A spin label study of the urinary bladder luminal membrane

Spin label experiments have been carried out on the urinary bladder luminal membrane of the bovine transitional epithelium employing the 5-, 7-, 12-, and 16-doxyl substituted stearic acid methyl esters, and compared for reference to similarly labeled bovine erythrocytes. The bladder membranes are significantly different from the bovine red blood cell membranes and show a lower order and polarity near the membrane surface. This fact and the general similarity of results for the bladder and isolated plaque membranes suggests that the highly organized proteins of the bladder membrane may act as a coat on the lipid bilayer and, while intrinsic in nature, do not significantly perturb the hydrophobic core of the lipid bilayer.     

The alteration of membrane proteins in human erythrocyte membranes induced by quinolinic acid,an endogenous neurotoxin Correlation of effect with structure

Quinolinic acid (2,3-pyridinedicarboxylic acid), an endogenous metabolite of l-tryptophan, reportedly via the kynurenine pathway, has been previously shown to possess neurotoxic properties when injected into rat striatum (Schwarcz R., Whetsell, W.O., Jr. and Mangano R.M. (1983) Science 219, 316–318) and to alter the physical state of human erythrocyte membrane proteins, as judged by ESR spectroscopy (Farmer, B.T., II and Butterfield, D.A. (1984) Life Sci. 35, 501–509). Both the morphologic and ESR studies employed nicotinic acid as one comparative control and found that the effect of quinolinic acid is significantly different from that of nicotinic acid. In the present study, we report that the effects of several structural analogues and positional isomers of quinolinic acid on the ESR parameter associated with the physical state of membrane proteins in human erythrocyte membranes suggest the following conclusions concerning the structure-effect relationship of quinolinic acid: The alteration in the conformation of membrane proteins: (1) requires the presence of two carboxylic acid groups; (2) is independent of their relationship to one another on the pyridine ring; (3) is slightly dependent on the presence of the pyridine nitrogen atom but is independent of the positional relationship of the two carboxylic acid moieties to the heteroatom; and (4) seems to depend upon the presence of restricted internal motion derived from the aromaticity in these compounds.     

Interaction of propranolol with model phospholipid membranes Monolayer,spin label and fluorescence spectroscopy studies

The interaction of propranolol with model phospholipid membranes was studied using various experimental techniques. The partition coefficient of propranolol in the negatively charged membranes of vesicles prepared from phosphatidylserine and phosphatidic acid was found to be more than 20-times higher than in neutral phosphatidylcholine membranes. Preferential interaction of propranolol with acidic phospholipid membranes was confirmed using the monolayer compression isotherm technique and the spin-labeling method. Phosphatidylserine monolayers were markedly expanded even at a relatively low drug concentration ($\text{5 · 10}{}^{\mathrm{?6}}$ M). In contrast, the effect of propranolol on phosphatidylcholine monolayers was much smaller, being detectable only at a higher concentration of the drug ($\text{1 · 10}{}^{\mathrm{?4}}$ M). Spin-labeling experiments show that propranolol exerts marked ordering effect on bilayers prepared from acidic phospholipids and does not change the order parameter of phosphatidylcholine membranes. The dependence of the propranolol fluorescence spectrum on the polarity of the solvent allowed us to identify the intercalation region of the drug in the membrane. The fluorophore moiety of propranolol was found to be localized in the lipid polar head groups region of the bilayer. The role of electrostatic and hydrophobic effects in propranolol-membrane interaction is discussed and the effect of propranolol on the ordering of phospholipid bilayers is compared with the effects of other anesthetic-like molecules.     

