Identification of uronic-acid-rich protein as urinary bikunin, the light chain of inter-alpha-inhibitor
Identification of uronic-acid-rich protein as urinary bikunin, the light chain of inter-alpha-inhibitor
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DOI：
10.1111/j.1432-1033.1996.00984.x
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发表时间：
1996-03-15
期刊：
影响因子：
--
通讯作者：
Khan, SR
中科院分区：
文献类型：
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作者：
Atmani, F;Mizon, J;Khan, SR
富尿酸蛋白（UAP）是一种尿中糖蛋白，在体外可抑制草酸钙结晶。它显示出与bikunin的结构相似性，bikunin是α间抑制剂（I α I）的一种组分，已知其抑制许多丝氨酸蛋白酶如胰蛋白酶和胰凝乳蛋白酶的作用。为了阐明这些大分子之间的关系，对UAP、I α I、尿bikunin和血浆bikunin进行了纯化和研究。用硫酸软骨素酶AC和链霉蛋白酶处理前后测定其草酸钙结晶抑制活性。用SDS/PAGE测定处理前后的分子量。用多克隆bikunin抗体进行免疫学鉴定。测定了硫酸软骨素酶处理前后不饱和蛋白的部分氨基酸序列。此外，还测定了UAP的抗胰蛋白酶活性，并与bikunin进行比较，bikunin负责I α I的抗蛋白酶活性。UAP具有较强的草酸钙结晶抑制活性。I α I和两种bikunins的抑制作用较小。软骨素酶AC对这些蛋白质的抑制活性没有影响，即使它们的分子量发生变化。然而，在链霉蛋白酶处理后，bikunins和UAP的抑制活性被完全破坏。测得UAP的抗胰蛋白酶活性为0.78 U/mg，低于bikunin的约1.9 U/mg。免疫印迹法显示bikunin抗体与尿及血浆中的bikunins和UAP均呈免疫反应。部分氨基酸序列分析证实了其为尿bikunin。
Uronic-acid-rich protein (UAP) is a urinary glycoprotein that inhibits calcium oxalate crystallization in vitro. It shows a structural similarity to bikunin, a component of inter-alpha-inhibitor (I alpha I) known for its inhibition of the action of many serine proteinases like trypsin and chymotrypsin. To clarify the relationship between these macromolecules, UAP, I alpha I, urinary bikunin, and plasma bikunin were purified and studied. Their calcium oxalate crystallization inhibitory activity was assayed before and after treatment with chondroitinase AC and pronase. Their molecular mass was determined by using SDS/PAGE before and after these treatments. Polyclonal bikunin antibody was used on Western blots for immunological identification. The partial amino acid sequence of UAP before and after chondroitinase treatment was determined. Also, the antitryptic activity of UAP was measured and compared to that of bikunin, which is responsible for the antiprotease activity of I alpha I. UAP exhibited a strong calcium oxalate crystallization inhibitory activity. I alpha I and both bikunins were less inhibitory. Chondroitinase AC had no effect on inhibitory activity of these proteins even when their molecular mass changed. However, after pronase treatment, the inhibitory activity of both bikunins and UAP was completely destroyed. The antitryptic activity of UAP was found to be 0.78 U/mg which is lower than that of bikunin which is about 1.9 U/mg. On Western blotting, bikunin antibody immunoreacted with UAP and both urinary and plasma bikunins. Partial amino acid sequence confirmed the identity of UAP as urinary bikunin.