AN IMPROVED ASSAY FOR MEASUREMENT OF NITRIC-OXIDE SYNTHASE ACTIVITY IN BIOLOGICAL TISSUES

While the L-arginine conversion assay has been utilized to measure nit ric oxide synthase (NOS) activity in isolated enzyme and pure cell pre parations, this method often fails to provide accurate measurements in whole tissues. Biological tissues contain variable amounts of unlabel ed substrate and enzymes are present which can compete for substrate o r independently form the product L-citrulline. NOS-independent convers ion 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 tiss ue, NOS-independent L-citrulline formation was observed which could no t be blocked by the NOS inhibitor L-NAME but was blocked by the argina se inhibitor L-ornithine, To eliminate the effect of arginase-mediated L-citrulline formation, KCl-washed membrane particulate fractions wer e obtained by high-speed centrifugation. While arginase-mediated nonsp ecific activity was 85% concentrated in the cytosol, 93% of NOS activi ty was localized within the particulate fraction of the heart. The rem aining arginase activity found in the crude pellet was mostly removed by a one step KCl wash purification and when incubation periods of 8 m in were utilized specific and accurate measurements of NOS activity we re obtained. NOS enzymatic properties were de fined for rat heart prep arations with a K-m of 2.9 mu M for L-arginine, All NOS activity detec ted was calcium-dependent suggesting it originated from the constituti ve endothelial isoform, Thus, NOS-independent activity can be largely eliminated from the heart tissue by assaying KCl-washed membrane parti culate fractions and this enables accurate quantitation of NOS activit y, (C) 1998 Academic Press.