Use of spin label and the flow-induced ESR spectral difference for studying erythrocyte deformation

ESR spin-labeling method is expanded to measure the macroscopic visco-elastic properties of erythrocytes. A suspension of erythrocytes with an incorporated fatty acid spin label was forced to flow through a flat ESR sample cells, and the ESR spectral change caused by the shear flow was utilized to assess the cell deformability. Chemical cross-linking or heat denaturation of membrane proteins to make the cells less deformable without any morphological change was found to reduce the relative spectral difference (delta h/h). This result indicates that the spectral difference is related to the cell deformation that accompanies the orientation of the cells in the shear flow. In addition, the average decay time (tau av) for the spectral difference observed when the flow was abruptly interrupted became shorter with an increase in the degree of cross-linking or heat-denaturation abruptly interrupted became shorter with an increase in the degree of cross-linking or heat-denaturation at 49 degrees C. Since the observed tau av is much shorter than the expected rotational correlation time for the erythrocyte, the decay is attributed to the deformation recovery process. It is demonstrated that the measurements of both delta h/h and tau av by ESR spectroscopy give qualitative information on the viscosity and the elasticity of the cell membrane system.     

Detergent solubilization of bovine erythrocytes. Comparison between the insoluble material and the intact membrane

Early works have shown that when biomembranes are extracted with the non-ionic detergent Triton X-100 at 4 degrees C, only a subset of the components is solubilized. The aim of this paper was to investigate the solubilization of a cell membrane at different Triton concentrations, and to compare the lipid composition and acyl chain order/mobility of the insoluble material with those of the original membrane. We choose bovine erythrocytes, because they have an uncommon composition, as they have a huge amount of sphingomyelin and phosphatidylcholine is almost absent. We determined the degree of order/mobility of the lipid acyl chains by EPR spectroscopy, using liposoluble spin labels. Incubation of bovine erythrocytes with increasing Triton X-100 concentrations yields decreasing amounts of insoluble material which is enriched in sphingomyelin and depleted in cholesterol. Complete lipid solubilization is achieved at a detergent/lipid ratio of about 60, which is much higher than the values reported for human erythrocytes, but is in line with results obtained in model systems. An insoluble pellet is still obtained at higher Triton concentrations, which seems to consist mainly of protein. A very high correlation is found between lipid chain mobility restrictions and sphingomyelin content in the lipid structures. The human erythrocyte membrane also fits well in this correlation, suggesting a significant role of sphingomyelin in determining acyl chain organization. The analogies and differences between our insoluble material and the detergent-resistant membranes (DRM) are discussed.     

ESR spectral changes induced by chlorpromazine in spin-labeled erythrocyte ghost membranes

chlorpromazine interacted preferentially with membrane proteins rather than membrane lipids in the initial incorporation into human erythrocyte ghosts, as demonstrated by means of the fluorescence quenching and a maleimide spin label. In this state the membrane fluidity increased. At higher concentrations of chlorpromazine, the membrane fluidity decreased and a motionally restricted signal from fatty acid spin labels appeared predominantly. However, no such signal appeared in protein-free vesicles. The temperature and pH dependences of the outer hyperfine splitting of this restricted signal were very similar to those of bovine serum albumin. On the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of chlorpromazine-treated and -untreated ghosts, it was found that there was no significant difference in membrane proteins between both samples except for the changes of a few bands which were not directly concerned with the occurrence of this restricted signal. These results suggest that the fatty acid spin labels bind preferably to membrane proteins as the lipid domain becomes packed with chlorpromazine.     

A spin label study of conformational changes in cytochrome c

Spin-labeled pig heart cytochromes c singly modified at Met-65, Tyr-74 and at one of the lysine residues, Lys-72 or Lys-73, were investigated by the ESR method under conditions of different ligand and redox states of the heme and at various pH values. Replacement of Met-80 by the external ligand, cyanide, was shown to produce a sharp increase in the mobility of all the three bound labels while reduction of the spin-labeled ferricytochromes c did not cause any marked changes in their ESR spectra. In the pH range 6-13, two conformational transitions in ferricytochrome c were observed which preceded its alkaline denaturation: the first with pK 9.3 registered by the spin label at the Met-65 position, and the second with pK 11.1 registered by the labels bound to Tyr-74 and Lys-72(73). The conformational changes in the 'left-hand part' of ferricytochrome c are most probably induced in both cases by the exchange of internal protein ligands at the sixth coordination site of the heme.     

