The rate of lateral diffusion of phospholipids in erythrocyte microvesicles

31P-NMR spectra of phospholipids in membranes of erythrocyte microvesicles isolated from outdated blood units were recorded in the temperature range 5 to 55 degrees C. Within that range the lineshape is strongly influenced by an increasing rate of lateral diffusion of phospholipids. At 36 degrees C a diffusion constant, D, of (2 +/- 1) X 10(-12) m2/s was obtained. The diffusion rate is by a factor of 3 to 10 greater than in erythrocyte membranes measured by the photobleaching technique and is comparable with values obtained for several lipid model membranes. The differences in lateral diffusion rates are probably connected with the depletion of microvesicle membranes in membrane proteins.     

The influence of fatty acid unsaturation and physical properties of microsomal membrane phospholipids on UDP-glucuronyltransferase activity.

The relationship between lipid composition, the physical properties of microsomal phospholipids and the kinetics of liver UDP-glucuronyltransferase was studied in microsomes from guinea pigs supplied with a normal or a fat-free diet for 28 days. Fatty acid deficiency did not modify either the cholesterol/phospholipid molar ratio or the polar head group composition, but exclusively redistributed the unsaturated fatty acid pattern, by partially exchanging oleic for linoleic acid. This phenomenon accounts for the decrease of both rotational and translational mobilities of the fluorescent probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and pyrene respectively. When the thermotropic behaviour of the different systems was assessed, no transition temperature (gel-liquid-crystalline) between 10 and 40 degrees C was seen as a consequence of the lower degree of unsaturation, either in the microsomal membranes or in the total lipid or total phospholipid extracts from the treated animals. In spite of this, the polarization ratio of trans-parinaric acid and the fluorescence intensity of merocyanine 540 revealed that a significant lateral phase separation occurred at 20-22 degrees C in the extracted phospholipids, which was smoother in the total lipid fractions and in the native microsomal membranes. Fatty acid deficiency caused an upward shift of the midpoint temperature of the lateral phase separation. Furthermore, the phosphatidylcholine extracted from the 'normal' microsomes showed a lateral phase separation centred at a lower temperature than that extracted from 'fat-deficient' microsomes. In contrast, the Arrhenius plot of UDP-glucuronyltransferase from 'normal' microsomes exhibited a change in slope at a higher temperature than that from treated microsomes. These results would suggest that fatty acid deficiency in guinea-pig liver microsomes, while rigidizing the bulk lipids, would segregate the most unsaturated phosphatidylcholine molecules towards the UDP-glucuronyltransferase microenvironment, in accordance with our previous results with cholesterol incorporation [Castuma & Brenner (1986) Biochemistry 25, 4733-4738].     

Structure and motion of phospholipids in human plasma lipoproteins. A 31P NMR study

The structure and motion of phospholipids in human plasma lipoproteins have been studied by using 31P NMR. Lateral diffusion coefficients, DT, obtained from the viscosity dependence of the 31P NMR line widths, were obtained for very low density lipoprotein (VLDL), low-density lipoprotein (LDL), high-density lipoproteins (HDL2, HDL3), and egg PC/TO microemulsions at 25 degrees C, for VLDL at 40 degrees C, and for LDL at 45 degrees C. At 25 degrees C, the rate of lateral diffusion in LDL (DT = 1.4 x 10(-9) cm2/s) is an order of magnitude slower than in the HDLs (DT = 2 x 10(-8) cm2/s). At 45 degrees C, DT for LDL increases to 1.1 x 10(-8) cm2/s. In contrast, DT for VLDL increases only slightly going from 25 to 40 degrees C. The large increase in diffusion rate observed in LDL occurs over the same temperature range as the smectic to disordered phase transition of the core cholesteryl esters, and provides evidence for direct interactions between the monolayer and core. In order to prove the orientation and/or order of the phospholipid head-group, estimates of the residual chemical shift anistropy, delta sigma, have been obtained for all the lipoproteins and the microemulsions from the viscosity and field dependence of the 31P NMR line widths. For VLDL and LDL, the anisotropy is 47-50 ppm at 25 degrees C, in agreement with data from phospholipid bilayers. For the HDLs, however, significantly larger values of 69-75 ppm (HDL2) and greater than 120 ppm (HDL3) were obtained.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mobility of ribosomes bound to microsomal membranes. A freeze-etch and thin-section electron microscope study of the structure and fluidity of the rough endoplasmic reticulum

