抄録
The calmodulin (CaM)-binding domain of rat neuronal nitric oxide synthase (nNOS) was analyzed using 3 synthetic peptides corresponding to different regions of the middle portion of the enzyme. One corresponding to nNOS 732-754 completely inhibited the NOS enzyme activity with an IC50 of about 1μM. Kinetic analysis indicated that the inhibition was not competitive with respect to L-arginine, and the peptide produced a Ca2+-dependent, electrophoretic mobility shift of CaM on 1M urea gels. A specific hydrophobic/basic amino acid cluster in the rat nNOS sequence, Lys732LysLeu, critical for its CaM binding was also identified. Addition of CaM to inactive nNOS, generated by mutation of (Lys732LysLeu) to (Asp732AspGlu), which maintains dimerization ability, resulted in no enzyme activity, even at a 30:1 molar excess of CaM. Since dimerization is necessary for the activation of the native NOS enzyme, application of a dominant negative mutant through NOS assembly might be a useful biochemical approach in terms of elucidating NOS function in site.
