Free fatty acids and esters can be immobilized by receptor rich membranes from Torpedo marmorata but not phospholipid acyl chains.

A long chain spin labeled fatty acid and the corresponding ester have been introduced into receptor rich membranes from Torpedo Marmorata. Superimposed to a mobile component, typical of the lipid phase, a strongly immobilized component is seen on the ESR spectra, both at low temperature (?4°C) and at room temperature. An estimation of the amount of immobilized signal as a function of the concentration of spin label in the membrane shows that a saturation is reached which corresponds to approximately twice the concentration of receptor protein. In the same membranes, a spin labeled phosphatidylcholine was introduced by the release of the phosphatidylcholine analog from purified phosphatidylcholine exchange protein, preloaded with this spin label. No immobilized component is seen in this latter case even at low temperatures. Therefore the immobilized component seen with the fatty acid cannot be considered as reporting on an immobilized boundary layer of phospholipids surrounding the proteins. We attribute the immobilized signal seen with fatty acids and esters to a particular interaction of amphiphilic molecules with the cholinergic receptor protein. Very likely this effect can be associated with the local anaesthetic effect detected previously with this fatty acid.     

Spin-label studies of the oligomeric structure of band 3 protein in erythrocyte membranes and in reconstituted systems

A spin-labeled fatty acid (16-doxylstearic acid), linked by an ester bond to a maleimide or a nitrene residue, was covalently attached to band 3 of erythrocyte membranes. The electron spin resonance spectrum of the spin-labeled protein was examined at different temperatures in: (a) whole erythrocyte ghosts; (b) ghosts depleted of spectrin and actin; (c) alkaline-treated ghosts; (d) vesicles made with purified band 3 reassociated with dimyristoylphosphatidylcholine. Most spectra are composite with a major component corresponding to a large overall splitting. The determination of the percentage of the immobilized component was carried out by pairwise subtraction. At low temperatures (1–7°C), the highest fraction of immobilized component was found in dimyristoylphosphatidylcholine vesicles (approx. 100%); alkaline-treated membranes had approx. 75% of the immobilized component at the same temperature; whole erythrocyte, spectrin/actin-depleted and spectrin/actin/ankyrin-depleted ghosts gave identical results (approx. 60% of immobilized component). The immobilized fraction decreased in all samples with increasing temperature or addition of a nonsolubilizing concentration of dodecyl octaethylene glycol monoether. In dimyristoylphosphatidylcholine vesicles, however, the modification in the ratio of the two components was obtained only above the lipid transition temperature (23°C). The strong immobilization of the spin-labeled lipid chain at all temperatures suggested trapping of the lipid chain between proteins. At low temperature, in dimyristoylphosphatidylcholine vesicles or in alkaline-treated ghosts, lipid-protein segregation is likely to take place. In whole erythrocyte ghosts, on the other hand, the large contribution of the motionally restricted component at physiological temperature indicates the oligomeric nature of band 3. Partial dissociation of the oligomers occurs as the temperature is increased, but the presence or absence of cytoskeletal proteins has no influence on the state of oligomerization of band 3.     

Distinct states of lipid mobility in bovine rod outer segment membranes Resolution of spin label results

Freely diffusable lipid spin labels in bovine rod outer segment disc membranes display an apparent two-component ESR spectrum. One component is markedly more immobilized than that found in fluid lipid bilayers, and is attributed to lipid interacting directly with rhodopsin. For the 14-doxyl stearic acid spin label this more immobilized component has an outer splitting of 59 G at 0°C, with a considerable temperature dependence, the effective outer splitting decreasing to 54 G at 24°C. Spin label lipid chains covalently attached to rhodopsin can also display a two-component spectrum in rod outer segment membranes. In unbleached, non-delipidated membranes the 16-doxyl stearoyl maleimide label shows an immobilized component which has an outer splitting of 59 G at 0°C and a considerable temperature dependence. This component which is not resolved at high temperatures (24–35°C), is attributed to the lipid chains interacting directly with the monomeric protein, as with the diffusable labels. In contrast, in rod outer segment membranes which have been either delipidated or extensively bleached, a strongly immobilized component is observed with the 16-doxyl maleimide label at all temperatures. This immobilized component has an outer splitting of 62–64 G at 0°C, with very little temperature dependence (61–62 G at 35°C), and is attributed to protein aggregation.     

