Biomedical subjects

J P Segrest

Publications and source records attributed to J P Segrest.

At least 37 records · Page 2Linked to original sources

PubMed1998

Potencies of lipoproteins in fasting and postprandial plasma to accept additional cholesterol molecules released from cell membranes.

To investigate the role of various lipoproteins in plasma to promote cholesterol efflux from cell membranes, potencies of lipoproteins in normolipidemic fasting and postprandial (PP) plasmas to accept additional cholesterol molecules from cell membranes were determined. We used red blood cells (RBCs) and lipoproteins in fresh blood as donors and acceptors of cell membrane cholesterol, respectively. When fresh fasting plasma (n=24) containing active lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester transfer proteins (CETP) was incubated with a 3-fold excess of autologous RBCs at 37 degrees C for 18 hours, plasma cholesterol levels increased by 19.6% (38.5+/-14.2 mg/dL) owing to an exclusive increase in the CE level. Very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) fractions retained 48.1%, 26.3%, and 25.6% of the net cholesterol mass increase in fasting plasma, resulting in 91%, 8%, and 21% increases in their cholesterol contents, respectively. The PP plasma was 1.3-fold more potent than fasting plasma in promoting cholesterol efflux from RBCs by associating excess cholesterol with chylomicrons, resulting in a 356% increase in the cholesterol content of chylomicrons. These increases in lipoprotein cholesterol content indicate that chylomicrons were about 3.9x, 44x, and 17x more potent than fasting VLDL, LDL, and HDL, respectively, in accepting additional cholesterol molecules released from RBCs. The capacity of PP plasma to promote cholesterol efflux from RBCs was significantly correlated with plasma cholesterol levels (r=0.60, P<0.005), triglycerides (r=0.68, P<0.001), chylomicrons (r=0.90, P<0.001), VLDL (r=0.65, P<0.001), and LDL (r=0.47, P<0.025) but not with the levels of HDL (r= -0.34, P<0.20). In fasting plasma containing a low level of VLDL and HDL, isolated chylomicrons supplemented to the plasma were approximately 9x more potent than HDL in boosting the capacity of plasma to promote cholesterol efflux from RBCs. This study indicates that chylomicrons in PP plasma are the most potent ultimate acceptors of cholesterol released from cell membranes and that a low HDL level is not a factor that limits the ability of PP plasma to promote cholesterol efflux from cell membranes. Our data obtained from an in-vitro system suggest that PP chylomicrons may play a major role in promoting reverse cholesterol transport in vivo, since the transfer of cholesterol from cell membranes to chylomicrons will lead to the rapid removal of this cholesterol by the liver. HDL in vivo may promote reverse cholesterol transport by enhancing the rapid removal of chylomicrons from the circulation, since the rate of clearance of chylomicrons is positively correlated with the HDL level in plasma.

Adult↗

PubMed1998

Apolipoprotein B-100: conservation of lipid-associating amphipathic secondary structural motifs in nine species of vertebrates.

Development of a computer program called LOCATE allowed us to show that human apolipoprotein B-100 is composed of five domains, NH2-alpha1-beta1-alpha2-beta2-alpha3-COOH, enriched, alternately, in amphipathic alpha helixes and amphipathic beta strands. Using updated versions of this program, here we compare the complete sequence of human apolipoprotein B-100 with partial sequences from eight additional species of vertebrates (chicken, frog, hamster, monkey, mouse, pig, rat, and rabbit). The lipid-associating amphipathic alpha helixes cluster in domains alpha2 (between residues 2075 +/- 25 and 2575 +/- 25) and alpha3 (between residues 4100 +/- 100 and 4550 +/- 50) in all species for which those regions have been sequenced but with little conservation of individual helixes. Lipid-associating amphipathic beta strands cluster in domains beta1 (approximately residues 827-2000) and beta2 (approximately residue 2571 to residue 4000 +/- 50) in all species for which these regions have been sequenced, with conservation of several individual amphipathic beta strands. Hydrophobic segments are present in apolipoprotein B-100 sequences of all nine species but the frequency of occurrence is no greater than generally found in beta sheet-containing proteins. We conclude that four alternating lipid-associating domains, -beta1-alpha2-beta2-alpha3-COOH, are common supramolecular features of apolipoprotein B-100 in nine vertebrate species.

