Studies with purified nitric oxide synthase from rat cerebellum have confirmed previous reports that product formation is enhanced by tetrahydrobiopterin [H4B; 6-(L-erythro-1,2-dihydroxypropyl)- 5,6,7,8-tetrahydropterin]. The effect of the natural isomer, (6R)-H4B, is observed at extremely low (<0.1 ,uM) concentrations and is remarkably selective. At these concentrations, only the diastereoisomer (6S)-H4B, the structural isomer 7-(L-eiythro-1,2-dihydroxypropyl) -5,6,7,8-tetrahydropterin, and 7,8-dihydrobiopterin showed detectable effects. Our observations are inconsistent with a stoichiometric role for H4B in the oxygenation of arginine [e.g., Stuehr, D. J., Kwon, N. S., Nathan, C. F.,Chem. 266, 6259-62631. Activity is initially independent of added H4B; enhanced product formation with H4B is observed only as incubation progresses. The effect of H4B is catalytic, with each mole of added H4B supporting the formation of >15 mol of product. Recycling of H4B was excluded by direct measurement during nitric oxide synthesis and by the demonstration that nitric oxide synthase is not inhibited by methotrexate. These combined results exclude R4B as a stoichiometric reactant and suggest that H4B enhances product formation by protecting enzyme activity against progressive loss. Preliminary studies indicate that the decreased activity in the absence of added H4B does not depend on catalytic turnover of the enzyme. The role of H4B may be allosteric or it may function to maintain some group(s) on the enzyme in a reduced state required for activity.Nitric oxide synthase (NOS) catalyzes the oxygenation of arginine in the presence of NADPH to form nitric oxide, citrulline, and NADP'. The enzyme is of great interest because nitric oxide appears to participate in a variety of physiological processes. In addition to its classic role as the endothelium-derived relaxing factor in mediating vasodilation (1-3), nitric oxide has been implicated in regulating macrophage antitumor and antimicrobial activity, platelet adhesion, and cerebellar signaling (4). Nitric oxide stimulates guanylate cyclase, yielding increased production of cyclic GMP that is proposed to mediate cerebellar signaling and possibly other physiological effects of nitric oxide (4). NOS has been reported in a variety of mammalian tissues (4).Differences in cofactor, substrate, and inhibitor specificities suggest that NOS may exist in at least three distinct forms (4-6).Tetrahydrobiopterin [H4B; 6-(L-erythro-1,2-dihydroxypropyl)-5,6,7,8-tetrahydropterin] causes a marked and specific stimulation of macrophage NOS (7,8). The original studies of Bredt and Snyder (9) of purified brain (cerebellar) NOS did not include the effects of H4B. However, recent studies show that activity of cerebellar NOS is also increased by H4B (10, 11). The biochemical basis for this effect is poorly understood. Studies of the macrophage enzyme (7, 12, 13) have been interpreted as showing that H4B participates stoichiometrically in the reaction; i.e., it provides reducing equivalents req...

Effects of inhibition of nitric oxide (NO) synthase by NG-nitro-L-arginine methyl ester (L-NAME) on the increases in local cerebral blood flow (LCBF) produced in the whisker-to-barrel sensory pathway by vibrissal stimulation were studied in conscious rats with the autoradiographic iodo[14C]antipyrine method. Unilateral whisker stroking increased LCBF in the ipsilateral trigeminal spinal and principal sensory nuclei, contralateral ventral posteromedial thalamic nucleus, and contralateral somatosensory barrel cortex. Intravenous L-NAME (30 mg/kg) lowered baseline LCBF without altering the percent increases due to stimulation. Intracisternal infusions of L-NAME in doses about 10 times the molar content of free arginine in brain inhibited brain NO synthesis activity by 88%, but the percent augmentations of LCBF by stimulation remained unchanged. Chronic treatment with L-NAME (50 mg/kg ip twice daily for 4 days) inhibited NO synthase activity in brain by 84% but also failed to reduce the percent increases in LCBF due to stimulation. These results indicate that NO does not mediate the increases in LCBF associated with functional activation.

The nitric oxide synthase-catalyzed conversion of L-arginine to L-citrulline and nitric oxide is known to be the sum of two partial reactions: oxygenation of arginine to N-hydroxyarginine, followed by oxygenation of N-hydroxyarginine to citrulline and nitric oxide. Whereas the conversion of N-hydroxyarginine to citrulline and nitric oxide has been the subject of a number of studies, the oxygenation of arginine to N-hydroxyarginine has received little attention. Here we show that substrate amounts of rat cerebellar nitric oxide synthase, in the absence of added NADPH, catalyze the conversion of arginine to N-hydroxyarginine as the dominant product. The product appears not to be tightly bound to the enzyme. A maximum of 0.16 mol of N-hydroxyarginine/mol of nitric oxide synthase subunit was formed. The reaction requires oxygen and the addition of Ca2+/calmodulin and is stimulated 3-fold by tetrahydrobiopterin. Upon addition of NADPH, citrulline is formed exclusively. Conversion of N-hydroxyarginine to citrulline, like the first partial reaction, requires Ca2+/calmodulin and is stimulated by tetrahydrobiopterin but differs from the first partial reaction in being completely dependent upon addition of NADPH. These results indicate that brain nitric oxide synthase contains an endogenous reductant that can support oxygenation of arginine but not of N-hydroxyarginine. The reductant is not NADPH, since the amount of nitric oxide synthase-bound NADPH is appreciably less than the amount required for N-hydroxyarginine synthesis. Possible candidates for this role are discussed in relation to proposed mechanisms of action of nitric oxide synthase.

Initial velocity and product inhibition kinetics of the histone acetyltransferase (EC 2.3.1.48) reaction indicate that the rat liver nuclear enzyme operates under a rapid equilibrium ordered bireactant mechanism. Histone adds first to the enzyme, and under the conditions of the experiment Ka = 0 as acetyl coenzyme A (CoA) concentration approaches saturating conditions. The Km for acetyl-CoA was 2.10 +/- 0.48 micrometer. Inhibition with acetyllysine resulted in a Kiq for the enzyme-acetyllysine complex of 1.96 +/- 0.30 mM. Inhibition with CoA yielded Kip for the ternary complex of 3.19 +/- 0.48 micrometer. These results indicate that the enzyme activity is comparatively independent of histone concentration, and, since the enzyme is sensitive only to acetyl-CoA and CoA concentrations, the enzyme will tend to maintain histones in the acetylated state.