INTERACTION OF CALMODULIN-BINDING DOMAIN PEPTIDES OF NITRIC-OXIDE SYNTHASE WITH MEMBRANE PHOSPHOLIPIDS - REGULATION BY PROTEIN-PHOSPHORYLATION AND CA2-CALMODULIN()

Endothelial nitric oxide synthase (eNOS) is unique among the NO syntha se isozymes in being modified with myristoyl group, which appears to b e necessary for its membrane association. However, the presence of myr istoylated eNOS in cytosolic fraction after the stimulation-dependent translocation of the enzyme from membrane to cytosol suggests that oth er regions may be involved in the eNOS-membrane interaction and its re gulation. In this study, we have synthesized a 20-amino acid peptide c orresponding to the putative calmodulin-binding domain of human eNOS a nd studied the interaction of the peptide with calmodulin and with var ious membrane phospholipids. The peptide formed a stoichiometric compl ex with calmodulin. Upon addition of various acidic phospholipids, the peptide showed a drastic conformational change from random coil to al pha-helix, as was evidenced by circular dichroism spectroscopy. These results suggest that the same domain of eNOS binds both calmodulin and membrane phospholipids. Furthermore, we found that the synthetic pept ide was phosphorylated in vitro by protein kinase C. Phosphorylation o f the peptide decreased its interaction with membrane phospholipids. T hus, our results raise the possibility that the calmodulin-binding dom ain is directly involved in the membrane association of eNOS and that phosphorylation of the domain and Ca2+-calmodulin may regulate the int eraction. Synthetic peptides corresponding to the calmodulin-binding d omains of macrophage and neuronal isozymes showed similar abilities to bind phospholipids, suggesting that the calmodulin-binding domains of NO synthase serve as the phospholipid-binding domains as well.