The long-term objective is to establish the protective/regulatory role of protein denitration enzymes, protein nitratases (or related enzymes), in reversing certain NO/peroxynitrate (PN)-mediated reactions (eg nitrotyrosine formation) and to elucidate a relationship between the deficiency of these enzymes and the pathogenesis of Alzheimer's and Parkinson's diseases, and prostate cancer.
The specific aims are: (I) To test pathogenesis of Alzheimer's and Parkinson's diseases, and prostate cancer.
The specific aims are: (I) To test substrate (PN-treated proteins: Histone H1, BSA, etc.) specificity for the novel protein-denitration activity of alpha, pi, psi isoforms of glutathione S-transferase (type I, redundant-dependent protein nitrases). The denitration activity can be monitored by nitrate production, and by the decreased anti-nitrotyrosine immunoreactivity in Western blot. (II) To compare subcellular (particulate and cytosolic) distribution of protein nitratases (type II, redundant independent) among bovine cerebral cortex, midbrain and cerebellum, and dog prostate. DEAE-cellulose chromatography, ammonium sulfate fractionation, molecular exclusion chromatography and affinity chromatography will be employed to purify the enzyme from crude extract. Characterization will include the effects of co-factors, pH, proteolysis (by m-calpain), phosphorylation (by protein kinases: PKA, PKG and PKC), nitration and auto-denitration, and the determination of molecular mass, subunits, substrate specificity, and isozymes. (IV) To study the effect of peroxynitrite-treatment on the activities of cAMP- dependent protein kinase (PKA), calmodulin and topoisomerase-1 (or other proteins/enzymes), and possible alteration/restoration of their activities after binding and DNAS electrophoretic mobility, respectively. The insight gained from this 4-year study might facilitate developing effective prevention/treatment of the above mentioned diseases in the future.

Project Start
2000-07-01
Project End
2001-06-30
Budget Start
Budget End
Support Year
27
Fiscal Year
2000
Total Cost
$48,534
Indirect Cost
Name
Bethune-Cookman College
Department
Type
DUNS #
City
Daytona Beach
State
FL
Country
United States
Zip Code
32114