Glutaraldehyde treatment of 25I-labeled low density lipoprotein (125I-native-LDL) produced a modified LDL ('lI-gut-LDL) with a molecular weight of 10 X 106 or more. Malondialdehyde treatment of 125I-hative-LDL produced a product (125I-MDA-LDL) with a molecular weight not appreciably different from that of the original lipoprotein. However, the electrophoretic mobility of MDA-LDL indicated a more negative charge than native-LDL. 125I-MDALDL was degraded by two processes: a high-affinity saturable process with maximal velocity at 10-15 pg of proteinper ml and a slower, nonsaturable process. The degradation of 12I-MDA-LDL was readily inhibited by increasing concentrations of nonradioactive MDA-LDL but was not inhibited by acetylated LDL or native-LDL even at concentrations as high as 1600 ptg of protein per ml. After exposure of native-LDL to blood platelet aggregation and release in vitro, 1.73 + 0.19 nmol of malondialdehyde per mg of LDL protein was bound to the platelet-modified-LDL. No detectable malondialdehyde was recovered from native-LDL that had-been treated identically except that the platelets were omitted from the reaction mixture.After incubation with glut-LDL, MDA-LDL, or plateletmodified-LDL for 3 days, human monocyte-macrophages showed a dramatic increase in cholesteryl ester content whereas the cholesteryl ester content of cells incubated with the same concentration of native-LDL did not. Based on these experiments we propose that modification of native-LDL may be a prerequisite to the accumulation of cholesteryl esters within the cells of the atherosclerotic reaction. We further hypothesize that one modification of LDL in vivo may result from malondialdehyde which is released from blood platelets or is produced by lipid peroxidation at the site of arterial injury. There is increasing evidence that the foam cells found in the atherosclerotic reaction are macrophages that are derived from blood-borne monocytes or from smooth muscle cells that have taken on many of the properties of macrophages (1-3). The hallmark of these cells is their high cholesteryl ester content (>50% of total cellular cholesterol) (4, 5). Our objective has been to define the conditions and mechanisms leading to cholesteryl ester accumulation within these cells. We learned from experiments to be reported elsewhere that human monocytes contain very little cholesteryl ester (approximately 2% of total cellular cholesterol), and the conversion of the monocytes into macrophages in vitro did not appreciably increase their cholesteryl ester content. Moreover, these cells did not accumulate cholesteryl esters when incubated in high concentrations of low density lipoprotein (LDL).The experiments reported here demonstrate that LDL must be modified before it will produce cholesteryl ester accumulation in human monocyte-macrophages. Based on these experiments we propose that one modification of LDL in vivo may result from an interaction with malondialdehyde which is released from blood platelets or is produced by lipid peroxidation...

These results indicate that 1) Section C, in particular Phe 288 , may be involved in the second step of catalysis, 2) Tyr 171 of Section A is essential for catalysis, most likely for the first reaction, 3) the two Asp residues in Section B are essential for the activity and most likely bind the substrate via magnesium salt bridges. Based on these results, a mechanism for the first reaction is proposed.

No abstract

Abstract-HDL mimetics have been constructed from a number of peptides and proteins with varying structures, all of which bind lipids found in HDL. HDL mimetics containing a peptide or protein have been constructed with as few as 4 and as many as 243 amino acid residues. Some HDL mimetics have been constructed with lipid but without a peptide or protein component. Some HDL mimetics promote cholesterol efflux, some have been shown to have a remarkable ability to bind oxidized lipids compared to human apolipoprotein A-I (apoA-I). Many of these peptides have been shown to have antiinflammatory properties. Based on studies in a number of animal models and in early human clinical trials, HDL mimetics appear to have promise as diagnostic and therapeutic agents. Separating HDL-Cholesterol Levels From HDL FunctionSimply increasing the amount of circulating HDL-cholesterol does not reduce the risk of coronary heart disease (CHD) events, CHD deaths, or total deaths. 1 Heinecke 2 has noted that HDL-cholesterol does not define the proteins associated with HDL and suggests that the HDL proteome is a marker, and perhaps a mediator, of CHD. Zheng et al 3 reported that apoA-I, the major protein in HDL, is a selective target for myeloperoxidase-catalyzed oxidation, which results in impairment of the ability of HDL to promote cholesterol efflux. Singh et al 4 suggested that HDL could be a therapeutic target by modifying its lipid and protein cargo to improve its antiinflammatory properties. One method that has been reported to modify the lipid and protein cargo of HDL involves treatment with apolipoprotein mimetic peptides. 5 The Development of Apolipoprotein Mimetic Peptides as Therapeutic AgentsThe efficacy of apoA-I in improving atherosclerosis in animal models 6,7 and in preliminary human studies 8 made it an attractive therapeutic candidate. However, human apoA-I has 243 amino acid residues, making it not only difficult and expensive to synthesize but necessitating that it be given intravenously. The initial promise 8 of therapeutic benefit from weekly intravenous doses for 5 to 6 weeks does not seem to have been borne out by subsequent larger clinical trials. 9 It is likely that longer periods of intravenous administration will be required, making this an unlikely therapy for the millions of patients with atherosclerosis.The laboratories of Segrest and Anantharamaiah designed an 18-aa peptide that did not have sequence homology with apoA-I but mimicked the class A amphipathic helixes contained in apoA-I. 10 -12 This peptide was called 18A because it contained 18 amino acids and formed a class A amphipathic helix. When the amino and carboxyl termini were blocked by addition of an acetyl group and amide group, respectively, stability and lipid-binding properties were improved and the peptide was called 2F because of the 2 phenylalanine residues on the hydrophobic face. The 2F peptide mimicked many of the lipid binding properties of apoA-I but failed to alter lesions in a mouse model of atherosclerosis. 13 Using a cell-based ...