The lateral mobility of ribosomes bound to rough endoplasmic reticulum (RER) membranes was demonstrated under experimental conditions. High- salt-washed rough microsomes were treated with pancreatic ribonuclease (RNase) to cleave the mRNA of bound polyribosomes and allow the movement of individual bound ribosomesmfreeze-etch and thin-section electron microscopy demonstrated that, when rough microsomes were treated with RNase at 4 degrees C and then maintained at this temperature until fixation, the bound ribosomes retained their homogeneous distribution on the microsomal surface. However, when RNase- treated rough microsomes were brought to 24 degrees C, a temperature above the thermotropic phase transition of the microsomal phospholipids, bound ribosomes were no longer distributed homogeneously but, instead, formed large, tightly packed aggregates on the microsomal surface. Bound polyribosomes could also be aggregated by treating rough microsomes with antibodies raised against large ribosomal subunit proteins. In these experiments, extensive cross-linking of ribosomes from adjacent microsomes also occurred, and large ribosome-free membrane areas were produced. Sedimentation analysis in sucrose density gradients demonstrated that the RNase treatment did not release bound ribosomes from the membranes; however, the aggregated ribosomes remain capable of peptide bond synthesis and were released by puromycin. It is proposed that the formation of ribosomal aggregates on the microsomal surface results from the lateral displacement of ribosomes along with their attached binding sites, nascent polypeptide chains, and other associated membrane proteins; The inhibition of ribosome mobility after maintaining rough microsomes at 4 degrees C after RNase, or antibody, treatment suggests that the ribosome binding sites are integral membrane proteins and that their mobility is controlled by the fluidity of the RER membrane. Examination of the hydrophobic interior of microsomal membranes by the freeze-fracture technique revealed the presence of homogeneously distributed 105-A intramembrane particles in control rough microsomes. However, aggregation of ribosomes by RNase, or their removal by treatment with puromycin, led to a redistribution of the particles into large aggregates on the cytoplasmic fracture face, leaving large particle-free regions.     

Insoluble complex formation between low density lipoprotein and heparin. A 31P-NMR study

31P-NMR has been used to probe the motions of the phosphate moiety of phospholipid head-groups in samples of human low density lipoprotein (LDL) in which particle tumbling has been greatly reduced by increasing the viscosity of the medium, by forming an LDL gel by ultracentrifugation, or by precipitation with heparin. The 31P-NMR spectra of LDL gel give broad "powder-like" lineshapes, with the sign and magnitude of the anisotropy characteristic of the bilayer mesophase, which narrow as the temperature is raised from 5 to 45 degrees C. This narrowing occurs over the same temperature range as the core cholesteryl ester liquid-crystalline to liquid phase transition, suggesting interactions between the surface and core. The 31P lineshapes of LDL-heparin insoluble complexes are also "powder-like", but are broader than native LDL at all temperatures studied. The spectra were simulated assuming an axially-symmetric shielding tensor motionally narrowed by Brownian isotropic diffusion [Burnell et al. (1980) Biochim. Biophys. Acta 603, 63-69], allowing determination of the lateral diffusion coefficients, DT, and the chemical shift anisotropy, delta sigma, of the monolayer phospholipids. Relative to LDL gel, the temperature-dependence of DT was reduced in the LDL-heparin insoluble complexes, and delta sigma was increased from 50 to 60 ppm. The results suggest that insoluble complex formation slows phospholipid lateral diffusion in the LDL monolayer and alters the orientation and/or order of the head-group.     

