Metabolism in rat liver microsomes of the nitroxide spin probe tempol.

Paramagnetic nitroxide spin labels have been extensively used to probe various biophysical and biochemical properties of the cellular environment. Recently nitroxides have been proposed as contrast enhancing agents in proton magnetic resonance imaging and contrast enhancement has been demonstrated in animal studies. Nitroxides, possessing a stable unpaired electron, increases the relaxation rates of protons, providing an enhancement of contrast. Nitroxides are metabolized intracellularly principally via reversible reduction to hydroxylamines. Rates of reduction depend on the physical characteristics of the nitroxides, in general 5-membered pyrrolidine ring are reduced more slowly than those with a 6-membered piperidine ring. Oxidation back to the nitroxide is relevant for lipid soluble hydroxylamines, while is low for water soluble ones. It is known that nitroxides are metabolized by subcellular fractions (cytosol, mitochondria, microsomes), though the enzymatic and non-enzymatic systems involved are poorly characterized. In the present study, the first of the necessary steps toward a systematic study of the metabolism of nitroxides by subcellular organelles, we have chosen to study the metabolism of 4-hydroxy 2,2,6,6-tetramethylpiperidine-N-oxyl in isolated rat liver microsomes. Microsomes were able to reduce Tempol slowly without any substrate addition; when NADPH was added, the reduction rate substantially increased. In phenobarbitone induced rats the reduction rate was significantly higher than in not-induced microsomes. NADPH-dependent reduction rate was inhibited by thallium chloride (an inhibitor of the flavin-centered cytochrome P-450 reductase), superoxide dismutase, and by N-ethylmaleimide; menadione increased it. The Tempol reduction rate was not significantly affected by various cytochrome P-450 inhibitors with the sole exception of metyrapone. A solution containing purified cytochrome P-450 reductase and NADPH readily reduced Tempol. Microsomes fortified with NADPH were able to reduce Tempol at an appreciable rate. In order to distinguish between reduction of nitroxides to hydroxylamine or destruction of nitroxides following nitroxide reduction, microsomal suspensions were treated with a mild oxidant (ferricyanide 0.5-10 mM). The recovery varied from 40 to 60%, indicating a process of probe destruction leading to as yet unknown metabolites. The present study clearly indicates that, in this model system, cytochrome c (P-450) reductase and not cytochrome P-450 is responsible for the observed Tempol metabolism; along with hydroxylamine formation, other Tempol derived metabolites are formed during the process.

Modification of fluidity and lipid-protein relationships in pig intestinal brush-border membrane by dietary essential fatty acid deficiency.

The effect of dietary essential fatty acid (EFA) deficiency on the dynamic molecular organization of pig intestinal brush-border membrane (BBM) was studied using purified BBM vesicles. A 6 week dietary treatment of weaning piglets induced a typical EFA-deficient pattern in the lipid composition of both plasma and epithelial membranes. In pigs fed on the EFA-deficient diet, the plasma 20:3(n - 9)/20:4(n - 6) ratio progressively increased and reached a stable value after 3 weeks of experiment, whereas it remained low (less than 0.2) in controls. In the intestinal BBM, the cholesterol/protein, phospholipid/protein and consequently the cholesterol/phospholipid ratios, as well as the phospholipid class distribution, were unchanged. In particular, the sphingomyelin/phosphatidylcholine (SM/PC) molar ratio was not affected. However, the fatty acid composition of phospholipid main classes was markedly modified, leading to decreased lipid fluidity and to a large change in membrane protein behaviour with EFA deficiency. These findings could be interpreted in terms of reduced lipid-protein interactions. Moreover, the increasing gradient of fluidity which took place within the lipidic matrix from its surface was modified by the dietary treatment, as fluidity was lowered by EFA deficiency at different depths of the layer.

Binding of spin-labeled clofibrate to lipoproteins.