An imidoester spin probe of membrane protein interactions: application to cytochrome c

The synthesis of an imidoester spin label, whose advantages relative to other spin labels include its water solubility, lysine specificity, and retention of positive charge at the reaction site is described. Cytochrome c is spin labeled and shown to exhibit spectral changes upon interacting with lipid vesicles and lipid-rich cytochrome oxidase preparations. Spin labeled cytochrome c in buffer or in the presence of mitochondria at high ionic strength had a correlation time of τ = 0.91 ± 10^?9 s; at low ionic strength the mitochondrial signal was more immobilized, τ = 2.27 ± 0.13 × 10^?9 s; and further immobilization was observed when cytochrome c was bound to the high-affinity site of purified oxidase containing 37% phospholipid (τ = 2.71 ± 0.22 × 10^?9). Cytochrome c-oxidase electron transfer rates were unaltered by spin labeling. The results suggest that this imidoester spin label will be useful for studies of protein-protein and protein-lipid interactions.     

Spin-label studies of membrane-associated denatured hemoglobin in normal and sickle cells

A maleimide spin label (N-(1-oxyl-2,2,5,5-tetramethylpyrrolidinyl)-maleimide) was reacted with oxyhemoglobin-free cell stromata of normal and sickle cells. The EPR spectrum of spin-labeled red cell membranes showed that the spin labels are attached to at least two different binding sites. There was a major signal, A, which characterized a strongly immobilized environment and a minor signal, B, which characterized a weakly immobilized environment. Quantitative EPR measurements using equal amounts of Hb AA and Hb SS red blood cells demonstrated that Hb SS red cell membranes had an approximately four times higher EPR signal intensity than Hb AA red cell membranes ((7.98 ± 1.14) · 10⁵ and (2.2 ± 1.2) · 10⁵ spin labels/cell, respectively). Moreover, the ratio of signal intensities A and B are different in these cells. Comparative spectrophotometric studies of membrane-associated denatured hemoglobins of Hb AA and Hb SS red cell membranes suggested that the EPR signal A is derived from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membranes. The combination of EPR spectrum of Hb AA membranes pretreated with N-ethyl-maleimide and that of spin-labeled precipitated hemoglobin further strengthened this conclusion.     

Effect of phospholipid substitution on the mobility of spin labels bound to the ATPase of sarcoplasmic reticulum

The mobility of spin labels covalently bound to the Ca2+-transport ATPase (ATP phosphohydrolase [EC 3.y.1.3]) was studied by electron spin-resonance spectroscopy in purified ATPase and reconstituted vesicles. The purified ATPase of sarcoplasmic reticulum of rabbit skeletal muscle was covalently labeled with maleimide spin-labels of different chain length and the phospholipids were exchanged for dipalmitoylphosphatidylcholine. The spectrum of the short-chain maleimide spin-label, bound to purified ATPase indicates reduced mobility after substitution of endogenous phospholipids with dipalmitoylphosphatidylcholine. With the long-chain maleimide derivative no difference was detected in the spectra, measured at 20-35 degrees C temperature before and after substitution with dipalmitoylphosphatidylcholine. Below 10 degrees C temperature the substitution with dipalmitoylphosphatidylcholine decreased the mobility of the prove, indicating that the microviscosity of environment in the vicinity of nitroxide groups was influenced by changes in the fatty acid composition. With both short and long chain spin-labels bound to purified ATPase adn sarcoplasmic reticulum vesicles the amplitude of weakly immobilized component sharply decreased in media containing 20-50% glycerol. Therefore, the mobility of covalently bound nitroxide group in short or long chain maleimide derivatives is also sensitive to the viscosity of the water phase.     