Amino Acid Sequence↗

PubMed1997

Transgenic mice expressing human ApoB95 and ApoB97. Evidence that sequences within the carboxyl-terminal portion of human apoB100 are important for the assembly of lipoprotein.

The structural features of apolipoprotein (apo) B that are important for its covalent linkage to apo(a) to form lipoprotein(a) (Lp(a)) are incompletely understood. Although apoB100 cysteine 4326 is required for the disulfide linkage with apo(a), other structural features, aside from a single free cysteine residue, must be important for apoB's initial interaction with apo(a) and for facilitating the formation of the disulfide bond. To determine if sequences carboxyl-terminal to cysteine 4326 affect the efficiency of Lp(a) formation, we used "pop-in, pop-out" gene targeting in a human apoB yeast artificial chromosome to introduce nonsense mutations into exon 29 of the apoB gene. The mutant yeast artificial chromosomes, which coded for the truncated versions of human apoB, apoB95, and apoB97, were then used to express these mutant forms of apoB in transgenic mice. As judged by in vitro assays of Lp(a) formation, apoB95 (4330 amino acids) formed a small amount of Lp(a) but did so slowly. In contrast, apoB97 (4397 amino acids) formed Lp(a) rapidly, although not quite as rapidly as the full-length apoB100 (4536 amino acids). These results were supported by an analysis of double-transgenic mice expressing both human apo(a) and either apoB95 or apoB97. In mice expressing both apoB95 and apo(a), there was only a trace amount of Lp(a) in the plasma, and most of the apo(a) was free, whereas in mice expressing both apoB97 and apo(a), virtually all of the apo(a) was bound to apoB97 in the form of Lp(a). These results show that sequences carboxyl-terminal to apoB95 (amino acids 4331-4536) are not absolutely required for Lp(a) formation, but this segment of the apoB molecule, particularly residues 4331-4397, is necessary for the efficient assembly of Lp(a).

Animals↗

PubMed1997

Role of lipids in the permeabilization of membranes by class L amphipathic helical peptides.

We studied the mechanism of membrane permeabilization by the 18L model peptide (GIKKFLGSIWKFIKAFVG), which features the consensus class L sequence averaged from the number of naturally occurring lytic peptides. Two aspects of membrane lipid composition significantly affected peptide-membrane interactions: the presence of acidic lipids and, in zwitterionic membranes, and the presence of nonbilayer forming lipids. In zwitterionic membranes, 18L peptide destabilizes the membrane, leading to a transient formation of large defects in the membrane which result generally in contents leakage, but in the presence of bilayer-bilayer contact can alternatively lead to vesicle fusion. In membranes containing acidic lipids (DOPC:DOPG, DOPG), 18L caused leakage but not fusion, probably due to mutual repulsion of acidic vesicles. While the extent of contents leakage was approximately the same as for zwitterionic membranes, the kinetics of leakage could be resolved only by using stopped-flow, leakage being essentially complete within the first minute. Previously, we reported that apolipoprotein (class A) and lytic (class L) peptide analogs have opposing effects on some properties of biological membranes. This reciprocal effect of 18L and Ac-18A-NH2, class A model peptide, is restricted to membranes with a high propensity for nonbilayer phase formation (DOPE, Me-DOPE, DOPC:DOPE, DOPC:Me-DOPE). The decrease in the content of nonbilayer phase forming lipid or the addition of acidic lipids reduces or eliminates the reciprocal effects. This suggests the importance of nonbilayer phase propensity for certain functions of biological membranes.

Amino Acid Sequence↗

PubMed1997

Interaction of class A amphipathic helical peptides with phospholipid unilamellar vesicles.

