Reductive Methylation Research Articles

Arsenic Methylation, Urinary Arsenic Metabolites and Human Diseases: Current Perspective

Arsenic can cause cancerous and non-cancerous human diseases. Inorganic arsenic from drinking water is the most common source of human exposure. Pentavalent arsenate can be reduced to trivalent arsenite in the blood, which is taken up mainly in the liver and metabolized by a sequence of reduction and oxidative methylation. A proportion of the inorganic arsenicals together with methylated metabolites are excreted in urine. Analyses of the urinary arsenic profile can give a hint to the methylation capacity of exposed individuals. All studies evaluating the association between urinary arsenic profiles and human diseases nowadays measure mainly the inorganic arsenate and arsenite and the two organic forms of methylated metabolites: the pentavalent form of monomethylarsonic acid (MMAV) and dimethylarsinic acid (DMAV). A review of the current literature suggests that reduced methylation capacity with increased MMAV percentage, decreased DMAV percentage, or decreased DMAV/MMAV is associated with skin lesions, skin cancer, bladder cancer, peripheral vascular disease, muscle cramps and structural chromosome aberrations in peripheral lymphocytes obtained from exposed subjects. The detection of the recently identified more toxic trivalent forms of methylated metabolites in urine awaits further confirmation.

Investigation of the detoxification mechanism of formaldehyde-treated tetanus toxin

The tetanus vaccine is based on the extremely potent tetanus neurotoxin (TeNT), which is converted by treatment with formaldehyde and lysine into the non-toxic, but still immunogenic tetanus toxoid (TTd). This formaldehyde-induced detoxification, which to a large extend determines the quality and properties of the vaccine component, occurs through partly unknown chemical modifications of the toxin. The aim of this study was to gain knowledge of the detoxification mechanism in the generation of the tetanus vaccine. Two approaches were chosen: (i) the effect of changes in the concentrations of lysine and formaldehyde in the detoxification process and (ii) characterisation of the chemically detoxified TTd. (i) We examined a number of TTd components that was produced by varying the concentrations of formaldehyde and lysine during the inactivation. Toxicity tests showed that the detoxification failed when the lysine or formaldehyde concentration was ≤1/5 or ≤1/10, respectively, of the standard level. Gel-electrophoretic analyses showed that inter-chain cross-linking was formaldehyde-dependent and, furthermore, revealed that inter-chain cross-linking was not the only requirement for the inactivation. In addition, the measurable amount of tyrosine correlated inversely with the degree of inter-chain cross-linking. (ii) To study the formaldehyde-induced chemical modifications, the TTd was investigated using protein chemical techniques in combination with mass spectrometry (MS). Using off-line liquid chromatography (LC)–MS, the most pronounced chemical modifications were characterised as unstable Schiff-bases (+12 Da) located on lysine residues and the N-termini of peptides throughout the molecule. Several arginine residues were also found with +12 Da modifications due to Schiff-base formation or as a consequence of degenerative fragmentation of lysine/formaldehyde adducts or cross-links during MS. A few tyrosine residues were similarly observed with a mass increase of 12 Da. Even though it cannot be ruled out that this is a residual mass of higher molecule adducts or cross-links to tyrosine, amino acid analysis and MS data indicated that the modification forms a ring structure from a carbon in the aromatic ring to the backbone N α. In addition, several mono-ɛ-methyllysines (+14 Da) were observed as a likely consequence of reductive methylation of the Schiff-bases. A substantial part (87%) of the known TeNT sequence, including the active site, was covered using the off-line LC–MS approach to investigate the tryptic digested TTd. In contrast to the results obtained from the gel-electrophoretic experiments, neither intra/inter-chain cross-links nor cross-links to external lysines were observed in the MS analysis. Instability of the cross-links during separation and/or MS is likely to explain their absence in the analyses. The biological relevance of the observed modifications is discussed in relation to 3D mapping analyses. Proposals for the TeNT detoxification are discussed, although no direct evidence for the exact mechanism could be obtained.

