RECOMBINANT ENDOTHELIAL NITRIC-OXIDE SYNTHASE - POSTTRANSLATIONAL MODIFICATIONS IN A BACULOVIRUS EXPRESSION SYSTEM

Nitric oxide synthesized by the endothelial isoform of nitric oxide sy nthase (ecNOS) is importantly involved in the homeostatic control of b lood pressure and platelet aggregation. The different members of the n itric oxide synthase protein family have several biochemical features in common but serve distinct physiological functions and are the produ cts of distinct genes, The ecNOS is further distinguished by its subce llular distribution in the endothelial cell membrane, and the enzyme u ndergoes several post-translational modifications, including myristoyl ation, palmitoylation, and phosphorylation. Overall, however, the ecNO S has remained less well characterized because of the challenges invol ved in isolating sufficient quantities of this membrane-associated pro tein from native or cultured endothelial cells. In this report, we des cribe the purification and characterization of ecNOS expressed in a he terologous system in recombinant baculovirus-infected insect Sf9 cells . Recombinant ecNOS is targeted to the Sf9 cell membrane and comprises similar to 10% of the total cellular protein, allowing purification t o homogeneity in a single-step procedure to yield a stable protein tha t retains the essential features of the native enzyme. Using biosynthe tic labeling and immunoprecipitation, we show that recombinant ecNOS i s myristoylated, palmitoylated, and phosphorylated when expressed in i nsect Sf9 cells. The interpretation of structural and enzymological st udies of recombinant ecNOS will be facilitated by the apparent fidelit y of its biosynthesis and post-translational modification in insect Sf 9 cells.