The exchangeable apolipoproteins are important in determining the structure/function properties of lipoproteins. These proteins typically contain varying amounts of amphipathic helices. Five model peptides, 18A, Ac-18A-NH2, Ac-18R-NH2, 37pA, and 37aA, have been designed to investigate variations of the amphipathic alpha-helix structural motif on their lipid-binding properties. These include the 18-residue peptides, 18A and Ac-18A-NH2, examples of class A helices, and Ac-18R-NH2, which has the positions of acidic and basic residues interchanged relative to 18A. Three larger peptides were also studied: 36A, a dimer of 18A, 37pA and 37aA, dimers of 18A coupled by Pro (18A-Pro-18A) and Ala (18A-Ala-18A), respectively. We report here the results of a thermodynamic characterization of the binding properties of these peptides to small unilamellar vesicles of POPC. Partition coefficients, Kp, were determined by fluorescence spectroscopy and binding enthalpies, deltaH, by titration calorimetry. These parameters were used to obtain the free energies, deltaG0, and entropies, deltaS0, of binding. The results of this study indicate Kp values on the order of 10(5), with interactions being enthalpically but not entropically favored in all cases. The presence of positively charged residues at the interface (18A and Ac-18A-NH2) enhances binding but has little effect on the extent of bilayer penetration. The presence of tandem repeats decreases lipid affinities for these small, highly curved bilayers. Our results are consistent with the idea that interaction appears to be confined largely to the surface, with some degree of penetration of the hydrophobic face of the helix into the interior of the bilayer.

Calorimetry↗

PubMed1997

Effect of vesicle size on their interaction with class A amphipathic helical peptides.

Throughout the life span of a lipoprotein particle, the type and number of exchangeable apolipoproteins on its surface varies with particle size, suggesting a role of surface curvature on the lipid-binding properties of these proteins. Peptides 18A, Ac-18A-NH2, Ac-18R-NH2, 37pA, and 37aA have been designed to investigate the lipid-binding properties of the amphipathic alpha-helix structural motif that appears to modulate the lipid-binding properties of the exchangeable plasma apolipoproteins. We report here the results of a quantitative thermodynamic characterization of the effects of modifying helix length and of varying both the location of charged residues about the polar face of the peptides and vesicle size on the lipid affinity and depth of bilayer penetration for model amphipathic alpha-helices. Partition coefficients, Kp, were determined by fluorescence spectroscopy, and binding enthalpies, deltaH, by titration calorimetry. The results indicate that Kp values are on the order of 10(5), with similar deltaG(o) values for the interactions of the peptides with vesicles of various sizes. It appears that a class A motif and increased alpha-helical content optimize binding for 18-residue peptides. The interactions of the model peptides with 20 nm SUV are enthalpically driven with small, negative entropy changes; however, interactions for larger vesicles are entropically driven, likely due to disordering of bilayer hydrocarbon chains. Thermodynamic data indicate that 37pA and 37aA induce greater disordering of bilayer hydrocarbon chains than Ac-18A-NH2. The results of this study suggest that the type of interaction, i.e., enthalpically or entropically driven, may be modulated by the lateral compressibility of the bilayer membrane.

Acrylamide↗

PubMed1997

Quantification of HDL2 and HDL3 cholesterol by the Vertical Auto Profile-II (VAP-II) methodology.

Of the several existing methods for quantification of major subspecies of high density lipoprotein (HDL), HDL2 and HDL3, the methods based upon double precipitation are particularly useful for large-scale studies or for routine assay because of their high speed and low cost. The Vertical Auto Profile-II (VAP-II) method developed in our laboratory primarily for the direct single test measurement of cholesterol (C) in all major lipoproteins, including Lp[a] and IDL, is rapid, highly sensitive, and suitable for large-scale studies. Here we describe the modification of this procedure so as to be able to quantify both HDL2- and HDL3-C in addition to all major lipoproteins without any additional assay steps, time, or cost. The VAP-II procedure was validated by comparison with four other methods using plasma samples obtained from 35 healthy subjects: 1) HDL-VAP-II (a variation of the VAP-II procedure designed specifically to separate HDL subspecies); 2) dextran sulfate (DS)/Mg2+ double precipitation method performed at Northwest Lipid Research Laboratories (NWLRL), Seattle, WA; 3) 4-30% polyacrylamide-agarose (4/30 PAA) nondenaturing gradient gel electrophoresis (GGE); and 4) analytical ultracentrifugation (AUC), with both GGE and AUC performed at the Donner Laboratory, University of California at Berkeley. Both HDL2- and HDL3-C measurements by VAP-II correlated well with the measurements by all comparison methods (r for HDL3-C: HDL-VAP-II, 0.948; NWLRL, 0.947; GGE, 0.861; and AUC, 0.706, and r for HDL2-C: HDL-VAP-II, 0.867; NWLRL, 0.854; GGE, 0.885; and AUC, 0.721). The measurements of HDL2- and HDL3-C by the VAP-II method are reproducible, with the long-term between-rotor CV of 5.0% for HDL3-C and 9.0% for HDL2-C.