Regiospecific hydroxylation of lauric acid at the (omega-1) position by hepatic and kidney microsomal cytochromes P-450 from rainbow trout

Microsomes from liver or kidney of untreated rainbow trout hydroxylated lauric acid specifically at the (omega-1) position. Turnover numbers for liver (2.72 min-1) and kidney (14.1 min-1) were decreased seven- and twofold, respectively, following treatment with beta-naphthoflavone. Laurate hydroxylation activity from untreated trout hepatic microsomes was sensitive to inhibition by SKF-525A, but was not sensitive to metyrapone and only partially inhibited by alpha-naphthoflavone. The temperature optimum of laurate (omega-1) hydroxylation in trout liver microsomes was 25-30 degrees C. The Km and Vmax for (omega-1)- hydroxylaurate formation was 50 microM and 1.63 nmol min-1 mg-1, respectively, in liver and 20 microM and 3.95 nmol min-1 mg-1, respectively, in kidney from untreated trout microsomes. (omega-1) Hydroxylation of laurate, in both liver and kidney microsomes, was sensitive to an antibody raised against a previously purified cytochrome P-450 isozyme (LM2) of trout liver microsomes, which has been shown to be active towards aflatoxin B1. Antibody to the major isozyme of cytochrome P-450 ( LM4b , active towards benzo(a)pyrene) induced by beta-naphthoflavone did not inhibit (omega-1) hydroxylation of laurate in microsomes from untreated or beta-naphthoflavone-treated trout.     

Binding of two spin-labelled derivatives of chlorpromazine to human erythrocytes.

The binding to human intact erythrocytes of two different spin-labelled derivatives of chlorpromazine has been studied. The influence of the positively charged side chain of the drug has been the focus of our attention. The positively charged amphiphilic compound (spin derivative I) is water-soluble up to 80 microM at pH values below 5.9. The apolar analogue (spin derivative II) aggregates in aqueous buffer from the lowest concentration tested. Both spin derivatives undergo a slow reduction inside the erythrocyte. The reduced nitroxides are readily reoxidized by adding a low, non-quenching, concentration of potassium ferricyanide to the intact erythrocytes. The fractions of spin label I and II bound to the erythrocyte membrane or to the erythrocyte-extracted lipids remain constant as a function of the temperature (3-42 degrees C) and as a function of the concentration of the spin label up to 150 microM. E.s.r. spectra of both spin labels show a two-component lineshape when they are bound to intact erythrocytes. Below 35 degrees C for the positively charged spin probe, and below 32 degrees C for the apolar spin probe, the simulation of the lineshape shows that more than 50% of the spectrum originates from a slow-motion component. This slow-motion component is also found in erythrocyte-extracted lipids probed by the positively charged spin label below 25 degrees C. In contrast, no slow-motion component is detected in the range 4-40 degrees C for the apolar spin label in erythrocyte-extracted lipids. In this environment the apolar probe experiences a single fast anisotropic motion with an exponential dependence on 1/temperature. Detailed lineshape simulations take into account the exchange frequency between binding sites where the probe experiences a fast motion and binding sites where it experiences a slow motion. The exchange frequency is strongly temperature-dependent. Characterization of the different motions experienced inside the different locations has been achieved and compared for whole erythrocytes and for the extracted lipids. The biochemical nature of the binding sites (membrane protein/acidic phospholipid) giving rise to the slow-motion component is discussed as a function of the polarity of the spin-labelled drug and as a function of the temperature controlling the fluidity of the lipid bulk and influencing the distribution of the drug inside the membrane.     

Protein and lipid lateral diffusion in normal and Rous sarcoma virus transformed chick embryo fibroblasts.

We measured the lateral diffusion of the fluorescent lipid analogue dioctadecylindocarbocyanine iodide (DiI) and of membrane glycoproteins labeled with tetramethylrhodamine (TRITC) succinyl concanavalin A (SConA) via fluorescence photobleaching recovery (FPR) at selected times during a temperature downshift experiment on transformation-defective temperature-sensitive (td-ts) Rous sarcoma virus (RSV) NY68-transformed chicken embryo fibroblasts (CEF) and on identically treated CEF and RSV-transformed CEF. There were no significant differences in the lateral diffusion in DiI at any of the times measured. The lateral diffusion of TRITC-SConA on the RSV-transformed CEF, (1.32 +/- 0.12).10(-10) cm2 s-1, was approximately two times faster than that observed in normal CEF, (0.61 +/- 0.06).10(-10) cm2 s-1. In the cells undergoing RSV NY68-mediated transformation, TRITC-SConA diffusion increased over a 24-h period from a value comparable to that observed in normal CEF, (0.72 +/- 0.13).10(-10) cm2 s-1 to a value comparable to the RSV-CEF transformed cells, (1.74 +/- 0.20).10(-10) cm2 s-1. All diffusion measurements reported were made at the permissive temperature for RSV-NY68 (35 degrees C) unless stated otherwise. The changes in the lateral diffusion of TRITC-SConA occurred between the fifth and twelfth hour of the downshift course and could be associated with cytoskeletal disruption and/or fibronectin degradation, both known to occur at this time in RSV-transformed cells. To assess the contribution of extracellular matrix (ECM) degradation, SConA mobility was measured in normal and RSV-transformed cells treated with trypsin. This treatment increased SConA mobility approximately 4-fold in the normal cells relative to untreated controls and only 2-fold in the RSV-CEF transformed cells. No significant difference in SConA mobility between trypsinized spherical normal and transformed cells was apparent.     

