Larry R. McLean

Ascorbate and phenolic antioxidant interactions in prevention of liposomal oxidation

Efficient prevention of membrane lipid peroxidation by vitamin E (α-tocopherol) may involve its regeneration by vitamin C (ascorbate). Conceivably, the efficacy of antioxidants designed as therapeutic agents could be enhanced if a similar regeneration were favorable; thus, a model membrane system was developed which allowed assessment of interaction of phenolic antioxidants with ascorbate and ascorbyl-6-palmitate. Ascorbate alone (50–200 μM) potentiated oxidation of soybean phosphatidylcholine liposomes by Fe2+/histidine-Fe3+, an effect which was temporally related to reduction of Fe3+ generated during oxidation. Addition of 200 μM ascorbate to α-tocopherol-containing liposomes (0.1 mol%) resulted in marked, synergistic protection. Accordingly, in the presence but not absence of ascorbate, α-tocopherol levels were maintained relatively constant during Fe2+/histidine-Fe3+ exposure. Probucol (4,4′-[(1-methylethylidine)bis(thio)]bis[2,6-bis(1,1-dimethylethyl)]phenol), and antioxidant which prevents oxidation of low density lipoproteins, and its analogues MDL 27,968 (4,4′-[(1-methylethylidene)bis(thio)]-bis[2,6-dimethyl]phenol) and MDL 28,881 (2,6-bis(1,1-dimethylethyl)-4-[(3,7,11-trimethyldodecyl)thio]phenol) prevented oxidation but exhibited no synergy with ascorbate. Ascorbyl-6-palmitate itself was an effective antioxidant but did not interact synergistically with any of the phenolic antioxidants. Differential scanning calorimetry revealed significant differences among the antioxidants in their effect on the liquid-crystalline phase transition of dipalmitoyl phosphatidylcholine (DPPC) liposomes. Both α-tocopherol and MDL 27,968 significantly reduced the phase transition temperature and the enthalpy of the transition. MDL 28,881 had no effect while probucol was intermediate. The potential for ascorbate or its analogues to interact with phenolic antioxidants to provide a more effective antioxidant system appears to be dictated by structural features and by the location of the antioxidants in the membrane.

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.

Cholesterol interaction with recombinant human sterol carrier protein-2

The interaction of human recombinant sterol carrier protein-2 (SCP-2) with sterols was examined. Two independent ligand binding methods, Lipidex 1000 binding of [3H]cholesterol and a fluorescent dehydroergosterol binding assay, were used to determine the affinity of SCP-2 for sterols. Binding analysis indicated SCP-2 bound [3H]cholesterol and dehydroergosterol with aKd of 0.3 and 1.7 μM, respectively, and suggested the presence of a single binding site. Phase fluorometry and circular dichroism were used to characterize the SCP-2 sterol binding site. Alterations in dehydroergosterol lifetime, SCP-2 tryptophan lifetime, and SCP-2 tryptophan quenching by acrylamide upon cholesterol binding demonstrated a shielding of the SCP-2 tryptophan from the aqueous solvent by bound sterol. Differential polarized phase fluorometry revealed decreased SCP-2 tryptophan rotational correlation time upon cholesterol binding. Circular dichroism of SCP-2 indicated that cholesterol elicited a small decrease in SCP-2 alpha helical content. The data suggest that SCP-2 binds sterols with affinity consistent with a lipid transfer protein that may act either as an aqueous carrier or at a membrane surface to enhance sterol desorption.

Promotion of β-structure by interaction of diabetes associated polypeptide (amylin) with phosphatidylcholine

The interaction of the diabetes associated polypeptide (amylin) with dimyristoylphosphatidylcholine (DMPC) was assessed by measurements of turbidity (absorbance at 400 nm) and secondary structure by CD spectroscopy. In trifluoroethanol, human amylin adopts a highly alpha-helical conformation while the rat peptide is less structured. In water, the rat peptide is largely disordered and the human peptide exhibits a combination of alpha- and beta-structures. Mixtures of DMPC and the rat peptide have no effect on either the turbidity of the DMPC or the CD spectrum of the peptide. By contrast, mixtures of the human peptide with DMPC form relatively clear mixtures similar to those observed with amphipathic alpha-helical peptides, but the structure adopted, based on the CD spectrum, is largely beta. These data demonstrate that fundamental differences in the structures adopted by amylins from human and rat species exist in mixtures with DMPC and suggest that these differences may be related to the formation of amyloid fibrils in the human amylin peptide which are not observed in the rat peptide.

