA的丧失外，中性糖组成和唾液酸含量保持不变。在4%聚丙烯酰胺凝胶中电泳后，汞处理的PG-ABC和PG-AC以单一主要条带迁移，但迁移率降低，这与电荷丧失一致。对特异性单克隆抗体的反应性丧失。处理后的聚集蛋白聚糖不结合透明质酸。与连接蛋白和透明质酸形成复合物并不能完全阻止这种丧失。然而，连接蛋白可以部分恢复透明质酸相互作用，因此乙酸汞对生物学功能的影响必须逐个评估。用乙酸汞处理是从软骨素酶ABC和ACII消化的蛋白聚糖中去除ΔGlcA的一种有效、快速、可重复的方法。
