The structure of human apolipoprotein E2, E3 and E4 in solution. 2. Multidomain organization correlates with the stability of apoE structure

The stabilities toward thermal and chemical denaturation of three recombinant isoforms of human apolipoprotein E (r-apoE2, r-apoE3 and r-apoE4), human plasma apoE3, the recombinant amino-terminal (NT) and the carboxyl-terminal (CT) domains of plasma apoE3 at pH 7 were studied using near and far ultraviolet circular dichroism (UV CD), fluorescence and size-exclusion chromatography. By far UV CD, thermal unfolding was irreversible for the intact apoE isoforms and consisted of a single transition. The r-apoE3 was found to be less stable as compared to the plasma protein and the stability of recombinant isoforms was r-apoE4相似文献   

Supermolecular organization of aminotransferases and dehydrogenases of alpha-ketoacid from rat brain mitochondria

A fraction of coarse mitochondria from the rat brain was deposited after short-term effect of supersound to obtain "metabolones". Activity of dehydrogenases of alpha-ketoacids, succinate dehydrogenase, aspartate-, alanine- and GABA-alpha-ketoglutarate amino-transferases has been determined in the supernatant liquid and in "metabolones". It is shown that dehydrogenase activity is mainly (93-100%) localized in "metabolones", while the level of aminotransferase activity in the latter is lower (72-94%). Nonproportionally high activity of aminotransferases in the supernatant liquid is found to considerably suprpass a decrease in activity of these enzymes in "metabolones" against a background of extremely scanty losses of protein (within 5%) induced by the supersound effect. A hypothetic model of a "metabolone" containing the enzymes of the Krebbs cycle and GABA-shunt is suggested.     

Preparation and characterization of monoclonal antibodies to human apolipoprotein E]

Monoclonal antibodies to human apolipoprotein E (apoE) were prepared and characterized. Antibodies of 3D12F11 clone were shown to be specific to apoE and belong to IgG1 subclass. Dissociation constant of antigen-antibody complex in solution was determined as 3.5 +/- 0.5 nM. The antibodies interacted neither heparin- nor lipid-binding sites of human apoE molecule. The obtained antibodies may be useful for metabolic and structural investigations of apoE as well as for clinical studies.     

The structure of human apolipoprotein E2, E3 and E4 in solution 1. Tertiary and quaternary structure

Three recombinant apoE isoforms fused with an amino-terminal extension of 43 amino acids were produced in a heterologous expression system in E. coli. Their state of association in aqueous phase was analyzed by size-exclusion liquid chromatography, sedimentation velocity and sedimentation equilibrium experiments. By liquid chromatography, all three isoforms consisted of three major species with Stokes radii of 4.0, 5.0 and 6.6 nm. Sedimentation velocity confirmed the presence of monomers, dimers and tetramers as major species of each isoform. The association schemes established by sedimentation equilibrium experiments corresponded to monomer-dimer-tetramer-octamer for apoE2, monomer-dimer-tetramer for apoE3 and monomer-dimer-tetramer-octamer for apoE4. Each of the three isoforms exhibits a distinct self-association pattern. The apolipoprotein multi-domain structure was mapped by limited proteolysis with trypsin, chymotrypsin, elastase, subtilisin and Staphylococcus aureus V8 protease. All five enzymes produced stable intermediates during the degradation of the three apoE isoforms, as described for plasma apoE3. The recombinant apoE isoforms, thus, consist of N- and C-terminal domains. The presence of the fusion peptide did not appear to alter the apolipoprotein tertiary organization. However, a 30 kDa amino-terminal fragment appeared during the degradation of the recombinant apoE isoforms resulting from cleavage in the 273-278 region. This region, not accessible in plasma apoE3, results from a different conformation of the C-terminal domain in the recombinant isoforms. A specific pattern for the apoE4 C-terminal domain was observed during the proteolysis. The region 230-260 in apoE4, in contrast to that of apoE3 and apoE2, was not accessible to proteases, probably due to the existence of a longer helix in this region of apoE4 stabilized by an interdomain interaction.     

Supermolecular organization of photosystem II and its associated light-harvesting antenna in Arabidopsis thaliana.