Blood Chemical Analysis↗

PubMed1996

Only the two end helixes of eight tandem amphipathic helical domains of human apo A-I have significant lipid affinity. Implications for HDL assembly.

Human apolipoprotein A-I (apo A-I) possesses multiple tandem repeating 22-mer amphipathic alpha-helixes. Computer analysis and studies of model synthetic peptides and recombinant protein-lipid complexes of phospholipids have suggested that apo A-I interacts with HDL surface lipids through cooperation among its individual amphipathic helical domains. To delineate the overall lipid-associating properties of apo A-I, the first step is to understand the lipid-associating properties of individual amphipathic helical domains. To this end, we synthesized and studied each of the eight tandem repeating 22-mer domains of apo A-I: residues 44-65, 66-87, 99-120, 121-142, 143-164, 165-186, 187-208, and 220-241. Among the 22-mers, only the N- and C-terminal peptides (44-65 and 220-241) were effective in clarifying multilamellar vesicles (MLVs) of dimyristoylphosphatidylcholine (DMPC). These two peptides also exhibited the highest partition coefficient into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine liposomes, the highest exclusion pressure for penetration into an egg yolk phosphatidylcholine monolayer, and the greatest reduction in the enthalpy of the gel-to-liquid crystalline phase transition of DMPC MLVs. These results suggest that the strong, lipid-associating properties of apo A-I are localized to the N- and C-terminal amphipathic domains. Although each of the eight peptides studied has an amphipathic structure, models based on changes in residual effective amino acid hydrophobicity resulting from differing depths of helix penetration into the lipid are best able to explain the high lipid affinity possessed by the two terminal domains. Differential scanning calorimetry (DSC) studies showed that on a molar basis, apo A-I is about 10 times more effective than the most effective peptide analyzed in reducing the enthalpy of the gel-to-liquid crystalline phase transition of DMPC MLVs. Because previous proteolysis experiments coupled with the present DSC results suggest that the lipid-associating domains of apo A-I are distributed throughout the length of the 243 amino acid residues, we propose that the terminal amphipathic helical domains are involved in the initial binding of apo A-I to the lipid surface to form HDL particles, followed by cooperative binding of the middle six amphipathic helical domains, perhaps aided by salt-bridge formation between adjacent helixes arranged in an antiparallel orientation.

Apolipoprotein A-I↗

PubMed1996

Probing structure and function of VLDL by synthetic amphipathic helical peptides.

Class A amphipathic helical peptides have been shown to mimic many properties of exchangeable apolipoproteins. The three analogs of the class A amphipathic peptides were used to probe the structure and function of human very low density lipoproteins (VLDL): 1) 18 residue peptide possessing a single helical domain (18A) with the sequence Asp-Trp-Leu-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-Leu-Lys-Glu-Ala-Phe; 2) two domains of 18A separated by a Pro (37pA); and 3) and 18A analog with the end groups protected to increase helicity (Ac-18A-NH2). Upon incubation of the peptides with VLDL at a peptide to VLDL, (protein) ratio of 1:1, the 37pA and Ac-18A-NH2 were able to displace most of apolipoprotein (apo) Cs and E from VLDL without alteration in its lipid composition and morphology while 18A had minimal effect. The extent of displacement was a function of the peptide to VLDL ratio. The rank order of displaceability of apolipoproteins on VLDL was apoE > C-III > C-II. The displacement of apoE and/or Cs from VLDL by peptides variably affected the ability of VLDL to interact with purified bovine milk lipoprotein lipase (LpL) and cultured macrophages. Treatment of VLDL with Ac-18A-NH2 markedly lowered its reactivity to LpL and its ability to induce lipid accumulation in cultured macrophages: however, treatment of VLDL with 37pA or 18A only minimally lowered their abilities. Ac-18A-NH2 treatment of VLDL resulted in the increase of apparent K(m) and a decrease of Vmax for lipoprotein lipase (LpL)-catalyzed hydrolysis of VLDL triglycerides. When an artificial triglyceride emulsion was used as a substrate of LpL, 37pA, but not Ac-18A-NH2, activated LpL. The above data indicate that 1) amphipathic helical peptides can alter the metabolic and functional properties of VLDL by dissociating the functionally important exchangeable apolipoproteins from VLDL as well as by acting as a functional element of VLDL after their incorporation; and 2) the class A amphipathic peptides having different lipid-associating properties exert significantly different effect on VLDL function.