Intra-albumin migration of bound fatty acid probed by spin label ESR

Conventional ESR spectra of 16-doxyl-stearic acid bound to bovine and human serum albumin were recorded at different temperatures in order to investigate the status of spin-labeled fatty acid in the interior of the protein globule. A computer spectrum simulation of measured spectra, performed by non-linear least-squares fits, clearly showed two components corresponding to strongly and weakly immobilized fatty acid molecules. The two-component model was verified on spectra measured at different pH. Thermodynamic parameters of the spin probe exchange between two spin probe states were analyzed. It was concluded that at physiological conditions, fatty acid molecules permanently migrate in the globule interior between the specific binding sites and a space among albumin domains.     

Spin-labeling studies of beef-liver glutamate dehydrogenase.

Bovine liver glutamate dehydrogenase was spin labeled with a nitroxide derivative of parachloromercuribenzoate. The ESR spectrum was of the immobilized type and the labeling yield 0.6 mole of spin label bound per mole of protomer under standard conditions. The specific activity of the labeled enzyme was not modified but the activation by ADP abolished. Inhibition by GTP was not altered but the ESR spectrum showed that the bound spin label was further immobilized in the presence of GTP and NADPH. In the presence of the coenzyme NADPH, the labeling yield decreased to half its initial value. Such a protection effect was observed neither with NADH nor with ADP.     

Phase transitions of phospholipid bilayers from an unsaturated fatty acid auxotroph of Escherichia coli

Total phospholipids were extracted from cells of temperature sensitive unsaturated fatty acid auxotrophs of Escherichia coli (K-12 UFA^ts) grown at 28°C (PL28), and at 42°C in the presence of 2% KCl as an osmotic stabilizer (PL42 (KCl)). From the analysis of fatty acids, it was shown that the content of unsaturated fatty acids of PL42 (KCl) is only 9% of the total fatty acids, while that of PL28 is 54%. The thermal phase transitions of the bilayers prepared from the phospholipid fractions were studied by proton magnetic resonance. The line widths of the methylene signals and the sums of the methylene and methyl signal intensities were plotted against reciprocal values of absolute temperature 1/T or temperature itself. From the plots phase transitions were detected at about 19°C for PL28 and at 43°C for PL42 (KCl). In spite of its complex composition of fatty acids a highly cooperative transition was observed in the case of PL42 (KCl). It was also suggested that the phospholipids bilayers in the biomembranes of this strain at the growth temperature (42°C) are in the state where the gel and liquid crystalline phases coexist.     

Enzymatic and Non-Enzymatic Esterification of long Polyunsaturated Fatty Acids and Lysophosphatidylcholine in Isooctane

Phosphatidylcholine containing large amounts of long polyunsaturated fatty acid, eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6), was synthesized in isooctane. Immobilized phospholipase A₂ was used as a catalyst. A parallel non-enzymatic esterification reaction was investigated in separate experiments.

The concentrations of lyso-phosphatidylcholine, polyunsaturated fatty acids, water and the enzyme were varied over wide ranges as were the temperature and the reaction time. The type of enzyme, carrier and immobilization procedure were held constant.

The yield of phosphatidylcholine was relatively high (about 21%) when the concentration of polyunsaturated fatty acids was high (300 mg/g of reaction mixture) and the water content was low (below 30% of the dry immobilized enzyme). The highest yield of phosphatidylcholine was found at 80 hours and 75°C. However, at this temperature an extensive non-enzymatic reaction between polyunsaturated fatty acids and lyso-phosphatidylcholine occurred. At 80°C the polyunsaturated fatty acids were partly oxidized. Therefore, a temperature of 45°C to 65°C is probably the optimum temperature for the reaction.     

The use of diaminodithiol for labeling small molecules with technetium-99m

To investigate the labeling of small molecules with ^99mTc by the bifunctional chelate approach, we have synthesized both a fatty acid and an estrone derivative containing a chelator of the N₂S₂ type. In the case of the fatty acid, this was a diaminodithiol (DADT) while for the estrone, a diaminodisulfide (DADS) was attached. The estrone derivative (5-(2-methylene estrone 3-methyl ether)-3,3,10,10-tetramethyl-1, 2-dithia-5,8-diazacyclodecane hydrochloride, DADS-E) was prepared by alkylation of DADS while the fatty acid derivative (N-(11-undecanoic acid)-N,N′-bis(2-methyl-2-mercaptopropyl) ethylenediamine hydrochloride, DADT-FA) was synthesized by alkylation of DADS followed by reduction. DADS-E was labeled in ethanol at elevated temperatures while DADT-FA was labeled at room temperature, both by stannous reduction. Paper chromatography showed both to be labeled and reverse-phase HPLC showed multiple peaks for both. Serum stability studies were performed by incubation at 37 °C with aliquots removed at 1 min and 1 day for analysis by size-exclusion HPLC. Initially, little pertechnetate or binding to serum proteins was observed whereas after 1 day the majority of activity in both cases was protein bound with 20 and 38% pertechnetate appearing for DADT-FA and DADS-E respectively. In conclusion, small biologically active molecules may be labeled with ^99mTc through an attached diaminodithiol or diaminodisulfide group.     