The organization of Arabidopsis thaliana photosystem II (PSII) and its associated light-harvesting antenna (LHCII) was studied in isolated PSII-LHCII supercomplexes and native membrane-bound crystals by transmission electron microscopy and image analysis. Over 4000 single-particle projections of PSII-LHCII supercomplexes were analyzed. In comparison to spinach supercomplexes [Boekema, E.J., van Roon, H., van Breemen, J.F.L. & Dekker, J.P. (1999) Eur. J. Biochem. 266, 444-452] some striking differences were revealed: a much larger number of supercomplexes from Arabidopsis contain copies of M-type LHCII trimers. M-type trimers can also bind in the absence of the more common S-type trimers. No binding of l-type trimers could be detected. Analysis of native membrane-bound PSII crystals revealed a novel type of crystal with a unit cell of 25.6 x 21.4 nm (angle 77 degrees ), which is larger than any of the PSII lattices observed before. The data show that the unit cell is built up from C2S2M2 supercomplexes, rather than from C2S2M supercomplexes observed in native membrane crystals from spinach [Boekema, E.J., Van Breemen, J.F.L., Van Roon, H. & Dekker, J.P. (2000) J. Mol. Biol. 301, 1123-1133]. It is concluded from both the single particle analysis and the crystal analysis that the M-type trimers bind more strongly to PSII core complexes in Arabidopsis than in spinach.     

The receptor-binding domain of human apolipoprotein E. Binding of apolipoprotein E fragments

To identify the domain of apolipoprotein E (apo-E) involved in binding to low density lipoprotein (LDL) receptors on cultured human fibroblasts, apo-E was cleaved and the fragments were tested for receptor binding activity. Two large thrombolytic peptides (residues 1-191 and 216-299) of normal apo-E3 were combined with the phospholipid dimyristoylphosphatidylcholine (DMPC) and tested for their ability to compete with 125I-LDL for binding to the LDL (apo-B,E) receptors on human fibroblasts. The NH2-terminal two-thirds (residues 1-191) of apo-E3 was as active as intact apo-E3 . DMPC, while the smaller peptide (residues 216-299) was devoid of receptor-binding activity. When apo-E3 was digested with cyanogen bromide (CNBr) and the four largest CNBr fragments were combined with DMPC and tested, only one fragment competed with 125I-LDL for binding to cultured human fibroblasts (CNBr II, residues 126-218). This fragment possessed binding activity similar to that of human LDL. The 125I-labeled CNBr II . DMPC complex also demonstrated high affinity, calcium-dependent saturable binding to solubilized bovine adrenal membranes. The binding of CNBr II . DMPC was inhibited by 1,2-cyclohexanedione modification of arginyl residues or diketene modification of lysyl residues. In addition, the CNBr II had to be combined with DMPC before it demonstrated any receptor-binding activity. Pronase treatment of the membranes abolished the ability of this fragment to bind to the apo-B,E receptors. This same basic region in the center of the molecule has been implicated as the apo-B,E receptor-binding domain not only by this study but also by other studies showing that 1) natural mutants of apo-E that display defective binding have single amino acid substitutions at residues 145, 146, or 158; and 2) the apo-E epitope of the monoclonal antibody 1D7, which inhibits apo-E binding, is centered around residues 139-146.     

Human apolipoprotein E3 in aqueous solution. I. Evidence for two structural domains

The stability and structure of human apolipoprotein (apo) E3 in aqueous solution were investigated by guanidine HCl denaturation and limited proteolysis. The guanidine HCl denaturation curve, as monitored by circular dichroism spectroscopy, was biphasic; the two transition midpoints occurred at 0.7 and 2.5 M guanidine HCl, indicating that there are stable intermediate structures in the unfolding of apoE. Limited proteolysis of apoE with five enzymes demonstrated two proteolytically resistant regions, an amino-terminal domain (residues 20-165) and a carboxyl-terminal domain (residues 225-299). The region between them was highly susceptible to proteolytic cleavage. Because of their similarity to the proteolytically resistant regions, the amino-terminal (residues 1-191) and carboxyl-terminal (residues 216-299) thrombolytic fragments of apoE were used as models for the two domains. Guanidine HCl denaturation of the carboxyl- and amino-terminal fragments gave transition midpoints of 0.7 and 2.4 M guanidine HCl, respectively. The results establish that the two domains identified by limited proteolysis correspond to the two domains detected by protein denaturation experiments. Therefore, the thrombolytic fragments are useful models for the two domains. The free energies of denaturation calculated from the denaturation curves of intact apoE or the model domains were approximately 4 and 8-12 kcal/mol for the carboxyl- and amino-terminal domains, respectively. The value for the carboxyl-terminal domain is similar to those of previously characterized apolipoproteins, whereas the value for the amino-terminal domain is considerably higher and resembles those of soluble globular proteins. These studies suggest that, in aqueous solution, apoE is unlike other apolipoproteins in that it contains two independently folded structural domains of markedly different stabilities: an amino-terminal domain and a carboxyl-terminal domain, separated by residues that may act as a hinge region.     