Lateral diffusion of epidermal growth factor complexed to its surface receptors does not account for the thermal sensitivity of patch formation and endocytosis

The patching and endocytosis of EGF (epidermal growth factor) bound to A-431 cells (a human epidermoid carcinoma line) are temperature-sensitive processes which are completely inhibited at 4 degrees C. Receptor-mediated endocytosis generally occurs through coated regions, and EGF bound to its membrane receptor must diffuse laterally to these points of internalization. In this work we investigated the thermal sensitivity of the lateral diffusion of EGF receptor complexes and the thermal sensitivity of the patching and endocytosis of the hormone receptor complexes. Using the fluorescence photobleach recovery technique, we measured the lateral diffusion coefficients of a fluorescent derivative of EGF as a function of temperature. The lateral diffusion coefficient (D) increased gradually from 2.8 X 10(-10) cm2/s at 5 degrees C to 8.5 X 10(-10) cm2/s at 37 degrees C, and no phase transition was detected. Neither was a phase transition detected when we measured the diffusion coefficient of fluorescent lipid probes over this temperature range. From a calculation of the collision frequency of the occupied EGF receptors with coated regions using our measured values of D at 5 and 37 degrees C, we conclude that diffusion is not the rate-limiting step for either endocytosis or patching.     

Factors affecting steroid and prostaglandin secretion by reproductive tissues of cycling and pregnant sows in vitro

Mitochondrial, microsomal and pellicular membranes were isolated from Tetrahymena cells grown at 39 degrees C or 15 degrees C, and phospholipids, in turn, were separated from total lipids extracted from these membranes. The effect of growth temperature on their solid-to-fluid phase transition temperature was examined by wide-angle X-ray diffraction. The transition temperatures of phospholipids from mitochondria, microsomes and pellicles were 21, 19 and 26 degrees C for cells grown at 39 degrees C and -8, -3 and 6 degrees C for cells grown at 15 degrees C, respectively. All phospholipids were found in a completely fluid state at these growth temperatures. From a comparison between the phospholipids and total lipids from pellicles of cells grown at 39 degrees C, a triterpenoid alcohol, tetrahymanol, caused the transition temperature to increase. The alignment of tetrahymanol in membranes was examined with pellicle'a total lipid oriented in a sample holder.     

Lateral diffusion of PEG-Lipid in magnetically aligned bicelles measured using stimulated echo pulsed field gradient 1H NMR

Lateral diffusion measurements of PEG-lipid incorporated into magnetically aligned bicelles are demonstrated using stimulated echo (STE) pulsed field gradient (PFG) proton (1H) nuclear magnetic resonance (NMR) spectroscopy. Bicelles were composed of dimyristoyl phosphatidylcholine (DMPC) plus dihexanoyl phosphatidylcholine (DHPC) (q = DMPC/DHPC molar ratio = 4.5) plus 1 mol % (relative to DMPC) dimyristoyl phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG 2000) at 25 wt % lipid. 1H NMR STE spectra of perpendicular aligned bicelles contained only resonances assigned to residual HDO and to overlapping contributions from a DMPE-PEG 2000 ethoxy headgroup plus DHPC choline methyl protons. Decay of the latter's STE intensity in the STE PFG 1H NMR experiment (g(z) = 244 G cm(-1)) yielded a DMPE-PEG 2000 (1 mol %, 35 degrees C) lateral diffusion coefficient D = 1.35 x 10(-11) m2 s(-1). Hence, below the "mushroom-to-brush" transition, DMPE-PEG 2000 lateral diffusion is dictated by its DMPE hydrophobic anchor. D was independent of the diffusion time, indicating unrestricted lateral diffusion over root mean-square diffusion distances of microns, supporting the "perforated lamellae" model of bicelle structure under these conditions. Overall, the results demonstrate the feasibility of lateral diffusion measurements in magnetically aligned bicelles using the STE PFG NMR technique.     

