Modification of proteins and polynucleotides by peroxynitrite.

Varied intensities of nitrotyrosine immunoreactivity were detected by Western blots after the reaction of proteins or enzymes with peroxynitrite (PN), a strong oxidant derived from nitric oxide. Intense immunoreactivity of cAMP-dependent protein kinase, calmodulin and most histones may depend on greater access to tyrosine residues in the reaction, whereas the absence of immunoreactivity of caspase-3, ubiquitin and S-100 proteins may reflect lack of accessibility. In addition, the changes in UV/visible absorbency were observed after PN-treatment of polynucleotides, polypeptides or proteins. Brief PN-treatment of invertase increased its enzymatic activity. Furthermore, PN-treatment of rabbit IgG decreased its recognition by anti-IgG. The results suggest that PN may chemically modify polypeptides, proteins and polynucleotides and may subsequently alter their biological activity.

Denitration of peroxynitrite-treated proteins by "protein nitratases" from dog prostate.

Putative "protein nitratases," which catalyze denitration of peroxynitrite (PN)-treated, proteins, were detected in the crude extract of dog prostate. Nitratase activity was monitored by the decreased intensity of nitrotyrosine immunoreactive-bands in Western blot and increased nitrate level in dialysate of incubation mixture, which contained prostate crude extract, protease inhibitors and a PN-treated substrate, such as treated histone (III-S), BSA, invertase, or polylysine. Nitratases were activated by preincubation with m-calpain/Ca2+. Furthermore, after denitration, the activity of PN/DTT-treated invertase decreased to the similar activity level of DTT-treated invertase. At least two different types of nitratases may occur: type I, reductant-dependent, and type II, reductant-independent.

Denitration of peroxynitrite-treated proteins by 'protein nitratases' from rat brain and heart.

Putative 'protein nitratases,' which catalyze denitration of peroxynitrite (PN)-treated proteins, were detected in the homogenate/crude extract of rat brains and hearts. Nitratase activity was monitored by the decreased intensity of nitrotyrosine immunoreactive-bands in Western blot and increased nitrate level in dialysate of incubation mixture, which contained homogenate/crude extract, protease inhibitors and a PN-treated substrate, such as treated histone (III-S), BSA or invertase. Enhanced activity of nitratases was noted by preincubating crude extract with Ca2+. In addition, at least two types of nitratases may occur: type I, reductant-dependent, and type II, reductant- independent. Furthermore, upon denitration, the activity of PN-treated invertase increased to the same activity level of the untreated invertase. The overall reaction catalyzed by nitratases for denitration of nitrotyrosine residues in protein could be as follows: Protein-Tyr-NO2 + H2O --> Protein-Tyr-H + H+ + NO3-. The nitration/denitration of protein-tyrosine may be crucial in regulating signal transduction.

Constitutive nitric oxide synthase in Saccharomyces cerevisiae.

After removing nonspecific immunoreactivities from crude extract by immunoaffinity chromatography, an immunoreactive-band at 60 kDa of constitutive nitric oxide synthase (cNOS) from Saccharomyces cerevisiae was detected by Western blot using mouse monoclonal anti-neuronal NOS (cNOS). The activity of yeast cNOS, which was prepared by either histone-agarose chromatography or anti-neuronal NOS immunoprecipitation, was monitored by the formation of citrulline. Yeast cNOS was activated in the presence of calmodulin and arginine, whereas it was inhibited by L-NAME, a mammalian NOS inhibitor. Moreover, actinomycin-D decreased the extracellular and the intracellular levels of nitrate and nitrite which had been converted from NO. The results suggest that cNOS occurs in unicellular eukaryotes and the enzyme activity can be regulated.

Soluble guanylate cyclase in Saccharomyces cerevisiae.

After removing nonspecific immunoreactivities from crude extract by immunoaffinity chromatography, an immunoreactive-band at 40kDa of soluble guanylate cyclase (SGC) from Saccharomyces cerevisiae was detected by Western blot using rabbit anti-beta 1 subunit of SGC. Cyclic GMP level and SGC activity was measured by ELISA. Immunoprecipitated yeast SGC was activated by sodium nitroprusside, whereas inhibited by 1H-(1,2,4)oxadiazolo(4,3-A)quinoxalin-1-one. Increased cyclic GMP level was also noted when intact yeast cells were incubated with s-nitrosoglutathione, a NO donor. The result implies that NO can be utilized intracellularly and extracellularly. Moreover, the presence of SGC suggests the significance of NO/cyclic GMP signaling in unicellular eukaryotes.

The presence/absence of Bcl-2, Ca2+/calmodulin-dependent protein kinase IV, calretinin and p53 in baker's yeast and wheat germ.

After removing the nonspecific immunoreactivities from crude extracts of Saccharomyces cerevisiae and wheat germ by immunoaffinity chromatography, the presence of Ca(2+)-related proteins was tested by Western blot analysis. Immunoreactivity for Bcl-2 was absent in the yeast, whereas the immunoreactivity was evident in wheat germ and remained unchanged after incubation for 4 h with or without actinomycin D. Such incubation caused the degradation of immunoreactive-peptides of Ca2+/calmodulin-dependent protein kinase IV (CaMPK IV) in the yeast and wheat germ. Calretinin and p53 were absent in the yeast and wheat germ. The level of cyclic AMP in the yeast increased 100% after incubation for 30 min with actinomycin D. These results suggest that actinomycin D may not affect intracellular levels of these calcium-related proteins in the yeast and wheat germ, and that Bcl-2 occurs in multicellular eukaryotes. Moreover, the cellular level of CaMPK IV may vary during the onset of cell division and differentiation.

Immunoreactivities of m-calpain, calpastatin, nitric oxide synthase, myelin basic protein and dynamin II in baker's yeast, wheat germ and lobster tail muscle.

Vertebrate m-calpain, calpastatin, constitutive nitric oxide synthase, myelin basic protein, and dynamin I are substrates of protein kinase C (PKC). The presence/absence of similar/related protein in nonvertebrate was investigated by immunological methods, including (1) affinity chromatography on agarose-secondary antibodies and agarose IgG for removal of nonspecific immunoreactivities from crude extracts; (2) omitting beta-mercaptoethanol treatment and boiling prior to SDS-PAGE to increase the immunoreactivity; (3) immunoreactivity comparisons of nonspecific IgG as controls with specific anti-(vertebrate PKC-substrates/related proteins) in Western blots. It was found that (a) m-calpain and dynamin I were absent in baker's yeast, wheat germ and lobster tail muscle, (b) m-calpain, nitric oxide synthase, myelin basic protein and dynamin II were present in all three samples, and (c) calpastatin was present in baker's yeast and lobster tail muscle. The presence and absence of these proteins suggest evolutionary conservation and divergence, respectively, of these PKC substrates.