Simultaneous effects of the apolipoprotein E polymorphism on apolipoprotein E, apolipoprotein B, and cholesterol metabolism.

Human apolipoprotein (apo) E is polymorphic. We have investigated the effect of the apo-E polymorphism on quantitative plasma levels of apo E, apo B, and total cholesterol in a sample of 563 blood-bank donors from Marburg and Giessen, West Germany. The relative frequencies of the epsilon 2, epsilon 3, and epsilon 4 alleles are .063, .793, and .144, respectively. The average effects of the epsilon 2 allele are to raise apo-E levels by 0.95 mg/dl, lower apo B levels by 9.46 mg/dl, and lower total cholesterol levels by 14.2 mg/dl. The average effects of the epsilon 4 allele are to lower apo-E levels by 0.19 mg/dl, to raise apo-B levels by 4.92 mg/dl, and to raise total cholesterol levels by 7.09 mg/dl. The average effects of the epsilon 3 allele are near zero for all three phenotypes. The apo-E polymorphism accounts for 20% of the variability of plasma apo-E levels, 12% of the variability of plasma apo-B levels, and 4% of the variability of total plasma cholesterol levels. The inverse relationship between the genotype-specific average apo-E levels and both the genotype-specific average apo-B and cholesterol levels is offset by a positive relationship between apo-E levels and both apo-B and cholesterol levels within an apo-E genotype. The apo-E polymorphism also has a direct effect on the correlation between apo-E and total cholesterol levels. The implication of these results on multivariate genetic analyses of these phenotypes is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human apolipoprotein E. Determination of the heparin binding sites of apolipoprotein E3

The interaction of human apolipoprotein (apo-) E3 with heparin was examined using heparin-Sepharose as a model system. The approach taken to determine the region of apo-E that is responsible for binding to heparin was to identify apo-E monoclonal antibodies that inhibited heparin binding, to determine the epitopes of the inhibiting antibodies, and finally to examine the heparin binding of fragments containing the inhibiting antibody epitopes. Three antibodies, designated 1D7, 6C5, and 3H1, were found to inhibit binding, suggesting that multiple heparin binding sites were present on apo-E. The epitopes of the inhibiting antibodies were determined by immunoblot analysis of synthetic or proteolytic fragments of apo-E. Measurement of the heparin binding activity of fragments containing epitopes of the inhibiting antibodies demonstrated that apo-E3 contains two heparin binding sites. The first site is located in the vicinity of residues 142-147 and coincides with the 1D7 epitope. The second binding site is contained in the carboxyl-terminal region of apo-E and is inhibited by 3H1, the epitope of which is located between residues 243 and 272. The epitope of the third inhibiting antibody, 6C5, is located at the amino terminus of apo-E; however, this antibody inhibits the second heparin binding site located in the carboxyl-terminal region. A head-to-tail association of apo-E, in which the 6C5 epitope and the second heparin binding site would be in close proximity, is proposed to account for this observation. In the lipid-free state both heparin binding sites on apo-E are expressed; however, when apo-E is complexed to phospholipid or on the surface of a lipoprotein particle, only the first binding site (residues 142-147) is expressed.     

Extrahepatic synthesis of apolipoprotein E

Apolipoprotein E (apoE) synthesis has been examined in rat and guinea pig tissues using in vitro translation and [35S]methionine labeling of tissue slices. A number of tissues not involved in lipoprotein synthesis synthesize a protein very similar to apoE, including the spleen, adrenal, kidney, testis, ovary, heart, and lung. Although the intestine is involved in lipoprotein synthesis, apoE synthesis could not be detected in intestinal mucosa. The protein synthesized by the extrahepatic tissues was identified as apoE by its electrophoretic mobility, its immunologic reactivity with a monospecific antibody and by limited proteolysis mapping with Staphylococcus aureus V8 protease. ApoE represented between 0.02 and 0.7% of the total protein synthesized in the extrahepatic tissues, indicating that apoE mRNA is a fairly abundant mRNA in these tissues. ApoE mRNA was also detected by hybridization with a rat apoE cDNA clone, which hybridized to a single mRNA 1250 nucleotides in length in rat liver and in extrahepatic tissues. Hybridization of the apoE clone to rat genomic DNA demonstrated that the apoE gene was more heavily methylated in intestinal mucosa, which did not synthesize apoE, than in liver, testis, or kidney. 35S labeling of peritoneal macrophages revealed that both rat and guinea pig macrophages synthesized and secreted apoE in vitro. Rhesus aortic smooth muscle cells also synthesized and secreted apoE. The possible functions of apoE synthesized in the peripheral tissues are considered.     