High pressure 2H nuclear magnetic resonance study of the gel phases of dipalmitoylphosphatidylcholine

The 2H-NMR lineshapes of dipalmitoylphosphatidylcholine perdeuterated in the acyl chains were studied in a 15% dispersion in water as a function of pressure from 1 bar to 5 kbar over the temperature range from 7 degrees C to 75 degrees C. Increasing pressure in the gel state had the same effect as lowering the temperature: the lineshape gradually changed from a motionally averaged to a rigid lattice type spectrum with much of the intensity in the shoulders at +/- 63 kHz. At very high pressures and low temperatures (7 degrees C, 2.5 kbar; 25 degrees C, 5 kbar) even the methyl portion of the spectrum became a rigid lattice type spectrum at +/- 21 kHz. In addition to the liquid crystalline phase, five gel phases were detected. Using different techniques to determine the phase transitions, a general pressure-temperature phase diagram was constructed.     

NMR studies of pig gastric microsomal H+,K+-ATPase and phospholipid dynamics. Effects of ethanol perturbation

The effects of ethanol on the gastric H+,K+-ATPase activity and the degree of mobility of various microsomal phospholipids were assessed using 31P and 1H NMR. This illuminated the role of lipid-protein association in the function of pig gastric microsomes. Treatment of gastric microsomes with 15% ethanol for 1 min at 37 degrees C inactivated the H+,K+-ATPase activity, which could largely be reconstituted by supplementation with phosphatidylcholine isolated from the gastric microsomes. Under similar conditions, the 1H NMR profile of the microsomal +N(CH3)3 choline moiety showed dramatic enhancement of peak intensity as well as a break point at 25 degrees C which was restored to the untreated control value after reconstitution. This break, together with the dramatic enhancement in the overall lipid profile, compared to the control and reconstituted microsomes, suggested a greater degree of freedom of movement of the microsomal lipids following ethanol perturbation. The data demonstrate the unique ability that a combined approach using 31P and 1H NMR holds as a noninvasive probe to study the structure-function relationship of biomembranes.     

19F NMR investigation of molecular motion and packing in sonicated phospholipid vesicles

Dimyristoylphosphatidylcholine (DMPC) labeled with a C19F2 group in the 4-, 8-, or 12-position of the 2-acyl chain has been investigated in sonicated unilamellar vesicles (SUV) by fluorine-19 nuclear magnetic resonance (NMR) at 282.4 MHz from 26 to 42 degrees C. The 19F NMR spectra exhibit two overlapping resonances with different line widths. Spin-lattice relaxation time measurements have been performed in both the laboratory frame (T1) and the rotating frame (T1 rho) in order to investigate the packing and dynamics of phospholipids in lipid bilayers. Quantitative line-shape and relaxation analyses are possible by using the experimental chemical shift anisotropy (delta nu CSA) and the internuclear F-F vector order parameter (SFF) values obtained from the 19F powder spectra of multilamellar liposomes. The following conclusions can be made: The 19F chemical shift difference between the inside and outside leaflets of SUV can be used to monitor the lateral packing of the phospholipid in the two SUV monolayers. The hydrocarbon chains in the outer layer are found to be more tightly packed than those of the inner one, and the differences between them become smaller near the chain terminals. The effective correlation time [(1-4) x 10(-7) s] obtained from either the motional narrowing of the line widths or off-resonance T1 rho measurements is shorter than that estimated from the Stokes-Einstein diffusion model (10(-6) s), on the basis of a hydrodynamic radius of 110 A for SUV.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular dynamics of 3,3',5-triiodo-L-thyronine in model membranes: a spin label study

