Molecular Cloning and Characterization of the Constitutive Bovine Aortic Endothelial Cell Nitric Oxide Synthase

Overview

Journal J Clin Invest

Specialty General Medicine

Date 1992 Nov 1

PMID 1385480

Citations 113

Authors

K Nishida

D G Harrison

J P Navas

A A Fisher

S P Dockery

M Uematsu

R M Nerem

R W Alexander

T J Murphy

Affiliations

Soon will be listed here.

Abstract

The constitutive endothelial cell nitric oxide synthase (NOS) importantly regulates vascular homeostasis. To gain understanding of this enzyme, a pEF BOS cDNA library of 5 x 10(5) clones was prepared from bovine aortic endothelial cells (BAEC) and screened with a 2.8-kb cDNA BamHI fragment of rat brain NOS. Clone pBOS13 was found to express NO synthase activity when transfected into COS-7 cells. Sequence analysis revealed sequences compatible with binding domains for calcium/calmodulin, flavin mononucleotide, flavin adenine nucleotide and NADPH. The deduced amino acid sequence revealed a protein with a relative mol mass of 133,286, which is 58% homologous to the rat cerebellar NOS and 51% homologous to the mouse macrophage NOS. The amino-terminal portion of the protein exhibits several characteristics peculiar to the endothelial cell NOS. These include a proline-rich region and several potential sites for proline-directed phosphorylation as well as a potential substrate site for acyl transferase. Northern hybridization to mRNA from cultured BAEC revealed an abundant 4.8-kb message, which was not increased by coincubation with tumor necrosis factor alpha, but was markedly increased by exposure to shear stress for 24 h. The unique features of the endothelial cell NO synthase, particularly in the amino terminal portion of the molecule, may provide for novel regulatory influences of enzyme activity and localization.

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References

Forstermann U, Pollock J, Schmidt H, Heller M, Murad F . Calmodulin-dependent endothelium-derived relaxing factor/nitric oxide synthase activity is present in the particulate and cytosolic fractions of bovine aortic endothelial cells. Proc Natl Acad Sci U S A. 1991; 88(5):1788-92. PMC: 51110. DOI: 10.1073/pnas.88.5.1788. View

Mizushima S, Nagata S . pEF-BOS, a powerful mammalian expression vector. Nucleic Acids Res. 1990; 18(17):5322. PMC: 332193. DOI: 10.1093/nar/18.17.5322. View

Stuehr D, Cho H, Kwon N, Weise M, Nathan C . Purification and characterization of the cytokine-induced macrophage nitric oxide synthase: an FAD- and FMN-containing flavoprotein. Proc Natl Acad Sci U S A. 1991; 88(17):7773-7. PMC: 52385. DOI: 10.1073/pnas.88.17.7773. View

Hall F, Vulliet P . Proline-directed protein phosphorylation and cell cycle regulation. Curr Opin Cell Biol. 1991; 3(2):176-84. DOI: 10.1016/0955-0674(91)90136-m. View

Kemp B, Pearson R . Protein kinase recognition sequence motifs. Trends Biochem Sci. 1990; 15(9):342-6. DOI: 10.1016/0968-0004(90)90073-k. View

Schmidt H, Seifert R, Bohme E . Formation and release of nitric oxide from human neutrophils and HL-60 cells induced by a chemotactic peptide, platelet activating factor and leukotriene B4. FEBS Lett. 1989; 244(2):357-60. DOI: 10.1016/0014-5793(89)80562-9. View

Tayeh M, Marletta M . Macrophage oxidation of L-arginine to nitric oxide, nitrite, and nitrate. Tetrahydrobiopterin is required as a cofactor. J Biol Chem. 1989; 264(33):19654-8. View

Towler D, Eubanks S, Towery D, Adams S, Glaser L . Amino-terminal processing of proteins by N-myristoylation. Substrate specificity of N-myristoyl transferase. J Biol Chem. 1987; 262(3):1030-6. View

Miller V, Vanhoutte P . Enhanced release of endothelium-derived factor(s) by chronic increases in blood flow. Am J Physiol. 1988; 255(3 Pt 2):H446-51. DOI: 10.1152/ajpheart.1988.255.3.H446. View

10.

Palmer R, Ferrige A, Moncada S . Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature. 1987; 327(6122):524-6. DOI: 10.1038/327524a0. View

11.

Levesque M, Nerem R . The elongation and orientation of cultured endothelial cells in response to shear stress. J Biomech Eng. 1985; 107(4):341-7. DOI: 10.1115/1.3138567. View

12.

SANGER F, Nicklen S, Coulson A . DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977; 74(12):5463-7. PMC: 431765. DOI: 10.1073/pnas.74.12.5463. View

13.

Xie Q, Cho H, Calaycay J, Mumford R, Swiderek K, Lee T . Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science. 1992; 256(5054):225-8. DOI: 10.1126/science.1373522. View

14.

Bredt D, Snyder S . Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc Natl Acad Sci U S A. 1990; 87(2):682-5. PMC: 53329. DOI: 10.1073/pnas.87.2.682. View

15.

Stuehr D, Kwon N, Nathan C . FAD and GSH participate in macrophage synthesis of nitric oxide. Biochem Biophys Res Commun. 1990; 168(2):558-65. DOI: 10.1016/0006-291x(90)92357-6. View

16.

Yui Y, Hattori R, Kosuga K, Eizawa H, Hiki K, Ohkawa S . Calmodulin-independent nitric oxide synthase from rat polymorphonuclear neutrophils. J Biol Chem. 1991; 266(6):3369-71. View

17.

Bredt D, Hwang P, Glatt C, Lowenstein C, Reed R, Snyder S . Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase. Nature. 1991; 351(6329):714-8. DOI: 10.1038/351714a0. View

18.

Pollock J, Forstermann U, Mitchell J, Warner T, Schmidt H, Nakane M . Purification and characterization of particulate endothelium-derived relaxing factor synthase from cultured and native bovine aortic endothelial cells. Proc Natl Acad Sci U S A. 1991; 88(23):10480-4. PMC: 52952. DOI: 10.1073/pnas.88.23.10480. View

19.

Myers P, Minor Jr R, Guerra Jr R, Bates J, Harrison D . Vasorelaxant properties of the endothelium-derived relaxing factor more closely resemble S-nitrosocysteine than nitric oxide. Nature. 1990; 345(6271):161-3. DOI: 10.1038/345161a0. View

20.

Hibbs Jr J, Taintor R, Vavrin Z . Macrophage cytotoxicity: role for L-arginine deiminase and imino nitrogen oxidation to nitrite. Science. 1987; 235(4787):473-6. DOI: 10.1126/science.2432665. View