Lipid-protein associations in chromatophores from the photosynthetic bacterium Rhodopseudomonas sphaeroides.

Lipid-protein interactions were examined in chromatophores isolated from the photosynthetic bacterium Rhodopseudomonas sphaeroides using lipid spin-labels. The chromatophores contain fluid bilayer and a significant amount of lipid immobilized by membrane proteins. For a typical preparation of cells grown under 600 ft-c illumination, 59% of the spin-labeled fatty acids were bound. Essentially the entire length of the 18-carbon fatty acid chain was immobilized, judging from results obtained with the spin-label at the 7, 12, and 16 positions. The amount immobilized varies directly with the bacteriochlorophyll content of the chromatophore material, suggesting that a significant fraction of the lipid spin-labels is immoblized on the hydrophobic surfaces of the chlorophyll-binding proteins. Changing the lipid spin-label head group from a negatively charged carboxyl group to a positively charged quarternary amine greatly decreased the amount of immobilized lipid. The changes in immobilized lipid with light level and polar head group suggest that the anntenna bacteriochlorophyll-binding proteins preferentially associate with negatively charged lipids.     

An electron spin resonance study of temperature-induced structural changes of the spin-labeled myosin head: an evidence for two conformational states of myosin head

Heavy meromyosin labeled at the SH1 thiol group with an iodoacetamide spin label was studied by electron spin resonance spectroscopy at various temperatures in the presence and absence of nucleotides and PPi. The electron spin resonance spectra of the spin label bound to myosin head showed temperature-dependent changes indicating changes of the structure around the SH1 thiol group of the myosin head. As the temperature was elevated, the bound spin label was more mobilized in all the systems examined. The mobilization of the bound spin label by the elevation of temperature was enhanced in the presence of nucleotides or PPi. The temperature-dependent spectral changes had isosbestic points indicating that the structural changes around the SH1 thiol group took place between two states of the bound spin label, a weakly immobilized and a strongly immobilized state.     

Strong interactions between a spin-labeled cholesterol analog and erythrocyte proteins in the human erythrocyte membrane

We have used a spin label analog of cholesterol bearing a nitroxide on the alkyl chain (26-nor-25-doxylcholestanol) to study cholesterol-protein interactions in the human erythrocyte membrane. As judged from the ESR spectrum, the spin label is readily incorporated into the membrane when added from a concentrated ethanolic solution to a cell or ghost suspension. With intact erythrocytes or white ghosts in isotonic buffer, the ESR spectrum is a superposition of a mobile component and a strongly immobilized component (outer hyperfine splitting 61–63 G). The latter corresponds to approx. 45% of the signal, a percentage which is barely affected by varying the temperature between 5 and 37°C. Removal of the cytoskeletal proteins spectrin and actin by low ionic strength treatment or of all extrinsic proteins by alkali treatment of ghosts reduces the immobilized fraction to approx. 25%. The effect of controlled proteolysis of intrinsic proteins was also tested. Pre-treatment of cells with chymotrypsin or pre-treatment of unsealed ghosts with trypsin has no effect on the ESR spectrum obtained with alkali-treated membranes. On the other hand, after chymotrypsin treatment of unsealed ghost, which reduces the band 3 protein to a 17.5 kDa membrane fragment, the strongly immobilized component is no longer observable. These data show that the cholesterol analog 26-nor-25-doxylcholestanol interacts strongly with one or several proteins of the erythrocyte membrane. That the intrinsic protein band 3 is involved is suggested by the disappearance of the immobilized fraction occurring upon chymotrypsin digestion of this protein. Our results are thus consistent with the proposal of a selective cholesterol-band 3 interaction in the erythrocyte membrane (Schubert, D. and Boss, K. (1982) FEBS Lett. 150, 4–8). Our data also suggest that this interaction is influenced by cytoskeletal proteins, an effect which can be explained considering the known linking of band 3 to the erythrocyte cytoskeleton via ankyrin. Experiments have also been carried out with 3-doxylandrostanol, a more commonly used cholesterol spin-label analog. With this spin label, at all temperatures investigated, we found it impossible to demonstrate unambiguously the existence of two separate spectral components. It is suggested that 26-nor-25-doxylcholestanol is a better reporter of cholesterol behavior in membranes.     