Synthesis of a regioisomeric analogue of the 3C-protease inhibitor thysanone via a Hauser annulation strategy

Hauser annulation of 3-cyano-5,7-dimethoxy-(3 H)-isobenzofuran-1-one 4 with ethyl acrylate as a method to access activated naphthoquinone 3, a key intermediate for the synthesis of thysanone 1, proved unreliable. In contrast to this, Hauser annulation of regioisomeric 3-cyano-4,6-dimethoxy-(3 H)-isobenzofuran-1-one 13 with ethyl acrylate proceeded readily affording ethyl 5,7-dimethoxy-1,4-naphthoquinone 12, after oxidation of the initial dihydroxynaphthalene 16. Allylation of naphthoquinone 12 followed by reductive methylation and Wacker oxidation afforded ketone 11 that underwent CBS reduction to (2′ S)-alcohol 19 followed by cyclisation to lactone 20. Reduction of the lactone followed by oxidative demethylation afforded (1 S,3 S)-6,8-dimethoxy-1-hydroxy-3-methylpyrano[2,3- c]-1,4-naphthoquinone 22, a regioisomeric analogue of the 3C-protease inhibitor thysanone 1.

Synthesis of 2,6‐diamino‐5‐[(2‐substituted phenylamino)ethyl]pyrimidin‐4(3h)‐one as inhibitors of folate metabolizing enzymes

Abstractmagnified imageA series of eleven novel 2,6‐diamino‐5‐[(2‐substituted phenylamino)ethyl]pyrimidin‐4(3H)‐one derivatives were synthesized as potential inhibitors of dihydrofolate reductase (DHFR) and thymidylate synthase (TS). The synthesis of analogues 2a‐f, 3a and 3e was achieved via an improved method. Commercially available anilines 12a‐f were used as starting materials which on reaction with chloroacetaldehyde followed by cyanoacetate and cyclocondensation with guanidine afforded 2,6‐diamino‐5‐[(2‐substituted phenylamino)ethyl]pyrimidin‐4(3H)‐one 2a‐f in three steps. The N‐methyl analogues 3a‐3e were prepared by reductive methylation. These compounds were evaluated against dihydrofolate reductase from Escherichia coli, Toxoplasma gondii, Pneumocystis carinii, human, and rat liver. Few compounds were marginally active against dihydrofolate reductase. The most potent inhibitor, (2c) which has a 1‐naphthyl substituent on the side chain, has an IC50 = 150 μM and 9.1 μM against Escherichia coli and Toxoplasma gondii DHFR, respectively.

Efficient and Mild Procedure for Reductive Methylation of Amines Using N‐Methylpiperidine Zinc Borohydride

A simple and efficient procedure for reductive methylation of primary and secondary amines using N‐methylpiperidine zinc borohydride (ZBNMPP), giving tertiary amines, is described. The reaction is carried out in methanol at room temperature under neutral conditions.

Lysine Methylation as a Routine Rescue Strategy for Protein Crystallization

Crystallization remains a critical step in X-ray structure determination. Because it is not generally possible to rationally predict crystallization conditions, commercial screens have been developed which sample a wide range of crystallization space. While this approach has proved successful in many cases, a significant number of proteins fail to crystallize despite being soluble and monodispersed. It is established that chemical modification can facilitate the crystallization of otherwise intractable proteins. Here we describe a method for the reductive methylation of lysine residues which is simple, inexpensive, and efficient, and report on its application to ten proteins. We describe the effect of methylation on the physico-chemical properties of these proteins, and show that it led to diffraction-quality crystals from four proteins and structures for three that had hitherto proved refractory to crystallization. The method is suited to both low- and high-throughput laboratories.

Concise preparation of N α-Fmoc-N ɛ-(Boc, methyl)-lysine and its application in the synthesis of site-specifically lysine monomethylated peptide

A concise preparation of N(alpha)-Fmoc-N(epsilon)-(Boc, methyl)-lysine and its application in the synthesis of site-specifically lysine monomethylated peptide is described. N (alpha)-Fmoc-N(epsilon)-(Boc, methyl)-lysine is obtained, via consecutive reductive benzylation and reductive methylation in a one-pot reaction, followed by debenzylation through catalytic hydrogenolysis and Boc protection in another one-pot reaction. A peptide containing monomethylated lysine is successfully synthesized by incorporating N(alpha)-Fmoc-N(epsilon)-(Boc, methyl)-lysine as a building block via solid-phase peptide synthesis.

