Limits...
Proteomic analysis of endogenous nitrotryptophan-containing proteins in rat hippocampus and cerebellum.

Uda M, Kawasaki H, Shigenaga A, Baba T, Yamakura F - Biosci. Rep. (2012)

Bottom Line: We also observed that the total amount of NO2Trp-containing proteins in the cerebellum was significantly greater than that in the hippocampus (P<0.05).The amounts of nNOS (neuronal nitric oxide synthase) and eNOS (endothelial nitric oxide synthase) were much greater in cerebellum than in hippocampus.This is the first evidence of several specific sites of nitrated tryptophan in proteins under physiological conditions in vivo.

View Article: PubMed Central - PubMed

Affiliation: Sportology Center, Juntendo University Graduate School of Medicine, Chiba, Japan.

ABSTRACT
Nitration of tryptophan residues is a novel post-translational modification. In the present study, we examined whether NO2Trp (nitrotryptophan)-containing proteins are produced in the hippocampus and cerebellum of the adult rat under physiological conditions in vivo. Using Western blot analysis with anti-6-NO2Trp-specific antibody, we found many similar immunoreactive spots in the protein extracts from both regions. These spots were subsequently subjected to trypsin digestion and LC-ESI-MS/MS (LC-electrospray ionization-tandem MS) analysis. We identified several cytoskeletal proteins and glycolytic enzymes as NO2Trp-containing proteins and determined the position of nitrated tryptophan residues with significant ion score levels (P<0.05) in several proteins in both regions. We also observed that the total amount of NO2Trp-containing proteins in the cerebellum was significantly greater than that in the hippocampus (P<0.05). Moreover, IP (immunoprecipitation) assays using anti-aldolase C antibody showed that the relative intensity of immunostaining for NO2Trp over aldolase C was much higher in cerebellum than in hippocampus. The amounts of nNOS (neuronal nitric oxide synthase) and eNOS (endothelial nitric oxide synthase) were much greater in cerebellum than in hippocampus. This is the first evidence of several specific sites of nitrated tryptophan in proteins under physiological conditions in vivo.

Show MeSH

Related in: MedlinePlus

Semi-quantitative studies of 6-NO2Trp-containing proteins(A) Representative result of Western blotting with an anti-6-NO2Trpantibody of hippocampus and cerebellum. (B) The difference in the relative amounts ofNO2Trp -containing proteins between hippocampus and cerebellum measured by densitometry.h, hippocampus; c, cerebellum.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3475453&req=5

Figure 4: Semi-quantitative studies of 6-NO2Trp-containing proteins(A) Representative result of Western blotting with an anti-6-NO2Trpantibody of hippocampus and cerebellum. (B) The difference in the relative amounts ofNO2Trp -containing proteins between hippocampus and cerebellum measured by densitometry.h, hippocampus; c, cerebellum.

Mentions: In order to compare gross amounts of 6-NO2Trp-containing proteins in hippocampus andcerebellum quantitatively, we measured total intensity of the bands on the membrane by Western blotanalysis using β-actin as a loading control. Figure 4(A)shows the difference in 6-NO2Trp-containing protein bands between hippocampus andcerebellum. The total intensity of 6-NO2Trp-containing proteins in the cerebellum wassignificantly greater than that in hippocampus (P<0.01, Figure 4B). We observed the appearance of several new bands and the increasedintensity of several bands for the cerebellum (Figure 4A). Inorder to clarify whether there is a protein with a higher rate of nitration in the cerebellum, wechose spots c9 and h9, which have been identified as aldolase C, as a target protein (Table 1), as the intensity of spot c9 in the Westernblotting of two-dimensional electrophoresis was also clearly higher than that of h9 (Figures 1A and 1B). We isolatedaldolase C by IP using anti-aldolase C antibody from the extracts of cerebellum and hippocampus andapplied the isolated samples to SDS/PAGE and Western blotting. The relative intensity ofimmunostaining for 6-NO2Trp over aldolase C was much higher in cerebellum than inhippocampus (Figure 5). Therefore the increase in the nitrationrate of the proteins, as shown in aldolase C, contribute, at least in part, to the increase in grossamounts of 6-NO2Trp-containing proteins shown in Figure4.


