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)     

Surface diffusion in human serum lipoproteins

From the viscosity dependence of the 31P NMR signals, the diffusion coefficients DT of phospholipid molecules in the surface monolayer of HDL, LDL and VLDL have been determined. DT for HDL3 and HDL2 are found to be 2.3 X 10(-8) cm2/s and 1.8 X 10(-8) cm2/s, respectively. These values are similar to values reported for diffusion of phospholipid molecules in phospholipid bilayers above the gel to liquid crystalline phase transition temperature. Viscosity dependence of [16,16,16-2H3]phosphatidylcholine incorporated into HDL2 yielded a value similar to that determined by 31P (DT = 1.9 X 10(-8) cm2/s). Slower diffusion coefficients were measured for LDL2 and VLDL. VLDL had a value DT = 9.1 X 10(-9) cm2/s. The diffusion coefficient for LDL2 was 1.4 X 10(-9) cm2/s. Thus, diffusion of phospholipids in LDL2 is a full order of magnitude slower at 25 degrees C than diffusion of phospholipids in the HDLs.     

Diffusion of nitric oxide into low density lipoprotein.

A key early event in the development of atherosclerosis is the oxidation of low density lipoprotein (LDL) via different mechanisms including free radical reactions with both protein and lipid components. Nitric oxide (( small middle dot)NO) is capable of inhibiting LDL oxidation by scavenging radical species involved in oxidative chain propagation reactions. Herein, the diffusion of ( small middle dot)NO into LDL is studied by fluorescence quenching of pyrene derivatives. Selected probes 1-(pyrenyl)methyltrimethylammonium (PMTMA) and 1-(pyrenyl)-methyl-3-(9-octadecenoyloxy)-22,23-bisnor-5-cholenate (PMChO) were chosen so that they could be incorporated at different depths of the LDL particle. Indeed, PMTMA and PMChO were located in the surface and core of LDL, respectively, as indicated by changes in fluorescence spectra, fluorescence quenching studies with water-soluble quenchers and the lifetime values (tau(o)) of the excited probes. The apparent second order rate quenching constants of ( small middle dot)NO (k(NO)) for both probes were 2.6-3.8 x 10(10) m(-1) s(-1) and 1.2 x 10(10) m(-1) s(-1) in solution and native LDL, respectively, indicating that there is no significant barrier to the diffusion of ( small middle dot)NO to the surface and core of LDL. Nitric oxide was also capable of diffusing through oxidized LDL. Considering the preferential partitioning of ( small middle dot)NO in apolar milieu (6-8 for n-octanol:water) and therefore a larger ( small middle dot)NO concentration in LDL with respect to the aqueous phase, a corrected k(NO) value of approximately 0.2 x 10(10) m(-1) s(-1) can be determined, which still is sufficiently large and consistent with a facile diffusion of ( small middle dot)NO through LDL. Applying the Einstein-Smoluchowsky treatment, the apparent diffusion coefficient (D(')NO) of ( small middle dot)NO in native LDL is on average 2 x 10(-5) cm(2) s(-1), six times larger than that previously reported for erythrocyte plasma membrane. Thus, our observations support that ( small middle dot)NO readily traverses the LDL surface accessing the hydrophobic lipid core of the particle and affirm a role for ( small middle dot)NO as a major lipophilic antioxidant in LDL.     

Interactions of nitric oxide and peroxynitrite with low-density lipoprotein

Nitric oxide (*NO) is a free radical species that diffuses and concentrates in the hydrophobic core of low-density lipoprotein (LDL) to serve as a potent inhibitor of lipid oxidation processes. Peroxynitrite (PN), the product of the diffusion-limited reaction between *NO and superoxide (O2*-) represents a relevant mediator of oxidative modifications in LDL. The focus of this review is the analysis of interactions between *NO and PN and its secondary reactions with oxygen radicals on LDL oxidation, which are relevant in the development of the early steps as well as progression of atherosclerosis. We propose that the balance between rates of PN and *NO production, which greatly depends on oxidative stress processes within the vascular wall, will critically determine the final extent of oxidative LDL modifications leading or not to scavenger receptor-mediated LDL uptake and foam cell formation.     