Synthesis and characterization of 1,1′-bis[( N-methyl- N-phenyl)aminomethyl(ethyl)]ferrocenes. Crystal structures of [Fe{( η5–C 5H 4)–C(C 6H 5) [dbnd]N–CH 2C 6H 4CH 3-4} 2] and 2[Fe{( η5–C 5H 4)–CH 2N(CH 3)–C 6H 4OCH 3-4} 2] · 1/4H 2O

Direct or catalytic condensation of diacylferrocenes (acyl = formyl, acetyl, and benzoyl) and anilines or benzylamines with titanium tetrachloride as a catalyst resulted in the corresponding diimines 1– 3, respectively. Reduction of these imines with sodium borohydride or lithium aluminum hydride/aluminum chloride in THF yielded 1,1′-bis[( N-phenyl)aminomethyl(ethyl)]ferrocenes ( 4, 5) and 1,1′-bis[( N-benzyl)aminobenzyl]ferrocenes ( 6), respectively. Reductive methylation of 4– 6 with aqueous formaldehyde, cyanoborohydride and acetic acid only afforded 1,1′-bis[( N-methyl- N-phenyl)aminomethyl(ethyl)]ferrocenes ( 7, 8). 1,1′-Bis[{( N-methyl- N-benzyl)amino}benzyl]ferrocenes ( 9) were not obtained, probably due to their debenzylation under the acidic conditions. The molecular structures of 3g and 7a were determined by single crystal X-ray analysis.

Active site directed chemical modification of α-galactosidase from Bacillus stearothermophilus (NCIM 5146): Involvement of lysine, tryptophan and carboxylate residues in catalytic site

The catalytic amino acid residues of the extracellular α-galactosidase (α- d-galactoside galactohydrolase; EC 3.2.1.22) from Bacillus stearothermophilus NCIM 5146 were investigated by pH dependence and chemical modification studies. These results suggested that carboxylate and a lysine residue take part in catalysis and only lysine residues were essential for substrate binding. Carbodiimide mediated chemical modification of the enzyme also supported that a carboxylate residue located in the active site act as a nucleophile base in substrate cleavage. Acylation and reductive methylation of lysine residues by acetic, citraconic anhydride and sodium borohydride suggested that four protonated lysine residues carrying positive charge on its ɛ-amino group provides the positive charge density for binding of the substrate. Additionally four tryptophan residues also found near to the active site and in a moderately hydrophobic environment. Kinetic and thermal inactivation study of modified enzyme indicated that these tryptophan residues might have a role in the catalytic site as well as in the thermal stabilization of active site conformation at higher temperature.

Antibacterial Agent Discovery Using Thymidylate Synthase Biolibrary Screening

Thymidylate synthase (TS, ThyA) catalyzes the reductive methylation of 2'-deoxyuridine 5'-monophosphate to 2'-deoxythymidine 5'-monophosphate, an essential precursor for DNA synthesis. A specific inhibition of this enzyme induces bacterial cell death. As a second round lead optimization design, new 1,2-naphthalein derivatives have been synthesized and tested against a TS-based biolibrary, including human thymidylate synthase (hTS). Docking studies have been performed to rationalize the experimentally observed affinity profiles of 1,2-naphthalein compounds toward Lactobacillus casei TS and hTS. The best TS inhibitors have been tested against a number of clinical isolates of Gram-positive-resistant bacterial strains. Compound 3,3-bis(3,5-dibromo-4-hydroxyphenyl)-1H,3H-naphtho[1,2-c]furan-1-one (5) showed significant antibacterial activity, no in vitro toxicity, and dose-response effects against Staphylococcus epidermidis (MIC=0.5-2.5 microg/mL) clinical isolate strains, which are resistant to at least 17 of the best known antibacterial agents, including vancomycin. So far this compound can be regarded as a leading antibacterial agent.

Dimethyl multiplexed labeling combined with microcolumn separation and MS analysis for time course study in proteomics

Stable-isotope labeling coupled with liquid-phase separation and MS analysis is a powerful technique for comparative proteomics. We developed a dimethyl labeling strategy (Anal. Chem. 2003, 75, 6843-6852 and J. Proteome Res. 2005, 4, 101-108) to label peptide N-terminus and epsilon-amino groups of Lys with water-soluble formaldehyde via reductive methylation, and an isotopic pair of formaldehyde is used for binary labeling on two sets of samples. In this study, this approach is extended to a four sample labeling by combining the binary isotopic reagents of formaldehyde (d0, d2) and the binary isotopic reducing reagents, sodium cyanoborohydride (d0, d3). To ensure sufficient mass difference, this multiplexed labeling is coupled with endoproteinase Lys-C instead of trypsin for digestion, resulting in at least two labeling sites with a mass difference of 4 Da for each pair of peptide digest. Moreover, multiplex dimethyl labeling was proved to have no significant isotopic effect during RP LC elution. This method was further applied for monitoring Lys-C digestion using hemoglobin as a model. Dimethyl labeled digests derived from seven time points (1-30 h) were grouped into two sets of sample mixtures, separated by nano-LC to reduce the complexity, and then analyzed by ESI-MS/MS. The temporal study reveals that Lys-C digestion was completed in 10-15 h for all detected peptides. The multiplex dimethyl method has not only provided a simultaneous detection mean for four sample sets but has also conserved all the advantages associated with the original binary method.

