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B S Masters

Publications and source records attributed to B S Masters.

At least 55 records · Page 3Linked to original sources
PubMed1997

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.

Base Sequence↗
PubMed1997

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.

Animals↗
PubMed1996

Binding of intermediate, product, and substrate analogs to neuronal nitric oxide synthase: ferriheme is sensitive to ligand-specific effects in the L-arginine binding site.

The electron paramagnetic resonance spectra of purified neuronal nitric oxide synthase indicates that the binding of ligands to the arginine site perturbs the environment of the high-spin ferriheme in a highly ligand-specific manner. Four categories of high-spin complex can be distinguished; all are five-coordinate, and all retain the axial thiolate ligand, but they differ in their ligation geometries. These spectroscopic species reveal distinct local conformations which can be stabilized individually by the binding of L-arginine, N omega-hydroxy-L-arginine, N omega-methyl-L-arginine, and N omega-nitro-L-arginine. Other arginine analog inhibitors stabilize one or more of these states, revealing patterns based on the nature of substituents at the terminal amino group.

Animals↗
PubMed1996

Neuronal nitric oxide synthase and dystrophin-deficient muscular dystrophy.

Neuronal nitric oxide synthase (nNOS) in fast-twitch skeletal muscle fibers is primarily particulate in contrast to its greater solubility in brain. Immunohistochemistry shows nNOS localized to the sarcolemma, with enrichment at force transmitting sites, the myotendinous junctions, and costameres. Because this distribution is similar to dystrophin, we determined if nNOS expression was affected by the loss of dystrophin. Significant nNOS immunoreactivity and enzyme activity was absent in skeletal muscle tissues from patients with Duchenne muscular dystrophy. Similarly, in dystrophin-deficient skeletal muscles from mdx mice both soluble and particulate nNOS was greatly reduced compared with C57 control mice. nNOS mRNA was also reduced in mdx muscle in contrast to mRNA levels for a dystrophin binding protein, alpha 1-syntrophin. nNOS levels increased dramatically from 2 to 52 weeks of age in C57 skeletal muscle, which may indicate a physiological role for NO in aging-related processes. Biochemical purification readily dissociates nNOS from the dystrophin-glycoprotein complex. Thus, nNOS is not an integral component of the dystrophin-glycoprotein complex and is not simply another dystrophin-associated protein since the expression of both nNOS mRNA and protein is affected by dystrophin expression.

Animals↗
PubMed1996

Electron paramagnetic resonance spectroscopy of the heme domain of inducible nitric oxide synthase: binding of ligands at the arginine site induces changes in the heme ligation geometry.

The electron paramagnetic resonance spectra of the heme domain of inducible nitric oxide synthase (iNOS) demonstrate a close relationship to the corresponding spectra of the neuronal isoform (nNOS). The binding of ligands to the iNOS arginine site perturbs the environment of the high-spin ferriheme in a highly ligand-specific manner. The iNOS forms five-coordinate, high-spin complexes with arginine analogs which are clearly related to the corresponding complexes of nNOS. Studies indicate that the binding of L-arginine, N(omega)-hydroxy-L-arginine (NHA), and N(omega)-methyl-L-arginine (NMA) produces various spectroscopic species closely corresponding to the equivalent complexes of nNOS, while N(omega)-nitro-L-arginine (NNA) binding produces a state which appears intermediate in character between the nNOS NNA and arginine complexes. These spectroscopic studies have permitted the determination of ligand-specific high-spin states which reveal similarities and differences between iNOS and nNOS.

Animals↗
PubMed1996

Characterization of bovine endothelial nitric oxide synthase expressed in E. coli.

Bovine endothelial constitutive nitric oxide synthase (eNOS) was expressed in E. coli as a soluble, catalytically active enzyme using the pCW expression vector coexpressed with a plasmid, pGroELS, encoding the chaperonins groEL and groES. The E. coli BL21 cultures reproducibly synthesized 6-10 mg of recombinant enzyme per liter of culture. The eNOS protein was purified using 2'5'-ADP Sepharose 4B and appeared as a single band of apparent molecular mass 135 kDa on SDS/PAGE. The recombinant resting enzyme is predominantly high spin with an absorbance maximum at 406 nm. The dithionite-reduced, CO-bound form shows an absorbance maximum at 444 nm. The spectral properties of recombinant eNOS from E. coli are identical to those observed with eNOS from stably transfected HEK 293 cells or from baculovirus expression systems. Enzymatic activity of eNOS from E. coli ranged between 68-135 nmol product formed/min/mg at 25 degrees C, using hemoglobin-NO capture or L-citrulline formation assays. The enzyme is replete with heme and flavins and both activity and [3H]-nitroarginine binding were largely dependent on tetrahydrobiopterin. The heterologous expression of eNOS offers a number of advantages over tissue sources of the protein.

