Objective: To establish a method for measuring the constitutive and inducible nitric oxide synthase (NOS) through the conversion of L-3H arginine to L-3H citrulline.
Methods: NOS extracted from cerebellar of porcine and rat was purificated through DEAE cellulose or/and 2', 5', -ADP agarose. NOS activity was assayed by monitoring the conversion of 3H-arginine to 3H-citrulline.
Results: A 4,460 fold purification of the porcine cerebellar NOS was obtained with an activity of 669 pmol mg-1 min-1 and a 2.7% recovery. For purification of rat cerebellar NOS, affinity chromatography with 2', 5'-ADP agarose column provided a 2646 fold purification of enzyme activity with a 688 pmol.ml-1.min-1 and 27% recovery. The purified rat and porcine cerebellar NOS constituted a single band on SDS/PAGE at about 160,000.
Conclusion: A stable and special method for measuring NOS activity was established.