Purification and localization of a 25-kD porcine renal puromycin aminonucleoside-binding protein.
BACKGROUND
Reactive oxygen species (ROS) are considered to have a role in the progression of puromycin aminonucleoside (PAN) nephrosis. However, the exact mechanism by which PAN induces ROS in this model is little known. In the present study, we attempted to purify a candidate for the target protein from PAN nephrotoxicity.


METHODS
Using PAN-affinity column chromatography, a series of PAN-binding proteins was isolated from porcine renal extracts. We produced a specific antibody against a 25-kD protein eluted from the PAN-affinity matrix, and then developed a method to purify this protein. A partial amino acid sequence of the 25-kD PAN-binding protein was determined, and its tissue distribution was examined by immunoblot and immunohistochemical studies.


RESULTS
The purified 25-kD PAN-binding protein was identified as a renal homolog of a new member of NAD(P)H:quinone oxidoreductases (NQOs, EC 1.6.99.2) that suppress the semiquinone and superoxide anion formation in cells, designated NQO2. Immunoblot analysis revealed a higher expression of the 25-kD PAN-binding protein in the kidney, brain, and liver among porcine major organs. Immunohistochemical studies showed an intrarenal distribution of this protein in epithelial cells of the glomeruli and tubules, mesangial cells, and vascular smooth muscle cells.


CONCLUSIONS
We have purified the renal homolog of NQO2 as a PAN-binding protein, and shown its unique tissue expression. PAN may bind to the NQO2 homolog and inhibit its function in the renal target cells. This is presumed to result in an increase of ROS in the kidney with PAN nephrosis.
Kodama
T
T
Third Department of Internal Medicine, Akita University School of Medicine, Japan.
Wakui
H
H
Komatsuda
A
A
Imai
H
H
Miura
A B
AB
Tashima
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D007668:Kidney / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000302:isolation & purification*; D011692:Puromycin Aminonucleoside / Q000378:metabolism*; D017382:Reactive Oxygen Species; D013552:Swine
D002352:Carrier Proteins; D017382:Reactive Oxygen Species; D011692:Puromycin Aminonucleoside; D016660:NAD(P)H Dehydrogenase (Quinone)