Amino Acid Sequence↗

PubMed1995

Identification and characterization of a new human gene (APOC4) in the apolipoprotein E, C-I, and C-II gene locus.

We have identified and characterized a previously unreported human gene that is found within the apolipoprotein (apo) E/C-I/C-II gene locus. On the basis of its location and its properties, this new gene has been designated APOC4. Nucleotide sequence analysis of genomic DNA and liver cDNA clones revealed a 3.3-kb gene consisting of three exons and two introns. Its 3' terminus lies 555 bp upstream of APOC2, giving both genes the same transcriptional orientation. The promoter of the APOC4 gene lacks a typical TATA box, consistent with an apparent heterogeneity in transcription start sites. RNase protection analysis indicated relatively low apoC-IV mRNA levels in human liver, compared to apoC-II mRNA levels. The predicted apoC-IV protein sequence, comprising 127 amino acid residues, contains a putative 25-residue signal peptide and two potential amphipathic alpha-helical domains. Amino acid sequence comparisons indicate a limited homology between apoC-IV and either apoC-I or apoC-II. Since its hepatic expression and predicted protein structure are characteristic of the other genes in this cluster, we propose that the APOC4 gene is a member of the apolipoprotein gene family.

Amino Acid Sequence↗

PubMed1995

Microenvironments of basic amino acids in amphipathic alpha-helices bound to phospholipid: 13C NMR studies using selectively labeled peptides.

The lipid-binding properties of serum apolipoproteins are mediated by class A amphipathic alpha-helices in which basic and acidic amino acid residues tend to be localized at the helix polar-nonpolar boundary and the center of the polar face, respectively. To better understand the role of the interfacial basic residues, the microenvironments and titration characteristics of lysine (Lys) residues have been examined using two 18-residue peptides. The parent 18A molecule (AspTrpLeuLysAlaPheTyrAspLysValAlaGluLysLeuLysGluAlaPhe) binds phospholipid relatively well because of its class A amphipathic helix, whereas 18R, which has the positions of basic and acidic residues reversed, binds to phospholipid relatively weakly. Lys residues were 13C-dimethylated, and NMR spectra were obtained of the peptides complexed with dimyristoylphosphatidylcholine (1/1 (w/w) DMPC/peptide). The four [13C]-epsilon-dimethyl-Lys in 18A gave four resonances at chemical shifts (delta) of 42-43 ppm, whereas only a single resonance at delta 42.70 ppm was observed from the four Lys in 18R. Measurements of delta as a function of pH gave pKa values. The four Lys in 18R had a common pKa of 10.3, indicating that the Lys in the polar face of the amphipathic helix have the pKa expected for noninteracting, fully hydrated groups. In contrast, the four Lys in Ac-18A-NH2 had pKa's in the range 9.4-11.0, indicating that they exist in various microenvironments at the polar-nonpolar boundary of the 18A helix. To examine individual Lys, 18A was synthesized with various Lys selectively labeled. Lys-4, -9, -13, and -15 in Ac-18A-NH2 associated with DMPC had pKa values of 11.0, 9.4, 9.4, and 10.3, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Efflux of cellular cholesterol and phospholipid to lipid-free apolipoproteins and class A amphipathic peptides.

