Bettie Sue Siler Masters

Dissecting the interaction between nitric oxide synthase (NOS) and caveolin. Functional significance of the nos caveolin binding domain in vivo.

Endothelial nitric oxide synthase (eNOS) is a dually acylated peripheral membrane protein that targets to the Golgi region and caveolae of endothelial cells. Recent evidence has shown that eNOS can co-precipitate with caveolin-1, the resident coat protein of caveolae, suggesting a direct interaction between these two proteins. To test this idea, we examined the interactions of eNOS with caveolin-1 in vitro and in vivo. Incubation of endothelial cell lysates or purified eNOS with glutathioneS-transferase (GST)-caveolin-1 resulted in the direct interaction of the two proteins. Utilizing a series of GST-caveolin-1 deletion mutants, we identified two cytoplasmic domains of caveolin-1 that interact with eNOS, the scaffolding domain (amino acids 61–101) and to a lesser extent the C-terminal tail (amino acids 135–178). Incubation of pure eNOS with peptides derived from the scaffolding domains of caveolin-1 and -3, but not the analogous regions from caveolin-2, resulted in inhibition of eNOS, inducible NOS (iNOS), and neuronal NOS (nNOS) activities. These results suggest a common mechanism and site of inhibition. Utilizing GST-eNOS fusions, the site of caveolin binding was localized between amino acids 310 and 570. Site-directed mutagenesis of the predicted caveolin binding motif within eNOS blocked the ability of caveolin-1 to suppress NO release in co-transfection experiments. Thus, our data demonstrate a novel functional role for caveolin-1 in mammalian cells as a potential molecular chaperone that directly inactivates NOS. This suggests that the direct binding of eNOS to caveolin-1, per se, and the functional consequences of eNOS targeting to caveolae are likely temporally and spatially distinct events that regulate NO production in endothelial cells. Additionally, the inactivation of eNOS and nNOS by the scaffolding domain of caveolin-3 suggests that eNOS in cardiac myocytes and nNOS in skeletal muscle are likely subject to negative regulation by this muscle-specific caveolin isoform.

Crystal Structure of Constitutive Endothelial Nitric Oxide Synthase: A Paradigm for Pterin Function Involving a Novel Metal Center

Nitric oxide, a key signaling molecule, is produced by a family of enzymes collectively called nitric oxide synthases (NOS). Here, we report the crystal structure of the heme domain of endothelial NOS in tetrahydrobiopterin (H4B)-free and -bound forms at 1.95 A and 1.9 A resolution, respectively. In both structures a zinc ion is tetrahedrally coordinated to pairs of symmetry-related cysteine residues at the dimer interface. The phylogenetically conserved Cys-(X)4-Cys motif and its strategic location establish a structural role for the metal center in maintaining the integrity of the H4B-binding site. The unexpected recognition of the substrate, L-arginine, at the H4B site indicates that this site is poised to stabilize a positively charged pterin ring and suggests a model involving a cationic pterin radical in the catalytic cycle.

[92] The preparation and properties of microsomal TPNH-cytochrome c reductase from pig liver

Publisher Summary This chapter discusses the preparation and properties of microsomal TPNH-cytochrome c reductase from pig liver. It is purified from microsomes both as a partially purified microsomal subparticle, and with lipase treatment and fractionation, as a soluble flavoprotein essentially homogeneous in the ultracentrifuge. The assay depends upon measurement of the rate of cytochrome c reduction at 550 mμ. Cytochrome c reductase activity in microsomes is associated with marked TPNH-neotetrazolium diaphorase activity. This activity is disproportionately lost upon lipase treatmeat of microsomes, and its loss may serve as an indication of loss of an intermediate cofactor in the intact microsome, or of loss of a specific environmental or configurational state of the enzyme. The steps of purification procedure described are: preparation of lipase, preparation of microsomes, lipase solubilization, pH precipitation, ammonium sulfate fractionation, column chromatography on hydroxylapatite, and calcium phosphate gel concentration. The prosthetic group of TPNH-cytochrome c reductase enzyme is flavin adenine dinucleotide, but the apoenzyme can be reactivated by FMN and FAD. The enzyme is specific for TPNH, but is relatively nonspecific for electron acceptor. TPNH-cytochrome c reductase has a pH optimum of 8.2, which appears to be independent of the buffer used. TPNH-cytochrome c reductase is markedly stimulated by low levels of p-chloromercuribenzoate; maximal activation is reached at 2 moles of PCMB per mole of flavin, and higher concentrations inhibit.

