Nitric oxide synthase from cerebellum catalyzes the formation of equimolar quantities of nitric oxide and citrulline from L-arginine.
This study examined whether constitutive nitric oxide (NO) synthase from rat cerebellum catalyzes the formation of equimolar amounts of NO plus citrulline from L-arginine under various conditions. Citrulline was determined by monitoring the formation of 3H-citrulline from 3H-L-arginine. NO was determined by monitoring the formation of total NOx (NO+nitrite [NO2-] + nitrate [NO3-]) by chemiluminescence after reduction of NOx to NO by acidic vanadium (III). Equal quantities of NO plus citrulline were generated from L-arginine and the formation of both products was linear for about 20 min at 37 degrees C provided L-arginine was present in excess to maintain a zero order reaction rate. Deletion of NADPH, addition of the calmodulin antagonist calmidazolium, or addition of NO synthase inhibitors (NG-methyl-L-arginine, NG-amino-L-arginine) abolished or markedly inhibited the formation of both NO and citrulline. The Km for L-arginine (14 microM; 18 microM) and the Vmax of the reaction (0.74 nmol/min/mg protein; 0.67 nmol/min/mg protein) were the same whether NO or citrulline formation, respectively, was monitored. These observations indicate clearly that NO and citrulline are formed in equimolar quantities from L-arginine by the constitutive isoform of NO synthase from rat cerebellum.
Bush
P A
PA
Department of Pharmacology, UCLA School of Medicine 90024.
Gonzalez
N E
NE
Griscavage
J M
JM
Ignarro
L J
LJ
D000594:Amino Acid Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D002531:Cerebellum / Q000201:enzymology*; D002956:Citrulline / Q000378:metabolism*; D007700:Kinetics; D008163:Luminescent Measurements; D008297:Male; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase; D011865:Radioisotope Dilution Technique; D051381:Rats; D011919:Rats, Inbred Strains; D014316:Tritium
D014316:Tritium; D002956:Citrulline; D009569:Nitric Oxide; D001120:Arginine; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases