Constitutive nitric oxide synthase in Saccharomyces cerevisiae

Abstract

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.