Animals↗
PubMed1996

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 IX 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 Ca(2+)-calmodulin and/or tetrahydrobiopterin (BH4).

Animals↗
PubMed1995

Inhibition of nitric oxide synthase activity by Zn2+ ion.

We have found neural nitric oxide synthase (nNOS) activity to be completely and reversibly inhibited by Zn2+ ion with an apparent Ki of 30 microM. Zn2+ blocks NADPH-dependent reduction of heme iron in nNOS and also blocks the calmodulin-dependent superoxide-mediated cytochrome c reductase activity exhibited by nNOS. However, Zn2+ ion has no apparent effect on the calmodulin-independent direct reduction of cytochrome c by nNOS. Zn2+ ion induces perturbation difference spectra in nNOS characterized by the appearance of a peak at approximately 430 nm and a trough at approximately 395 nm, with an apparent spectral binding constant of 50 microM. These spectral changes are consistent with a Zn(2+)-dependent change in the spin-state equilibrium of the heme iron in nNOS. The spectral binding constant for L-arginine binding to nNOS (approximately 1.5 microM) is not significantly affected by the presence of 50 microM Zn2+, indicating that Zn(2+)-dependent inhibition of nNOS activity is not due to interference with substrate binding. The estimated maximal change in nNOS absorbance at approximately 418 nm caused by the L-arginine-dependent conversion of the ferric heme iron from hexacoordinate low-spin to pentacoordinate high-spin is increased by 50% in the presence of 50 microM Zn2+, which reflects the increased initial amount of low-spin ferric heme iron present. These data indicate that Zn(2+)-dependent inhibition of nNOS activity is due to binding of Zn2+ to the hemoprotein domain in the enzyme and that inhibition is associated with perturbations in the environment of the heme iron that appear to block its ability to mediate oxygen reduction.

Animals↗
PubMed1995

Characterization by electron paramagnetic resonance of the interactions of L-arginine and L-thiocitrulline with the heme cofactor region of nitric oxide synthase.

Nitric oxide synthase (NOS) catalyzes sequential NADPH- and O2-dependent mono-oxygenase reactions converting L-arginine to N omega-hydroxy-L-arginine and N omega-hydroxy-L-arginine to citrulline and nitric oxide. The homodimeric enzyme contains one heme/monomer, and that cofactor is thought to mediate both partial reactions. Here we show by electron paramagnetic resonance spectroscopy that binding of substrate L-arginine to neuronal NOS perturbs the heme cofactor binding pocket without directly interacting as a sixth axial heme ligand; heme iron is exclusively high spin. In contrast, binding of L-thiocitrulline, a NOS inhibitor, produces both high and low spin iron spectra; L-thiocitrulline sulfur is a sixth axial heme ligand in one, but not all, of the low spin forms. The high spin forms of the L-thiocitrulline NOS complex display a distortion in the opposite direction to that caused by L-arginine binding. The findings elucidate the binding interactions of L-arginine and L-thiocitrulline to neuronal NOS and demonstrate that each causes a unique perturbation to the heme cofactor pocket of NOS.

Animals↗
PubMed1995

High-level expression of functional rat neuronal nitric oxide synthase in Escherichia coli.

The neuronal nitric oxide synthase (nNOS) has been successfully overexpressed in Escherichia coli, with average yields of 125-150 nmol (20-24 mg) of enzyme per liter of cells. The cDNA for nNOS was subcloned into the pCW vector under the control of the tac promotor and was coexpressed with the chaperonins groEL and groES in the protease-deficient BL21 strain of E. coli. The enzyme produced is replete with heme and flavins and, after overnight incubation with tetrahydrobiopterin, contains 0.7 pmol of tetrahydrobiopterin per pmol of nNOS. nNOS is isolated as a predominantly high-spin heme protein and demonstrates spectral properties that are identical to those of nNOS isolated from stably transfected human kidney 293 cells. It binds N omega-nitroarginine dependent on the presence of bound tetrahydrobiopterin and exhibits a Kd of 45 nM. The enzyme is completely functional; the specific activity is 450 nmol/min per mg. This overexpression system will be extremely useful for rapid, inexpensive preparation of large amounts of active nNOS for use in mechanistic and structure/function studies, as well as for drug design and development.

Amino Acid Oxidoreductases↗
PubMed1995

Modular structure of neuronal nitric oxide synthase: localization of the arginine binding site and modulation by pterin.

A putative dihydrofolate reductase (DHFR) module has been identified in neuronal nitric oxide synthase, consisting of amino acids 558-721, and is proposed to be the site of tetrahydrobiopterin (BH4) binding. This polypeptide has been expressed in E. coli as a fusion protein with glutathione S-transferase (GST), using the plasmid pGEX-4T1. The protein binds N omega-nitro-L-arginine (NNA) tightly, but this binding is not stimulated by BH4. cDNAs for Module II (residues 220-557) and Module III (residues 220-721) have been expressed as fusion proteins with GST. Module II does not bind NNA. However, Module III does bind NNA and binding is significantly stimulated by BH4. These observations are taken as strong evidence that the DHFR module contains the L-arginine binding site and, presumably, the BH4 binding site by analogy to its homology with DHFR, but that tight binding of BH4 requires amino acids 220-577.

