Protein nitration

Various proteins/enzymes obtained commercially were tested for the presence of endogenously nitrated tyrosine by Western blot analysis omitting reduci ng agent in the step of SDS-PAGE. Histones II-S and VIII-S, IgG, cAMP-depen dent protein kinase (PKA), phosphorylase b, and phosphorylase kinase exhibi ted strong immunoreactive bands. Histone VI-S, glycogen synthase, lactate d ehydrogenase, actin, thyroglobulin, and macroglobulin exhibited moderate im munoreactivity. Histone III-S, casein, acetyl cholinesterase, DNase I, and lipase had only traceable immunoreactivity. Whereas histone VII-S, pyruvate kinase, trypsin, pepsin, chymotrypsin, protease IV, and protease XIII, and glutathione S-transferase lacked immunoreactivity. A variation of immunore activity between hypertensive and normaltensive rat hearts was found in the histone-agarose fractions of crude extracts. Additionally, nitrotyrosine i mmunoreactivity was observed in non-mammalian organisms including Escherici a coli, Saccharomyces cerevisiae and Triticum vulgaris. Upon the treatment of 15 muM peroxynitrite (PN), strong oxidant derived from nitric oxide (NO) , the apparent K-m of PKA for cAMP increased from approximately 10(-8) to 1 0(-6) M. The results imply that the varied nitration of tyrosine residues i n proteins/enzymes may occur as a post-translational modification in vivo, and such discriminative nitration may be vital in PN/NO-regulated signal tr ansduction cascade.