Karen A. Hagaman

Effect of lipid physical state on the rate of peroxidation of liposomes

The effect of cholesterol on the rate of peroxidation of arachidonic acid and 1-palmitoyl-2-arachidonoyl phosphatidylcholine (PAPC) in dimyristoylphosphatidylcholine (DMPC) liposomes was examined above and below the phase transition temperature (Tm) of the lipid. The rate of peroxidation of arachidonic acid was more rapid below the phase transition temperature of the host lipid. At a temperature below the Tm (4 degrees C), increasing concentrations of cholesterol reduced the rate of peroxidation of arachidonic acid as judged by the production of thiobarbituric acid reactive substances. Above Tm (37 degrees C), cholesterol increased the rate of peroxidation of the fatty acid. Similarly, PAPC was peroxidized more rapidly at 4 degrees C than at 37 degrees C. However, cholesterol had little effect on the rate of peroxidation of PAPC at 4 degrees C. The rate of peroxidation of arachidonic acid was related to the lipid bilayer fluidity as judged by fluorescence anisotropy measurements of diphenylhexatriene. The rate of peroxidation increased slowly with increasing rigidity of the probe environment when the bilayer was relatively fluid and more rapidly as the environment became more rigid. The increase in the rate of peroxidation of arachidonic acid in the less fluid host lipid was unrelated to differences in iron binding or to transfer of arachidonic acid to the aqueous phase. Decreasing the concentration of arachidonic acid in DMPC to less than 2 mol% dramatically decreased the rate of peroxidation at 4 degrees C, suggesting that formation of clusters of fatty acids at 4 degrees C is required for rapid peroxidation.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of lipid structure in the activation of phospholipase A2 by peroxidized phospholipids

The time course of hydrolysis of a mixed phospholipid substrate containing bovine liver 1,2-diacyl-sn-glycero-3-phosphocholine (PC) and 1,2-diacyl-sn-glycero-3-phosphoethanolamine (PE) catalyzed byCrotalus adamanteus phospholipase A2 was measured before and after peroxidation of the lipid substrate. The rate of hydrolysis was increased after peroxidation by an iron/adenosine diphosphate (ADP) system; the presence of iron/ADP in the assay had a minimal inhibitory effect. The rate of lipid hydrolysis was also increased after the substrate was peroxidized by heat and O2. Similarly, peroxidation increased the rate of hydrolysis of soy PC liposomes that did not contain PE. In order to minimize interfacial factors that may result in an increase in rate, the lipids were solubilized in Triton X-100. In mixtures of Triton with soy PC in the absence of PE, peroxidation dramatically increased the rate of lipid hydrolysis. In addition, the rate of hydrolysis of the unoxidizable lipid 1-palmitoyl-2-[1-14C]oleoyl PC incorporated into PC/PE liposomes was unaffected by peroxidation of the host lipid. These data are consistent with the notions that the increase in rate of hydrolysis of peroxidized PC substrates catalyzed by phospholipase A2 is due largely to a preference for peroxidized phospholipid molecules as substrates and that peroxidation of host lipid does not significantly increase the rate of hydrolysis of nonoxidized lipids.

Short model peptides having a high α-helical tendency: Design and solution properties

Secondary structure is not typically observed for small peptides in solution. Several of the properties of α‐helical peptides are known which lead to the stabilization of the structure. The utilization of all the known factors important for α‐helical stabilization in the design of model α‐helical peptides (MAP) is reported. The peptides are based on the repeating eleven amino acid sequence, Glu‐Leu‐Leu‐Glu‐Lys‐Leu‐Leu‐Glu‐Lys‐Leu‐Lys (MAP1–11). The CD spectra of these peptides give evidence for more α‐helical content than has been reported for any short peptide (< 18 amino acids) to date. This α‐helical tendency does not require the presence of lipid or reduced temperature. For instance, Suc‐[Trp9]MAP9‐3″ amide (5), a seventeen amino acid peptide has 100% and 80% α‐helical contents at 1.7 × 10−4 M and 1.7 × 10−5 M, respectively. Suc‐[Trp9]MAP2‐11 amide (3), merely ten amino acids in length, is 51% α‐helical at 1.7 × 10−4 M in 0.1 M phosphate buffer at room temperature. In the presence of lipid or trifluoroethanol, the α‐helical content of these peptides is increased. This series of peptides demonstrates the complimentarity of various secondary structure design principles and the extent to which structure can be induced in small linear peptides.

