5-nitroxide Stearate Research Articles

Vitamin D-mediated intestinal calcium transport: Effects of essential fatty acid deficiency and spin label studies of enterocyte membrane lipid fluidity

Vitamin D-3 and its metabolites regulate the transport of calcium across the intestinal epithelial cell via a mechanism which is as yet unknown. The purpose of this study was to evaluate the effect of an essential fatty acid deficiency on vitamin D-stimulated intestinal calcium transport as measured by both in vivo and in vitro techniques. We also describe in this report a procedure for the isolation of chick intestinal epithelial cell brush border and basal lateral membranes and an assessment of the effect of dietary vitamin D on the lipid fluidity of these membranes. An essential fatty acid deficiency in both vitamin D-replete and deficient chicks resulted in a decrease in intestinal mucosal levels of linoleic acid, with a compensatory increase in the levels of the short chain fatty acid, myristic acid, and the unsaturated fatty acids, palmitoleic and eicosatrienoic acids. An essential fatty acid deficiency did not affect the ability of vitamin D-deficient chicks to respond to vitamin D with a 2-fold increase in serum calcium and a 4–5-fold increase in intestinal calcium transport, measured in vivo. However, an essential fatty acid deficiency resulted in an inability of vitamin D to increase calcium flux in vitamin D-deficient chick ileum as measured under in vitro conditions. Dietary vitamin D resulted in no detectable change in the protein composition in either the brush border or basal lateral membranes as evidenced by SDS-polyacrylamide electrophoresis. In addition, vitamin D did not alter the levels of brush border membrane cholesterol or lipid phosphorus (0.27 ± 0.03 and 0.19 ± 0.01 μmol/mg protein, respectively). Brush border and basal lateral membranes were labeled with the 5-nitroxide stearate spin probe I(12,3). The polarity of the environment of the probe in the brush border membranes is much greater than that of the basal lateral membranes. In addition, the lipid environment of the brush border membrane is much less fluid ( S = 0.650) than that of the basal lateral membrane ( S = 0.583). The data concerning membrane lipid fluidity is qualitatively similar to fluorescence polarization studies of rat intestinal epithelial cell membranes and confirms the concept that a given cell may contain plasma membrane regions having discrete lipid structures/fluidities. Dietary vitamin D had no detectable effect on the lipid fluidity or polarity in either the brush border or basal lateral membranes. The results do not support a role for an alteration in essential fatty acid composition or gross changes in the lipid fluidity of the brush border or basal lateral membranes as mechanisms by which vitamin D regulates intestinal calcium transport.

Effects of calcium, lanthanum, and temperature on the fluidity of spin-labeled human platelets.

Previous platelet studies have shown that calcium plays important roles in stimulus-secretion coupling, aggregation, and other membrane-associated functions. In addition, lanthanum induces platelet aggregation and the platelet release reaction and also influences platelet responsiveness to various stimuli. The spin-label results presented here suggest that one mechanism through which calcium and lanthanum mediate their effects on platelet functions may be by decreasing the lipid fluidity of the surface membrane. The structure of platelet membrane lipids was examined with the spin-label method. Washed human platelets were labeled with the 5-, 12- and 16-nitroxide stearic acid spin probes. Order parameters which measure the fluidity of the lipid environment of the incorporated probe may be calculated from the electron spin resonance (ESR) spectra of 5-nitroxide stearate [I(12,3)]-labeled cells. Evidence is presented which indicates that these spectra principally reflect properties of the platelet surface membrane lipids. The membrane fluidity increased with temperature for the range 17 to 37 degrees C. Either calcium or lanthanum additions to intact cells increased the rigidity of the platelet membranes at 37 degrees C, although the La3+ effect was larger and occurred at lower concentrations than that of Ca2+. For example, addition of 1 mM La3+ or 4 mM Ca2+ increased the order parameter of I(12,3)-labeled platelets by 4.3 +/- 1.7% or 2.1 +/- 0.5%. Preliminary studies conducted on purified platelet plasma membranes labeled with I(12,3) indicated that 1 mM LaCl3 or 4 mM CaCl2 additions similarly decreased the lipid fluidity at 37 degrees C. The above cation-induced effects on the fluidity of whole platelets were reversed by the use of the divalent cation-chelating agent ethylene glycol-bis-(beta-aminoethyl ether)-N,N'-tetra-acetic acid (EGTA). Lastly, lanthanum (0.2-1 mM) caused rapid aggregation of platelets which were suspended in a 50-mM Tris buffer pH 7.4 that did not contain adenosine.