Spin label studies on the interactions of bovine adrenodoxin with NADPH-adrenodoxin reductase and with cytochrome P-450scc.

Adrenodoxin of bovine adrenocortical mitochondria was spin-labeled with two different spin-labeling reagents, N-(2,2,5,5-tetramethyl-3-carbonylpyrroline-1-oxyl)imidazole (I) and N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide (II), without major loss of its activity for electron transport from NADPH to cytochrome c. The EPR spectrum of adrenodoxin spin-labeled with either of the reagents showed a pattern typical of a moderately immobilized spin label. When adrenodoxin was treated with (I), approximately two amino acid residues per molecule were spin-labeled, whereas a single residue was labeled by (II). While assition of NADPH to adrenodoxin spin-labeled with (I) did not diminish the EPR signal intensity, addition of the reductant to the labeled adrenodoxin in the presence of adrenodoxin reductase caused slow reduction of the spin label, the rate of which was dependent on the aerobicity. Addition of adrenodoxin reductase to adrenodoxin spin-labeled with (I) or (II) resulted in the appearance of a more immobilized component in the EPR spectrum. The ratio of the more immobilized component to the less immobilized component was saturated at a molar ratio of one to one. Addition of cytochrome P-450scc to adrenodoxin labeled with (I) had similar effects on the EPR spectrum.     

Fatty acid binding to vesicular stomatitis virus glycoprotein: a new type of post-translational modification of the viral glycoprotein.

The glycoprotein (G) of vesicular stomatitis virus (VSV) binds 1–2 moles of fatty acid per mole of protein. The fatty acids cannot be released by repeated extractions of the protein with organic solvents, nor can they be released by denaturing the protein with ionic or nonionic detergents. Pronase digestion of G yields an organic extractable fragment that contains bound fatty acid. The fatty acid is quantitatively released from this fragment and from intact G by mild alkali treatment in methanol and is identified by gas-liquid and thin-layer chromatography as, predominantly, the methyl ester of palmitic acid. Insignificant amounts of phosphate are found in G, thus ruling out the presence of bound phospholipid. Chicken embryo fibroblast pre-labeled with ³H-palmitate and then infected with VSV for 4 hr show the presence of ³H label in G but not in other viral structural proteins. The ³H label is present only in the fatty acid moiety of the protein. Much smaller amounts of ³H fatty acid are bound to G protein formed by the VSV mutant ts045 grown at the nonpermissive temperature, and no ³H fatty acid is bound to G synthesized at 37°C in cells pretreated with tunicamycin, an inhibitor of glycosylation. However, infection with the VSV-Orsay strain at 30°C in the presence of tunicamycin allows for production of VSV particles with nonglycosylated G (Gibson, Schlesinger and Kornfeld, 1979), and this G has the same proportion of the fatty acid as does the normal glycosylated G. These data indicate that fatty acids become covalently attached to the G polypeptide chain during maturation of the protein—perhaps as the glycoprotein moves to the cell's plasma membrane.     