Probucol reduces the rate of association of apolipoprotein C-III with dimyristoylphosphatidylcholine

The effect of low concentrations of probucol and cholesterol on the association of dimyristoylphosphatidylcholine with human plasma apolipoprotein C-III was studied. Liposomes of dimyristoylphosphatidylcholine with or without probucol or cholesterol were prepared by swelling the lipids in buffer at 37 degrees C. The association of apolipoprotein C-III with the liposomes was determined at 24 degrees C by measuring the rate of clearing of turbidity at 400 nm following addition of protein. At a weight ratio of probucol/dimyristoylphosphatidylcholine of 1:25 (5 mol% probucol), the rate of clearing of liposomes was decreased by 60%; 5 mol% cholesterol had no effect on the clearing rate. Liposomes were then added to the preformed apolipoprotein C-III/lipid micelles. In the absence of probucol, the added liposomes cleared rapidly regardless of the presence or absence of cholesterol. With 5 mol% probucol, almost no decrease in absorbance was noted on addition of liposomes to the micelles. These data show that probucol reduces the rate of association of an apolipoprotein with lipid and suggests that the interaction of probucol with lipid may modify the assembly and/or metabolism of lipoproteins.

Antioxidant activity of probucol and its effects on phase transitions in phosphatidylcholine liposomes

The effect of probucol on the phase behavior of dimyristoylphosphatidylcholine (DMPC) was examined by fluorescence polarization and differential scanning calorimetry (DSC). Probucol broadens and shifts the temperature of the main phase transition of DMPC liposomes as measured by fluorescence polarization with diphenylhexatriene and trimethyl-ammonium-diphenylhexatrine at concentrations as low as 5 mole%. As measured by DSC, probucol reduces the transition temperature of the gel----liquid-crystalline phase transition of DMPC by approx. 2 C degrees at all concentrations above about 5 mole% probucol and eliminates the pretransition at less than 1 mole%. In addition, the phase transition of DMPC is broadened and the enthalpy of the transition reduced by approx. 50%. Even at high concentrations of probucol, the gel----liquid-crystalline phase transition of DMPC is not eliminated. Similar effects are observed with dipalmitoylphosphatidylcholine liposomes. Based on these DSC measurements, measurements of the melting of probucol in dry mixtures with DMPC and observations of probucol mixtures with DMPC under polarizing optics, the maximum solubility of probucol in DMPC is approx. 10 mole%. This concentration exceeds that required (approx. 0.5 mole%) to prevent peroxidation of 10 mole% arachidonic acid in DMPC liposomes for 30 min in the presence of 0.05 mM Fe(NH4)(SO4)2 at 4 degrees C. Thus, probucol has a limited solubility in saturated phosphatidylcholine bilayers, but is an effective antioxidant at concentrations lower than its maximum solubility.

Amphipathic α-helical peptides based on surfactant apoprotein SP-A

Abstract Three peptides based on the putative amphipathic helical region of the major pulmonary surfactant apoprotein (SP-A) were synthesized by solid-phase techniques, mixed with DPPC and tested for efficacy as lung surfactants in an in vitro adult rat lavaged lung model. The peptides correspond to residues 81–102 (SP-A 81–102 ) and 78–101 (SP-A 78–101 ) of the native human sequence and an analog with increased hydrophobicity, Leu 84,90 SP-A 78–101 . Neither native sequence was effective in simple mixtures with DPPC. However, substitution of leucine residues for Asp 84 and Thr 90 of SP-A 81–102 yielded a peptide which was active in mixtures with DPPC, restoring quasi-static lung compliance to 90% of the unlavaged value. In the absence of peptide, DPPC had no effect on the P-V curve of the lavaged lung. The activity of the Leu 84,90 analog correlated with an increased amphipathic α-helical potential and an improvement in several predictive parameters for lipid-binding. The similarities between this active peptide and other active amphipathic α-helical peptides lend support to the hypothesis that amphipathic α-helical potential and the size of the hydrophobic face are critical for functional synthetic surfactant peptides in simple mixtures with dipalmitoylphosphatidylcholine.