A spin label study of the erythrocyte membranes in duchenne muscular dystrophy

Red blood cells and freshly prepared erythrocyte membranes of 15 patients with Duchenne muscular dystrophy (DMD) as well as age-matched controls were studied by the spin label method. No significant modifications appeared for spin-labelled proteins of ghost membranes. With the two fatty acid spin labels, 5-nitroxide stearate and 16-nitroxide stearate, we have confirmed previous results of Sato et al. concerning the thermal behaviour of the erythrocyte membranes, i.e. no change near the polar part probed by 5-nitroxide stearate and a linearization of the fluidity versus temperature variation around 12°C, as explored by 16-nitroxide stearate. Furthermore we studied in the whole erythrocyte the amplitude of the 5-nitroxide stearate electron spin resonance signal as a function of the microwave power. This saturation effect was observed in 12 out of 15 controls and only in 1 out of 13 DMD cases studied. In erythrocyte membranes labelled with 16-nitroxide stearate the penetration of the label inside membranes was statistically different between DMD and controls. These new findings furnish further arguments in favour of a structural alteration of the phospholipid organization of erythrocyte membranes in DMD. Associated together, these different sets of tests obtained by spin labelling permit good statistical discrimination between DMD and normal subjects.

Differences in membrane unsaturated fatty acids and electron spin resonance in different types of myeloid leukemia cells

The fatty acid composition and some physical properties of intact cells and isolated plasma membranes of two types of mouse myeloid leukemia cell clone grown in culture have been examined. One clone type, MGI +D +, can be induced by the macrophage and granulocyte-inducing protein (MGI) to differentiate into mature macrophages and granulocytes. The other clone type, MGI +D −, could not be induced to differentiate into mature cells. A two-fold increase in the ratio of saturated fatty acid to unsaturated fatty acid was found in the MGI +D − compared to the MGI +D + clones. The MGI +D − clones produced an unusual polyunsaturated C 20:5 fatty acid at 28°C, whereas the MGI +D + clones did not grow at this temperature. The cells and their isolated plasma membranes were studied by electron spin resonance. The motion of the 5-nitroxide stearate spin label was found to be higher in the intact cells and in the membranes of MGI +D − clones than of the MGI +D + clones. The cells of MGI +D + clones showed a similar freedom of motion to normal myeloblasts from the bone marrow. The results indicate that myeloid leukemia cells which differ in their competence to be induced to differentiate into mature cells have different physical properties of their plasma membranes and that this is correlated with their fatty acid acyl chain composition.

Binding of nitroxide stearate spin labels to bovine serum albumin

1. 1. 12-Nitroxide stearate binds to bovine serum albumin at about four independent and equivalent binding sites with an association constant of about 10 6 M −1. The binding at these high-affinity binding sites is significantly reduced by addition of unlabeled stearate. These data suggest that nitroxide stearates probe the high affinity binding sites for long-chain fatty acids. 2. 2. Qualitative analyses of the ESR spectra of 5-, 12- and 16-nitroxide stearate bound to bovine serum albumin and measurements of the interaction of these compounds so bound with ferricyanide ion provide a rough description of the binding site as follows: the polar headgroup of the spin-labeled fatty acid is rigidly fixed, but fairly accessible to paramagnetic ions. The middle part of the hydrocarbon chain of bound stearate spin label also is rigidly fixed but differs in being shielded from the solvent, presumably by a hydrophobic cleft. The methyl terminus shows greater motion, appearing to move within a narrow cone, and also appears to be somewhat accessible to paramagnetic ions.

