Characterization of fibronectin metabolites in normal rat urine

Fibronectin fragments showing immunoreactivity against an anti-rat plasma fibronectin antibody were detected in normal rat urine. Untreated urine showed immunoreactivity and this reactivity could not be dialyzed. These rat urinary fibronectin-related substances (RUF) were adsorbed on heparin-immobilized gel but hardly at all on gelatin-immobilized gel. They were separated into three fractions by DEAE-Sephadex ion exchange chromatography and each fraction was further purified by heparin-Sepharose affinity chromatography. The partially purified preparations, RUF-1, RUF-2 and RUF-3, were analyzed by the sandwich-type enzyme-linked immunosorbent assay (ELISA) method, and it was found their maximum reactivities were about 30%, 45% and 20% that of the intact plasma fibronectin, respectively. Plasma fibronectin was digested with elastase and then analyzed by the ELISA method to elucidate the patterns shown by the fragments. The fragments showed almost the same patterns on ELISA analysis, and the maximum immunoreactivity decreased in parallel with the degradation. On SDS-polyacrylamide gel electrophoresis, the RUF preparations were found to be composed of multiple forms of molecules, Mr 15,000-100,000, which reacted with the antibody. These results suggest that various forms of fibronectin metabolites are excreted in normal rat urine.