Synthesis of Chiral Resorcinarene-based Hosts and a Mass Spectrometric Study of their Chemistry in Solution and the Gas Phase

The syntheses and characterization of new chiral tetrabenzoxazine and tetrakis-(dialkylaminomethyl) resorcinarenes can be achieved through the reaction of resorcinarene with chiral amines and formaldehyde. In order to examine their host–guest chemistry, chiral quaternary ammonium guests were synthesized by methylation of different amines and amino acid methyl esters through a reductive methylation followed by addition of methyl iodide. Subsequent anion exchange of the iodide against tetraphenylborate helps to improve solubility of the salts in organic solvents. After characterization in solution, mass spectrometry is used to examine the resorcinarenes’ chemistry in the gas phase. Interesting implications of the fragmentation behavior for their solution phase chemistry arise, for which a first example is presented. Ammonium ion binding is indicated by mass spectrometry. Nevertheless, chiral recognition between the chiral hosts and pseudoracemic 1:1 mixtures of appropriately deuterium-labeled chiral guest cations is however not observed.

Catalytic Mechanism and Structure of Viral Flavin-dependent Thymidylate Synthase ThyX

By using biochemical and structural analyses, we have investigated the catalytic mechanism of the recently discovered flavin-dependent thymidylate synthase ThyX from Paramecium bursaria chlorella virus-1 (PBCV-1). Site-directed mutagenesis experiments have identified several residues implicated in either NADPH oxidation or deprotonation activity of PBCV-1 ThyX. Chemical modification by diethyl pyrocarbonate and mass spectroscopic analyses identified a histidine residue (His53) crucial for NADPH oxidation and located in the vicinity of the redox active N-5 atom of the FAD ring system. Moreover, we observed that the conformation of active site key residues of PBCV-1 ThyX differs from earlier reported ThyX structures, suggesting structural changes during catalysis. Steady-state kinetic analyses support a reaction mechanism where ThyX catalysis proceeds via formation of distinct ternary complexes without formation of a methyl enzyme intermediate.

Trivalent Arsenicals Are Bound to Proteins during Reductive Methylation

Inorganic arsenic is converted to methylated metabolites, and most is excreted in urine as dimethylarsinic acid in humans and animals. The present study was conducted to investigate the metabolism of arsenic and identify hepatic and renal metabolites of arsenic after an intravenous injection of arsenite (0.5 mg As/kg body weight) in rats. Similar levels of arsenic were found in the soluble (SUP) and nonsoluble sediment (SED) fractions of both organs after 1 h. More than 80% of the SUP arsenic was bound to high molecular weight (HMW) proteins in both organs. Arsenic bound to the HMW and SED proteins were oxidized with H(2)O(2) and released in the pentavalent forms (arsenate, monomethylarsonic, and dimethylarsinic acids). The relative ratios of the three arsenicals changed depending on organ, fraction (HMW and SED), and time. Since the arsenic metabolites/intermediates were liberated from proteins by oxidation with H(2)O(2) and recovered in the pentavalent forms, and only tri- but not pentavalent arsenicals were bound to proteins in vitro, it was deduced that arsenic metabolites bound to proteins during the successive methylation pathway are in the trivalent forms; that is, successive methylation reaction takes place with simultaneous reductive rather than stepwise oxidative methylation. Thus, on the basis of the present observations, it was proposed that inorganic arsenic was successively methylated reductively in the presence of glutathione, rather than a stepwise oxidative methylation, and pentavalent arsenicals (MMA(V) and DMA(V)) were present as end products of metabolism, rather than intermediates. We also discussed the in vitro formation of dimethylthioarsenicals after incubating dimethylarsinous acid with liver homogenate.