The binding of spin-labeled clofibrate to native and partially delipidated lipoproteins is a rapid, linear and non-saturable process observed up to the critical micellar concentration of the drug. Low-density lipoproteins (LDL) display a lower affinity for the drug than very-low-density lipoproteins (VLDL) and high-density lipoproteins (HDL) relative to their respective specific volume. Unlike various lipophilic drugs, uptake of spin-labeled clofibrate does not correlate with lipoprotein lipid volume. Spin-labeled clofibrate binding to LDL is enhanced when the temperature increases above 25 degrees C. The binding to HDL and VLDL is less temperature-sensitive. The simulation of the ESR spectra has shown that two types of motion should be superimposed for the spin-labeled clofibrate in HDL, in LDL or in partially delipidated LDL. From 40 down to 25 degrees C for HDL and LDL, a fast anisotropic motion is observed. From 25 degrees C down to 5 degrees C, a two-component motion takes place, including a slow isotropic motion of the probe tumbling in a highly hydrophobic environment. Interactions of spin-labeled clofibrate with the apolipoproteins in HDL and LDL are assumed from the emergence of this strongly immobilized component observed when the temperature decreases. In contrast, for spin-labeled clofibrate inserted in the apolar core of VLDL, ESR shows only one component in the whole temperature range (5-40 degrees C). The location of the spin-labeled drug inside the various lipoprotein particles is discussed as a function of temperature.

Measurement of outward translocation of phospholipids across human erythrocyte membrane.

Spin-labeled phospholipids have been used to study the outside----inside and inside----outside transport of phospholipids across the human erythrocyte membrane at 37 degrees C. As already shown, inward transport is much faster for aminophospholipids than for phosphatidylcholine. In addition, we show here that outward transport of the phosphatidylserine and phosphatidylethanolamine analogues is three to four times faster than that of phosphatidylcholine. Magnesium depletion of the erythrocytes considerably decreases the outward rate of both aminophospholipids to values close to that of phosphatidylcholine. These results suggest that the outward aminophospholipid translocation is, at least partly, protein mediated. The protein involved could be identical to the inward Mg-ATP-dependent aminophospholipid carrier.

Influence of lipid headgroup on the specificity and exchange dynamics in lipid-protein interactions. A spin-label study of myelin proteolipid apoprotein-phospholipid complexes.

The pH and salt dependences of the interaction of phosphatidic acid, phosphatidylserine, and stearic acid with myelin proteolipid apoprotein (PLP) in dimyristoylphosphatidylcholine (DMPC) recombinants have been studied by electron spin resonance spectroscopy, using spin-labeled lipids. The two-component spin-label spectra have been analyzed both by spectral subtraction and by simulation using the exchange-coupled Bloch equations to give the fraction of lipids motionally restricted by the protein and the rate of lipid exchange between the fluid and motionally restricted lipid populations. For stearic acid, phosphatidic acid, and phosphatidylserine, the fraction of motionally restricted spin-label increases with increasing pH, with pKa's of 7.7, 7.6, and ca. 9.4, respectively. The corresponding pKa's for the bulk lipid regions of the bilayer are estimated, from changes in the ESR spectra, to be 6.7, 7.4, and 11, respectively. In the dissociated state at pH 9.0, the fraction of motionally restricted component decreases with increasing salt concentration, reaching an approximately constant value at [NaCl] = 0.5-1.0 M for all three negatively charged lipids. The net decreases for stearic acid and phosphatidic acid are considerably smaller (by ca. 30%) than those obtained on protonating the two lipids, whereas for phosphatidylserine the fraction of motionally restricted lipid in high salt is reduced to that corresponding to phosphatidylcholine. For a fixed lipid/protein ratio, the on-rate for exchange at the lipid-protein interface is independent of the degree of selectivity and has a shallow temperature dependence, as expected for a diffusion-controlled process.(ABSTRACT TRUNCATED AT 250 WORDS)

Spin-label and fluorescence labeling studies of the thioester bonds in human alpha 2-macroglobulin.

