Dependent Inhibition of NO Formation

In a recent study carried out in our laboratory, it was demonstrated that preincubation of brain cytosolic preparations in a complete reaction mixture led to a time-dependent loss of NOS activity (Mittal and Jadhav, 1994). This inhibition was not attributable to denaturation or proteolysis, since the preparations contained protease inhibitors and the rates of l-citrulline formation were linear up to 8 min at 37°C (Fig. 5). The omission of individual ingredients of the reaction mixture from the preincubation phase revealed the requirement of calcium ion for the inhibitory phenomenon (Table I). However, the concentrations required for the optimal inhibitory effect were significantly higher (1 mM) than those required for the catalytic activity (100 /zM) of NOS. 

Because of the known requirement of calmodulin for the catalytic activity of NOS, the possibility of alteration in the interaction of calmodulin with NOS during preincubation was tested. The exogenous addition of bovine brain calmodulin during the preincubation did not reverse NOS 

FIGURE 5 Effects of preincubation on nitric oxide synthase activity. Rat brain cytosol was preincubated for 0 min (O) and for 3 min ( ) at 37°C prior to addition of 100 /uM (each) [ H]L-arginine and NADPH. (Adapted from Mittal and Jadhav, 1994.) 

TABLE I Effect of Omission of Different Reaction Ingredients on Preincubation-Dependent Nitric Oxide Synthase (NOS) Inhibition 

Ingredient omitted NOS activity (pmol L-citrulline/mg protein/min) 

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