Purification, crystallization and preliminary X-ray diffraction analysis of the phage T4 vertex protein gp24 and its mutant forms

The study of bacteriophage T4 assembly has revealed regulatory mechanisms pertinent not only to viruses but also to macromolecular complexes. The capsid of bacteriophage T4 is composed of the major capsid protein gp23, and a minor capsid protein gp24, which is arranged as pentamers at the vertices of the capsid. In this study the T4 capsid protein gp24 and its mutant forms were overexpressed and purified to homogeneity. The overexpression from plasmid vectors of all the constructs in Escherichia coli yields biologically active protein in vivo as determined by assembly of active virus following infection with inactivated gene 24 mutant viruses. The gp24 mutant was subjected to surface entropy reduction by mutagenesis and reductive alkylation in order to improve its crystallization properties and diffraction quality. To determine if surface mutagenesis targeting would result in diffractable crystals, two glutamate to alanine mutations (E89A,E90A) were introduced. We report here the biochemical observations and consequent mutagenesis experiment that resulted in improvements in the stability, crystallizability and crystal quality of gp24 without affecting the overall folding. Rational modification of the protein surface to achieve crystallization appears promising for improving crystallization behavior and crystal diffracting qualities. The crystal of gp24(E89A,E90A) diffracted to 2.6 Å resolution compared to wild-type gp24 at 3.80 Å resolution under the same experimental conditions. Surface mutation proved to be a better method than reductive methylation for improving diffraction quality of the gp24 crystals.

The Role of Protein Dynamics in Thymidylate Synthase Catalysis: Variants of Conserved 2‘-Deoxyuridine 5‘-Monophosphate (dUMP)-Binding Tyr-261,

The enzyme thymidylate synthase (TS) catalyzes the reductive methylation of 2'-deoxyuridine 5'-monophosphate (dUMP) to 2'-deoxythymidine 5'-monophosphate. Using kinetic and X-ray crystallography experiments, we have examined the role of the highly conserved Tyr-261 in the catalytic mechanism of TS. While Tyr-261 is distant from the site of methyl transfer, mutants at this position show a marked decrease in enzymatic activity. Given that Tyr-261 forms a hydrogen bond with the dUMP 3'-O, we hypothesized that this interaction would be important for substrate binding, orientation, and specificity. Our results, surprisingly, show that Tyr-261 contributes little to these features of the mechanism of TS. However, the residue is part of the structural core of closed ternary complexes of TS, and conservation of the size and shape of the Tyr side chain is essential for maintaining wild-type values of kcat/Km. Moderate increases in Km values for both the substrate and cofactor upon mutation of Tyr-261 arise mainly from destabilization of the active conformation of a loop containing a dUMP-binding arginine. Besides binding dUMP, this loop has a key role in stabilizing the closed conformation of the enzyme and in shielding the active site from the bulk solvent during catalysis. Changes to atomic vibrations in crystals of a ternary complex of Escherichia coli Tyr261Trp are associated with a greater than 2000-fold drop in kcat/Km. These results underline the important contribution of dynamics to catalysis in TS.

Determination of the Orientation of Adsorbed Cytochromecon Carboxyalkanethiol Self-Assembled Monolayers by In Situ Differential Modification

The contact domain utilized by horse cytochrome c when adsorptively bound to a C(10)COOH self-assembled monolayer (SAM) was delineated using a chemical method based on differential modification of surface amino acids. Horse cytochrome c was adsorbed at low ionic strength (pH 7.0, 4.4 mM potassium phosphate) onto 10 microm diameter gold particles coated with HS(CH(2))(10)COOH SAMs. After in situ modification of lysyl groups by reductive Schiff-base methylation, the protein was desorbed, digested using trypsin, and the peptide mapped using LC/MS. Relative lysyl reactivities were ascertained by comparing the resulting peptide frequencies to control samples of solution cytochrome c modified to the same average extent. The least reactive lysines in adsorbed cytochrome c were found to be 13, 72, 73, 79, and 86-88, consistent with a contact region located up and to the left (Met-80 side) of the solvent-exposed heme edge (conventional front face view). The most reactive lysines were 39, 53, 55, and 60, located on the lower backside. The proposed orientation features a heme tilt angle of approximately 35-40 degrees with respect to the substrate surface normal. Factors that can complicate or distort data interpretation are discussed, and the generality of differential modification relative to existing in situ methods for protein orientation determination is also addressed.

