Abstract
Endothelial cell nitric oxide synthase (NOS) is known to have a N-myristoylation consensus sequence. Such a consensus sequence is not evident in the macrophage, smooth muscle and neuronal NOS. A functional role for this N-terminal myristoylation is not clear yet. In the present study, we examined the effect of N-terminal myristoylation on the NOS activity determined by the conversion of L-[3H]arginine to L-[3H]citrulline and extracellular NO release determined by nitrite production in the conditioned medium from the COS-7 cells transfected with wild type bovine aortic endothelial cell (BAEC) NOS cDNA or nonmyristoylated BAEC-NOS mutant cDNA. NOS activity of wild type BAEC-NOS in COS-7 cells was localized in the particulate fraction and that of mutant NOS was in the cytosolic fraction. In contrast, nitrite production from COS-7 cells transfected with wild type BAEC-NOS cDNA was greater than that of mutant cDNA in a time dependent and a concentration dependent manner. These results suggest that membrane localization of NOS with myristoylation facilitates extracellular transport of NO and leads to enhanced NO signaling on the vascular smooth muscle cells and the intravascular blood cells including neutrophils, macrophages and platelets.
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References
Nathan CF: Nitric oxide as a secretory product of mammalian cells. FASEB J 6: 3051–3064, 1992
Dinerman JL, Lowenstein CJ, Snyder SH: Molecular mechanisms of nitric oxide regulation. Potential relevance to cardiovascular disease. Circ Res 73: 217–222, 1993
Nishida K, Harrison DG, Navas JP, Fisher AA, Dockery SP, Uematsu M, Nerem RM, Alexander RW, Murphy TJ: Molecular cloning and characterization of the constitutive bovine aortic endothelial cell nitric oxide synthase. J Clin Invest 90: 2097–2101, 1992
Nunokawa Y, Ishida N, Tanaka S: Cloning of inducible nitric oxide synthase in rat vascular smooth muscle cells. Biochem Biophys Res Commun 191: 89–94, 1993
Pollock JS, Klinghofer U, Förstermann U, Murad F: Endothelial nitric oxide synthase is myristylated. FEBS Lett 309: 402–404, 1992
Busconi L, Michel T: Endothelial nitric oxide synthase N-terminal myristoylation determines subcellular localization. J Biol Chem 268: 8410–8413, 1993
Sessa WC, Barber CM, Lynch KR: Mutation of N-myristoylation site converts endothelial cell nitric oxide synthase from a membrane to a cytosolic protein. Circ Res 72: 921–924, 1993
Takebe Y, Seiki M, Fujisawa J, Hoy P, Yokota K, Arai K, Yoshida M, Arai N: SRα promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Mol Cell Biol 8: 466–472, 1988
Pollock JS, Nakane M, Buttery LDK, Martinez A, Springall D, Polak JM, Forstermann U, Murad F: Characterization and localization of endotherial nitric oxide synthase using specific monoclonal antibodies. Am J Physiol 265: C1379–1387, 1993
Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning, a Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1989
Sakoda T, Kaibuchi K, Kishi K, Kishida S, Doi K, Hoshino M, Hattori S, Takai Y:smg/rap 1/Krev-1 p21s inhibit the signal pathway to the c-fos promoter/enhancer from c-Ki-ras p21 but not from c-raf-1 kinase in NIH3T3 cells. Oncogene 7: 1705–1711, 1992
Bredt DS, Snyder SH: Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc Natl Acad Sci USA, 87: 682–685, 1990
Ohashi Y, Katayama M, Hirata K, Suematsu M, Kawashima S, Yokoyama M: Activation of nitric oxide synthase from cultured aortic endothelial cells by phospholipids. Biochem Biophys Res Commun 195: 1314–1320, 1993
Myers PR, Guerra R, Harrison DG: Release of NO and EDRF from cultured bovine aortic endothelial cells. Am J Physiol 256: H1030-H1037, 1989
Kim S, Mizoguchi A, Kikuchi A, Takai Y:smg-p21/rap 1/Krev-1 proteins which are partly distinct from those of c-ras p21s. Mol Cell Biol 10: 2645–2652, 1990
Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976
Kamps MP, Buss JE, Setton BM: Mutation of NH2-terminal glycine of p6o8rc prevents both myristoylation and morphological transformation. Proc Natl Acad Sci USA 82: 4625–4628, 1985
Buss JE, Kamps MP, Gould K, Sefton BM: The absence of myristic acid decreases membrane binding of p60src but does not affect tyrosine protein kinase activity. J Virol 58: 468–474, 1986
Jones TLZ, Simonds WF, Merendino JJJr, Brann MR, Apiegel AM Myristoylation of an inhibitory GTP-binding protein a subunit is essential for its membrane attachment. Proc Natl Acad Sci USA 87: 568–572, 1990
Kaplan JM, Mardon G, Bishop JM, Vermus HE: The first seven amino acids encoded by the v-src oncogene act as a myristoylation signal: Lysine 7 is a clitical determinant. Mol Cell Biol 8: 2435–2441, 1988
Palmiter RD, Gagnon J, Vogt VM, Ripley S, Eisenman, RN: The NH2-terminal sequence of the avian oncovirus gag precursor polyprotein (Pr76gag). Virology 91: 423–433, 1978
Förstermann U, Mülsch A, Böhme E, Busse R: Stimulation of soluble guanylate cyclase by an acetylcholine-induced endothelium-derived factor from rabbit and canine arteries. Circ Res 58: 531–538, 1986
Michel T, Gordon KL, Busconi L: Phosphorylation and subcellular translocation of endothelial nitric oxide synthase. Proc Nalt Acad Sci USA 90: 6252–6256, 1993
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Sakoda, T., Hirata, Ki., Kuroda, R. et al. Myristoylation of endothelial cell nitric oxide synthase is important for extracellular release of nitric oxide. Mol Cell Biochem 152, 143–148 (1995). https://doi.org/10.1007/BF01076076
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DOI: https://doi.org/10.1007/BF01076076