Upon cleavage of the reactive thioester bonds (Cys-949-Glx-952) of tetrameric human alpha 2-macroglobulin (alpha 2M) by methylamine, one sulfhydryl group per alpha 2M subunit is exposed. These identical sulfhydryl group sites were labeled with the thiol-specific nitroxide spin-labels (1-oxy-2,2,5,5-tetramethyl-3-pyrrolin-3-yl)methyl methanethiosulfonate and (1-oxy-2,2,6,6-tetramethyl-4-piperidinyl)methyl methanethiosulfonate, a homologous series of maleimide spin-labels, and the thiol-specific fluorescent probe 2-[(4-maleimidophenyl)amino]naphthalene-6-sulfonic acid sodium salt (MANS). The ESR and fluorescence results showed that these sulfhydryl group sites were at the base of a narrow crevice that is greater than or equal to 8 A deep. Although the bound MANS fluorophore was slightly blue shifted with an enhanced quantum yield vs the free label in water, the environment of the sulfhydryl site appeared to be of a polar nature when compared with the emission maxima in several solvents of varying polarity. The Glx residue participating in the thioester linkage in the intact protein was labeled with 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl. The distance between the Glx and Cys moieties was estimated at greater than or equal to 10-25 A from double spin-labeling experiments.

Noncompetitive inhibitors reach their binding site in the acetylcholine receptor by two different paths.

Electron spin resonance was used to contrast the accessibility of tertiary and quaternary local anesthetics to their high affinity binding site in the desensitized acetylcholine receptor (AChR). The time dependence of agonist addition on the association of spin-labeled local anesthetics with the nicotinic AChR-enriched membranes from Torpedo californica was studied. Preincubation of AChR-enriched membranes with agonist for more than a few minutes before the addition of C6SLMel, a quaternary amine local anesthetic, resulted in substantial reduction in the initial association of the label with the receptor. The time-dependent reduction in the initial association of the label with the receptor is modeled by an exponential function having a rate constant of approximately 0.2 min-1. In contrast, agonist preincubation did not produce a comparable decrease in the association of C6SL, a tertiary amine analog, with the AChR. These findings show that whereas the affinity of either anesthetic for the AChR is dependent on the presence of agonist, for C6SLMel the timing of agonist addition is an important factor in determining the rate of anesthetic association with the receptor. Our results are concerned with the desensitized receptor at an early phase, when the average open-channel time limits the anesthetic binding to the receptor. We interpret our results by a model in which the cationic local anesthetic reaches its high affinity binding site in the receptor by an aqueous path that is accessible only when the channel is open. On the other hand, anesthetic in its uncharged form is not restricted only to the aqueous path of access. An additional path, probably through the lipid bilayer, allows uncharged forms of anesthetics to reach the high affinity binding site in the AChR even when the aqueous path is closed. During the "open state" of the receptor both cationic and uncharged anesthetics have access to the high affinity site through the aqueous path. However, after this open state, the channel opens only intermittently. The rapidly decreasing open time results in the time-dependent reduction in the binding of cationic anesthetics. This model is consistent with the open channel hypothesis of anesthetic binding to the AChR immediately after agonist stimulation; however, our model also includes an additional hydrophobic path of access for uncharged and reversibly charged anesthetics.

Interaction of spin-labeled tryptamine with monoamine oxidase: probing the microenvironment of the active site by spin probe-spin label techniques.

The spin-labeled substrate, tryptamine, was used as a structural probe of the active site of bovine liver monoamine oxidase B (amine:oxygen oxidoreductase (deaminating) (flavin-containing), EC 1.4.3.4). When the reaction was monitored by electron spin resonance (ESR), line broadening effects indicative of binding with an apparent relation to substrate specificity of the highly purified enzyme were observed. The spectrum indicated that the bound tryptamine was 'partially immobilized' with a dissociation constant of 39 microM and 2.2 mol bound per enzyme dimer. The correlation time, reflecting the environment of the tryptamine binding site, was determined to be 6.2 ns. The topology of the active site was investigated by using dual spin-label methodology in which the spin-labeled substrate, tryptamine, and the 15N-substituted and deuterated maleimide spin label covalently bound to the essential sulfhydryl groups were used. The ESR spectral data suggested that the essential sulfhydryl groups are at least 14 A away from the tryptamine-binding site. The environment surrounding both spin-labeled substrates, tryptamine and [2H,15N]MSL, and the motional properties of the enzyme are discussed.