A spin-labeled derivative of 3,3',5-triiodo-L-thyronine, 3-[( alpha-carboxy-4-(4-hydroxy-3-iodophenoxy)-3,5-diiodophenethyl++ +] carbamoyl)-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy (SL-T3) has been synthesized. Evaluation of its binding to nuclei after incubation with rat pituitary tumor GH3 cells at 37 degrees C showed that it bound to nuclei with a 18% potency of that of T3. The dynamic interaction of SL-T3 with multilamellar vesicles prepared from dimyristoylphosphatidylcholine (DMPC) was investigated using electron spin resonance techniques. At 31 degrees C, the lateral diffusion constant of SL-T3 in DMPC membranes was found to be 3.0 X 10(-8) cm2/s as determined by the ESR line-broadening method. The temperature dependency of the ESR spectrum of SL-T3 in DMPC multilamellar vesicles showed a break at 23.5 degrees C, which is close to the main phase-transition temperature, 23.7 degrees C, of DMPC membranes. This suggests that the motion of the probe reflects the motion of phospholipids in DMPC membranes, and that the probe itself does not perturb the membrane structure. SL-T3 appears to be a useful probe for studying the motion of thyroid hormone in the plasma membrane of responsive cells.     

Effect of prolactin on the phospholipid composition and cholesterol level in liver microsomes of the carp during acclimation to different temperatures

The phospholipid composition, content of cholesterol and its esters in the carp (Cyprinus carpio L.) liver microsomes depend on the environmental temperature. The free cholesterol amount and cholesterol/phospholipids ratio in microsomes decrease after the lowering of temperature from 20 to 5 degrees C. The temperature elevation to 30 degrees C results in an increase of the cholesterol ester content. The relative proportions of phosphatidyl choline, phosphatidyl ethanolamine, sphingomyelin, phosphatidyl inositol, phosphatidyl serine, phosphatidic acid increase with a significant decrease of the unidentified phospholipids amount at 30 degrees C. Prolactin affects the cholesterol content and phospholipid composition of liver microsomes. The hormone has a more pronounced effect at subextremal temperatures (5 and 30 degrees C). The actions of prolactin and temperature on the cholesterol content are similar. The hormone influence on the membrane phospholipid composition is opposite to the effect of the temperature acclimation. The possible role of prolactin in the temperature adaptation of the membrane lipids metabolism in poikilotherms is discussed.     

Lateral diffusion of gramicidin C in phospholipid multibilayers. Effects of cholesterol and high gramicidin concentration

We have measured the lateral diffusion coefficient (D), of active dansyl-labeled gramicidin C (DGC), using the technique of fluorescence photobleaching recovery, under conditions in which the cylindrical dimer channel of DGC predominates. In pure, hydrated, dimyristoylphosphatidylcholine (DMPC) multibilayers (MBL), D decreases from 6 X 10(-8) cm2/s at 40 degrees C to 3 X 10(-8) cm2/s at 25 degrees C, and drops 100-fold at 23 degrees C, the phase transition temperature (Tm) of DMPC. Above Tm, addition of cholesterol decreases D; a threefold stepwise drop occurs between 10 and 20 mol %. Below Tm, increasing cholesterol increases D; a 10-fold increase occurs between 10 and 20 mol % at 21 degrees C, between 20 and 25 mol % at 15 degrees C, and between 25 and 30 mol % at 5 degrees C. In egg phosphatidylcholine (EPC) MBL, D decreases linearly from 5 X 10(-8) cm2/s at 35 degrees C to 2 X 10(-8) cm2/s at 5 degrees C; addition of equimolar cholesterol reduces D by a factor of 2. Thus this transmembrane polypeptide at low membrane concentrations diffuses quite like a lipid molecule. Its diffusivity in lipid mixtures appears to reflect predicted changes of lateral composition. Increasing gramicidin C (GC) in DMPC/GC MBL broadened the phase transition, and the diffusion coefficient of the lipid probe N-4-nitrobenzo-2-diazole phosphatidylethanolamine (NBD-PE) at 30 degrees C decreases from 8 X 10(-8) cm2/s below 5 mol % GC to 2 X 10(-8) cm2/s at 14 mol % GC; D for DGC similarly decreases from 4 X 10(-8) cm2/s at 2 mol % GC to 1.4 X 10(-8) cm2/s at 14 mol % GC. Hence, above Tm, high concentrations of this polypeptide restrict the lateral mobility of membrane components.     