INDUCIBLE NITRIC-OXIDE SYNTHASE GENERATES SUPEROXIDE FROM THE REDUCTASE DOMAIN

In the absence of l-arginine, the heme center of the oxygenase domain of neuronal nitric-oxide synthase reduces molecular oxygen to superoxide (O⨪2). Our recent work has provided evidence that inducible NOS (iNOS) may also catalyze O⨪2 formation in macrophages. However, there has been a lack of direct evidence of superoxide generation from the purified iNOS, and it was previously hypothesized that significant O⨪2production does not occur. Moreover, the mechanism and enzyme site responsible for O⨪2 generation is unknown. To determine whether iNOS produces O⨪2 and to identify the mechanism of this process, we performed electron paramagnetic resonance measurements on purified iNOS using the spin trap 5,5-dimethyl-1-pyrrolineN-oxide. In the presence of NADPH, prominent O⨪2adduct signals were detected from iNOS. These signals were totally abolished by superoxide dismutase but not affected by catalase. High concentrations of l-arginine decreased this O⨪2formation, whereas its enantiomer d-arginine did not. Pre-incubation of iNOS with the flavoprotein inhibitor diphenyleneiodonium totally blocked these O⨪2 signals. Conversely, pretreatment of the enzyme with the heme blocker cyanide had no effect on O⨪2 generation. Furthermore, strong O⨪2 generation was directly detected from the isolated iNOS reductase domain. Together, these data demonstrate that iNOS does generate O⨪2, and this mainly occurs at the flavin-binding sites of the reductase domain.

20-Hydroxyeicosatetraenoic acid is an endogenous vasoconstrictor of canine renal arcuate arteries

Recent studies have indicated that renal arteries can produce 20-hydroxyeicosatetraenoic acid (20-HETE) and suggest the potential involvement of a P450 metabolite of arachidonic acid in the myogenic activation of canine renal arteries. In the present study, the effects of 20-HETE on isolated canine renal arcuate arteries were studied. Administration of 20-HETE to the bath or the lumen at concentrations of 0.01-1 microM produced a graded reduction in the diameter of these vessels. In contrast, 19(R)-HETE was a vasodilator, whereas 19(S)-HETE was relatively inactive. The vasoconstrictor response to 20-HETE was not altered by the cyclooxygenase inhibitor indomethacin, endoperoxide/thromboxane receptor antagonist SQ29548, or combined blockade of the cyclooxygenase, lipoxygenase, and P450 pathways using indomethacin, baicalein, and 7-ethoxyresorufin. The response to 20-HETE was associated with depolarization and a sustained increase in the intracellular calcium concentration in renal vascular smooth muscle cells. Patch-clamp studies indicated that 20-HETE significantly reduced mean open time, the open-state probability, and the frequency of opening of a 117-pS K+ channel recorded from renal vascular smooth muscle cells in the cell-attached mode. Microsomes prepared from the renal cortex of dogs produced 20-HETE and 20-carboxyarachidonic acid when incubated with [14C]arachidonic acid. These results indicate that 20-HETE is an endogenous constrictor of canine renal arcuate arteries. The vasoconstrictor response to 20-HETE resembles the myogenic activation of these vessels after elevations in transmural pressure and suggests a potential role for this substance in the regulation of renal vascular tone.

An Autoinhibitory Control Element Defines Calcium-regulated Isoforms of Nitric Oxide Synthase

Nitric oxide synthases (NOSs) are classified functionally, based on whether calmodulin binding is Ca2+-dependent (cNOS) or Ca2+-independent (iNOS). This key dichotomy has not been defined at the molecular level. Here we show that cNOS isoforms contain a unique polypeptide insert in their FMN binding domains which is not shared with iNOS or other related flavoproteins. Previously identified autoinhibitory domains in calmodulin-regulated enzymes raise the possibility that the polypeptide insert is the autoinhibitory domain of cNOSs. Consistent with this possibility, three-dimensional molecular modeling suggested that the insert originates from a site immediately adjacent to the calmodulin binding sequence. Synthetic peptides derived from the 45-amino acid insert of endothelial NOS were found to potently inhibit binding of calmodulin and activation of cNOS isoforms. This inhibition was associated with peptide binding to NOS, rather than free calmodulin, and inhibition could be reversed by increasing calmodulin concentration. In contrast, insert-derived peptides did not interfere with the arginine site of cNOS, as assessed from [3H]N G-nitro-l-arginine binding, nor did they potently effect iNOS activity. Limited proteolysis studies showed that calmodulin’s ability to gate electron flow through cNOSs is associated with displacement of the insert polypeptide; this is the first specific calmodulin-induced change in NOS conformation to be identified. Together, our findings strongly suggest that the insert is an autoinhibitory control element, docking with a site on cNOSs which impedes calmodulin binding and enzymatic activation. The autoinhibitory control element molecularly defines cNOSs and offers a unique target for developing novel NOS activators and inhibitors.

Neuronal nitric oxide synthase, a modular enzyme formed by convergent evolution: structure studies of a cysteine thiolate-liganded heme protein that hydroxylates L-arginine to produce NO. as a cellular signal.

