Functional Expression of Three Isoforms of Human Nitric Oxide Synthase in Baculovirus-Infected Insect Cells

Nakane, Masaki; Pollock, Jennifer S.; Klinghofer, Vered; Basha, Fatima Z.; Marsden, Philip A.; Hokari, Atsushi; Ogura, Tsutomu; Esumi, Hiroyasu; Carter, G.W.

doi:10.1006/bbrc.1995.1073

Cited by 41 publications

(27 citation statements)

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“…The rank order of potency of NOS inhibitors on crude and semipurified neutrophil iNOS is consistent with that seen with human recombinant iNOS activity (19) where L-NMMA and L-NIO are equipotent and both are much more potent than aminoguanidine. L-NNA is a less potent inhibitor of human recombinant iNOS than of human recombinant eNOS or neuronal NOS.…”

Section: Discussionsupporting

confidence: 71%

Bacterial infection induces nitric oxide synthase in human neutrophils.

Wheeler¹,

Smith²,

et al. 1997

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The identification of human inflammatory cells that express inducible nitric oxide synthase and the clarification of the role of inducible nitric oxide synthase in human infectious or inflammatory processes have been elusive. In neutrophilenriched fractions from urine, we demonstrate a 43-fold increase in nitric oxide synthase activity in patients with urinary tract infections compared with that in neutrophilenriched fractions from noninfected controls. Partially purified inducible nitric oxide synthase is primarily membrane associated, calcium independent, and inhibited by arginine analogues with a rank order consistent with that of purified human inducible nitric oxide synthase. Molecular, biochemical, and immunocytochemical evidence unequivocally identifies inducible nitric oxide synthase as the major nitric oxide synthase isoform found in neutrophils isolated from urine during urinary tract infections.

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Section: Discussionsupporting

confidence: 71%

Bacterial infection induces nitric oxide synthase in human neutrophils.

Wheeler¹,

Smith²,

et al. 1997

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“…3C). This result suggests that the subcellular localization of native salivary-gland NOS in the salivary glands may be in the cytosol, since other recombinant NOS (neuronal NOS, endothelial NOS, inducible NOS) expressed in the same system showed the same subcellular distribution patterns as native proteins [28]. This result was compatible with the absence of a N-myristoylation membranebinding sequence in the N-terminus of salivary-gland NOS.…”

Section: -supporting

confidence: 61%

cDNA Cloning, Expression and Characterization of Nitric‐oxide Synthase from the Salivary Glands of the Blood‐Sucking Insect Rhodnius prolixus

Yuda

Hirai

Miura

et al. 1996

European Journal of Biochemistry

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Rhodnius prolixus, a blood-sucking bug, is a unique insect that is known to produce nitric oxide (NO) in the salivary glands to use as a vasodilator for blood sucking. We report here the cloning of the NO synthase (NOS) cDNA from these salivary glands and its expression in a baculovirus system. This cDNA encodes a protein of 1174 amino acids with a calculated molecular mass of 132331 Da. The primary structures of mammalian NOS, including the putative cofactor-recognition sites for heme, tetrahydrobiopterin (BH,), calmodulin, FMN, FAD and NADPH are all conserved in salivary-gland NOS. Recombinant salivary-gland NOS differed from nerve NOS and endothelial NOS in that it lacked a large N-terminal domain and an N-terminal myristylation sequence, respectively. Salivary-gland NOS produced in a baculovirus system showed NOS activity and demonstrated that salivary-gland NOS was soluble and was Ca2+ and calmodulin dependent, similarly to mammalian constitutive NOS isoforms. Recombinant salivary-gland NOS was purified to near homogeneity and migrated at 130 kDa on SDS/PAGE.Keywords : insect; salivary gland ; nitric-oxide synthase; CaZ+ and calmodulin dependence; baculovirus.NO is produced in various mammalian tissues where it plays physiologically important roles [I]. NO acts as neurotransmitter in the peripheral and the central nervous systems and is involved in brain development, memory formation and learning. In the circulatory system, NO is produced in endothelial cells, and acts as a regulator of vascular tone and platelet aggregation. NO is induced in macrophages, neutrophils and liver cells, and acts as an important effector of the cell-mediated immune system. The enzyme critical for NO production, NO synthase (NOS), is found in these different tissues, and various isofoms have been purified and characterized, and some have been cloned [2, 31. Mammalian NOS have been grouped into two general categories: constitutive (Ca*' kalmodulin dependent, e.g. endothelial NOS and neuronal NOS) and inducible (Ca"/calmodulin independent, e.g. macrophage) types.By contrast, in invertebrates, the production and utilization of NO is not as well understood, although similar physiological functions are probably present. In some insects and molluscs, the existence of NOS in brain has been demonstrated, and the function of NO as a neurotransmitter was suggested [4, 51. In the silkworm, Bombyx mori, NOS activity in the fat body was induced by lipopolysaccharide, an agent that mimics bacterial assault, which suggested the involvement of NO in the insect immune defense system [ 3 ] . This activity required Ca2+, calmodulin, NADPH, FAD, FMN and tetrahydrobiopterin (BH,), similarly to mammalian NOS. Correspondence to M . Yuda, Department of Medical Zoology,Fux: f 8 l 592 31 5215. Abhreviufions. NO, nitric oxide; NOS, nitric-oxide synthase; RACE, rapid amplification of cDNA ends ; BH,, tetrahydrobiopterin ; MOI, multiplicity of infection.Note. The nucleotide sequence data published here have been submitted to the GenBank database and ar...

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“…, and aminoguanidine (101.2 Ϯ 4.8 and 86.5 Ϯ 6.3 M) were similar for partially purified rH-iNOS and native human iNOS (respectively), indicating that the active sites of both enzymes interacted similarly with each inhibitor. As reported previously (25), aminoguanidine also inhibited rH-iNOS and native human iNOS much less potently than murine iNOS (IC 50 ϭ 6.6 Ϯ 1.0 M). We tested the effect of EGTA on the recombinant and native enzymes at concentrations ranging from 0.03 to 2 mM.…”

Section: Expression Of Active Rh-inos-rh-inos Was Expressed Insupporting

confidence: 85%

“…However, rodent and human iNOS differ in several respects. For example, unlike cNOS enzymes, which are 90 -95% identical among mammalian species, the amino acid identity of murine and human iNOS is approximately 80% (14); human iNOS is significantly more sensitive to EGTA inhibition than murine iNOS (14,24); and the inhibitor, aminoguanidine, has been reported to be 20-fold more active against murine iNOS as compared with human iNOS (25). Since sources of native human iNOS are limited, the expression of a recombinant human iNOS that faithfully represents its native counterpart is essential for the characterization of inhibitors intended for clinical use.…”

mentioning

confidence: 99%