Deuterium nuclear magnetic resonance study of the interaction of branched chain compounds (phytanic acid, phytol) with a phospholipid model membrane

Deuterium nuclear magnetic resonance (2H-NMR) spectra have been determined for 50 wt% aqueous dispersions of 1-palmitoyl(stearoyl)-2-[2H31]palmitoyl-sn-glycero-3-phosphocho lin e (PC-d31) containing 20 mol% of the isoprenoid compounds phytol or phytanic acid over the temperature range -5-55 degrees C. Concentration effects of the isoprenoid compounds are also reported. First moments (M1) and order parameters were calculated from the spectra. 20 Mol% of either branched chain compound causes an approximate 9% increase in the mean order parameter SCD. Significant effects are seen on the PC-d31 phase behavior. 20 Mol% of either branched chain compound causes the gel to liquid crystalline onset temperature (Ts) to drop to 28 degrees C from 38 degrees C for PC-d31 alone, as seen from the temperature dependent M1 values. The melting range ([Tl--Ts]) is congruent to 1.5 degrees C for PC-d31 and congruent to 11 degrees C for PC-d31 containing 20 mol% of the branched chain compounds. This is in direct contrast to their straight chain analogues, hexadecanol and palmitic acid, which have been shown to elevate the phase transition temperature. The isoprenoid compounds cause significant disruption of the gel phase, forcing nearest neighbor phospholipid chains apart. Transverse relaxation times (T2e, the time constant for decay of the quandrupolar echo) have been determined over the temperature range -5-50 degrees C. Possible explanation for the effect of the isoprenoid compounds on the dynamic structure of phospholipids in the bilayer are proffered.     

Quantitation of lateral stress in lipid layer containing nonbilayer phase preferring lipids by frequency-domain fluorescence spectroscopy.

Frequency-resolved fluorescence measurements have been performed to quantitate the lateral stress of the lipid layer containing nonbilayer phase preferring dioleoylphosphatidylethanolamine (DOPE). On the basis of a new rotational diffusion model, the wobbling diffusion constant (Dw), the curvature-related hopping diffusion constant (DH), and the two local orientational order parameters ([P2] and [P4]) of 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl] carbonyl]-3-sn-phosphatidylcholine (DPH-PC) in fully hydrated DOPE and DOPE/dioleoylphosphatidylcholine (DOPC) mixtures were calculated from the frequency-domain anisotropy data. The values of [P2], [P4], and DH for DOPE were found to increase significantly at approximately 12 degrees C, the known lamellar liquid crystalline (L alpha) to inverted hexagonal (HII) phase transition temperature of DOPE. Similar features as well as a decline of Dw were detected in the DOPE/DOPC mixtures as the DOPE content was increased from 85% to 90% at 23 degrees C, corresponding to the known lyotropic phase transition of the DOPE/DOPC. In contrast, for DOPC (0-40 degrees C) and DOPE/DOPC (0-100% DOPE at 3 degrees C), which remained in the L alpha phase, these changes were not detected. The most probable local orientation of DPH-PC in the DOPE/DOPC mixtures shifted progressively toward the normal of the lipid/water interface as the content of DOPE increased. We concluded that the curvature-related lateral stress in the lipid layer increases with the content of the nonbilayer phase preferring lipids.     

The mechanism of hydroperoxide-dependent reactions with participation of cytochrome P-450

Cytochrome P-450 destruction kinetics by cumene hydroperoxide (CHP) has been studied at 25 degrees C in phosphate buffer, pH 7.25-7.50, in various systems: intact and induced rat or rabbit microsomes, highly purified LM2- and LM2- and LM4-forms of cytochrome P-450 from rabbit liver microsomes. The destruction kinetics is characterized by three phases in all systems. The CHP-influenced cytochrome P-450 destruction is a radical chain process with linear termination of the chains. The acidic phospholipids, phosphatidylserine and phosphatidylinositol and total microsomal phospholipids containing the acidic lipid components activate cytochrome P-450 in the hydroxylation of aniline and naphthalene by CHP. Phosphatidylcholine and sphingomyelin have no effect upon the cytochrome P-450 activity in the type I and II substrates oxidation by CHP. The phase transitions of the microsomal phospholipids influence the interaction of cytochrome P-450 with its reductase, altering the activation energy of type I substrates oxidation. The type II substrate oxidation is not affected by phase transitions in the full microsomal hydroxylating system.