Molecular biology of apolipoprotein E

Apolipoprotein E, first identified 26 years ago as a serum protein that mediates extracellular cholesterol transport, is now known to regulate multiple additional metabolic pathways. Several clinically important disorders of the vasculature and brain are differentially caused, or modified, by the three isoforms of this protein. Apolipoprotein E was previously believed to traffic exclusively through binding cell surface receptors, endocytosis, and hydrolysis. However, recent studies reveal a variety of additional physiologically important roles for apolipoprotein E that are mediated through interactions with different families of receptors, through binding other proteins, and through other intracellular trafficking pathways and second messengers. Much research is now directed toward identifying those pathways of apolipoprotein E metabolism that are differentially regulated by the various isoforms of apolipoprotein E, with the goal of identifying the particular molecular pathways that result in vascular and neurologic disorders.     

Recycling of apolipoprotein E in mouse liver

Following the internalization of low density lipoprotein (LDL) by the LDL receptor within cells, both the lipid and the protein components of LDL are completely degraded within the lysosomes. Remnant lipoproteins are also internalized by cells via the LDL receptor as well as other receptors, but the events following the internalization of these complexes, which use apolipoprotein E (apoE) as their ligand for receptor capture, have not been defined. There is evidence that apoE-containing beta-very low density lipoproteins follow differential intracellular routing depending on their size and apoE content and that apoE internalized with lipoproteins can be resecreted by cultured hepatocytes and fibroblasts. In the present studies, we addressed the question of apoE sparing or recycling as a physiologic phenomenon. Remnant lipoproteins (d < 1.019 g/ml) from normal mouse plasma were iodinated and injected into normal C57BL/6 mice. Livers were collected at 10, 30, 60, and 120 min after injection, and hepatic Golgi fractions were prepared for gel electrophoresis analysis. Golgi preparations were analyzed for galactosyltransferase enrichment (>40-fold above cell homogenate) and by appearance of the Golgi stacks and vesicles on electron microscopy. Iodinated apoE was consistently found in the Golgi fractions peaking at 10 min and disappearing by 2 h after injection. Although traces of apoB48 were present in the Golgi fractions, the apoE/apoB ratio in the Golgi was 50-fold higher compared with serum. Quantitatively similar results were obtained when the very low density lipoprotein remnants were injected into mice deficient in either apoE or the LDL receptor, indicating that the phenomenon of apoE recycling is not influenced by the production of endogenous apoE and is not dependent on the presence of LDL receptors. In addition, radioactive apoE in the Golgi fractions was part of d = 1.019-1.21 g/ml complexes, indicating an association of recycled apoE with either newly formed lipoproteins or the internalized complexes. These studies show that apoE recycling is a physiologic phenomenon in vivo and establish the presence of a unique pathway of intracellular processing of apoE-containing remnant lipoproteins.     

Astrocytes synthesize apolipoprotein E and metabolize apolipoprotein E-containing lipoproteins

We have previously demonstrated that astrocytes synthesize and secrete apolipoprotein E in situ. In the present work, primary cultures of rat brain astrocytes were used to study apolipoprotein E synthesis, secretion, and metabolism in vitro. The astrocytes in culture contained immunoreactive apolipoprotein E in the area of the Golgi apparatus. Incubation of the astrocytes with [35S]methionine resulted in the secretion of labeled immunoprecipitable apolipoprotein E, which constituted 1-3% of the total secreted proteins. The apolipoprotein E secreted in culture and the apolipoprotein E in rat brain extracts differed from serum apolipoprotein E in two respects: both had a slightly higher apparent molecular weight (approx. 36,000) and more acidic isoforms than serum apolipoprotein E. Sialylation of the newly secreted apolipoprotein accounted for the difference in both the apparent molecular weight and isoelectric focusing pattern of newly secreted apolipoprotein E and plasma apolipoprotein E. The astrocytes possessed apolipoprotein B,E(LDL) receptors capable of binding and internalizing apolipoprotein E-containing lipoproteins. The uptake of lipoproteins by the cells led to a reduction in the number of cell surface receptors and to the intracellular accumulation of cholesteryl esters. Since apolipoprotein E is present within the brain, and since brain cells can express apolipoprotein B,E(LDL) receptors, apolipoprotein E-containing lipoproteins may function to redistribute lipid and regulate cholesterol homeostasis within the brain.     

