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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.

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Comparison of the protein amounts of NOS isoforms between the hippocampus andcerebellum(A) nNOS, (B) eNOS, (C) iNOS. Upper part: the differencein the amounts of the proteins estimated by densitometry. h, hippocampus; c, cerebellum.
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Figure 6: Comparison of the protein amounts of NOS isoforms between the hippocampus andcerebellum(A) nNOS, (B) eNOS, (C) iNOS. Upper part: the differencein the amounts of the proteins estimated by densitometry. h, hippocampus; c, cerebellum.

Mentions: Finally, we compared relative amounts of NOS proteins in the extracts from cerebellum andhippocampus. Figure 6 shows the results of Western blotanalysis using anti-nNOS, -eNOS and -iNOS antibodies. The amounts of nNOS and eNOS weresignificantly larger in cerebellum than in hippocampus. We did not observe any difference in proteinexpression of iNOS between these regions.


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)

Comparison of the protein amounts of NOS isoforms between the hippocampus andcerebellum(A) nNOS, (B) eNOS, (C) iNOS. Upper part: the differencein the amounts of the proteins estimated by densitometry. h, hippocampus; c, cerebellum.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Comparison of the protein amounts of NOS isoforms between the hippocampus andcerebellum(A) nNOS, (B) eNOS, (C) iNOS. Upper part: the differencein the amounts of the proteins estimated by densitometry. h, hippocampus; c, cerebellum.
Mentions: Finally, we compared relative amounts of NOS proteins in the extracts from cerebellum andhippocampus. Figure 6 shows the results of Western blotanalysis using anti-nNOS, -eNOS and -iNOS antibodies. The amounts of nNOS and eNOS weresignificantly larger in cerebellum than in hippocampus. We did not observe any difference in proteinexpression of iNOS between these regions.

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