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Expression of rat brain nitric oxide synthase in baculovirus-infected insect cells and characterization of the purified enzyme.

ABSTRACT: Rat brain nitric oxide synthase was expressed to a high level in baculovirus-infected insect cells and purified to apparent homogeneity by affinity chromatography. The enzyme had a specific activity of approximately 1 mumol of citrulline.min-1.mg of protein-1 and contained 0.93, 0.45, 0.18 and 0.23 mol of haem, (6R)-5,6,7,8-tetrahydro-L-biopterin (H4biopterin), FAD and FMN per mol of subunit respectively.

SUBMITTER: Harteneck C

PROVIDER: S-EPMC1137388 | biostudies-other | 1994 Dec

REPOSITORIES: biostudies-other

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Absence of nitric-oxide synthase in sequentially purified rat liver mitochondria.

Project description:Data, both for and against the presence of a mitochondrial nitric-oxide synthase (NOS) isoform, is in the refereed literature. However, irrefutable evidence has not been forthcoming. In light of this controversy, we designed studies to investigate the existence of the putative mitochondrial NOS. Using repeated differential centrifugation followed by Percoll gradient fractionation, ultrapure, never frozen rat liver mitochondria and submitochondrial particles were obtained. Following trypsin digestion and desalting, the mitochondrial samples were analyzed by nano-HPLC-coupled linear ion trap-mass spectrometry. Linear ion trap-mass spectrometry analyses of rat liver mitochondria as well as submitochondrial particles were negative for any peptide from any NOS isoform. However, recombinant neuronal NOS-derived peptides from spiked mitochondrial samples were easily detected, down to 50 fmol on column. The protein calmodulin (CaM), absolutely required for NOS activity, was absent, whereas peptides from CaM-spiked samples were detected. Also, l-[(14)C]arginine to l-[(14)C]citrulline conversion assays were negative for NOS activity. Finally, Western blot analyses of rat liver mitochondria, using NOS (neuronal or endothelial) and CaM antibodies, were negative for any NOS isoform or CaM. In conclusion, and in light of our present limits of detection, data from carefully conducted, properly controlled experiments for NOS detection, utilizing three independent yet complementary methodologies, independently as well as collectively, refute the claim that a NOS isoform exists within rat liver mitochondria.

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Beta-actin association with endothelial nitric-oxide synthase modulates nitric oxide and superoxide generation from the enzyme.

Project description:Protein-protein interactions represent an important post-translational mechanism for endothelial nitric-oxide synthase (eNOS) regulation. We have previously reported that beta-actin is associated with eNOS oxygenase domain and that association of eNOS with beta-actin increases eNOS activity and nitric oxide (NO) production. In the present study, we found that beta-actin-induced increase in NO production was accompanied by decrease in superoxide formation. A synthetic actin-binding sequence (ABS) peptide 326 with amino acid sequence corresponding to residues 326-333 of human eNOS, one of the putative ABSs, specifically bound to beta-actin and prevented eNOS association with beta-actin in vitro. Peptide 326 also prevented beta-actin-induced decrease in superoxide formation and increase in NO and L-citrulline production. A modified peptide 326 replacing hydrophobic amino acids leucine and tryptophan with neutral alanine was unable to interfere with eNOS-beta-actin binding and to prevent beta-actin-induced changes in NO and superoxide formation. Site-directed mutagenesis of the actin-binding domain of eNOS replacing leucine and tryptophan with alanine yielded an eNOS mutant that exhibited reduced eNOS-beta-actin association, decreased NO production, and increased superoxide formation in COS-7 cells. Disruption of eNOS-beta-actin interaction in endothelial cells using ABS peptide 326 resulted in decreased NO production, increased superoxide formation, and decreased endothelial monolayer wound repair, which was prevented by PEG-SOD and NO donor NOC-18. Taken together, this novel finding indicates that beta-actin binding to eNOS through residues 326-333 in the eNOS protein results in shifting the enzymatic activity from superoxide formation toward NO production. Modulation of NO and superoxide formation from eNOS by beta-actin plays an important role in endothelial function.

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2023-06-01 | PXD040726 | Pride

Role of nitric oxide synthase in insect cell radioresistance: an in-silico analysis.

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Correction: Characterization of Calmodulin-Free Murine Inducible Nitric-Oxide Synthase.

Project description:[This corrects the article DOI: 10.1371/journal.pone.0121782.].

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Brain nitric oxide synthase is a haemoprotein.

Project description:Brain nitric oxide (NO) synthase showed pyridine haemochrome spectra typical of ferroprotoporphyrin IX-containing enzymes. The haem content of purified NO synthase was in the range 0.7-0.9 mol/mol of 160 kDa subunit. In the presence of CO, NO, KCN and miconazole, the L-citrulline-forming activity of NO synthase was markedly diminished, demonstrating that enzyme-bound haem is involved in enzymic NO synthesis.

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Traumatic brain injury disrupts cerebrovascular tone through endothelial inducible nitric oxide synthase expression and nitric oxide gain of function.

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Project description: Not available

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