The mechanism(s) by which lipid-free apolipoprotein (apo) AI is able to stimulate efflux of cholesterol and phospholipid from cells in cultures has (have) been examined. This process was found to be enhanced when macrophages were enriched with cholesterol. There were 12- and 4-fold increases in cholesterol and phospholipid efflux, respectively, from cholesterol-enriched mouse macrophages when compared to cells not loaded with cholesterol. This enhancement in cholesterol efflux to lipid-free apo AI from macrophages enriched with cholesterol was found to be controlled by the level of free cholesterol in the cells. When cholesterol-enriched mouse macrophages were exposed to lipid-free apo AI at 20 micrograms/mL (706 nM), there was significant efflux of [14C]cholesterol and [3H]phospholipid (20% +/- 0.5%/24 h and 6% +/- 0.3%/24 h, respectively). In comparison, HDL at equivalent protein concentrations only stimulated 11% and 4% efflux of cholesterol and phospholipid, respectively. Synthetic peptides containing amphipathic helical segments that mimic those present in apo AI were used to examine the structural features of the apoprotein which stimulate lipid efflux. Peptides containing only one (18A) or two (37pA) amphipathic helical segments stimulated as much cholesterol efflux from both mouse macrophages and L-cells as apo AI. The order of efficiency, as assessed by the mass concentration at which half-maximal efflux was reached (EC50), was apo AI > 37pA > 18A, indicating that acceptor efficiency was dependent on the number of amphipathic helical segments per molecule. When the helical content of 18A was increased by neutralizing the charges at the ends of the peptide (Ac-18A-NH2), there was a substantial increase in the efficiency for cholesterol efflux (EC50 18A = 17 micrograms/mL vs Ac-18A-NH2 = 6 micrograms/mL). In contrast, when the amphipathicity of the helix in 18A was decreased by scrambling the amino acid sequence, thereby reducing its lipid affinity, cholesterol and phospholipid efflux were not stimulated. The efficiency with which the peptides stimulated cholesterol efflux was in order of their lipid affinity (37pA > Ac-18A-NH2 > 18A), and this order was similar for phospholipid efflux. The time course of lipid release from mouse macrophages and L-cells indicated that phospholipid appeared in the extracellular medium before cholesterol. These results suggest that the apo AI or peptides first interacted with the cell to form protein/phospholipid complexes, that could then accept cholesterol.

Amino Acid Sequence↗

PubMed1995

Molecular basis for prokaryotic specificity of magainin-induced lysis.

Magainins and mastoparans are examples of peptide antibiotics and peptide venoms, respectively. They have been grouped together as class L amphipathic helixes [Segrest, J.P., et al. (1990) Proteins 8, 103-117] because of similarities in the distribution of Lys residues along the polar face of the helix. Class L venoms lyse both eukaryotic and prokaryotic cells whereas class L antibiotics specifically lyse bacteria. The structural basis for the specificity of class L antibiotics is not well understood. Sequence analysis showed that class L antibiotics have a Glu residue on the nonpolar face of the amphipathic helix; this is absent from class L venoms. We synthesized three model class L peptides with or without Glu on the nonpolar face: 18LMG (LGSIWKFIKAFVGGIKKF), [E14]18LMG and [G5,E14]18LMG. Hemolysis, bacteriolysis, and bacteriostasis studies using these peptides showed that the specificity of lysis is due to both the presence of a Glu residue on the nonpolar face of the helix and the bulk of the nonpolar face. Studies using large unilamellar phospholipid vesicles showed that the inclusion of cholesterol greatly inhibited leakage by the two Glu-containing peptides. These results cannot be attributed to changes in the phase behavior of the lipids caused by the inclusion of cholesterol or to differences in the secondary structure of the peptides. These results suggest that eukaryotic cells are resistant to lysis by magainins because of peptide-cholesterol interactions in their membranes that inhibit the formation of peptide structures capable of lysis, perhaps by hydrogen bonding between Glu and cholesterol. Bacterial membranes, lacking cholesterol, are susceptible to lysis by magainins.

Amino Acid Sequence↗

PubMed1995

Effect of the cholesterol content of reconstituted LpA-I on lecithin:cholesterol acyltransferase activity.