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.

Mechanism of action of lipoprotein lipase

Publisher Summary This chapter describes several of the methods used to determine the mechanism of action of lipoprotein lipase purified from bovine milk and the effect of apoC-II on lipoprotein lipase (LpL) catalysis. LpL is located primarily in adipose tissue, lung, muscle, and lactating mammary gland. The chapter discusses the characteristic properties of the enzyme regardless of tissue source: (1) inhibition of enzyme activity by 1 M NaCI and protamine sulfate; (2) enhancement of enzyme activity by apolipoprotein C-II (apoC-II), a protein constituent of triglyceride-rich lipoproteins and high-density lipoproteins (HDL); and (3) a pH optimum for triacylglycerol substrates. The chapter also gives the range of molecular weights of LpL purified from several sources; the values range from 34 to 78,000. Although the primary physiological substrates for LpL are long chain triacylglycerols and phospholipids, the enzyme also catalyzes the hydrolysis of water-soluble short chain phosphatidylcholines, triacylglycerols, and p-nitrophenyl esters. The enzyme is specific for the sn -1 ester bond of triacylglycerols and the sn -1 ester bond of phospholipids.

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.

Lipid and membrane interactions of neuropeptide Y

The interactions of neuropeptide Y with dimyristoylphosphatidylcholine and cell membranes were examined by several physical techniques to probe the potential role of its putative C-terminal amphipathic alpha-helix. Neuropeptide Y binding was demonstrated by a rapid release of entrapped 6-carboxyfluorescein and a rapid decrease in the turbidity of dimyristoylphosphatidylcholine liposomes. In addition, an increase in tyrosine fluorescence intensity and an increase in the anisotropy of diphenylhexatriene in dimyristoylphosphatidylcholine liposomes was observed. In isolated, aortic smooth muscle cell membranes, the anisotropy of diphenylhexatriene increased as a function of added neuropeptide Y. The concentration range (low microM) over which neuropeptide Y increases the polarization of diphenylhexatriene in cell membranes is similar to the range in which it inhibits isoproterenol-stimulated cAMP accumulation. This inhibition is not affected by pertussis toxin, nor does neuropeptide Y cause the release of preloaded [3H]adenine from cells into the medium. These data suggest that neuropeptide Y contains an amphipathic alpha-helical region which interacts with lipids in much the same way as the amphipathic alpha-helical regions of the plasma apolipoproteins and that the inhibition of isoproterenol-stimulated cAMP accumulation at low microM concentrations of peptide may be the result of an alteration in the cell membrane bilayer structure.

Human postheparin plasma lipoprotein lipase and hepatic triglyceride lipase.

Publisher Summary Lipoprotein lipase (LpL) and hepatic triglyceride lipase (H-TGL) are the major lipolytic enzymes responsible for the metabolism of lipoproteins in the circulation. These enzymes catalyze the hydrolysis of the sn-1 ester bond of lipoprotein di- and triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines. With lipoprotein substrates, the phospholipase A1 activity is approximately 1 % of that for triacylglycerols. The major lipoprotein substrates for LpL are chylomicrons and very low-density lipoproteins (VLDL), whereas lipoproteins of intermediate-density (IDL) and highdensity (HDL), particularly the HDL2 subfraction, are the preferred lipoprotein substrates for H-TGL. These lipolytic enzymes are anchored to the plasma membrane of endothelial cells by electrostatic interactions with heparan sulfate proteoglycans. Intravenous heparin administration releases LpL and H-TGL from these sites, resulting in lipoprotein triglyceride hydrolysis, thus the term postheparin plasma lipolytic activity (PHLA). The major tissue sources of LpL are adipose tissue and muscle. H-TGL is synthesized in the periportal hepatocyte. The primary structures of human LpL and H-TGL are deduced from the respective complementary DNAs (cDNAs). LpL contains 448 amino acids, whereas H-TGL has 477 residues; the proteins are 47% homologous. The enzymes belong to a superfamily of lipases that also includes pancreatic lipase.