Probing of sulfhydryl groups in the adenosine 5'-diphosphate/adenosine 5'-triphosphate carrier by maleimide spin-labels.

Binding of spin-labeled maleimides to the mitochondrial ADP/ATP carrier was investigated both in mitochondria and in the detergent-solubilized carrier protein. In mitochondria, spin-label binding to the carrier was evaluated by preincubation with the inhibitor carboxyatractyloside. The membrane sidedness of SH groups in the carrier molecule was determined by chemical reduction of nitroxides on the cytosolic membrane surface by Fe2+ or by pretreatment of the mitochondria with impermeant SH reagents. These experiments suggest that each subunit of the dimeric carrier incorporates one spin-labeled maleimide. Roughly half of the carrier-bound spin-labels were found on either side of the mitochondrial membrane. The detergent-solubilized carrier protein was labeled with a series of maleimide derivatives containing a spacer of increasing length between the maleimide and nitroxide moieties. A total spin-label binding of 2-3 mol/mol of protein dimer, depending on the spin-label length, was found. The electron spin resonance spectra of the spin-labeled protein invariably showed strongly and weakly immobilized components. Increasing the distance of the nitroxide from the maleimide ring resulted in a strong increase of the contribution of the weakly immobilized component. These observations led to the conclusions that the geometrical constraint of spin-label mobility changes at a distance of about 10 A from the maleimide binding site.

Orientation dependence and motional properties of spin labels in cardiac and skeletal muscle fibres.

Orientation dependence and rotational motion of maleimide spin labels attached to the fast reacting thiol sites of myosin were studied in glycerinated cardiac and skeletal muscle fibres in rigor and in relaxing medium. The probe order in skeletal muscle was shown to be about one order of magnitude higher than that in cardiac muscle. In skeletal muscle in rigor the orientational order is static on the time scale of the saturation transfer electron paramagnetic resonance measurement (ST EPR, rotational correlation time of the label is greater than 1 ms), but in cardiac muscle fibres, a disorder was observed which was at least partly dynamical, the rotational correlation time being about 100 microseconds. In relaxing solution the degree of order of probe molecules in both types of muscle was strongly reduced at and above the resting length. The disorder was at least partly dynamical on the ST EPR time scale, the apparent rotational correlation times being 200 microseconds for skeletal muscle and 60 microseconds for cardiac muscle, respectively. According to the results of ST EPR the rotational behavior of cross-bridges was identical in cardiac and skeletal muscle in relaxing medium.

Oxidant stress in malaria as probed by stable nitroxide radicals in erythrocytes infected with Plasmodium berghei. The effects of primaquine and chloroquine.

Erythrocytes from normal mice and mice infected with the malarial parasite Plasmodium berghei reduce the water-soluble spin probes 2,2,6,6-tetramethylpiperidine-4-hydroxy-N-oxyl (TEMPOL), 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) and 2,2,6,6-tetramethylpiperidine-4-keto-N-oxyl (TEMPONE) at similar rates under both air and N2 atmospheres. The ESR signal of the lipid-soluble spin probe 5-doxyl-stearate is stable on incorporation into erythrocytes from normal mice. In contrast, parasitized red cells reduce this nitroxide probe, at a rate which increases with the level of parasitemia. Inhibitors of electron transport such as KCN and NaN3, increase the rate of reduction. We propose that nitroxide reduction occurs via the electron transport chain in the parasite. The antimalarial drug primaquine causes reduction of both water-soluble and lipid-soluble spin probes. This action of primaquine is independent of its ability to release H2O2 from oxyhemoglobin, and is ascribed to the ability of primaquine to accelerate flux through the hexose monophosphate shunt. The increased production of NADPH results in increased rates of reduction of the nitroxide radicals. Methylene blue, which also increases flux through the shunt, is even more effective than primaquine at reducing the nitroxides. Chloroquine has no such effect. Parasitized mice treated with chloroquine six hours prior to ESR measurements show less nitroxide reducing capacity than do untreated mice. Chloroquine is known to decrease flux through the hexose monophosphate shunt. The metabolic influences of the two antimalarial drugs are, thus, quite different.