Diffusion and partition of solutes in cartilage under static load

We describe experimental apparatus, methodology and mathematical algorithms to measure diffusion and partition for typical small ionic solutes and inulin (a medium size solute) in statically loaded cartilage. The partition coefficient based on tissue water (K(H(2)O)) of Na(+) increased from 1.8 to 4.5 and for SO(4)(-2) decreased from 0.5 to 0.1, when the applied pressure was raised from zero to 22 atm K(H(2)O) of inulin decreased from 0.3 to 0.05, for an increase in pressure from zero to 11 atm. Our theoretical interpretation of the results is that the partition coefficient can be expressed as a function of fixed charge density (FCD) for both loaded and unloaded cartilage. The partition coefficient shows good agreement with the ideal Gibbs-Donnan equilibrium, particularly when FCD is based on extrafibrillar water (EFW). The diffusion coefficients, D also decreased with an increase in applied pressure; raising the pressure from 0 to 22 atm resulted in the following changes in the values of D: for Na(+) from 2.86 x 10(-6) to 1.51 x 10(-6) cm(2)/s, for SO(4)(-2) from 1.58 x 10(-6) to 7.5 x 10(-7) cm(2)/s, for leucine from 1.69 x 10(-6) to 8.30 x 10(-7) cm(2)/s and for inulin from 1.80 x 10(-7) to 3.30 x 10(-8) cm(2)/s. For the three small solutes (two charged and one neutral) the diffusion coefficient D is highly correlated with the fraction of fluid volume in the tissue. These experimental results show good agreement with the simple model of Mackie and Meares: hence solute charge does not affect the diffusion of small solutes under load. For inulin D & K show some agreement with a modified Ogston model based on two major components, viz., glycosaminoglycans (GAG) and core protein. We conclude that the changes in the partition and diffusion coefficients of small and medium size solutes in statically loaded cartilage can be interpreted as being due to the reduction in hydration and increase in FCD. The change in the latter affects the partition of small ionic solutes and the partition and diffusion of larger molecules. Our results throw light on the ionic environment of chondrocytes in loaded cartilage as well as on the transport of solutes through the matrix.     

Salicylate inhibits LDL oxidation initiated by superoxide/nitric oxide radicals

Simultaneously produced superoxide/nitric oxide radicals (O2*-/NO*) could form peroxynitrite (OONO-) which has been found to cause atherogenic, i.e. oxidative modification of LDL. Aromatic hydroxylation and nitration of the aspirin metabolite salicylate by OONO- has been reported. Therefore we tested if salicylate may be able to protect LDL from oxidation by O2*-/NO* by scavenging the OONO reactive decomposition products. When LDL was exposed to simultaneously produced O2*-/NO* using the sydnonimine SIN-1, salicylate exerted an inhibitory effect on LDL oxidation as measured by TBARS and lipid hydroperoxide formation and alteration in electrophoretic mobility of LDL. The cytotoxic effect of SIN-1 pre-oxidised LDL to endothelial cells was also diminished when salicylate was present during SIN-1 treatment of LDL. Spectrophotometric analysis revealed that salicylate was converted to dihydroxybenzoic acid (DHBA) derivatives in the presence of SIN-1. 2,3- and 2,5-DHBA were even more effective to protect LDL from oxidation by O2*-/NO*. Because O2*-/NO* can occur in vivo, the results may indicate that salicylate could act as an efficacious inhibitor of O2*-/NO* initiated atherogenic LDL modification, thus further supporting the rationale of aspirin medication regarding cardiovascular diseases.     

Comparison of the effects of O2*-/HO* free radical- and copper ions-oxidized LDL or lipoprotein(a) on the endothelial cell releases of tissue plasminogen activator and plasminogen activator inhibitor-1.

We investigated the effects of low density-lipoproteins (LDL) and lipoprotein(a) [Lp(a)] oxidized by O2*-/HO* free radicals generated by gamma radiolysis of water, on the release of tissue Plasminogen Activator (tPA) and of its main inhibitor Plaminogen Activator Inhibitor-1 (PAI-1) by human umbilical vein endothelial cells (HUVEC). These effects were compared to those of lipoproteins issued from the same preparations but oxidized by the classical copper ions procedure. The results showed that O2*-/HO* free radical oxidized LDL and Lp(a) led to a dramatic decrease of PAI-1 release but did not affect tPA release, whereas copper oxidation of lipoproteins resulted in an increase in PAI-1 release and a decrease in tPA release. Chemical analysis revealed that O2*-/HO* free radical oxidized lipoproteins exhibited very much lower levels of phosphatidylcholine hydroperoxides, lysophosphatidylcholine and oxysterols (7-ketocholesterol, 7beta-hydroxycholesterol, 5,6beta-epoxycholesterol) than copper oxidized LDL. Thus, the discordant effects of O2*-/HO* oxidized and copper oxidized LDL and Lp(a) on the endothelial releases of PAI-1 and tPA appeared to be due to qualitatively and/or quantitatively different formation of oxidized components by the two oxidation processes.     

