IDENTIFICATION OF URONIC-ACID-RICH PROTEIN AS URINARY BIKUNIN, THE LIGHT-CHAIN OF INTER-ALPHA-INHIBITOR

Uronic-acid-rich protein (UAP) is a urinary glycoprotein that inhibits calcium oxalate crystallization in vitro. It shows a structural simil arity to bikunin, a component of inter-alpha-inhibitor (I alpha I) kno wn for its inhibition of the action of many serine proteinases like tr ypsin and chymotrypsin. To clarify the relationship between these macr omolecules, UAP, I alpha I, urinary bikunin, and plasma bikunin were p urified 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 be fore and after these treatments. Polyclonal bikunin antibody was used on Western blots for immunological identification. The partial amino a cid sequence of UAP before and after chondroitinase treatment was dete rmined. Also, the antitryptic activity of UAP was measured and compare d to that of bikunin, which is responsible for the antiprotease activi ty of I alpha I. UAP exhibited a strong calcium oxalate crystallizatio n inhibitory activity. I alpha I and both bikunins were less inhibitor y. Chondroitinase AC had no effect on inhibitory activity of these pro teins even when their molecular mass changed. However, after pronase t reatment, the inhibitory activity of both bikunins and UAP was complet ely 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 Wes tern blotting, bikunin antibody immunoreacted with UAP and both urinar y and plasma bikunins. Partial amino acid sequence confirmed the ident ity of UAP as urinary bikunin.