The nitric oxide synthases (NOS‐I, neuronal, NOS‐II, inducible, and NOS‐III, endothelial) are the most recent additions to the large number of heme proteins that contain cysteine thiolate‐liganded protoporphyrin EX heme prosthetic groups. This group of oxygenating enzymes also includes one of the largest gene families, that of the cytochromes P450, which have been demonstrated to be involved in the hydroxylation of a variety of substrates, including endogenous compounds (steroids, fatty acids, and prostaglandins) and exogenous compounds (therapeutic drugs, environmental toxicants, and carcinogens). The substrates for cytochromes P450 are universally hydrophobic while the physiological substrate for the nitric oxide synthases is the amino acid L‐arginine, a hydrophilic compound. This review will discuss the approaches being used to study the structure and mechanism of neuronal nitric oxide synthase in the context of its known prosthetic groups and regulation by Ca2+‐calmodulin and/or tetrahydrobiopterin (BH4).—Masters, B. S. S., McMillan, K., Sheta, E. A., Nishimura, J. S., Roman, L. J., Martasek, P. Neuronal nitric oxide synthase, a modular enzyme formed by convergent evolution: structural studies of a cysteine thiolate‐li‐ganded heme protein that hydroxylates L‐arginine to produce NO· as a cellular signal. FASEB J. 10, 552‐558 (1996)

Inhibitors of cytochrome P-450 attenuate the myogenic response of dog renal arcuate arteries.

The role of cytochrome P-450 in the myogenic response of isolated, perfused renal arcuate arteries of dogs to elevations in transmural pressure was examined. The phospholipase A2 inhibitor oleyloxyethylphosphorylcholine (1 and 10 microM) inhibited the greater than threefold increase in active wall tension in these arteries after an elevation in perfusion pressure from 80 to 160 mm Hg. Inhibition of cyclooxygenase activity with indomethacin (1 or 10 microM) had no effect on this response. The cytochrome P-450 inhibitors ketoconazole (10 and 100 microM) and beta-diethyl-aminoethyldiphenylpropylacetate (SKF 525A, 10 and 100 microM) also inhibited the myogenic response. At a pressure of 160 mm Hg, SKF 525A (10 microM) and ketoconazole (100 microM) reduced active wall tension in renal arteries by approximately 70%. Partial inhibition of the myogenic response was obtained after perfusion of the vessels with mechanism-based inhibitors of P-450, 1-aminobenzotriazole (75 microM) and 12-hydroxy-16-heptadecynoic acid (20 microM). The thromboxane receptor antagonist SQ 29,548 (1 or 10 microM) had no effect on the pressure-induced increase in active wall tension in renal arteries. Arachidonic acid (50 microM) constricted isolated perfused renal arteries and potentiated the myogenic response in the presence of indomethacin. This response was completely reversed by ketoconazole (100 microM) or SKF 525A (100 microM). Microsomes (1 mg/ml) prepared from small renal arteries (200-500 microns) and incubated with [1-14C]arachidonic acid (0.5 mu Ci, 50 microM) produced a metabolite that coeluted with 20-hydroxyeicosatetraenoic acid (20-HETE) during reversed-phase high-performance liquid chromatography. The formation of this product was inhibited by both ketoconazole and SKF 525A at concentrations of 10 and 100 microM. These results are consistent with the involvement of the vasoconstrictor 20-HETE and other cytochrome P-450 metabolites of endogenous fatty acids in the myogenic response.

The oxidative metabolism of arachidonic acid by purified cytochromes P-450☆

Abstract Arachidonic acid is catalytically oxidized using either of two types of purified cytochrome P-450 reconstituted with the purified flavo-protein, NADPH-cytochrome P-450 reductase. The reaction is dependent on the presence of cytochrome P-450, NADPH, and oxygen. The patterns of products formed are unique for the type of cytochrome P-450 used. This suggests an enzyme-directed specificity of the site of attack on the unsaturated fatty acid by the hemeprotein. Additional experiments show a possible role for cytochrome b5 since the addition of purified cytochrome b5 enhances the rate of metabolism of arachidonic acid 2 to 3 fold.

The C termini of constitutive nitric-oxide synthases control electron flow through the flavin and heme domains and affect modulation by calmodulin

The sequences of nitric-oxide synthase flavin domains closely resemble that of NADPH-cytochrome P450 reductase (CPR). However, all nitric-oxide synthase (NOS) isoforms are 20–40 residues longer in the C terminus, forming a “tail” that is absent in CPR. To investigate its function, we removed the 33 and 42 residue C termini from neuronal NOS (nNOS) and endothelial NOS (eNOS), respectively. Both truncated enzymes exhibited cytochrome c reductase activities without calmodulin that were 7–21-fold higher than the nontruncated forms. With calmodulin, the truncated and wild-type enzymes reduced cytochrome c at approximately equal rates. Therefore, calmodulin functioned as a nonessential activator of the wild-type enzymes and a partial noncompetitive inhibitor of the truncated mutants. Truncated nNOS and eNOS plus calmodulin catalyzed NO formation at rates that were 45 and 33%, respectively, those of their intact forms. Without calmodulin, truncated nNOS and eNOS synthesized NO at rates 14 and 20%, respectively, those with calmodulin. By using stopped-flow spectrophotometry, we demonstrated that electron transfer into and between the two flavins is faster in the absence of the C terminus. Although both CPR and intact NOS can exist in a stable, one-electron-reduced semiquinone form, neither of the truncated enzymes do so. We propose negative modulation of FAD-FMN interaction by the C termini of both constitutive NOSs.