Formation of lipid-protein adducts in low-density lipoprotein by fluxes of peroxynitrite and its inhibition by nitric oxide.

Peroxynitrite (PN), the product of the diffusion-limited reaction between nitric oxide (*NO) and superoxide (O*-(2)), represents a relevant mediator of oxidative modifications in low-density lipoprotein (LDL). This work shows for the first time the simultaneous action of low-controlled fluxes of PN and *NO on LDL oxidation in terms of lipid and protein modifications as well as oxidized lipid-protein adduct formation. Fluxes of PN (e.g., 1 microM min(-1)) initiated lipid oxidation in LDL as measured by conjugated dienes and cholesteryl ester hydroperoxides formation. Oxidized-LDL exhibited a characteristic fluorescent emission spectra (lambda(exc) = 365 nm, lambda(max) = 417 nm) in parallel with changes in both the free amino groups content and the relative electrophoretic mobility of the particle. Physiologically relevant fluxes of *NO (80-300 nM min(-1)) potently inhibited these PN-dependent oxidative processes. These results are consistent with PN-induced adduct formation between lipid oxidation products and free amino groups of LDL in a process prevented by the simultaneous presence of *NO. The balance between rates of PN and *NO production in the vascular wall will critically determine the final extent of LDL oxidative modifications leading or not to scavenger receptor-mediated LDL uptake and foam cell formation.     

Antioxidant effect of probucol on RO2*/O2(*-)-induced peroxidation of human low-density lipoproteins

This study was designed to evaluate the antioxidant effect of probucol on peroxidation of low-density lipoproteins (LDLs) initiated by oxygenated free radicals (O2*-) and ethanol-derived peroxyl radicals (RO2*) generated by gamma radiolysis. Initial radiolytic yields related to the markers of lipid peroxidation [i.e. decrease in endogenous alpha-tocopherol, formation of thiobarbituric acid-reactive substances (TBARS) and conjugated dienes] were determined as a function of LDL concentration (1.5 and 3 g l(-1), expressed as total LDL) and in the absence or the presence of probucol at different concentrations (2.3 x 10(-6), 3.5 x 10(-6), 9 x 10(-6) and 20.5 x 10(-6) mol l(-1)). Our results showed that probucol was able to decrease not only the yields of TBARS and conjugated dienes but also the levels of these peroxidation products obtained at high doses (2500 Gy) compared to LDLs without probucol. Under our conditions, probucol displayed an optimal antioxidant effect for an initial concentration in LDLs equivalent to 15 probucol molecules per LDL particle, which corresponded to a pharmacologically relevant concentration of probucol. Moreover, our data showed that probucol was unable to react with RO2* and thus did not protect LDL vitamin E from free radical attack. In addition, the scavenging capacity of probucol on O2*- appeared to be very poor, and probucol more likely reacted with LDL intermediate radical products. Finally, a very significant steady-state level of probucol remained in LDLs at high doses (up to 2500 Gy), equivalent to at least 40% of the initial concentration of probucol. This addressed the question of a mechanism for regeneration of probucol in LDLs. Our results as a whole suggested that the antioxidant effect of probucol in vivo could not be explained by its scavenging capacity with regard to RO2*/O2*- free radicals.     

Nitric oxide and carbon monoxide equilibria of horse myoglobin and (N-methylimidazole)protoheme. Evidence for steric interaction with the distal residues

The Soret absorption maxima and extinction coefficients of the CO and NO complexes of horse myoglobin and (NMeIm)protoheme (NMeIm = 1-methylimidazole) have been determined. The partition coefficient N, equal to the ratio P1/2 (CO)/P1/2(NO), has been determined spectrophotometrically for horse myoglobin and (NMeIm)protoheme. P1/2-(NO) values calculated from the partition coefficients are 5.7 x 10(7) mmHg for (NMeIm)protheme and 1.1 x 10(6) mmHg for horse myoglobin. The ratio of P1/2(NO) values for protein and model is 1.9 which is similar to a value of 1.6 reported for the ratio of P1/2(O2) values. These values may be compared to a ratio of 15 for CO binding to protein and model complexes. This different ratio for CO provides further evidence for steric interaction of the bound CO with the protein based on a consideration of the preferred nonlinear geometry of Fe-NO and Fe-O2 and the linear geometry of Fe-CO.     

