Inter-isoformal regulation of nitric oxide synthase through heteromeric dimerization.
Biochemical characterization of neuronal nitric oxide synthase (nNOS) has demonstrated a unique complexity with the native protein being a homodimer. To cast light on its enzyme structure-activity relationship, inactive nNOS, generated by mutation of (Lys732-Lys-Leu) to (Asp732-Asp-Glu) (Watanabe et al., FEBS Lett., 403 (1997) 75-78), and active nNOS were co-expressed using the Sf9 and COS-7 cell expression system. Co-transfectants of active and inactive nNOS resulted in attenuation of Ca2+/calmodulin (CaM) dependent NOS enzyme activity to a level 46.4+/-4.30% as much as active homomeric enzyme. Dimerization between active and inactive nNOS was observed by low temperature SDS-PAGE in the co-transfectants. The dimerization and attenuation of Ca2+/CaM dependent activity were not observed when active and inactive nNOS were combined in vitro, indicating that nNOS dimerization occurs intracellularly to form an active enzyme. Furthermore, we co-expressed inactive nNOS with active inducible NOS (iNOS) to analyze inter-isoformal regulation of NOSs. Interestingly, inactive nNOS also showed similar effects on enzyme activity and heteromeric dimerization against iNOS, thus indicating that inter-isoformal regulation of the two isoforms might also be involved in control of neuron function.
Watanabe
Y
Y
Department of Pharmacology, Nagoya University School of Medicine, Showa-ku, Nagoya 466, Japan.
Nishio
M
M
Hamaji
S
S
Hidaka
H
H
D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009474:Neurons / Q000201:enzymology; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009841:Oligonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction; D013329:Structure-Activity Relationship; D014162:Transfection
D002147:Calmodulin; D007527:Isoenzymes; D009841:Oligonucleotides; D019001:Nitric Oxide Synthase; D002118:Calcium