The Study On The Role Of NO Synthase In The Injury Of The Nervous System

Nitric Oxide Synthase (NOS) specifically catalyzes the conversion from L-arginine to L-hydroxyarginine, which is further oxidized into L-citrulline and nitric oxide. Nitric oxide is then again oxidized into the stable terminal products as nitrite and nitrate. As a cellular signaling molecule, NO plays an important role in the regulatory mechanism of the neural, immunal and cardiovascular systems. Being the critical enzyme in synthesizing NO, NOS directly regulates the production of NO and its biological effects. Since NO has a very short half-life, the technical difficulty in its direct measurement has made it necessary to measure NOS activity in our experiments on rat brain ischemia model. NOS consists of a constitutive NOS, which is Ca²⁺/ CaM dependent and an inducible NOS, which is not. In order to investigate the role of NMDA-NO-cGMP pathway in ischemic brain injury, We have established a method of measuring the constitutive and inducible NOS through the conversion of L-³H-arginine to L-³H-citrulline, and a method of measuring NMDA receptor activity through ³H-MK801 binding. Furthermore, we have successfully transfected the full length rat bralin NOS cDNA into NG108-15 cells, and have selected the stable clones of high expression. NOS in porcine and rat brains have also been purified.1. Extraction, purification and characterization of NO synthase.We found the activity of the crude rat and bovine cerebellar NOS preparations were time and dose dependent. The Km of rat cerebellar NOS to its substrate L-arginine was 1. 6μM and V_max was 23. 9pmol/mg/min. Rat cerebellar NOS was dependent on Ca²⁺,calmodulin,NADPH and BH₄; their respective EC₅₀ were 0. 55mM,33nM,32.5μM and 0. 43μM. EGTA 3 to 5 mM could completely inhibit the activity of crude rat cerebellar NOS preparations; the calmodulin antagonist TFP inhibited its activity completely at...