An improved assay for measurement of nitric oxide synthase activity in biological tissues.
While the L-arginine conversion assay has been utilized to measure nitric oxide synthase (NOS) activity in isolated enzyme and pure cell preparations, this method often fails to provide accurate measurements in whole tissues. Biological tissues contain variable amounts of unlabeled substrate and enzymes are present which can compete for substrate or independently form the product L-citrulline. NOS-independent conversion of radiolabeled L-arginine to L-citrulline occurs due to arginase- and ornithine transcarbamylase-mediated reactions and this limits the accuracy of this assay for measurement of NOS activity. In heart tissue, NOS-independent L-citrulline formation was observed which could not be blocked by the NOS inhibitor L-NAME but was blocked by the arginase inhibitor L-ornithine. To eliminate the effect of arginase-mediated L-citrulline formation, KCl-washed membrane particulate fractions were obtained by high-speed centrifugation. While arginase-mediated nonspecific activity was 85% concentrated in the cytosol, 93% of NOS activity was localized within the particulate fraction of the heart. The remaining arginase activity found in the crude pellet was mostly removed by a one-step KCl wash purification and when incubation periods of 8 min were utilized specific and accurate measurements of NOS activity were obtained. NOS enzymatic properties were defined for rat heart preparations with a Km of 2.9 microM for L-arginine. All NOS activity detected was calcium-dependent suggesting it originated from the constitutive endothelial isoform. Thus, NOS-independent activity can be largely eliminated from the heart tissue by assaying KCl-washed membrane particulate fractions and this enables accurate quantitation of NOS activity.
Molecular and Cellular Biophysics Laboratories, Department of Medicine, Division of Cardiology, The Johns Hopkins Medical Institutions, Johns Hopkins Bayview Medical Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224, USA.
R R
RR
Giraldez
J L
JL
Zweier
D000818:Animals; D001119:Arginase / Q000037:antagonists & inhibitors / Q000378:metabolism; D001120:Arginine / Q000378:metabolism; D002250:Carbon Radioisotopes; D002458:Cell Fractionation; D002852:Chromatography, Ion Exchange; D002956:Citrulline / Q000096:biosynthesis; D003600:Cytosol / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D066298:In Vitro Techniques; D007700:Kinetics; D008566:Membranes / Q000201:enzymology; D008722:Methods; D009206:Myocardium / Q000201:enzymology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D009952:Ornithine / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012588:Scintillation Counting
D002250:Carbon Radioisotopes; D004791:Enzyme Inhibitors; D002956:Citrulline; D001120:Arginine; D009952:Ornithine; D019001:Nitric Oxide Synthase; D001119:Arginase; D019331:NG-Nitroarginine Methyl Ester