TY - JOUR
T1 - Subunit interactions of endothelial nitric-oxide synthase
T2 - Comparisons to the neuronal and inducible nitric-oxide synthase isoforms
AU - Venema, Richard C.
AU - Ju, Hong
AU - Zou, Kong
AU - Ryan, James W.
AU - Venema, Virginia J.
PY - 1997
Y1 - 1997
N2 - Endothelial nitric-oxide synthase (eNOS) is comprised of two identical subunits. Each subunit has a bidomain structure consisting of an N-terminal oxygenase domain containing heme and tetrahydrobiopterin (BH4) and a C- terminal reductase domain containing binding sites for FAD, FMN, and NADPH. Each subunit is also myristoylated and contains a calmodulin (CaM)-binding site located between the oxygenase and reductase domains. In this study, wild-type and mutant forms of eNOS have been expressed in a baculovirus system, and the quaternary structure of the purified enzymes has been analyzed by low temperature SDS-PAGE. eNOS dimer formation requires incorporation of the heme prosthetic group but does not require myristoylation or CaM or BH4 binding. In order to identify domains of eNOS involved in subunit interactions, we have also expressed 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. Comparative analysis of NOS domain interactions shows that subunit association of eNOS and nNOS involves not only head to head interactions of oxygenase 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.
AB - Endothelial nitric-oxide synthase (eNOS) is comprised of two identical subunits. Each subunit has a bidomain structure consisting of an N-terminal oxygenase domain containing heme and tetrahydrobiopterin (BH4) and a C- terminal reductase domain containing binding sites for FAD, FMN, and NADPH. Each subunit is also myristoylated and contains a calmodulin (CaM)-binding site located between the oxygenase and reductase domains. In this study, wild-type and mutant forms of eNOS have been expressed in a baculovirus system, and the quaternary structure of the purified enzymes has been analyzed by low temperature SDS-PAGE. eNOS dimer formation requires incorporation of the heme prosthetic group but does not require myristoylation or CaM or BH4 binding. In order to identify domains of eNOS involved in subunit interactions, we have also expressed 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. Comparative analysis of NOS domain interactions shows that subunit association of eNOS and nNOS involves not only head to head interactions of oxygenase 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.
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U2 - 10.1074/jbc.272.2.1276
DO - 10.1074/jbc.272.2.1276
M3 - Article
C2 - 8995432
AN - SCOPUS:0031030864
SN - 0021-9258
VL - 272
SP - 1276
EP - 1282
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 2
ER -