SUBUNIT INTERACTIONS OF ENDOTHELIAL NITRIC-OXIDE SYNTHASE - COMPARISONS TO THE NEURONAL AND INDUCIBLE NITRIC-OXIDE SYNTHASE ISOFORMS

Endothelial nitric-oxide synthase (eNOS) is comprised of two identical subunits. Each subunit has a bidomain structure consisting of an N-te rminal oxygenase domain containing heme and tetrahydrobiopterin (BH,) and a C-terminal reductase domain containing binding sites for FAD, FM N, and NADPH. Each subunit is also myristoylated and contains a calmod ulin (CaM)-binding site located between the oxygenase and reductase do mains. In this study, wild-type and mutant forms of eNOS have been exp ressed in a baculovirus system, and the quaternary structure of the pu rified enzymes has been analyzed by low temperature SDS-PAGE. eNOS dim er formation requires incorporation of the heme prosthetic group but d oes not require myristoylation or CaM or BH, binding. In order to iden tify domains of eNOS involved in subunit interactions, we have also ex pressed eNOS oxygenase and reductase domain fusion proteins in a yeast two-hybrid system. Corresponding human neuronal NOS (nNOS) and murine inducible NOS (iNOS) fusion proteins have also been expressed. Compar ative analysis of NOS domain interactions shows that subunit associati on of eNOS and nNOS involves not only head to head interactions of oxy genase domains but also tail to tail interactions of reductase domains and head to tail interactions between oxygenase and reductase domains . In contrast, iNOS subunit association involves only oxygenase domain interactions.