Spin label study of the perturbation effect of the local anaesthetics tetracaine and dibucaine on synaptosomes at pharmacological concentrations.

The method of electron spin resonance spectroscopy of spin probes was used to determine the lowest concentrations of the local anaesthetics dibucaine and tetracaine exerting perturbations on synaptosome membranes. The perturbation depends on the temperature and the membrane depth, as well as on the concentration and the structure of the anaesthetics. Using spin labelled stearic acid at the 5th carbon position a negligible effect of the anaesthetics on the order parameter was found in the membrane both at 1 degree and 22 degrees, within the buffer concentration 0.01-10 mmol/l, but at 37 degrees and concentrations higher than 0.1 mmol/l the disordering effect was significant and of comparable efficiency for dibucaine and tetracaine. Employing stearic acid labelled at the 16th carbon position, disordering of the hydrocarbon core of the membrane caused by tetracaine or dibucaine was detected at 1 degree and 22 degrees, as well as at 37 degrees. The disordering effect occurred at buffer concentrations higher than 0.01 mmol/l for dibucaine, and higher than 0.1 mmol/l for tetracaine. At equal anaesthetic membrane concentrations and at the 16th carbon membrane depth, dibucaine was approximately twice as effective as tetracaine in perturbing synaptosomes. Tetracaine induced nonlamellar phases in the rat brain lipid membrane as detected by 31P NMR spectroscopy. The dynamic and structural perturbation effects of the local anaesthetics was found in that concentration range at which the anaesthetics influence various activities of biological membranes.

A spin-label study of membrane proteins and internal microviscosity of erythrocytes in hereditary spherocytosis.

Electron spin resonance (ESR) spectra of erythrocyte membranes of patients with hereditary spherocytosis (HS) and of healthy controls labeled with a maleimide spin label did not differ significantly both before and after prolonged incubation at 37 degrees C. It suggests that the different behavior of spin-labeled HS erythrocyte membranes upon incubation at a higher temperature reported previously is due indeed to structural abnormalities of HS red cell membranes and not to alterations in their proteolytic activity. Measurements of the rotational correlation time of Tempamine spin probe demonstrated a significant elevation of internal microviscosity of erythrocytes in HS, more pronounced in non-splenectomized patients.

Selective outside-inside translocation of aminophospholipids in human platelets.

Spin-labeled analogues of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin were added to human platelet suspensions. Due to the partial water solubility of these spin-labeled lipids which possess a relatively short beta-chain (C5), they incorporate rapidly in membranes. The orientation of the spin-labels within the platelet plasma membrane was assessed by following the spontaneous reduction at 37 and 4 degrees C due to endogenous reducing agents present in the cytosol. The rate of spontaneous reduction showed unambiguously that the labels incorporated initially in the outer leaflet of the plasma membrane and that the rate of outside-inside translocation of the aminophospholipids was faster than that of the choline derivatives. For example, at 37 degrees C, the half-time for the transverse diffusion of a phosphatidylcholine analogue was found to be of the order of 40 min, while it was less than 7 min for the phosphatidylserine analogue. At low temperatures, a fraction of the labels gave rise to a strongly immobilized ESR component. This fraction, which corresponded to 20-30% of the initial spin-label concentration, was found resistant to chemical reduction from the inner side of the membrane and also to externally added reducing agents such as ascorbate. Presumably these immobilized lipids are trapped in a gel phase formed in the outer leaflet at 4 degrees C. Cell aging, which depletes the cells of ATP, resulted in the progressive inhibition of the fast transport of the aminophospholipids from the outer to inner leaflet. Treatment of the cells with iodoacetamide completely blocked the transverse diffusion of the spin-labels.(ABSTRACT TRUNCATED AT 250 WORDS)

Phospholipid outside-inside translocation in lymphocyte plasma membranes is a protein-mediated phenomenon.