Variations of lipid-protein interactions in erythrocyte ghosts as a function of temperature and pH in physiological and non-physiological ranges: A study using paramagnetic quenching of protein fluorescence by nitroxide lipid analogues

1. 1.|Incorporation of stearic acid nitroxides into erythrocyte ghosts markedly depresses the fluorescence of membrane protein tryptophan residues. 5-Nitroxide stearate quenches fluorescence more efficiently than 16-nitroxide stearate. Both compounds exhibit dynamic (diffusion-limited) quenching above 0.28 μmol/mg protein and static quenching at lower nitroxide protein ratios. Static quenching can be attributed to high affinity binding of nitroxide stearates by membrane protein. The dynamic phase represents distribution of the stearate analogues into a fluid lipid system. 2. 2.|Protein fluorophores accessible to quenching by a cholesterol analogue, androstane nitroxide, are saturated at low nitroxide/protein ratios ( < 0.14 μ mol/mg protein), without resolution of a static quenching phase. This suggests that sterols are segregated away from protein, probably in “clusters”. 3. 3.|Paramagnetic quenching by stearate nitroxides increases abruptly between 35 and 50 °C. This discontinuous enhancement of quenching by temperature is reversible up to 41 °C but irreversible at higher temperatures. The discontinuity is also diminished by lowering pH from 7.3 through 6.5 to 6.0. Quenching by androstane nitroxide increases linearly with temperature up to approx. 41 °C and then rises exponentially. We attribute the reversible quenching thermotropism detected by stearate derivatives to reversible, thermotropic unfolding and/or depolymerisation of membrane proteins. The irreversible phase, detected also by the sterol derivative can be attributed to non-reversible protein denaturation. 4. 4.|Paramagnetic quenching of membrane tryptophan fluorescence by stearate derivatives is minimal at approx. pH 7.1 (35 °C) and increases sharply at lower and higher pH values, suggesting that two categories of protein residues, titrating between pH 6 and 8, profoundly influence the association of fatty acyl chains and penetrating protein segments. Quenching by androstane nitroxide exhibits no significant variation between pH 6 and 8, consistent with other data indicating that erythrocyte membrane sterols are segregated from membrane proteins, probably in clusters. 5. 5.|Our new approach confirms previous suggestions of a boundary layer of lipid in close association with some proteins in erythrocyte membranes, as well as experiments indicating that the lipid status in this boundary layer depends on that state of membrane proteins. However, sterols appear to be largely excluded from this boundary domain. Our data further show that lipid-protein interactions in erythrocyte membranes can vary significantly with fluctuations of temperature and pH in the physiological range.

Fluorescence quenching in lecithin and lecithin/cholesterol liposomes by paramagnetic lipid analogues. Introduction of a new probe approach

1. 1. Perylene, whether incorporated into lecithin or lecithin/cholesterol (1:1) liposomes, exhibits identical fluorescence spectra, but fluorescence in the presence of cholesterol is enhanced by 30–50 %. 2. 2. The fluorescence of perylene in pure dipalmitoyllecithin vesicles increases sharply at the transition temperature ( T t = 41 °C ). No such fluorescence jump is observed in lecithin/cholesterol (1:1) micelles. 3. 3. In lecithin liposomes maximal quenching of perylene fluorescence at 25 °C is effected by cholestane spin label (80 %) followed by androstane spin label (70 %), 5-nitroxide stearate (60 %)and 16-nitroxide stearate (50 %). 4. 4. In liposomes containing 50 mol % cholesterol these differences are reduced; however, the sequence of quenching efficiencies is the same except for the nitroxide stearates, which interchange their positions. 5. 5. Paramagnetic quenching of perylene fluorescence is stable below 35 °C and above 45 °C, but decreases sharply about the phase-transition temperature of dipalmitoyllecithin. 6. 6. In lecithin/cholesterol (1:1, molar ratio) liposomes fluorescence quenching diminishes linearly, but only slightly, with increasing temperature. 7. 7. Cholestane spin label and androstane spin label at concentrations of 20 mol % themselves suppress the quenching discontinuity at T t , indicating a cholesterol-like structural effect. 8. 8. The quenching phenomena observed are attributed to a non-random accommodation of fluorophore and quencher molecules (co-clustering) below the phase transition and a statistical distribution of both impurities above T t . 9. 9. In the presence of cholesterol the clustering tendencies are reduced or even eliminated; this is compatible with the concept that cholesterol fluidizes the phosphatide acyl chains below the transition temperature.