Nitric oxide induced oxidative changes in erythrocyte membrane components

The effects of NO in its environment may vary considerably depending on various factors. This study shows oxidative mechanism of cellular membrane alterations, which is not associated with triggering of ONOOH generation but is induced by pure NO. Our investigation examined the influence of low concentration of NO (0.1; 0.2 mmol/l) on the qualitative changes of structure and dynamics of erythrocyte membrane. NO causes a statistically significant increase in membrane fluidity on different depths of lipid bilayer that is correlated with increase of lipids peroxidation. Statistically significant changes in the conformational state of cytoskeleton proteins were also detected. NO can be considered as a molecule responsible for determining rheological properties of erythrocytes membrane. Therefore, we propose that NO acts as pro-oxidant molecule at concentrations for which membrane appeared to be the first target before it entered the cytosol.     

A comparison of the mobilities and thermal transitions of retrovirus lipid envelopes and host cell plasma membranes by electron spin resonance spectroscopy

The lipid bilayers of seveal type-C retroviruses and selected host cells were spin labeled with 5-doxyl stearic acid, and intact viruses and cells were subjected to electron spin resonance spectroscopy in order to measure lipid mobility. Thermal transition profiles generated for four different retroviruses were dissimilar; differences in the values of the hyperfine splitting constant 2T_∥ and in the positions of thermal break points reflect variations in mobility which can be correlated with the phospholipid/cholesterol molar ratios of the viral envelopes. Moreover, removal of virion surface projections by protease digestion altered the mobility of the envelope and the positions of thermal break points, but the effect observed depended upon the particular retrovirus examined. Studies on retrovirus-infected and uninfected host cells have revealed that persistent virus infection can elicit changes in host plasma membrane mobility and in the positions of thermal break points, the direction and magnitude of which are highly dependent upon the particular retrovirus-host cell system under consideration.     

Occurrence of creatine kinase activity in human erythrocyte membrane

Some evidences for creatine kinase activity in normal human erythrocyte membrane were presented. The creatine kinase was indicated to be a constituent of the integral proteins of erythrocyte membrane or to be tightly bound to the membrane, and was contrasted to the results obtained with adenylate kinase. Isoenzyme distribution of the erythrocyte creatine kinase by electrophoresis was identical to MM-creatine kinase from rabbit muscle.     

Electron spin resonance study of the isolated lipid components from Blastocladiella emersonii zoospores

The physical properties of the plasma membrane of the aquatic phycomycete Blastocladiella emersonii were investigated, in particular the effects of cations on membrane structure. Intact zoospores and lipid extracts were labelled with the spin-labels 5-nitroxystearate (5-NS), 12-nitroxystearate (12-NS), and 2,2,6,6-tetramethylpiperidine-1-oxyl (Tempo). Electron spin resonance spectroscopy indicated a total of three breaks in plots of the hyperfine splitting parameter, 2T_|, order parameter, S, and the partition coefficient, f, vs. temperature. The first and third break points (T_L and T_H) were found to be independent of the external K⁺, Ca²⁺, or Mg²⁺ concentrations. They were similar to the break points found in aqueous dispersions of lipid extracts and correlate well with the temperature limits for zoospore viability. In contrast, the middle break point (T_M) was markedly influenced by the external Ca²⁺ concentration. Ca²⁺ increased T_M from 12°C (no Ca²⁺ added) to 22°C (10 mM Ca²⁺), i.e., growth temperature. K⁺ reversed this Ca²⁺ effect, downshifting T_M from 22°C to 10°C. A comparison of the physico-chemical effects of these ions on the membrane, as revealed by the cation-induced shift in T_M, is closely correlated with the temperature dependence and physiological effects of cations on zoospore differentiation. This suggests that cations may modify the physical state of the plasma membrane and be involved in regulating the initial changes during zoospore encystment.     

Effect of lipid membranes on the apparent pK of the local anesthetic tetracaine spin label and titration studies

Electrometric titrations and spin label data demonstrate changes in the experimentally determined apparent pK of an ionizable drug in the presence of membranes. This effect is attributed to the difference in partition coefficients for the charged and uncharged forms of the drug. Investigation of the binding of a local anesthetic, tetracaine, to egg phosphatidylcholine membranes indicates that the drug apparent pK decreases in the presence of membranes, the decrease being a function of membrane concentration. The agreement between titration and spin label studies is very good and could be simulated by calculating membrane-bound and free populations of charged and uncharged tetracaine from the independently-measured partition coefficients for the two forms.     