We have measured the transbilayer diffusion of spin-labeled analogs of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine in pig lymphocyte plasma membrane. At 4 degrees C and 37 degrees C the aminophospholipids are rapidly transported from the outer to the inner leaflet of the membrane, whereas the choline-containing phospholipids experience a slower diffusion. This selectivity is abolished after cell treatment by SH-group reagents indicating that the aminophospholipid translocation is protein-dependent and must be driven by a system analogous to the one existing in the human red cell membrane. The fact that the selectivity exists at low temperature, that it does not depend on cytoskeleton integrity and that there is a competition between the two aminophospholipids show that this translocation is not purely an endocytic process.

Orientation of spin-labeled nucleotides bound to myosin in glycerinated muscle fibers.

Electron paramagnetic resonance (EPR) spectroscopy of paramagnetic derivatives of ATP has been used to probe the angular distribution of myosin in glycerinated muscle fibers. Three nucleotide spin labels have been prepared with the nitroxide free radical moiety attached, via an ester linkage to either: the 2' or 3' positions of the ribose unit of ATP (SL-ATP), the 2' position of 3' deoxy ATP (2'SL-dATP), or the 3' position of 2' deoxy ATP (3'SL-dATP). In muscle fibers, these nucleotides are quickly hydrolyzed to their diphosphate forms. All three diphosphate analogues bind to the nucleotide site of myosin with similar affinities: rabbit psoas fibers, 7 X 10(3)/M; insect flight muscle, 5 X 10(3)/M; and rabbit soleus muscle, 2 X 10(4)/M. Analysis of the spectra showed that the principal z-axis of the nitroxide attached to bound nucleotides was oriented with respect to the filament axis. The principal axes of 3'SL-dADP and 2'SL-dADP appeared to be preferentially aligned at mean angles of 67 degrees +/- 4 degrees and 55 degrees +/- 5 degrees, respectively. The distribution of probes about these angles can be described by Gaussians with widths of 16 degrees +/- 4 degrees and 13 degrees +/- 5 degrees, respectively. The spectrum of bound SL-ADP was a linear combination of the spectra of the two deoxy analogues. These orientations were the same in the three muscle types examined, indicating a high degree of homology in the nucleotide binding site. Applying static strains as high as 0.2 N/mm2 to muscle fibers caused no change in the orientation of myosin-bound, spin-labeled nucleotides. When muscle fibers were stretched to decrease actin and myosin filament overlap, bound SL-ADP produced EPR spectra indicative of probes with a highly disordered angular distribution. Sodium vanadate and SL-ATP caused fiber stiffness to decrease, and the EPR spectrum of the bound analogue indicated an increase in the fraction of disoriented probes with a concomitant decrease in the fraction of oriented probes. These findings indicate that when myosin is bound to actin its nucleotide site is highly oriented relative to the fiber axis, and when this interaction is removed the orientation of the nucleotide site becomes highly disordered.

Dynamic fluorescence quenching studies on lipid mobilities in phosphatidylcholine-cholesterol membranes.

Bimolecular collision rate of 8-anilinonaphthalene-1-sulfonic acid (ANS) and the nitroxide doxyl group attached to various carbons on stearic acid spin labels (n-SASL) in phosphatidylcholine-cholesterol membranes in the fluid phase was studied by observing dynamic quenching of ANS fluorescence by n-SASL's. The excited-state lifetime of ANS and its reduction by the n-SASL doxyl group were directly measured by the time-correlated single photon counting technique to observe only dynamic quenching separately from static quenching and were analyzed by using Stern-Volmer relations. The collision rate of ANS with the n-SASL doxyl group ranges between 1 X 10(7) and 6 X 10(7), and the extent of dynamic quenching by n-SASL is in the order of 5-much much greater than 6- greater than 7- less than 9- less than 10- less than 12- less than 16-SASL (less than 5-SASL) in dimyristoylphosphatidylcholine (DMPC) membranes. Collision rate of 16-SASL is only 10% less than that of 5-SASL. Since the naphthalene ring of ANS is located in the near-surface region of the membrane, these results indicate that the methyl terminal of SASL appears in the near surface area frequently, probably due to extensive gauche-trans isomerism of the methylene chain. The presence of 30 mol% cholesterol decreases the collision rate of ANS with 12- and 16-SASL doxyl groups but not with the 5-SASL doxyl group in DMPC membranes. On the other hand, in egg-yolk phosphatidylcholine membranes, inclusion of 30 mol% cholesterol does not affect the collision of ANS with either 5-SASL or 16-SASL doxyl groups, in agreement with our previous observation that alkyl chain unsaturation moderates cholesterol effects on lipid motion in the membrane (Kusumi et al., Biochim. Biophys. Acta 854, 307-317). It is suggested that dynamic quenching of ANS fluorescence by lipid-type spin labels is a useful new monitor of membrane fluidity that reports on various lipid mobilities in the membrane; a class of motion can be preferentially observed over others by selecting a proper spin label, i.e., rotational diffusion of lipid about its long axis and translational diffusion by using 5-SASL, wobbling motion of the lipid long axis by using 7-SASL or androstane spin label, and gauche-trans isomerism by using 16-SASL.

