The stimulatory effects of Hofmeister ions on the activities of neuronal nitric-oxide synthase. Apparent substrate inhibition by l-arginine is overcome in the presence of protein-destabilizing agents.

A variety of monovalent anions and cations were effective in stimulating both calcium ion/calmodulin (Ca2+/CaM)-independent NADPH-cytochrome c reductase activity of, and Ca2+/CaM-dependent nitric oxide (NO.) synthesis by, neuronal nitric oxide synthase (nNOS). The efficacy of the ions in stimulating both activities could be correlated, in general, with their efficacy in precipitating or stabilizing certain proteins, an order referred to as the Hofmeister ion series. In the hemoglobin capture assay, used for measurement of NO. production, apparent substrate inhibition by L-arginine was almost completely reversed by the addition of sodium perchlorate (NaClO4), one of the more effective protein-destabilizing agents tested. Examination of this phenomenon by the assay of L-arginine conversion to L-citrulline revealed that the stimulatory effect of NaClO4 on the reaction was observed only in the presence of oxyhemoglobin or superoxide anion (generated by xanthine and xanthine oxidase), both scavengers of NO. Spectrophotometric examination of nNOS revealed that the addition of NaClO4 and a superoxide-generating system, but neither alone, prevented the increase of heme absorption at 436 nm, which has been attributed to the nitrosyl complex. The data are consistent with the release of autoinhibitory NO. coordinated to the prosthetic group of nNOS, which, in conjunction with an NO. scavenger, causes stimulation of the reaction.

Inhibition of NADPH-cytochrome P450 reductase and glyceryl trinitrate biotransformation by diphenyleneiodonium sulfate.

We reported previously that the flavoprotein inhibitor diphenyleneiodonium sulfate (DPI) irreversibly inhibited the metabolic activation of glyceryl trinitrate (GTN) in isolated aorta, possibly through inhibition of vascular NADPH-cytochrome P450 reductase (CPR). We report that the content of CPR represents 0.03 to 0.1% of aortic microsomal protein and that DPI caused a concentration- and time-dependent inhibition of purified cDNA-expressed rat liver CPR and of aortic and hepatic microsomal NADPH-cytochrome c reductase activity. Purified CPR incubated with NADPH and GTN under anaerobic, but not aerobic conditions formed the GTN metabolites glyceryl-1,3-dinitrate (1,3-GDN) and glyceryl-1,2-dinitrate (1,2-GDN). GTN biotransformation by purified CPR and by aortic and hepatic microsomes was inhibited > 90% after treatment with DPI and NADPH. DPI treatment also inhibited the production of activators of guanylyl cyclase formed by hepatic microsomes. We also tested the effect of DPI on the hemodynamic-pharmacokinetic properties of GTN in conscious rats. Pretreatment with DPI (2 mg/kg) significantly inhibited the blood pressure lowering effect of GTN and inhibited the initial appearance of 1,2-GDN (1-5 min) and the clearance of 1,3-GDN. These data suggest that the rapid initial formation of 1,2-GDN is related to mechanism-based GTN biotransformation and to enzyme systems sensitive to DPI inhibition. We conclude that vascular CPR is a site of action for the inhibition by DPI of the metabolic activation of GTN, and that vascular CPR is a novel site of GTN biotransformation that should be considered when investigating the mechanism of GTN action in vascular tissue.

The influence of chaotropic reagents on neuronal nitric oxide synthase and its flavoprotein module. Urea and guanidine hydrochloride stimulate NADPH-cytochrome c reductase activity of both proteins.

Changes in flavin and protein fluorescence of neuronal nitric oxide synthase (nNOS) and its flavoprotein module were studied in the presence of urea and compared with those previously reported for cytochrome P450 reductase (CPR) [R. Narayanasami, P. M. Horowitz, and B. S. S. Masters (1995) Arch. Biochem. Biophys. 316, 267-274]. As in the case of CPR, FMN was relatively loosely bound to nNOS and the flavoprotein module, but FAD remained bound at concentrations of up to 2 M urea Protein fluorescence increased progressively with increasing urea concentration, but could not be correlated with changes in flavin binding. NADPH-cytochrome c reductase activity of both nNOS and the flavoprotein module, but not that of CPR, was stimulated at early time points by both urea and guanidine hydrochloride (GnHCl), with levels of initial activity returning to baseline values within 60 min after addition of the chaotropic agent. Thus, at 3-4 M urea, enhancements of reductase activities of 20- and 5-fold with nNOS and the flavoprotein module, respectively, were obtained. Comparable enhancements of 12- and 6- to 7-fold, respectively, were obtained with calmodulin (CaM)/ CaCl2 and 0.5 M GnHCl. Thus, the effects of urea and GnHCl mimicked the stimulating effects of CaM. Separate preincubations of nNOS and cytochrome c with urea or GnHCl prior to initiation of the reductase assay showed that sensitivity to chaotropic agent under these conditions was a property of nNOS and not of cytochrome c. Moreover, when the nonprotein electron acceptor 2,6-dichlorophenolindophenol was employed in place of cytochrome c, comparable stimulation of reductase activity was observed in the presence of either urea or GnHCl. Fluorescence of 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfate in the presence of either nNOS or the flavoprotein module was increased optimally between 3 and 4 M urea, consistent with simultaneous exposure of hydrophobic regions of both proteins to solvent and optimization of reductase activity. FMN release from nNOS, but not from the flavoprotein module, was enhanced by CaM. Addition of FMN or FMN + FAD to nNOS, in the presence or absence of urea, brought about a doubling of initial cytochrome c reductase activity, but did not prevent the eventual decline in activity to basal levels. These data are consistent with conformational changes which favor increased electron transfer similar to that achieved with nNOS in the presence of CaM.