Amphiphile dependency of the monomeric and dimeric forms of acetylcholinesterase from human erythrocyte membrane

Human erythrocyte membrane-bound acetylcholinesterase was converted to a monomeric species by treatment of ghosts with 2-mercaptoethanol and iodoacetic acid. After solubilization with Triton X-100, the reduced and alkylated enzyme was partially purified by affinity chromatography and separated from residual dimeric enzyme by sucrose density gradient centrifugation in a zonal rotor. Monomeric and dimeric acetylcholinesterase showed full enzymatic activity in presence of Triton X-100 whereas in the absence of detergent, activity was decreased to approx. 20% and 15%, respectively. Preformed egg phosphatidylcholine vesicles fully sustained activity of the monomeric species whereas the dimer was only 80% active. The results suggest that a dimeric structure is not required for manifestation of amphiphile dependency of membrane-bound acetylcholinesterase from human erythrocytes. Furthermore, monomeric enzyme appears to be more easily inserted into phospholipid bilayers than the dimeric species.     

Permeability of human erythrocyte membrane vesicles to alkali cations

The permeability of inside-out and right-side-out vesicles from erythrocyte membranes to inorganic cations was determined quantitatively. Using ⁸⁶Rb as a K analog, we have measured the rate constant of ⁸⁶Rb efflux from vesicles under equilibrium exchange conditions, using a dialysis procedure. The permeability coefficients of the vesicles to Rb are only about an order of magnitude greater than that of whole erythrocytes. Furthermore, we have measured many of the specialized transport systems known to exist in erythrocytes and have shown that glucose, sulfate, ATP-dependent Ca and ATP-dependent Na transport activities are retained by the vesicle membranes. These results suggest that inside-out and right-side-out vesicles can be used effectively to study transport properties of erythrocyte membranes.     

Detection of cholesterol-rich microdomains in the inner leaflet of the plasma membrane

The C-terminal domain (D4) of perfringolysin O binds selectively to cholesterol in cholesterol-rich microdomains. To address the issue of whether cholesterol-rich microdomains exist in the inner leaflet of the plasma membrane, we expressed D4 as a fusion protein with EGFP in MEF cells. More than half of the EGFP-D4 expressed in stable cell clones was bound to membranes in raft fractions. Depletion of membrane cholesterol with beta-cyclodextrin reduced the amount of EGFP-D4 localized in raft fractions, confirming EGFP-D4 binding to cholesterol-rich microdomains. Subfractionation of the raft fractions showed most of the EGFP-D4 bound to the plasma membrane rather than to intracellular membranes. Taken together, these results strongly suggest the existence of cholesterol-rich microdomains in the inner leaflet of the plasma membrane.     

Determination of the electric potential at the external and internal bilayer-aqueous interfaces of the human erythrocyte membrane using spin probes

Partitioning of oppositely charged amphipathic spin probes indicates that the electric potential at the external bilayer-aqueous interface of the human erythrocyte is insignificant, and that protruding sialic acids do not contribute to this potential. This potential at the surface is distinguished from the electrokinetic potential due to all charged groups within the hydrodynamic surface of shear. By contrast, using inside-out erythrocyte membrane vesicles, a substantial potential is observed at the cytoplasmic membrane surface. This can be attributed to the asymmetric distribution of acidic phospholipids on the two sides of the erythrocyte membrane bilayer.     

Aging of the erythrocyte IV. Spin-label studies of membrane lipids,proteins and permeability

Spin-label studies demonstrated age-related alterations of the erythrocyte membrane concerning both lipid and protein components. Decrease in fluidity of membrane lipids correlated with decreased membrane permeability to a hydrophobic spin label TEMPO, permeability to a more hydrophilic TEMPOL being less affected. The rigidification of membrane lipids was much more pronounced in whole membranes than in liposomes composed of membrane lipids, suggesting changes in lipid-protein interactions as an important factor in the decrease of lipid fluidity in aged red cells. ESR spectra of membrane-bound maleimide spin label evidenced alterations in the state of membrane proteins during cell aging in vivo.     