Antioxidant properties of melatonin: a pulse radiolysis study

Various one-electron oxidants such as OH*, tert-BuO*, CCl3OO*, Br2*- and N3*, generated pulse radiolytically in aqueous solutions at pH 7, were scavenged by melatonin to form two main absorption bands with lambda(max) = 335 nm and 500 nm. The assignment of the spectra and determination of extinction coefficients of the transients have been reported. Rate constants for the formation of these species ranged from 0.6-12.5x10(9) dm3 mol(-1) s(-1). These transients decayed by second order, as observed in the case of Br2*- and N3* radical reactions. Both the NO2* and NO* radicals react with the substrate with k = 0.37x10(7) and 3x10(7) dm3 mol(-1) s(-1), respectively. At pH approximately 2.5, the protonated form of the transient is formed due to the reaction of Br2*- radical with melatonin, pKa ( MelH* <=> Mel* + H+) = 4.7+/-0.1. Reduction potential of the couple (Mel*/MelH), determined both by cyclic voltammetric and pulse-radiolytic techniques, gave a value E(1)7 = 0.95+/-0.02 V vs. NHE. Repair of guanosine radical and regeneration of melatonin radicals by ascorbate and urate ions at pH 7 have been reported. Reactions of the reducing radicals e(aq)- and H* atoms with melatonin have been shown to occur at near diffusion rates.     

The kinetics of the reaction between NO and O2 as studied by a novel approach

The kinetics of the reaction between NO and O2 was determined by measuring the time course of the decrease in the concentration of NO with a quench-flow technique. NO and O2 were mixed rapidly and reacted for periods of time varying from 10 to 50 s. A second rapid mixing with a solution containing an excess of deoxyhemoglobin and sodium hydrosulfite trapped free NO as nitrosylhemoglobin and reduced O2. The spectrum of the mixture of deoxy- and nitrosylhemoglobin was recorded within 30 s from the second mixing, before any appreciable dissociation of NO from the protein, by means of a flow-cell mounted on-line with the quench-flow apparatus. The amount of NO not consumed in the auto-oxidation reaction was calculated from the proportion of nitrosylhemoglobin in the mixture. As NO and O2 bind deoxyhemoglobin at comparable rates and NO is oxidized to nitrate by oxyhemoglobin, the ratio of hemoglobin/(NO + O2) had to be optimized to avoid the interference of this oxidation reaction. The kinetics was first and second order with respect to O2 and NO, respectively and third order overall with a rate constant k = 4 x kaq = 4 x 2.23 (+/- 0.26) x 10(6) M-2 s-1 at 20 degrees C, invariant in the pH range 7-9, in agreement with published values obtained by different methodologies.     

Micellar catalysis for oxidation of nitric oxide (NO) in the multi-phase systems in vivo.

The equation of the dependence of the third-order reaction acceleration due to concentrating the reagents in a small volume of the hydrophobic phase on the partition coefficients of reagents (Q) and on the lipophilic phase fraction (x), [k(app)/ k2 = H(Q(NO),Q(O2),x)] was analyzed. It was demonstrated that the numeric value of dH/dx at x-->0 could not be used in order to calculate the efficiency of catalysis from the experimental data. It was shown that, unlike in two-phase systems (with an aqueous and a hydrophobic phase), the dependence of H on Q in multi-phase systems, that include all in vivo systems, is different. The multiple phase state of the systems has a determining role for a regulation of NO-dependent processes and in the realization of conditions of 'NO catastrophes'.     

Oxygen diffusion in biological and artificial membranes determined by the fluorochrome pyrene

Quenching of pyrene fluorescence by oxygen was used to determine oxygen diffusion coefficients in phospholipid dispersions and erythrocyte plasma membranes. The fluorescence intensity and lifetime of pyrene in both artificial and natural membranes decreases about 80% in the presence of 1 atm O2, while the fluorescence excitation and emission spectra and the absorption spectrum are unaltered. Assuming the oxygen partition coefficient between membrane and aqueous phase to be 4.4, the diffusion coefficients for oxygen at 37 degrees C are 1.51 X 10(-5) cm2/s in dimyristoyl lecithin vesicles, 9.32 X 10(-6) cm2/s in dipalmitoyl lecithin vesicles, and 7.27 X 10(-6) cm2/s in erythrocyte plasma membranes. The heats of activation for oxygen diffusion are low (less than 3 kcal/degree-mol). A dramatic increase in the diffusion constant occurs at the phase transition of dimyristoyl and dipalmitoyl lecithin, which may result from an increase in either the oxygen diffusion coefficient, partition coefficient, or both. The significance of the change in oxygen diffusion below and above the phase transition for biological membranes is discussed.     