Relationships between NADPH diaphorase staining and neuronal, endothelial, and inducible nitric oxide synthase and cytochrome P450 reductase immunoreactivities in guinea-pig tissues.

The presence of NADPH diaphorase staining was compared with the immunohistochemical localization of four NADPH-dependent enzymes-neuronal (type I), inducible (type II), and endothelial (type III) nitric oxide synthase (NOS) and cytochrome P450 reductase. Cell types that were immunoreactive for the NADPH-dependent enzymes were also stained for NADPH diaphorase, suggesting that endothelial and neuronal NOS and cytochrome P450 reductase all show NADPH diaphorase activity in formaldehyde-fixed tissue. However, in some tissues, the presence of NADPH diaphorase staining did not coincide with the presence of any of the NADPH-dependent enzymes we examined. In vascular endothelial cells, the punctate pattern of staining observed with NADPH diaphorase histochemistry was identical to that seen following immunohistochemistry using antibodies to endothelial NOS. In enteric and pancreatic neurons and in skeletal muscle, the presence of NADPH diaphorase staining correlated with the presence of neuronal NOS. In the liver, sebaceous glands of the skin, ciliated epithelium, and a subpopulation of the cells in the subserosal glands of the trachea, zona glomerulosa of the adrenal cortex, and epithelial cells of the lacrimal and salivary glands, the presence of NADPH diaphorase staining coincided with the presence of cytochrome P450 reductase immunoreactivity. In epithelial cells of the renal tubules and zona fasciculata and zona reticularis of the adrenal cortex, NADPH diaphorase staining was observed that did not coincide with the presence of any of the enzymes. Inducible NOS was not observed in any tissue. Thus, while tissues that demonstrate immunoreactivity for neuronal and endothelial NOS also stain positively for NADPH diaphorase activity, the presence of NADPH diaphorase staining does not reliably or specifically indicate the presence of one or more NOS isoforms.

Flavin-binding and protein structural integrity studies on NADPH-cytochrome P450 reductase are consistent with the presence of distinct domains.

NADPH-cytochrome P450 reductase (reductase) contains FMN and FAD in 1:1 stoichiometry as tightly bound cofactors. Electrons from NADPH are transferred to cytochrome P450 through the intermediacy of reductase. A knowledge of the interactions which must occur to allow the intermolecular and intramolecular transfer of electrons is not only of intrinsic interest but is necessary to understand the regulation of the overall oxidation-reduction processes in which cytochromes P450 participate in the endoplasmic reticulum of many organs. In the present study, urea has been employed as a chaotropic agent to study the dissociation of flavins from NADPH-cytochrome P450 reductase. The results show that dissociation of FMN occurs at concentrations of urea between 0 and 1 M and that, as the concentrations of urea approach 1 M, the intrinsic protein fluorescence increases, indicating a change in protein conformation. Above 2 M urea protein fluorescence increases, reaching a plateau at 3 M urea, and FAD begins to dissociate from the enzyme. In the range of 0-1 M urea, a completely reversible dissociation of FMN occurs and, at 3 M urea, the fluorescence values representing flavin dissociation and protein conformation changes have reached a maximum. Thus, the definition of various states of the flavoprotein with both, one, or no flavins bound and the ability to remove the flavins reversibly under specific conditions have permitted the construction of a simple model to explain the various unfolding intermediates of this enzyme. Our experiments suggest that reductase is composed of distinct domains which can be examined independently by the application of chaotropic agents.

31P NMR spectroscopic studies on purified, native and cloned, expressed forms of NADPH-cytochrome P450 reductase.

31P NMR spectroscopy has been utilized in conjunction with site-directed mutagenesis and phospholipid analysis to determine structural aspects of the prosthetic flavins, FAD and FMN, of NADPH-cytochrome P450 reductase. Comparisons are made among detergent-solubilized and protease (steapsin)-solubilized preparations of porcine liver reductases, showing unequivocally that the 31P NMR signals at approximately 0.0 ppm in the detergent-solubilized, hydrophobic form are attributable to phospholipids. By extraction and TLC analysis, the phospholipid contents of detergent-solubilized rat liver reductase, both tissue-purified and Escherichia coli-expressed, have been determined to reflect the membranes from which the enzyme was extracted. In addition, the cloned, wild-type NADPH-cytochrome P450 reductase exhibits an additional pair of signals downfield of the normal FAD pyrophosphate resonances reported by Otvos et al. [(1986) Biochemistry 25, 7220-7228], but these signals are not observed with tissue-purified or mutant enzyme preparations. The Tyr140----Asp140 mutant, which exhibits only 20% of wild-type activity, displays no gross changes in 31P NMR spectra. However, the Tyr178----Asp178 mutant, which has no catalytic activity and does not bind FMN, exhibits no FMN 31P NMR signal and a normal, but low intensity, pair of signals for FAD. The latter experiments, taking advantage of mutations in residues putatively on either side of the FMN isoalloxazine ring, suggest subtle to severe changes in the binding of the flavin prosthetic groups and, perhaps, cooperative interactions of flavin binding to NADPH-cytochrome P450 reductase.