Curvature-dependent lateral distribution of raft markers in the human erythrocyte membrane

The distribution of raft markers in curved membrane exvaginations and invaginations, induced in human erythrocytes by amphiphile-treatment or increased cytosolic calcium level, was studied by fluorescence microscopy. Cholera toxin subunit B and antibodies were used to detect raft components. Ganglioside GM1 was enriched in membrane exvaginations (spiculae) induced by cytosolic calcium and amphiphiles. Stomatin and the cytosolic proteins synexin and sorcin were enriched in spiculae when induced by cytosolic calcium, but not in spiculae induced by amphiphiles. No enrichment of flotillin-1 was detected in spiculae. Analyses of the relative protein content of released exovesicles were in line with the microscopic observations. In invaginations induced by amphiphiles, the enrichment of ganglioside GM1, but not of the integral membrane proteins flotillin-1 and stomatin, was observed. Based on the experimental results and theoretical considerations we suggest that membrane skeleton-detached, laterally mobile rafts may sort into curved or flat membrane regions dependent on their intrinsic molecular shape and/or direct interactions between the raft elements.     

Effects of plasmenylethanolamine on the dynamic properties of the hydrocarbon region of mixed phosphatidylcholine-phosphatidylethanolamine aqueous dispersions. A spin label study

Spin-labeled aqueous dispersions of total phospholipid extracts from whole brains of hibernating hamsters and rats chronically consuming ethanol were compared with dispersions from control animals. Order parameter values and approximate rotational correlation times for the nitroxide spin labels indicated that ethanol consumption results in an adaptive decrease in bilayer membrane fluidity, while hibernation produces increases in fluidity. Since it has been proposed that changes in plasmenylethanolamine such as those seen with hibernation play a role in the homeoviscous adaptation of brain membranes, electron spin resonance studies using aqueous phospholipid dispersions containing equimolar mixtures of rat brain phosphatidylethanolamine and phosphatidylcholine, or synthetic dioleylphosphatidylcholine and dioleylphosphatidylethanolamine, and brain plasmenylethanolamine were performed. The molar amount of plasmenylethanolamine was varied within the ethanolamineglycerophospholipid fraction of each dispersion. Order parameter values of spin labels in liposomes containing brain phosphatidylcholine and phosphatidylethanolamine increased in parallel with increases in plasmenylethanolamine concentrations, indicating that fluidity was decreasing. Liposomes composed of synthetic dioleyl phospholipids exhibited biphasic changes in order parameter (S) values as plasmenylethanolamine replaced the diacyl form. Below 30% (mol%) plasmenylethanolamine, S values decreased, while above 30%, S values were seen to increase; indicating an initial fluidization, followed by a decrease in fluidity.     

Effect of factors of favism on the protein and lipid components of rat erythrocyte membrane

Erythrocytes prepared from riboflavin- and tocopherol-deficient (RT^?) and from control rats were used to investigate the mechanism of oxidative hemolysis by the factors of favism. RT^? erythrocytes have a defense system against the oxidative stress which is blocked either where regeneration of GSH occurs or the scavenging of the radicals from the membrane is prevented. The oxidative factors used were isouramil, divicine and diamide. When RT^? erythrocytes were treated with isouramil, GSH decreased to undetectable levels and was not regenerated. Complete hemolysis occurred, but no oxidation of SH groups of membrane proteins or formation of spectrin polymers was detected. A similar effect was observed with diamide. However, SH groups of membrane proteins were completely oxidized and spectrin polymers were formed. Extensive lipid peroxidation was also detected together with a 30% fall in the arachidonic acid level. Control erythrocytes treated with either isouramil or diamide were not hemolyzed. When treated with isouramil, after a fall in the first few minutes, the GSH level was completely regenerated after 20 min. Incubation with diamide caused extensive oxidation of SH groups of membrane proteins and formation of spectrin polymers. No lipid peroxidation was detected after treatment with isouramil, but the same decrease of arachidonic acid occurred as in RT^? erythrocytes. These results support the hypothesis that oxidative hemolysis by the factors of favism is caused by uncontrolled peroxidation of membrane lipids.