Tissue-specific expression of genes encoding apolipoprotein E and apolipoprotein A-I in rabbits

We determined the site of synthesis of apolipoprotein (apo) E and apo-A-I in rabbit by measuring in vitro translational activity of their mRNAs from the liver and from the intestine. Poly(A+) RNA isolated from liver and intestinal epithelium of rabbits fed either a chow diet or a cholesterol-rich diet was translated in vitro in the rabbit reticulocyte lysate system using [35S] methionine as the labeled precursor. Newly synthesized apolipoproteins were immunoprecipitated with specific antisera and quantitated after electrophoresed on 10% polyacrylamide slab gels in the presence of 0.2% sodium dodecyl sulfate. The levels of liver apo-E and apo-A-I mRNAs from chow-fed rabbits are 0.41 and 0.002% of total translatable mRNA, respectively. The level of liver apo-A-I mRNA in the rabbit is approximately 500-fold lower than the reported level of apo-A-I mRNA in rat and human livers. Rabbit intestinal apo-E and apo-A-I mRNAs levels are 0.0036 and 0.67%, respectively. Our results indicate that in rabbits apo-E is synthesized primarily in the liver and that apo-A-I is synthesized primarily in the intestine. When rabbits are fed a cholesterol-rich diet, liver and intestinal apo-E in mRNA levels and intestinal apo-A-I mRNA levels are not changed. In contrast, the liver apo-A-I mRNA level increases 5-fold in response to the cholesterol-rich diet. However, because the intestinal liver apo-A-I mRNA level is so low, the 5-fold induction only increases liver mRNA levels to 2.7% of the corresponding intestinal apo-A-I mRNA level.     

Environmental enrichment stimulates neurogenesis in apolipoprotein E3 and neuronal apoptosis in apolipoprotein E4 transgenic mice

Neurodegeneration in Alzheimer's disease (AD) is associated with the activation of neurogenesis. The mechanisms underlying this crosstalk between neuronal death and birth and the extent to which it is affected by genetic risk factors of AD are not known. We employed transgenic mice expressing human apolipoprotein E4 (apoE4), the most prevalent genetic risk factor for AD, or expressing human apoE3 (an AD-benign allele), in order to examine the hypothesis that apoE4 tilts the balance between neurogenesis and neuronal cell death in favor of the latter. The results showed an isoform-specific increase in neurogenesis in the hippocampal dentate gyrus (DG) under standard conditions in apoE4-transgenic mice. Environmental stimulation, which increases neurogenesis in the DG of apoE3-transgenic and wild-type mice, had the opposite effect on the apoE4 mice, where it triggered apoptosis while decreasing hippocampal neurogenesis. These effects were specific to the DG and were not observed in the subventricular zone, where neurogenesis was unaffected by either the apoE genotype or the environmental conditions. These in vivo findings demonstrate a linkage between neuronal apoptosis and the impaired neuronal plasticity and cognition of apoE4-transgenic mice, and suggest that similar interactions between apoE4 and environmental factors might occur in AD.     

Human apolipoprotein E3 in aqueous solution. II. Properties of the amino- and carboxyl-terminal domains

Hydrodynamic, chromatographic, and spectroscopic techniques were used to study the aqueous solution properties of the two structural domains of human apolipoprotein (apo) E3. An amino-terminal thrombolytic fragment of apoE (22 kDa, residues 1-191) and a carboxyl-terminal thrombolytic fragment of apoE (10 kDa, residues 216-299) were used as models for the two domains. Sedimentation equilibrium ultracentrifugation showed that apoE and the 10-kDa model domain self-associated predominantly as tetramers. The 22-kDa model domain was primarily monomeric. Molecular weights calculated from the weight average sedimentation and diffusion coefficients or from the sedimentation coefficients and Stokes radii were in agreement with the sedimentation equilibrium results. Derived frictional coefficients suggest larger axial ratios and/or more extensive hydration for the apoE and the 10-kDa domain tetramers as compared with the 22-kDa domain. Proteolysis of apoE followed by high performance liquid chromatography showed rapid production of free 22-kDa domain, whereas the free 10-kDa domain appeared as a tetramer late in the course of the hydrolysis. Assessment by circular dichroism demonstrated that both model domains and apoE had over 54% alpha-helical content, which changed little in a detergent (octyl-beta-D-glucopyranoside) or lipid (dimyristoylphosphatidylcholine) environment. In contrast to the circular dichroism results, apoE and the 10-kDa domain showed a marked blue shift in the fluorescence maximum in a lipid environment. The results suggest that the self-association of apoE in solution as a tetramer is mediated by the carboxyl-terminal domain and that the amino- and carboxyl-terminal domains do not associate with one another. The amino-terminal domain is most likely compact and globular, whereas the carboxyl-terminal domain is probably elongated. The isolated model domains appear to have structures that are similar to those of the domains in the intact protein.     

