Characterization of uronic-acid-rich inhibitor of calcium oxalate crystallization isolated from rat urine.
Human urine contains several macromolecules which inhibit calcium oxalate crystallization. Uronic-acid-rich protein (UAP), a glycoprotein with a molecular weight of approximately 35 kDa, is one such inhibitor. Here we report the characterization of UAP extracted from rat urine using three chromatographic steps including diethylaminoethanol (DEAE)-Sephacel, Sephacryl S-300 and Mono Q column and compare it with human UAP. The molecular weight of rat UAP (UAPr) is similar to that of human UAP (UAPh), being approximately 35 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Their amino acid compositions are identical, they contain a high percentage of aspartic and glutamic acids and they react positively in the carbazole reaction, suggesting that they contain uronic acid. The inhibitory activities of UAPh and UAPr were assayed on a calcium oxalate crystallization system in vitro using [45Ca]calcium chloride. Both exert a strong inhibition, suggesting that UAPr, like UAPh, plays an important role in preventing and reducing calcium oxalate crystallization in the urine. On Western blot analysis, both UAPh and UAPr immunoreact with inter-alpha-trypsin inhibitor (ITI) antibody. Nevertheless, using the Ouchterlony immunodiffusion technique, there was no precipitation line between ITI antibody and UAP. Therefore, we hypothesize that UAP is related to ITI and that they may have the same epitope but are not completely identical. We conclude that UAP belongs to the ITI superfamily of macromolecules which contribute to the regulation of the calcium oxalate crystallization process.
Atmani
F
F
University of Florida, College of Medicine, Department of Pathology and Laboratory Medicine, Gainesville 32610, USA.
Khan
S R
SR
D000595:Amino Acid Sequence; D000818:Animals; D002129:Calcium Oxalate / Q000037:antagonists & inhibitors*; D002845:Chromatography / Q000379:methods; D003460:Crystallization; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D017386:Sequence Homology, Amino Acid
D010446:Peptide Fragments; D002129:Calcium Oxalate