Conditions required for the measurement of nitric oxide synthase activity in a myenteric plexus/smooth muscle preparation from the rat ileum.
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.
Department of Anatomy and Developmental Biology, University College London, UK.
J
J
Lincoln
W A
WA
Messersmith
D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D001120:Arginine / Q000378:metabolism; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002956:Citrulline / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D007082:Ileum / Q000201:enzymology; D066298:In Vitro Techniques; D009130:Muscle, Smooth / Q000201:enzymology*; D009197:Myenteric Plexus / Q000201:enzymology*; D009249:NADP / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000032:analysis* / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
D000975:Antioxidants; D002147:Calmodulin; D005182:Flavin-Adenine Dinucleotide; D001708:Biopterins; D002956:Citrulline; D009249:NADP; D005486:Flavin Mononucleotide; D001120:Arginine; D019001:Nitric Oxide Synthase; C003402:sapropterin; D002118:Calcium