The production of cholesteryl ester (CE) by lecithin: cholesterol acyl transferase (LCAT) is elevated significantly in hyperlipidemic subjects at high risk for coronary artery disease. To elucidate the molecular events involved, the relationship between LCAT activation and apolipoprotein (apo) A-I charge and structure in high density lipoproteins (HDL) has been studied in both native HDL and homogeneous recombinant HDL (Lp2A-I) particles containing apoA-I, palmitoyloleoyl phosphatidylcholine and cholesterol. Increasing the cholesterol content of discoidal Lp2A-I from 4 to 26 molecules/particle raises the maximum rate of cholesterol esterification by LCAT (Vmax) from 3.1 to 9.2 nmol CE/h/unit of LCAT and increases the apparent Km from 0.5 to 3.5 microM cholesterol. Similarly, increasing the cholesterol content in triolein core-containing Lp2A-I (4-18 molecules/particle) and in native HDL3 (12-21 molecules/particle) also significantly increases the Vmax for LCAT (2.8-7.7 and 0.5-3.6 nmol CE/h, respectively) and raises the Km values (7.6-36.9 and 7.3-8.5 microM cholesterol, respectively). In contrast, changes in the cholesterol content of native and recombinant HDL have no significant effect on the apparent Km values when expressed in terms of the concentration of either apoA-I or palmitoyloleoyl phosphatidylcholine. This appears to indicate that interfacial cholesterol content has no effect on the binding affinity of LCAT to different LpA-I particles but directly affects catalysis by modulating the interaction of cholesterol molecules with the active site of LCAT. Increasing the cholesterol content of the different HDL particles progressively increases the particle net negative charge, and these changes in apoA-I charge are strongly correlated with both the Vmax and apparent Km values for LCAT. This suggests that the conformation and charge of apoA-I play a central role in LCAT activation and that these parameters are influenced by the amount of cholesterol in the surface of HDL particles.

Apolipoprotein A-I↗

PubMed1995

Effect of the arrangement of tandem repeating units of class A amphipathic alpha-helixes on lipid interaction.

Exchangeable apolipoproteins possess tandem repeating units of class A amphipathic helical segments and many of them are linked together by proline residues. To understand the optimal arrangement of the amphipathic helixes for lipid association, we have studied the interactions of three model class A amphipathic helical peptides with lipids. The three peptides are: 37pA, a dimer of 18A (DWLKAFYDKVAEKLKEAF) linked together by a Pro (18A-Pro-18A); 37aA, a dimer of 18A linked together by an Ala (18A-Ala-18A); and 36A, a dimer of 18A without any linker residue (18A-18A). Circular dichroism (CD) spectra showed that the peptides are predominantly alpha-helical in aqueous and lipid environments. Temperature dependent CD studies indicated that in buffer helix stability decreases in the order 36A > 37aA > 37pA; however, in the presence of dimyristoyl phosphatidylcholine (DMPC), the above order is reversed. The retention times of the peptides on a C18 reversed-phase high performance liquid chromatography column decreased in the order 36A > 37aA > 37pA, consistent with the lengths of the nonpolar faces of the alpha-helixes being in the same order; the retention time of the parent 18A was shorter than 37pA. While 37pA adsorbed to egg phosphatidylcholine monolayers most strongly, the degree and rate of association of 36A were significantly lower. Differential scanning calorimetry indicated that, while 37pA was most effective in reducing the enthalpy of the gel to liquid-crystalline phase transition of DMPC multilamellar vesicles, 36A was least effective; 36A was even less effective than 18A. Fluorescence quenching experiments with iodide and acrylamide indicated that, in the presence of DMPC, Trp residues in 36A are most exposed to the quenchers while in 37pA they are least exposed. In the presence of DMPC, shielding of Trp in 18A from the quenchers was more than that observed with Trp residues in 36A. The results of this study suggest that the arrangement of tandem repeating amphipathic helical units which results in the formation of a class A amphipathic helix with a nonpolar face longer than five or six turns reduces the ability of the helix to associate with phospholipid.

Amino Acid Sequence↗

PubMed1995

Mechanisms for the modulation of membrane bilayer properties by amphipathic helical peptides.

The amphipathic helix, in which hydrophobic and hydrophilic residues are grouped on opposing faces, is a structural motif found in many peptides and proteins that bind to membranes. One of the physical properties of membranes that can be altered by the binding of amphipathic helices is membrane monolayer curvature strain. Class A amphipathic helices, which are present in exchangeable plasma lipoproteins, can stabilize membranes by reducing negative monolayer curvature strain; proline-punctuated class A amphipathic helical segments are particularly effective in this regard. This property is suggested to be associated with some of the beneficial biological effects of this protein. On the other hand, lytic amphipathic helical peptides can act by increasing negative curvature strain or by forming pores composed of helical clusters. Thus, different amphipathic helical peptides can be membrane stabilizing or be lytic to membranes, depending on the structural motif of the helix, which in turn determines the nature of its association with membranes. Features of these peptides that are responsible for their specific properties are discussed.

