Allosteric inhibition of rat neuronal nitric-oxide synthase caused by interference with the binding of calmodulin to the enzyme.
A sigmoid-type dependence on the inhibitor concentration was observed in the cytochrome c reductase activity for peptide inhibitors (mastoparan and melittin), calmodulin antagonists (W-7 and tamoxifen) and monobutyltin in a reconstituted system comprised of recombinant rat neuronal nitric-oxide synthase (nNOS) and calmodulin (CaM). The increase in the concentration of CaM in the system induced a decrease in the inhibitory effect, indicating that the inhibitors might interfere with the interaction between nNOS and CaM. The changes in the fluorescence spectra of dansylated CaM caused by the addition of mastoparan, melittin and monobutyltin indicated complex formation between CaM and those compounds, which led to the decrease in the effective concentration of CaM available to nNOS. The sigmoid-type inhibition of mastoparan and melittin fit the theoretical equations quite well, assuming that two CaM molecules bind cooperatively to one nNOS homodimer. Monobutyltin, tamoxifen and W-7 were found to inhibit nNOS activity by binding to the CaM binding site of the nNOS homodimer, in addition to the binding of the inhibitors to calmodulin. These compounds inhibited the L-citrulline formation of nNOS from L-arginine, and the inhibitory effects were abrogated by raising the concentration of calmodulin. It became clear that the binding of calmodulin to nNOS can be interfered with in two ways: (1) via a decrease in the effective concentration of calmodulin caused by complex formation between the inhibitor and calmodulin, and (2) via the inhibition of the binding of calmodulin to nNOS caused by the occupation of the binding site by the inhibitor.
Ohashi
Koji
K
Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi-cho, Hiroshima 734-8551, Japan.
Yamazaki
Takeshi
T
Kitamura
Shigeyuki
S
Ohta
Shigeru
S
Izumi
Shunsuke
S
Kominami
Shiro
S
D000494:Allosteric Regulation; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D002956:Citrulline / Q000378:metabolism; D003579:Cytochrome Reductases / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D008555:Melitten / Q000494:pharmacology; D052248:Nitric Oxide Synthase Type I / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification; D010455:Peptides / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D014862:Wasp Venoms / Q000494:pharmacology
D002147:Calmodulin; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D011994:Recombinant Proteins; D014862:Wasp Venoms; D008555:Melitten; D002956:Citrulline; C037500:mastoparan; D052248:Nitric Oxide Synthase Type I; D003579:Cytochrome Reductases