A newly isolated fungal strain used as whole‐cell biocatalyst for biodiesel production from palm oil

A strain of Aspergillus niger isolated from atmospherically exposed bread and Jatropha curcas seed was utilized as a whole‐cell biocatalyst for palm oil methanolysis to produce fatty acid methyl esters (FAME), or biodiesel. The A. niger strain had a lipase activity of 212.58 mU mL^?1 after 144 h incubation at 25 °C with an initial pH value of 6.5, using 7% polypeptone (w/w on basal medium) as the nitrogen source and 3% olive oil (w/w on basal medium) as a carbon source. The A. niger cells spontaneously immobilized within polyurethane biomass support particles (BSPs) during submerged fermentation. Thereafter, the methanolysis of palm oil was achieved via a three‐step addition of methanol in the presence of BSPs‐immobilized with A. niger cells. The influence of water content, reaction temperature and enzyme concentration on reaction rate was investigated. An 8% water content and a temperature of 40 °C in the presence of 30 immobilized BSPs, resulted in an 87% FAME yield after 72 h.     

A study on the reaction of human erythrocytes with hydrogen peroxide

Membrane fluidity of human erythrocytes treated with H2O2 (1--20 mM) was studied using three kinds of fatty acid spin labels. A strongly immobilized signal appeared on exposure of erythrocytes to H2O2 but was not observed in either H2O2- or Fenton's reagent-treated ghosts or lipid vesicles prepared from H2O2-treated erythrocytes, indicating that the appearance of this signal necessitates the reaction of hemoglobin with H2O2 and is not due to lipid peroxidation. The ESR spectrum of maleimide-prelabeled erythrocytes showed an isotropic signal and the rotational correlation time (tau c) increased as the concentration of H2O2 was increased. Furthermore, maleimide labeling of H2O2-pretreated erythrocytes showed a strongly immobilized component, in addition to a weakly immobilized component. From the relative ratio of the signal intensity of hemoglobin and membrane proteins, it was found that label molecules bound predominantly to hemoglobin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of H2O2-treated erythrocytes demonstrated globin aggregation. Therefore, the changes in the ESR signal observed on H2O2 treatment may be due to some change in hemoglobin, such as globin aggregation or its binding to the membranes. The ESR spectrum of H2O2-treated erythrocytes at -196 degrees C is characterized by signals of nonheme ferric iron type (g equal to 4.3), low spin ferric iron, and free radical type at g equal to 2.00. At higher H2O2 concentrations, the ESR lines due to low spin ferric iron became broad and their peak heights decreased, compared with that at g equal to 2.00 or 4.3. These results indicate that oxidative stress such as decrease of membrane fluidity, lipid peroxidation, and globin aggregation in H2O2-treated erythrocytes is dependent on the reaction of hemoglobin with H2O2.     

Synergistic effect of high-light and low temperature on cell growth of the Δ12 fatty acid desaturase mutant in Synechococcus sp. PCC 7002

The effect of polyunsaturated fatty acids on photosynthesis and the growth of the marine cyanobacterium Synechococcus sp. PCC 7002 was examined using wild-type and Δ12 fatty acid desaturase mutant strains. Under a light intensity of 250 μmol m⁻² s⁻¹, wild-type cells could grow exponentially in a temperature range of 20–38 °C, but growth was non-exponential below 20 °C and ceased at 12 °C. The Δ12 desaturase mutant cells lacking polyunsaturated fatty acids had the same growth rate as wild-type cells in a temperature range of 25–38 °C but grew slowly at 22 °C, and no cell growth took place below 18 °C. Under a very high-light intensity of 2.5 mmol m⁻² s⁻¹, wild-type cells could grow exponentially in a temperature range of 30–38 °C, although the high-light grown cells became chlorotic because of nitrogen limitation. The temperature sensitive phenotype in the Δ12 desaturase mutant was enhanced in cells grown under high-light illumination; the mutant cells could grow at 38 °C, but were killed at 30 °C. The decrease of oxygen evolution and nitrate consumption by whole cells as a function of temperature was similar in both wild type and the Δ12 desaturase mutant. No differences were observed in either light-induced damage of oxygen evolution or recovery from this damage. No inactivation of oxygen evolution took place at 22 °C under the normal light intensity of 250 μmol m⁻² s⁻¹. These results suggest that growth of the Δ12 desaturase mutant at low temperature is not directly limited by the inactivation of photosynthesis, and raise new questions about the functions of polyunsaturated membrane lipids on low temperature acclimation in cyanobacteria. This revised version was published online in June 2006 with corrections to the Cover Date.