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Purification and localization of a 25-kD porcine renal puromycin aminonucleoside-binding protein
T Kodama1, H Wakui, A Komatsuda
1Third Department of Internal Medicine, Akita University School of Medicine, Japan.
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.
Insights
Researchers identified a novel NAD(P)H:quinone oxidoreductase 2 (NQO2) homolog as a puromycin aminonucleoside (PAN) binding protein. This discovery sheds light on the mechanism of PAN nephrosis by linking PAN to increased reactive oxygen species (ROS).
Area of Science:
- Nephrology
- Biochemistry
- Toxicology
Background:
- Reactive oxygen species (ROS) are implicated in puromycin aminonucleoside (PAN) nephrosis progression.
- The precise mechanism of PAN-induced ROS generation remains largely unknown.
- This study aimed to identify the target protein responsible for PAN nephrotoxicity.
Purpose of the Study:
- To isolate and identify the protein that binds to puromycin aminonucleoside (PAN).
- To investigate the role of this protein in the development of PAN-induced nephrosis.
- To understand the mechanism of ROS generation in PAN nephrosis.
Main Methods:
- Puromycin aminonucleoside (PAN)-affinity column chromatography was used to isolate PAN-binding proteins from porcine renal extracts.
- A specific antibody was generated against a 25-kD PAN-binding protein for purification.
- Partial amino acid sequencing, immunoblot, and immunohistochemical analyses were performed.
Main Results:
- A 25-kD PAN-binding protein was purified and identified as a renal homolog of NAD(P)H:quinone oxidoreductase 2 (NQO2).
- NQO2 is known to suppress semiquinone and superoxide anion formation.
- The protein was highly expressed in porcine kidney, brain, and liver, with intrarenal localization in glomerular and tubular epithelial cells, mesangial cells, and vascular smooth muscle cells.
Conclusions:
- The renal homolog of NQO2 was purified as a PAN-binding protein with a distinct tissue expression pattern.
- PAN may bind to and inhibit the function of the NQO2 homolog in renal target cells.
- This interaction is presumed to lead to increased ROS in the kidney, contributing to PAN nephrosis.