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 similarity to bikunin, a component of inter-alpha-inhibitor (IalphaI) known for its inhibition of the action of many serine proteinases like trypsin and chymotrypsin. To clarify the relationship between these macromolecules, UAP, IalphaI, 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 IalphaI. UAP exhibited a strong calcium oxalate crystallization inhibitory activity. IalphaI 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.
Atmani
F
F
University of Florida, College of Medicine, Department of Pathology and Laboratory Medicine, Gainesville FL 32610-0275, USA.
Mizon
J
J
Khan
S R
SR
D000510:Alpha-Globulins / Q000737:chemistry / Q000494:pharmacology / Q000652:urine*; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002129:Calcium Oxalate; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology / Q000652:urine*; D006801:Humans; D046911:Macromolecular Substances; D008297:Male; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000652:urine*; D017386:Sequence Homology, Amino Acid; D014360:Trypsin Inhibitor, Kunitz Soybean; D014361:Trypsin Inhibitors / Q000652:urine
D000510:Alpha-Globulins; D006023:Glycoproteins; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D011480:Protease Inhibitors; C109148:SPINT2 protein, human; D014361:Trypsin Inhibitors; D002129:Calcium Oxalate; C011877:inter-alpha-inhibitor; D014360:Trypsin Inhibitor, Kunitz Soybean; D010450:Endopeptidases