Purification to Homogeneity and Characterisation of Rat Brain Recombinant Nitric Oxide Synthase

Abstract

We have previously demonstrated high expression of rat neuronal nitric oxide synthase (NO synthase) in a baculovirus system [Charles, I. G., Chubb, A., Gill, R., Clare, J., Lowe, P. N., Holmes, L. S., Page, M., Keeling, J. G., Moncada, S. & Riveros-Moreno, V. (1993) Biochem. Biophys. Res. Commun. 196, 1481-1489], where a small proportion of the expressed enzyme was soluble and active, but the majority was insoluble (approximately 15% of the total insoluble proteins). NO synthase is a complex enzyme, requiring several cofactors for full activity. These include tightly bound FAD, FMN, heme and tetrahydrobiopterin, in addition to calmodulin and NADPH. Here, we report that a substantial proportion of the total NO synthase produced becomes soluble following addition of hemin (2.5 micrograms/ml) to the culture medium. However, the enzyme purified under these conditions had very low specific activity, 50 nmol.min-1.mg-1, after ADP-Sepharose affinity purification. Full activity (approximately 800 nmol.min-1.mg-1) could, however, be obtained by including precursors for the cofactors, nicotinic acid, riboflavin, and sepiapterin in the culture medium. We demonstrate that the enzyme activity is exclusively associated with the dimeric form of the enzyme, which had the following molar ratios for the cofactors: heme, 0.92; FAD, 0.57; FMN, 0.34; H4biopterin, 0.32, with a specific activity of 1500 nmol.min-1.mg-1. The provision of substantial quantities of good quality enzyme, as described here, will facilitate the studies on the relationship between enzyme structure and its mechanism of catalysis.

Antifungal Imidazoles Block Assembly of Inducible NO Synthase into an Active Dimer

“…Some general features have emerged. For example, various derivatives of L-Arg and guanidine, various pteridines, ethylisothiourea, and 7-nitroindazole all promote NOS subunit dimerization (41,47,51,56,67) or stabilize the dimer (42,43,58). This suggests structural constraints are minimal, and the promoting effect is not limited to molecules acting as substrates or cofactors for NO synthesis.…”

Mechanistic Studies with Potent and Selective Inducible Nitric-oxide Synthase Dimerization Inhibitors

“…Dimerization of iNOS is required for fully coupled enzyme activity because the flow of electrons during catalysis occurs in trans from the reductase domain of one monomer to the oxygenase domain of the other monomer (25). Dimerization of NOS monomers is initiated by heme insertion, which results in rapid conformational changes (18,19,26). The heme-containing iNOS monomer is an intermediate in dimerization and, in the presence of H 4 B and L-arginine, forms a stable active dimer (18,24,(27)(28)(29).…”

Association of β-amyloid peptide fragments with neuronal nitric oxide synthase: Implications in the etiology of Alzheimers disease

“…All amyloid peptides [Aβ [17][18][19][20][21][22][23][24][25][26][27][28] , Aβ [22][23][24][25][26][27][28][29][30][31][32][33][34][35] , Aβ [32][33][34][35] , Aβ [25][26][27][28][29][30][31][32][33][34][35] and Aβ ], acrylamide; bis-acrylamide, bromophenol blue, N, N, N' , N'-tetramethylethylenediamine, ammonium persulphate, β-mercaptoethanol, Coomassie Blue commercial stain, Nα-benzoyl-l-arginine ethyl ester hydrochloride, DL-citrulline, calcium chloride, tetrahydro-l-biopterin dichloride, β-nicotinamide adenine dinucleotide phosphate reduced tetrasodium salt, thiosemicarbazide, 2,3 butanedione monoxime, DEAE-Sepharose ® , bovine serum albumin, Bradford reagent, ethylenediaminetetracetic acid and 4-(2-hydroxyethyl) piperazine-N'-(2-ethane-sulphonic acid monosodium salt [Hepes] were obtained from Sigma-Aldrich (Johannesburg, South Africa). Commercial nNOS obtained from rat brain, dithiothreitol (DTT), sodium dodecyl sulphate (SDS), glycerol (50%), Tris (hydroxymethyl) aminomethane, phenylmethylsulphonylfluoride (PMSF), ferric chloride hexahydrate (FeCl 3 ), sodium chloride (NaCl), perchloric acid, sulphuric acid, ortho-phosphoric ac...…”

“…Kinetic analysis Partially purified nNOS (5 μl) in Tris HCl buffer (50 mM, pH 7.6) was treated with amyloid peptides Aβ 1-40 , Aβ [22][23][24][25][26][27][28][29][30][31][32][33][34][35] , Aβ [17][18][19][20][21][22][23][24][25][26][27][28] , Aβ [32][33][34][35] and Aβ [25][26][27][28][29][30][31][32][33][34][35] at 2 µM in a total volume of 1.0 ml and in the presence of benzoyl arginine ethyl ester (0-10 µM). The nNOS activity was assayed, at each substrate concentration.…”

“…It is relatively well known 8,9 that there are a number of amyloidogenic proteins and short peptide fragments, including the Aβ [25][26][27][28][29][30][31][32][33][34][35] fragment itself, that are capable of producing amyloid fibrils 10 and neurotoxic oligomers 11 . This present study uses a series of readily available different sized Aβ peptides [Aβ [17][18][19][20][21][22][23][24][25][26][27][28] , Aβ [22][23][24][25][26][27][28][29][30][31][32][33][34][35] and Aβ 32-3 5] along with the neurotoxic elements Aβ [25][26][27][28][29][30][31]…”