Novel hydrogel obtained by chitosan and dextrin-VA co-polymerization

A novel hydrogel was obtained by reticulation of chitosan with dextrin enzymatically linked to vinyl acrylate (dextrin-VA), without cross-linking agents. The hydrogel had a solid-like behaviour with G′ (storage modulus) >> G″ (loss modulus). Glucose diffusion coefficients of 3.9 × 10⁻⁶ ± 1.3 × 10⁻⁶ cm²/s and 2.9 × 10⁻⁶ ± 0.5 × 10⁻⁶ cm²/s were obtained for different substitution degrees of the dextrin-VA (20% and 70% respectively). SEM observation revealed a porous structure, with pores ranging from 50 μm to 150 μm.     

Pulsed high-field gradient in vivo NMR spectroscopy to measure diffusional water permeability in Corynebacterium glutamicum

Pulsed high-field gradient in vivo NMR spectroscopy was used to measure diffusional water permeability in cell suspensions of the Gram-positive bacterium Corynebacterium glutamicum. Two different regions of H2O mobility were detected. One was characterized by the apparent coefficient of self-diffusion, D(1 app) = (4.6-12.7)x10(-8) cm(2) s(-1), depending on the observation time t. The other region was characterized by D(2) = 1.4x10(-5) cm(2) s(-1). The value of D(2) was similar to the diffusion coefficient of H2O in free water and in extracellular biological fluids. Restricted diffusion could be demonstrated for the slower process (D(1)). It was attributed to the cytoplasm of the cells. The membrane permeability, P(d H2O), for C. glutamicum was (4.8+/-0.4)x10(-3) cm s(-1). It compared favorably with values reported for human erythrocytes and was higher by a factor of about 100 compared to the diffusional permeability for ethanol, P(d ethanol), in Zymomonas mobilis. Addition of HgCl2, a water channel inhibitor in eukaryotes, decreased P(d H2O) in C. glutamicum by a factor of approximately 8. To our knowledge, these are the first functional studies of water transport in prokaryotes that yielded quantitative data, viz., transmembrane water permeability expressed through D(H2O) and P(d H2O).     

Membrane insertion and lateral diffusion of fluorescence-labelled cytochrome c oxidase subunit IV signal peptide in charged and uncharged phospholipid bilayers.

The synthetic 25-residue signal peptide of cytochrome c oxidase subunit IV was labelled with the fluorophor 7-nitrobenz-2-oxa-1,3-diazole (NBD) at its single cysteine residue. Addition of small unilamellar vesicles of 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC) to the labelled peptide resulted in a shift of the NBD excitation and emission spectra to shorter wavelengths. Binding of the peptide to the vesicles was measured by the increase in the fluorescence emission yield. A surface partition constant of (3.9 +/- 0.5) x 10(3) M-1 was derived from these titrations. When the membrane contained, in addition to POPC, negatively charged 1-palmitoyl 2-oleoyl phosphatidylglycerol (POPG), the NBD fluorescence spectra were further shifted to shorter wavelengths and exhibited increased quantum yields. The apparent partition constants were increased to 10(4)-10(5) M-1 for vesicles with 20 or 100 mol% POPG. Lateral diffusion of the peptide was measured by fluorescence recovery after photobleaching in multibilayers of POPC, POPG, POPC/POPG (4:1) and 1,2-dimyristoyl phosphatidylcholine. The lateral diffusion coefficients of the peptide in bilayers of POPC (8 x 10(-8) cm2/s at 21 degrees C) were 1.5-1.6-fold greater than those of NBD-labelled phospholipids (5 x 10(-8) cm2/s at 21 degrees C), but 1.5-1.8-fold smaller (3 x 10(-8) cm2/s in 20% POPG and at 21 degrees C) than the lipid diffusion coefficients in the negatively charged bilayers. It is concluded that the signal peptide associates with phospholipid bilayers in two different forms, which depend on the lipid charge. The experiments with POPC bilayers are well explained by a model in which the peptide partitions into the region of the phospholipid head-groups and diffuses along the membrane/water interface. If POPG is present in the membrane, electrostatic attractions between the basic residues of the peptide and the acidic lipid head-groups result in a deeper penetration of the bilayer. For this case, two models that are both consistent with the experimental data are discussed, in which the peptide either forms an oligomer of three to six partially helical membrane-spanning monomers, or inserts into the bilayer with its amphiphilic helical segment aligned parallel to the plane of the membrane and located near the head-group and outer hydrocarbon region of the bilayer.     