Fragmentation of Peptides with N-terminal Dimethylation and Imine/Methylol Adduction at the Tryptophan Side-Chain

The reaction between formaldehyde and the side-chain of tryptophan results in a methylol adduct. This methylol adduct formation also occurs during reductive methylation reactions. In the current study, we investigate the fragmentation pattern of peptides with N-terminal dimethylation and methylol adduction at the tryptophan side-chain. Once formed, the methylol group can easily undergo water loss to form an imine. The peptides with imine or methylol adduct on tryptophan exhibit similar MS/MS fragmentation patterns. We observed ions resulting from an intramolecular reaction between the dimethylamino group at the peptide N-terminus or the lysine side-chain and the imine group. This reaction reduces the imine to a methyl group. We also observed the loss of the imine adduct on tryptophan. This reaction is likely to occur through the reaction of an amino or hydroxyl group with the imine adduct followed by subsequent loss of methylenimine or formaldehyde.

Thymidylate synthase gene promoter polymorphism in patients with advanced head and neck cancer treated by radio- and 5-fluorouracil chemotherapy

Thymidylate synthase (TS) is a rate-limiting enzyme in the DNA synthetic pathway, and represents a cellular target of antimetabolite drug 5-fluorouracil. It catalyzes the reductive methylation of deoxyuridine-5'-monophosphate to deoxythymidine-5'-monophosphate. Inhibition of TS will result in depletion of both dTMP and, subsequently, dTTP. As a consequence, dUTP is misincorporated into DNA, resulting in DNA breakage and cell death. Developing resistance against 5-fluorouracil (FURA) based drugs might be due to the failure of inhibition of thymidylate synthase enzyme function. In the promoter region of the TS gene there is a tandem repeat sequence (2R or 3R), which was found to be polymorphic and influences the gene expression. Effectiveness and tolerability of Tegafur treatment might be influenced by expression of the TS gene. Our purpose was to determine the effectiveness and tolerability of concomitant radiotherapy and low-dose chemotherapy using Tegafur in respect of promoter polymorphism of thymidilate synthase gene. TS promoter polymorphism (2R/2R, 2R/3R, 3R/3R) was determined by polymerase chain reaction using genomic DNA, in 47 patients with advanced head and neck cancer. Thirty patients out of 47 showed complete response, and genotyping of these patients revealed 2R/2R in 22 (73.3%), 2R/3R in 2 (6.7%) and 3R/3R in 6 (20%). Seventeen out of 47 patients reacted with partial response, and 2R/2R or 2R/3R were revealed in 5 (29.4%) and 3 (17.6%) patients, respectively, and 3R/3R genotype was identified in 9 patients (53%). We did not find any correlation between patient's data and response to therapy, but strong correlation was found between the latter and the patient's genotype. This facts indicate that the analysis of promoter polymorphism of thymidylate synthase gene might be a useful target to examine before FURA-based chemotherapy, and might allow to go into the direction of individualized treatment of head and neck cancer. We suppose that tumor response depends on genomic features of the patients.

Characterization of nascent HDL particles and microparticles formed by ABCA1-mediated efflux of cellular lipids to apoA-I

The nascent HDL created by ABCA1-mediated efflux of cellular phospholipid (PL) and free (unesterified) cholesterol (FC) to apolipoprotein A-I (apoA-I) has not been defined. To address this issue, we characterized the lipid particles released when J774 mouse macrophages and human skin fibroblasts in which ABCA1 is activated are incubated with human apoA-I. In both cases, three types of nascent HDL containing two, three, or four molecules of apoA-I per particle are formed. With J774 cells, the predominant species have hydrodynamic diameters of approximately 9 and 12 nm. These discoidal HDL particles have different FC contents and PL compositions, and the presence of acidic PL causes them to exhibit alpha-electrophoretic mobility. These results are consistent with ABCA1 located in more than one membrane microenvironment being responsible for the production of the heterogeneous HDL. Activation of ABCA1 also leads to the release of apoA-I-free plasma membrane vesicles (microparticles). These larger, spherical particles released from J774 cells have the same PL composition as the 12 nm HDL and contain CD14 and ganglioside, consistent with their origin being plasma membrane raft domains. The various HDL particles and microparticles are created concurrently, and there is no precursor-product relationship between them. Importantly, a large fraction of the cellular FC effluxed from these cells by ABCA1 is located in microparticles. Collectively, these results show that the products of the apoA-I/ABCA1 interaction include discoidal HDL particles containing different numbers of apoA-I molecules. The cellular PLs and cholesterol incorporated into these nascent HDL particles originate from different cell membrane domains.