Evidence for constrained lipid mobility in the erythrocyte ghost. A spin label study

Our data show that the ESR spectrum of 5-nitroxide stearate bound to erythrocyte membranes varies with the amount of label bound and suggest that, at high binding, a significant proportion of label molecules lie within ≈ 15 · 10 −8 cm ; this gives rise to spin-exchange (ambient temperature) and dipole-dipole interactions. We find that these spectral manifestations due to label clustering can be abolished by reduction of pH and the conjoint action of lysolecithin and trypsin, although both perturbations increase 5-nitroxide stearate binding. Both perturbations are known to mobilize intramembranous particles by modifying or extracting some membrane proteins. We accordingly suggest that the lipids and proteins of erythrocyte membranes exist in a relatively mosaic, and that the mobility of both components is restricted by some membrane-associated protein framework.

Studies on Tetrahymena membranes: : Temperature-induced alterations in fatty acid composition of various membrane fractions in Tetrahymena pyriformis and its effect on membrane fluidity as inferred by spin-label study

The fatty acid distribution pattern of lipids extracted from different subcellular components of Tetrahymena pyriformis was found to be significantly different from one type of membrane to another. The growth-temperature shift caused alterations in fatty acid composition. The ratio of palmitoleic to palmitic acid, especially, showed a sharp linear decline with increase of temperature in all of the membrane fractions. The spin labels were rapidly incorporated into Tetrahymena membranes. The order parameter of 5-nitroxide stearate spin label incorporated into various membrane fractions was found to be different for the different membrane fractions, suggesting the following order of the fluidity; microsomes > pellicles > cilia. The fluidity of the surface membranes, cilia and pellicles isolated from Tetrahymena cells grown at 15°C was noticeably higher than that of the membranes from cells grown at 34°C but was not so different with microsomal fractions. The motion of the spin label in the pellicular membrane was more restricted than in its extracted lipids, thus indicating the assumption that in Tetrahymena membranes the proteins influence the fluidity. It was also suggested that a sterol-like triterpenoid compound, tetrahymanol, which is principally localized in the surface membranes, would be involved in the membrane fluidity.

Hydrophobic binding sites in bovine serum albumin and erythrocyte ghost proteins. Study by spin-labelling, paramagnetic fluorescence quenching and chemical modification

1. 1. The binding of 5- and 16-nitroxide stearates to bovine serum albumin and erythrocyte ghosts has been measured by paramagnetic quenching of protein fluorescence. 2. 2. Bovine serum albumin exhibits saturable binding only, while erythrocyte ghosts show both saturable and unsaturable components. 3. 3. When bovine serum albumin saturated with 5- or 16-nitroxide stearate is reacted with o- phthaldehyde , a lipophilic dialdehyde, all the bound spin-label becomes displaced into aqueous solution. 4. 4. o- Phthaldehyde produces a similar effect with erythrocyte ghosts equilibrated at similar level of spin label. However, about 5% of the spin label remains membrane bound. 5. 5. Sulphydryl blockers, such as p- chloromercuribenzoate , reduce the action of o- phthaldehyde on ghosts by about 70%, but have no effect in the case of serum albumin. 6. 6. The reaction of o- phthaldehyde with the phosphatidylethanolamine in erythrocyte membrane lipid does not produce displacement of nitroxide stearates. 7. 7. We suggest that certain erythrocyte membrane proteins are surrounded by a shell of closely associated lipid, which is perturbed with certain protein modifications. We further point out analogies between lipid protein interactions in erythrocyte membranes and serum albumin.