NOSIP, Control Peptide (eNOS interacting protein)

Synthetic peptide (Ac-CAEKSRPVMQA-NH2) (linear) corresponding to aa292-301 of human NOSIP with a N-terminal cysteine for conjugation. The peptide has an acetyl group blocking the amino terminus and an amide group blocking the carboxyl terminus.\n\nProduction of nitric oxide (NO) in endothelial cells is regulated by direct interactions of endothelial nitric oxide synthase (eNOS) with effector proteins such as Ca2+-calmodulin, by posttranslational modifications such as phosphorylation via protein kinase B, and by translocation of the enzyme from the plasma membrane caveolae to intracellular compartments. Reversible acylation of eNOS is thought to contribute to the intracellular trafficking of the enzyme; however, protein factor(s) that govern the translocation of the enzyme are still unknown. Here we have used the yeast two-hybrid system and identified a novel 34kD protein, termed NOSIP (eNOS interacting protein), which avidly binds to the carboxyl-terminal region of the eNOS oxygenase domain. Coimmunoprecipitation studies demonstrated the specific interaction of eNOS and NOSIP in vitro and in vivo, and complex formation was inhibited by a synthetic peptide of the caveolin-1 scaffolding domain. NO production was significantly reduced in eNOS-expressing CHO cells (CHO-eNOS) that transiently overexpressed NOSIP. Stimulation with the calcium ionophore A23187 induced the reversible translocation of eNOS from the detergent-insoluble to the detergent-soluble fractions of CHO-eNOS, and this translocation was completely prevented by transient coexpression of NOSIP in CHO-eNOS. Immunofluorescence studies revealed a prominent plasma membrane staining for eNOS in CHO-eNOS that was abolished in the presence of NOSIP. Subcellular fractionation studies identified eNOS in the caveolin-rich membrane fractions of CHO-eNOS, and coexpression of NOSIP caused a shift of eNOS to intracellular compartments. We conclude that NOSIP is a novel type of modulator that promotes translocation of eNOS from the plasma membrane to intracellular sites, thereby uncoupling eNOS from plasma membrane caveolae and inhibiting NO synthesis.\n\nTheoretical Molecular Weight (M+H+): \n1261.5 D\n\nApplications: \nSuitable for use in Western Blot. Other applications not tested. \n\nStorage and Stability: \nLyophilized powder may be stored at 4 degrees C for short-term only. Stable for 12 months at -20 degrees C. Reconstitute to nominal volume (see reconstitution instructions for peptides) and store at -20 degrees C. For maximum recovery of product, centrifuge the original vial prior to removing the cap. Further dilutions can be made in assay buffer.\n\n\n

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