Orientation and vertical fluctuations of spin-labeled analogues of cholesterol and androstanol in phospholipid bilayers.

We have used ESR and NMR linewidth broadening by spin-labels to determine the overall orientation of spin-labeled analogues of cholesterol and androstanol in egg lecithin bilayers. While the cholesterol analogues were found to have a single orientation in each monolayer, with the acyl chain pointing towards the center of the bilayer, the androstanol analogue appeared, at least in sonicated vesicles, to experience two opposite orientations in the same monolayer, very likely with a rapid reorientation. The possibility of rapid vertical fluctuations of the sterol molecules within the phospholipid bilayer is also discussed.

Spin trapping of superoxide and hydroxyl radicals with substituted pyrroline 1-oxides.

The synthesis of three nitrones, 5-butyl-5-methyl-1-pyrroline 1-oxide (BMPO), 5,5-dipropyl-1-pyrroline 1-oxide (DPPO), and 2-aza-2-cyclopentenespirocyclopentane 2-oxide (CPPO), was conducted with use of the zinc/ammonium chloride reduction of appropriately substituted gamma-nitrocarbonyl compounds. The lipophilicity of these nitrones was estimated by determining their partition coefficients in a 1-octanol/water system. These nitrones were found to possess more lipophilic character than the most frequently used cyclic nitrone, 5,5-dimethyl-1-pyrroline 1-oxide (DMPO), which exhibits a partition coefficient of only 0.02. Hyperfine coupling constants for the spin trapping of superoxide and hydroxyl radical by the various nitrones were determined. The rate of spin trapping of superoxide with each nitrone was conducted by competitive kinetics with superoxide dismutase (SOD). In addition, the ability of DPPO and BMPO to spin trap free radicals generated during the metabolism of menadione by rat enterocyte cells was investigated. From these studies, DPPO and BMPO appear to be more suitable spin traps than DMPO when one is interested in monitoring free radicals generated intracellularly.

Binding of local anesthetics to reconstituted acetylcholine receptors: effect of protein surface potential.

Nicotinic acetylcholine receptor isolated from Torpedo californica electric organ is reconstituted into lipid bilayers of zwitterionic dioleoylphosphatidylcholine. These membranes are labeled with a spin-labeled quaternary amine local anesthetic (C6SLMeI), which has been shown previously to be a noncompetitive blocker of acetylcholine receptor-ion channel function in the micromolar concentration range. The electron spin resonance spectral component corresponding to protein-immobilized anesthetic spin-label can be resolved from the composite data spectrum by using spectral subtraction of lipid components. This protein-immobilized component is shown to represent C6SLMeI bound to a finite number of sites on the receptor. We demonstrate that C6SLMeI binds to the receptor as a function of the surface potential on the protein and suggest that the acetylcholine receptor reconstituted into zwitterionic phospholipid, which has no surface potential of its own, provides an excellent model system with which to study effects of protein surface charge. We hypothesize that the primary pathway of interaction of C6SLMeI with the acetylcholine receptor is via the aqueous medium.