Isoform-specific effects of salts on nitric oxide synthase activity.
We investigated the effects of salts on the properties of the neuronal, endothelial, and inducible isoforms of nitric oxide synthase (nNOS, eNOS, and iNOS), and found pronounced isoform-specific effects on NOS-catalyzed L-citrulline formation. Salts inhibited iNOS monotonously, whereas nNOS and eNOS were stimulated up to 3-fold at low, and inhibited at high (>/=0.1-0.2 M) salt concentrations. The effectivities of different ions mostly followed the Hofmeister series, indicating that the effects can for a large part be ascribed to changes in protein solvation. Km(Arg) increased in the presence of NaCl, demonstrating the importance of charge interactions for substrate binding. The coupling of NADPH oxidation to NO production was not affected by KCl. Salts (</=1 M) had no major impact on the tertiary and quaternary structure, or on the state of the heme. Extrapolation of these results to commonly applied experimental conditions for in vitro activity assays suggests that true specific activities of nNOS and eNOS may, in some cases, be underestimated as much as 3-fold.
Schrammel
A
A
Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, A-8010 Graz, Austria.
Gorren
A C
AC
Stuehr
D J
DJ
Schmidt
K
K
Mayer
B
B
D000818:Animals; D000838:Anions / Q000494:pharmacology; D001120:Arginine / Q000378:metabolism; D002412:Cations / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D009249:NADP / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000145:classification / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects; D051381:Rats; D012492:Salts / Q000494:pharmacology*
D000838:Anions; D002412:Cations; D007527:Isoenzymes; D012492:Salts; D009569:Nitric Oxide; D009249:NADP; D011189:Potassium Chloride; D001120:Arginine; D019001:Nitric Oxide Synthase