Kinetics of the interaction of amphipathic α-helical peptides with phosphatidylcholines

Abstract The rate of association of three amphipathic α-helical peptides with phosphatidylcholine liposomes was examined to provide more detailed information on the relationship between peptide length and the kinetics of lipid interactions. When added to dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC) liposomes from a guanidine-HCl solution, a ten residue peptide rapidly decreased the turbidity of the liposomes. However, a related 17-residue peptide had only a minimal effect on liposome turbidity. A 14-residue peptide was intermediate in effectiveness. Similarly, when liposomes were added to peptides dissolved in an aqueous buffer, the ten residue peptide but not the 17-residue peptide cleared the turbid liposomes and the 14-residue peptide was intermediate in efficacy. The rate of binding to the liposomes was compared with the three peptides by measurements of the kinetics of energy transfer from the single tryptophan residue of the peptides to a fluorescent probe in the bilayer interior. The trytophan residue of the ten residue peptide effectively transferred energy to the probe, while that of the 14-residue peptide was less effective. Little or no energy transfer was observed with the 17-residue peptide. The binding of the 10 residue peptide was rapid and complete within

Interactions of MDL 29,311 and probucol metabolites with cholesteryl esters.

The hypothesis that the efficacy of hydrophobic antioxidants in animal models of atherogenesis may, in part, be related to physical effects on cholesteryl esters in cells was probed with analogs and metabolites of probucol. The interactions of an effectivebis-thiomethane analog (MDL 29,311) and selected metabolites of probucol with cholesteryl oleate were examined by differential scanning calorimetry and polarized light microscopy. Like probucol, MDL 29,311 and the bisphenol metabolite decrease the liquid-crystalline phase transition enthalpy of cholesteryl oleate with increasing concentrations. At 20 mol%, no transition is detectable. By contrast, the spiroquinone metabolite of probucol and the diphenoquinone metabolite common to both molecules have minimal effects on the liquid-crystalline transitions of cholesteryl oleate. At 20 mol%, neither compound has as great an effect as 1 mol% MDL 29,311. Consistent with their effects on dry cholesteryl oleate, MDL 29,311 and the bisphenol metabolite convert lipid inclusions in cells supplemented with cholesterol to an isotropic physical state similar to that observed with probucol. The number of anisotropic inclusions in the cells decreases with increasing concentration in the medium in the range of 50 to 250 μg/mL. In cells fed with the spiroquinone or diphenoquinone metabolites, the lipid inclusions are liquid-crystalline and resemble those observed with cholesterol-fed controls. These data are interpreted in terms of a model in which hydrophobic antioxidants closely related to probucol disrupt the packing of cellular cholesteryl esters.

Fluorescence Quenching And Multiple-Frequency Phase Fluorometry In Peptide-Lipid Micelles

The conformation and environment of the single tryptophan residue of a model amphipathic helical peptide (MAP) in mixed micelles with dimyristoylphosphatidylcholine (DMPC) at different molar ratios was examined by circular dichroism (CD) and fluorescence spectroscopy. The peptide was synthesized by solid-phase techniques and micelles were formed by incubation. The radius of the disk-shaped micelles (measured by electron microscopy) decreases with increasing concentrations of MAP within the micelles. The alpha-helix content (measured by CD) of MAP increases and the fluorescence spectrum shifts to shorter wavelengths with larger diameter micelles. In acrylamide quenching experiments, the rate decreases and the activation energy increases with increasing micelle diameter. Multiple-frequency phase fluorometry data are consistent with either two environments or a Lorentzian distribution of a single environment for the tryptophan residue in the mixed micelles. The distribution depends upon the size of the micelle. With increasing micelle diameter, the relative fraction of the longer lifetime component or the width of the lifetime distribution is increased. These data suggest that the tryptophan residue environment within the model peptide-lipid micelle varies as a function of micellar size. A model for the lipid-peptide interaction in which the conformation of the peptide depends upon the degree of curvature at the edge of the micelle will be presented..