Amino Acid Sequence↗

PubMed1995

A prospective, randomized trial of phenytoin in nonepileptic subjects with reduced HDL cholesterol.

Observational studies have demonstrated a positive association between phenytoin use and HDL cholesterol (HDL-C). Our goal was to determine whether phenytoin raises HDL-C in nonepileptic subjects at risk for coronary artery disease. We performed a double-blind, placebo-controlled, parallel-group study in 41 subjects with reduced levels of HDL-C. Subjects were placed on an American Heart Association Step I diet and were randomized to receive either phenytoin or placebo for 3 months. Serum levels of phenytoin were monitored and adjusted to between 7.5 and 15 micrograms/mL. Fasting levels of lipids and lipoproteins were determined twice at baseline (weeks -2 and -1) and during the treatment phase of the study (weeks 11 and 12). Compared with dietary baseline, phenytoin-treated subjects experienced significant paired percent increases in total HDL-C (12.4%; P < .01), an effect confined to the HDL2 subfraction (137%; P < .01). The paired percent increases in HDL-C and HDL2 levels remained significant after adjustment for placebo (P < .05, P < .025, respectively). There were no significant differences in the paired percent changes from dietary baseline in total cholesterol, triglyceride, or LDL cholesterol levels between placebo and phenytoin-treated groups. The significant paired percent increases in total HDL-C and HDL2 from dietary baseline suggest a potential role for phenytoin in subjects with reduced levels of HDL-C.

Adult↗

PubMed1995

Lipolysis-induced partitioning of free fatty acids to lipoproteins: effect on the biological properties of free fatty acids.

Free fatty acids (FFA) released during the lipolysis of triglyceride (TG)-rich lipoproteins in vivo are generally believed to be bound to serum albumin. When hypertriglyceridemic (HTG) sera were lipolyzed in vitro by purified bovine milk lipoprotein lipase (LpL), there was an 11- to 18-fold increase in serum FFA levels, and a major portion (> 80%) of the FFA in serum was partitioned to lipoprotein fractions. The greatest portion (33%) of FFA in lipolyzed HTG serum was associated with newly formed flocculent remnants that banded just below low density lipoproteins (LDL) in the density gradient tube. Very low density lipoprotein (VLDL), LDL, and high density lipoprotein (HDL) fractions in lipolyzed HTG serum contained 18- to 29-times more FFA molecules than those in prelipolysis serum. Analysis of the fatty acyl chain composition of FFA in lipolyzed HTG serum showed that the extent of partitioning of saturated FFA into the lipoprotein fractions relative to that of polyunsaturated FFA was about 4.5- to 11-times greater than that partitioned into the free protein fraction; most (84%) of FFA partitioned into flocculent remnants were saturated fatty acids. In vivo lipolysis of TG-rich lipoproteins in HTG subjects, induced by heparinization, resulted in only a small (2.8-fold) increase in serum FFA and little or no increase in the partitioning of FFA to lipoproteins. However, in vitro incubation of the postheparin serum at 37 degrees C for 90 min resulted in a 2.9- to 6.8-fold increase in the serum FFA level and the partitioning of > 66% of total serum FFA into lipoprotein fractions. Studies of the interaction of various plasma fractions from control and in vitro lipolyzed HTG serum with cultured mouse peritoneal macrophages (MPM) showed that FFA partitioned to lipoprotein fractions were highly cytotoxic to cultured MPM, whereas FFA partitioned to albumin at a 10 x greater concentration were not cytotoxic. The cytotoxic potencies of FFA bound to lipoproteins and albumin were further compared after in vitro incorporation of FFA (oleic acids) into LDL and to albumin. FFA bound to LDL but not to albumin were cytotoxic to cultured MPM; the cytotoxicity of FFA bound to LDL was more closely related to the FFA to LDL-cholesterol molar ratio than to the total FFA concentration in the culture dish. The ability of FFA bound to LDL and albumin to induce foam cell formation was studied in THP-1 monocyte-derived macrophages, which were less susceptible to cytotoxicity produced by FFA bound to LDL than MPM.(ABSTRACT TRUNCATED AT 400 WORDS)

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