Permeation of tetracyclines through membranes of liposomes and Escherichia coli.

Uptake of tetracycline (tc), 2-tetracyclinonitrile (CN-tc), and 9-(N, N-dimethylglycylamido)-6-demethyl-6-deoxytetracycline (DMG-DMDOT) by liposomes containing Tet repressor (TetR) and by Escherichia coli cells overexpressing TetR was examined. TetR specifically binds to tetracyclines, enhances their fluorescence and thereby allows selective detection of tetracyclines that have crossed the membranes. Analysis of the diffusion of tc and DMG-DMDOT into liposomes yielded permeation coefficients of (2.4 +/- 0.6) x 10-9 cm.s-1 and (3.3 +/- 0.8) x 10-9 cm.s-1, respectively. Similar coefficients were obtained for uptake of these tetracyclines by E. coli, indicating that diffusion through the cytoplasmic membrane is the rate-limiting step. The permeation coefficients translate into half-equilibration times of approximately 35 +/- 15 min and explain how efflux pumps can mediate resistance against tetracyclines. Furthermore, diffusion of CN-tc into liposomes was at least 400-fold slower than that of tc, indicating that the carboxamide group at position C2 is required for efficient permeation of tc through lipid membranes and thereby explaining the lack of antibiotic activity of CN-tc.     

Diffusion of low density lipoprotein-receptor complex on human fibroblasts

Diffusion of the complex consisting of low density lipoprotein (LDL) bound to its receptor on the surface of human fibroblasts has been measured with the help of an intensely fluorescent, biologically active LDL derivative, dioctadecylindocarbocyanine LDL (dil(3)-LDL). Fluorescence photobleaching recovering and direct video observations of the Brownian motion of individual LDL-receptor complexes yielded diffusion coefficients for the slow diffusion on cell surfaces and fast diffusion on membrane blebs, respectively. At 10 degrees C, less that 20 percent of the LDL-receptor complex was measurably diffusible either on normal human fibroblasts GM-3348 or on LDL-receptor- internalization-defective J.D. cells GM-2408A. At 21 degrees and 28 degrees C, the diffusion fractions of approximately 75 and 60 percent, respectively, on both cell lines. The lipid analog nitrobenzoxadiazolephosphatidylcholine (NBD-PC) diffused in the GM-2408A cell membrane at 1.5x10(-8) cm(2)/sec at 22 degrees C. On blebs induced in GM-2408A cell membranes, the dil(3)-LDL receptor complex diffusion coefficient increased to approximately 10(-9) cm(2)/s, thus approaching the maximum theoretical predictions for a large protein in the viscous lipid bilayer. Cytoskeletal staining of blebs with NBD- phallacidin, a fluorescent probe specific for F-actin, indicated that loss of the bulk of the F-actin cytoskeleton accompanied the release of the natural constraints on later diffusion observed on blebs. This work shows that the internalization defect of J.D. is not due to immobilization of the LDL-receptor complex since its diffusibility is sufficient to sustain even the internalization rates observed in the native fibroblasts. Nevertheless, as with many other cell membrane receptors, the diffusion coefficient of the LDL-receptor complex is at least two orders of magnitude slower on native membrane than the viscous limit approached on cell membrane blebs where it is released from lateral constraints. However, LDL-receptor diffusion may not limit LDL internalization in normal human fibroblasts.     

Major differences in oxysterol formation in human low density lipoproteins (LDLs) oxidized by *OH/O2*- free radicals or by copper.

The aim of our study was to determine the oxysterol formation in low density lipoproteins (LDLs) oxidized by defined oxygen free radicals (*OH/O2*-). This was compared to the oxysterol produced upon the classical copper oxidation procedure. The results showed a markedly lower formation of oxysterols induced by *OH/O2*- free radicals than by copper and thus suggested a poor ability of these radicals to initiate cholesterol oxidation in LDLs. Moreover, the molecular species of cholesteryl ester hydroperoxides produced by LDL copper oxidation seemed more labile than those formed upon *OH/O2*(-)-induced oxidation, probably due to their degradation by reaction with copper ions.