Proteomic analysis of endogenous nitrotryptophan-containing proteins in rat hippocampus and cerebellum.

Uda M, Kawasaki H, Shigenaga A, Baba T, Yamakura F - Biosci. Rep. (2012)

Semi-quantitative studies of 6-NO2Trp-containing proteins(A) Representative result of Western blotting with an anti-6-NO2Trpantibody of hippocampus and cerebellum. (B) The difference in the relative amounts ofNO2Trp -containing proteins between hippocampus and cerebellum measured by densitometry.h, hippocampus; c, cerebellum.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3475453&req=5

Figure 4: Semi-quantitative studies of 6-NO2Trp-containing proteins(A) Representative result of Western blotting with an anti-6-NO2Trpantibody of hippocampus and cerebellum. (B) The difference in the relative amounts ofNO2Trp -containing proteins between hippocampus and cerebellum measured by densitometry.h, hippocampus; c, cerebellum.
Mentions: In order to compare gross amounts of 6-NO2Trp-containing proteins in hippocampus andcerebellum quantitatively, we measured total intensity of the bands on the membrane by Western blotanalysis using β-actin as a loading control. Figure 4(A)shows the difference in 6-NO2Trp-containing protein bands between hippocampus andcerebellum. The total intensity of 6-NO2Trp-containing proteins in the cerebellum wassignificantly greater than that in hippocampus (P<0.01, Figure 4B). We observed the appearance of several new bands and the increasedintensity of several bands for the cerebellum (Figure 4A). Inorder to clarify whether there is a protein with a higher rate of nitration in the cerebellum, wechose spots c9 and h9, which have been identified as aldolase C, as a target protein (Table 1), as the intensity of spot c9 in the Westernblotting of two-dimensional electrophoresis was also clearly higher than that of h9 (Figures 1A and 1B). We isolatedaldolase C by IP using anti-aldolase C antibody from the extracts of cerebellum and hippocampus andapplied the isolated samples to SDS/PAGE and Western blotting. The relative intensity ofimmunostaining for 6-NO2Trp over aldolase C was much higher in cerebellum than inhippocampus (Figure 5). Therefore the increase in the nitrationrate of the proteins, as shown in aldolase C, contribute, at least in part, to the increase in grossamounts of 6-NO2Trp-containing proteins shown in Figure4.

Bottom Line: We also observed that the total amount of NO2Trp-containing proteins in the cerebellum was significantly greater than that in the hippocampus (P<0.05).The amounts of nNOS (neuronal nitric oxide synthase) and eNOS (endothelial nitric oxide synthase) were much greater in cerebellum than in hippocampus.This is the first evidence of several specific sites of nitrated tryptophan in proteins under physiological conditions in vivo.

View Article: PubMed Central - PubMed

Affiliation: Sportology Center, Juntendo University Graduate School of Medicine, Chiba, Japan.

ABSTRACT
Nitration of tryptophan residues is a novel post-translational modification. In the present study, we examined whether NO2Trp (nitrotryptophan)-containing proteins are produced in the hippocampus and cerebellum of the adult rat under physiological conditions in vivo. Using Western blot analysis with anti-6-NO2Trp-specific antibody, we found many similar immunoreactive spots in the protein extracts from both regions. These spots were subsequently subjected to trypsin digestion and LC-ESI-MS/MS (LC-electrospray ionization-tandem MS) analysis. We identified several cytoskeletal proteins and glycolytic enzymes as NO2Trp-containing proteins and determined the position of nitrated tryptophan residues with significant ion score levels (P<0.05) in several proteins in both regions. We also observed that the total amount of NO2Trp-containing proteins in the cerebellum was significantly greater than that in the hippocampus (P<0.05). Moreover, IP (immunoprecipitation) assays using anti-aldolase C antibody showed that the relative intensity of immunostaining for NO2Trp over aldolase C was much higher in cerebellum than in hippocampus. The amounts of nNOS (neuronal nitric oxide synthase) and eNOS (endothelial nitric oxide synthase) were much greater in cerebellum than in hippocampus. This is the first evidence of several specific sites of nitrated tryptophan in proteins under physiological conditions in vivo.

Show MeSH
Related in: MedlinePlus