PURIFICATION AND LOCALIZATION OF A 25-KD PORCINE RENAL PUROMYCIN AMINONUCLEOSIDE-BINDING PROTEIN

Background. Reactive oxygen species (ROS) are considered to have a rol e in the progression of puromycin aminonucleoside (PAN) nephrosis. How ever, the exact mechanism by which PAN induces ROS in this model is li ttle known. In the present study, we attempted to purify a candidate f or the target protein from PAN nephrotoxicity. Methods. Using PAN-affi nity column chromatography, a series of PAN-binding proteins was isola ted from porcine renal extracts. We produced a specific antibody again st a 25-kD protein eluted from the PAN-affinity matrix, and then devel oped a method to purify this protein. A partial amino acid sequence of the 25-kD PAN-binding protein was determined, and its tissue distribu tion was examined by immunoblot and immunohistochemical studies. Resul ts. The purified 25-kD PAN-binding protein was identified as a renal h omolog of a new member of NAD(P)H:quinone oxidoreductases (NQOs, EC 1. 6.99.2) that suppress the semiquinone and superoxide anion formation i n cells, designated NQO(2). Immunoblot analysis revealed a higher expr ession of the 25-kD PAN-binding protein in the kidney, brain, and live r among porcine major organs. Immunohistochemical studies showed an in trarenal distribution of this protein in epithelial cells of the glome ruli and tubules, mesangial cells, and vascular smooth muscle cells. C onclusions. We have purified the renal homolog of NQO(2) as a PAN-bind ing protein, and shown its unique tissue expression. PAN may bind to t he NQO(2) homolog and inhibit its function in the renal target cells. This is presumed to result in an increase of ROS in the kidney with PA N nephrosis.