Heterogeneity of apolipoprotein E epitope expression on human lipoproteins: importance for apolipoprotein E function

The conformations of apolipoproteins on the surfaces of lipoprotein particles affect their physiologic functions. The conformations of apoE on plasma lipoproteins were examined using a panel of eight anti-apoE monoclonal antibodies (MAbs). The antibodies, which reacted with the major isoforms of apoE (E2, E3, and E4), defined at least five epitopes on apoE. Proteolytic fragments and synthetic peptides of apoE were used in binding assays to assign antibody epitopes; the epitopes were all localized to the middle third of the apoE molecule. The expression of apoE epitopes on isolated apoE and on lipoproteins was probed in competitive microtiter plate immunoassays using the anti-apoE MAbs, 125I-labeled apoE as tracer, and isolated apoE, intermediate density (IDL), very low density (VLDL1-3), and high density (HDL2 and HDL3) lipoproteins as competitors. The antibodies determined the patterns of competition exhibited by the lipoprotein preparations. Antibodies of the IgM class (WU E-1, WU E-2, WU E-3) defined two sets of conformation-dependent epitopes that were assigned towards the middle and the carboxyl terminal of the middle third of apoE. Competition curves using these antibodies, apoE, and lipoproteins showed a large variability in ED50 values. MAbs WU E-4, WU E-7, and WU E-10 defined epitopes near the receptor recognition site on apoE. Competition curves demonstrated small ranges of ED50 values. MAbs WU E-11 and WU E-12, which defined epitopes toward the amino-terminal region of apoE, exhibited competition curves for apoE and lipoproteins that had consistent, but wider ranges of ED50 values. There was no strict relationship between lipoprotein flotation rates and epitope expression for any of the MAbs. Immunoaffinity chromatography of VLDL subfractions on four different MAb columns indicated that the differences in the competitive abilities of VLDL subfractions were partly due to heterogeneity of apoE epitope expression within any population of particles. VLDL particles specifically retained on two different anti-apoE MAb columns were better competitors than unretained fractions for 125I-labeled LDL binding to the apoB, E-receptor of cultured human fibroblasts, suggesting that increased accessibility of apoE on the surface of VLDL is associated with increased receptor recognition. These data suggest that individual epitopes of apoE can be modulated; epitope expressions are not determined solely by the sizes and/or densities of lipoprotein particles; and differences in apoE conformation have significant metabolic consequences.     

In vitro metabolism of apolipoprotein E

Apolipoprotein E plays a major role in the uptake of chylomicrons and of very-low-density lipoprotein (VLDL) remnants by the liver. It has also been clearly demonstrated that apolipoprotein E rapidly and spontaneously exchanges between lipoproteins. To assess whether all lipoprotein-bound apolipoprotein E is available to participate in spontaneous transfer and/or exchange, the present study followed the fate of radiolabeled apolipoprotein E in an in vitro system. The results show that in vitro, apolipoprotein E can be considered as having both a spontaneously exchangeable pool and a nonexchangeable pool. Based upon specific radioactivity data, only a limited amount of apolipoprotein E originating in VLDL or in high-density lipoproteins (HDL) was capable of in vitro exchange with that in other lipoprotein fractions. Lipolysis of VLDL triacylglycerol by milk lipoprotein lipase, however, resulted in complete transfer of VLDL apolipoprotein E mass and radioactivity to HDL, supporting the potential for transformation of exchangeable apolipoprotein to a transferable pool in vivo. The results of these studies indicate that during the course of lipoprotein metabolism, conformational changes occur which alter the accessibility of apolipoprotein E. Such dynamic heterogeneity may have implications for the regulation of lipoprotein metabolism.