Amino Acid Oxidoreductases↗
PubMed1995

S-alkyl-L-thiocitrullines. Potent stereoselective inhibitors of nitric oxide synthase with strong pressor activity in vivo.

Nitric oxide synthase catalyzes the oxidation of a guanidino nitrogen of L-arginine to nitric oxide with concomitant formation of citrulline. Enzyme activity is inhibited by a variety of N omega-monosubstituted L-arginine analogs including N omega-alkyl-, N omega-amino-, and N omega-nitro-L-arginine derivatives. We report here that both constitutive and inducible isoforms of nitric oxide synthase are strongly inhibited by S-alkyl-L-thiocitrullines (N delta-(S-alkyl)isothioureido-L-ornithines) with n-alkyl groups of one to three carbons. These compounds represent a novel class of inhibitors and are the most potent nitric oxide synthase-inhibiting amino acids described to date. Inhibition is reversible, stereoselective, and competitive with L-arginine. Spectral studies show no direct interaction of inhibitor sulfur with heme iron, a result in contrast to that seen previously with the parent compound, L-thiocitrulline. The S-alkyl-L-thiocitrullines have strong pressor activity in normotensive control rats; S-methyl-L-thiocitrulline reverses hypotension in a rat model of septic peritonitis and in dogs administered endotoxin. These latter findings suggest that the inhibitors may have therapeutic utility in treating hypotension due to the overproduction of nitric oxide.

Amino Acid Oxidoreductases↗
PubMed1995

Crystallization and preliminary x-ray studies of NADPH-cytochrome P450 reductase.

NADPH-cytochrome P450 reductase (CPR; NADPH:ferrihemoprotein reductase, EC 1.6.2.4) catalyzes the transfer of electrons to all known microsomal cytochromes P450. CPR is unique in that it is one of only two mammalian enzymes known to contain both flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), the other being the various isoforms of nitric oxide synthase. Similarities in amino acid sequence and in functional domain arrangement with other key flavoproteins, including nitric oxide synthase, make CPR an excellent prototype for studies of interactions between two flavin cofactors. We have obtained diffraction-quality crystals of rat liver CPR, expressed in Escherichia coli and solubilized by limited proteolysis with trypsin. The crystals were grown in Hepes buffer (pH 7.0), containing polyethylene glycol 4500 and NaCl. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 103.3 A, b = 116.1 A, and c = 120.4 A. If we assume that there are two molecules of the 72-kDa CPR polypeptide per asymmetric unit, the calculated value of Vm is 2.54 A3/Da.

Amino Acid Sequence↗
PubMed1995

Prokaryotic expression of the heme- and flavin-binding domains of rat neuronal nitric oxide synthase as distinct polypeptides: identification of the heme-binding proximal thiolate ligand as cysteine-415.

The heme- and flavin-binding domains of constitutive rat neuronal nitric oxide synthase (NOS) were expressed in Escherichia coli as distinct polypeptides with properties characteristic of the intact enzyme. The amino-terminal heme-binding domain (residues 1-714) was expressed using the expression vector pCW. The denatured molecular mass of the expressed protein was 80 kDa, and the protein was shown to be immunoreactive to rabbit anti-NOS IgG. The NOS hemoprotein exhibited a ferrous-carbon monoxide difference spectrum with a wavelength maximum at 445 nm. Spectral perturbation with L-arginine and BH4 elicited a type I difference spectrum, confirming the presence of binding sites for these molecules within the N-terminal NOS polypeptide. Site-directed mutagenesis was applied to the putative axial heme ligand, cysteine-415, generating the histidine mutant, which confirmed the identity of the proximal ligand. NOS flavoproteins, with (C1, residues 715-1429) and without (C2, residues 749-1429) an amino-terminal calmodulin-binding motif, were expressed using the vector pPROK-1. The C1 and C2 flavoproteins were immunoreactive to anti-NOS IgG and were sized at approximately 80 kDa. Both of the purified flavoproteins exhibited optical absorbance properties typical of a flavin prosthetic group, with wavelength maxima at 380 and 450 nm, and were competent in NADPH-dependent electron transfer to cytochrome c, with observed rates of approximately 2-4 mumol/min/mg. The bacterial expression of the NO synthase heme-binding oxygenase and flavoprotein oxidoreductase domains as isolated proteins with specific properties of the intact enzyme represents an important development in structure-function studies of this complex enzyme.

Amino Acid Oxidoreductases↗