Calcium-dependent inhibition of constitutive nitric oxide synthase.
The objective of these investigations was to study the regulatory properties of brain constitutive NO synthase. NOS activity was determined in 18,000 X g supernatant by conversion of 3H-L-arginine to 3H-L-citrulline in the presence of NADPH. The expression of catalytic activity of NOS required the presence of calcium ion and calmodulin. The preincubation of enzyme preparations at 37 degrees C in standard reaction mixture led to time-dependent inhibition of L-citrulline formation. This inhibition also required the presence of calcium ion during preincubation phase, and the enzyme remained calmodulin-dependent as exhibited by sensitivity to calmodulin antagonists trifluoperazine (TFP) and calcineurin. The modified enzyme showed significant decrease in the Vmax with NADPH and L-arginine without any change in apparent Km. Inclusion of protease inhibitors, leupeptin, pepstatin A, PMSF and soyabean trypsin inhibitor to the preparations did not alter preincubation-dependent inhibition of NO synthase. Thus, the calcium-dependent inhibitory phenomenon was not due to either the denaturation or proteolysis or the loss of calmodulin sensitivity of NO synthase. These observations indicate that cytosolic isoform of constitutive NO synthase undergoes dual regulation by physiological concentrations of calcium ion.
Mittal
C K
CK
Division of Pharmaceutical Science, College of Pharmacy and Health Science, Houston 77004.
Jadhav
A L
AL
D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D001120:Arginine / Q000378:metabolism; D001921:Brain / Q000201:enzymology*; D019703:Calcineurin; D002122:Calcium Chloride / Q000494:pharmacology*; D002147:Calmodulin / Q000494:pharmacology; D002148:Calmodulin-Binding Proteins / Q000494:pharmacology; D002956:Citrulline / Q000378:metabolism; D007700:Kinetics; D019001:Nitric Oxide Synthase; D010749:Phosphoprotein Phosphatases / Q000494:pharmacology; D011865:Radioisotope Dilution Technique; D051381:Rats; D017207:Rats, Sprague-Dawley; D014268:Trifluoperazine / Q000494:pharmacology; D014316:Tritium
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D014316:Tritium; D014268:Trifluoperazine; D002956:Citrulline; D001120:Arginine; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D002122:Calcium Chloride