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Conditions required for the measurement of nitric oxide synthase activity in a myenteric plexus/smooth muscle preparation from the rat ileum.

Lincoln, J; Messersmith, WA; (1995) Conditions required for the measurement of nitric oxide synthase activity in a myenteric plexus/smooth muscle preparation from the rat ileum. J Neurosci Methods , 59 (2) pp. 191-197.

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Abstract

Nitric oxide synthase (NOS) activity was measured, by the conversion of arginine to citrulline, in a preparation from the rat ileum consisting of the myenteric plexus and smooth muscle layers. A variety of incubating media were used in order to establish the optimal conditions required for the assay. NOS activity was present in the soluble fraction and was Ca(2+)- and calmodulin-dependent, characteristic of neuronal NOS. Exogenous Ca2+ was required for activity to be detectable but NOS activity progressively decreased with Ca2+ concentrations above 1.25 mM. Activity varied with arginine concentration, reaching saturation at 6 microM, and required the addition of the co-substrate NADPH. Endogenous levels of co-factors in the crude soluble fraction were not sufficient to maintain NOS activity. Omission of flavin adenine dinucleotide and tetrahydrobiopterin from the incubation medium reduced activity by 90%, and both co-factors had to be present for maximal activity to occur. These results emphasize the need to control assay conditions when measuring NOS activity in crude preparations from peripheral tissue.

Type: Article
Title: Conditions required for the measurement of nitric oxide synthase activity in a myenteric plexus/smooth muscle preparation from the rat ileum.
Location: Netherlands
Keywords: Animals, Antioxidants, Arginine, Biopterin, Calcium, Calmodulin, Citrulline, Flavin Mononucleotide, Flavin-Adenine Dinucleotide, Ileum, In Vitro Techniques, Muscle, Smooth, Myenteric Plexus, NADP, Nitric Oxide Synthase, Rats, Rats, Wistar
UCL classification: UCL > School of Life and Medical Sciences
UCL > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > School of Life and Medical Sciences > Faculty of Life Sciences > Biosciences (Division of) > Cell and Developmental Biology
URI: http://discovery.ucl.ac.uk/id/eprint/124453
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