Opsonization with Antimyelin Antibody Increases the Uptake and Intracellular Metabolism of Myelin in Inflammatory Macrophages

In most demyelinating diseases, macrophages are believed to be active agents of myelin destruction. In experimental encephalomyelitis, these cells appear to strip off and ingest the myelin lamellae, and myelin debris has been observed within the cell body. We show here in vitro conditions in which rat peritoneal macrophages phagocytose and metabolize CNS myelin lipids. Purified rat myelin, prelabeled in vivo with [14C]acetate, was incubated with preimmune serum or rabbit antiserum to rat CNS myelin and added to macrophage monolayers. Myelin opsonized with antimyelin antibodies was more readily phagocytosed and metabolized by cultured macrophages than untreated myelin or that preincubated with preimmune serum. In the presence of macrophages, levels of myelin polar lipids and cholesterol decreased, whereas radioactive cholesterol ester and triglyceride accumulated. Up to five times as much radioactive cholesterol ester and about twice as much triglyceride accumulated in macrophage cultures containing antibody-treated myelin as in cultures fed preimmune serum-treated myelin or in those incubated with untreated myelin. Both the fatty acid and the cholesterol from cholesterol ester contained radioactive label; therefore, both were derived at least partly from the radioactive myelin lipid. Antiserum to myelin purified from peripheral nerve was almost as effective as that to CNS myelin in stimulating cholesterol metabolism, whereas antiserum to galactocerebroside was about 70% as active. Antiserum to basic protein had less effect, whereas antiserum to the myelin-associated glycoprotein and proteolipid protein was inactive. Of the polar lipids, ethanolamine phosphatide was most degraded in both the antiserum- and preimmune serum-treated myelin, with the diacyl form and plasmalogen form degraded about equally. These experiments indicate that myelin-specific antibodies in inflammatory CNS lesions may participate in and stimulate macrophage-mediated demyelination.     

The hydrolysis of cholesterol esters in plasma lipoproteins by hormone-sensitive cholesterol esterase from adipose tissue.

Adipose tissue contains a high level of neutral esterase active against emulsions of cholesteryl oleate. The present studies show that this enzyme can also effectively hydrolyze the cholesterol esters in native rat plasma high density lipoproteins (HDL) and low density lipoproteins (LDL). The hydrolysis of lipoprotein cholesterol esters by a pH 5.2 isoelectric precipitate fraction from the freshly prepared 100,000 X g supernatant of chicken adipose tissue was low, but increased more than 50-fold on activation with cyclic AMP-dependent protein kinase. Rat adipose tissue homogenates were also very active against lipoprotein cholesterol esters, hydrolyzing as much as 60% of the total labeled cholesterol ester in HDL or LDL in 1 h. Activity was optimal at pH 7 and very low at pH 4. No protease activity was detected at pH 7 and, since assays were done in 2 mM EDTA, phospholipase A activity was presumably negligible. The results show that hormone-sensitive cholesterol esterase of adipose tissue has ready access to the neutral lipid core of plasma lipoproteins, either because the enzyme penetrates the polar shell or because the cholesterol ester in the core is exposed, at least intermittently, to allow enzyme-substrate complex formation. Whether or not this enzyme activity plays a role in lipoprotein degradation by adipose tissue remains to be determined.     

Using photolabile protecting groups for the controlled release of bioactive volatiles

To develop their biological activity, bioactive volatile compounds, such as pheromones or fragrances, have to evaporate from surfaces. Because these surfaces are usually exposed to natural daylight, the preparation of non-volatile precursors using photoremovable protecting groups is an ideal tool to control the release of caged volatile molecules from various surfaces by light-induced covalent bond cleavage. Many photoreactions occur under mild environmental conditions and are highly selective. To break covalent bonds under typical application conditions, the photoreaction has to proceed at ambient daylight, to tolerate the presence of oxygen and to run in polar media (e.g. in water). The amount of volatiles generated from photochemical delivery systems depends on the light intensity to which the systems are exposed. Both photoisomerisations and photofragmentations have successfully been investigated for the slow release of caged pheromones and fragrances from their corresponding precursors.     

The metabolism of cholesterol in the presence of liver mitochondria from normal and thyroxine-treated rats

1. [26-(14)C]- and [4-(14)C]-Cholesterol were incubated with liver mitochondria from normal and thyroxine-treated rats, and the radioactivity was measured in the carbon dioxide evolved during the incubation, in a butanol extract of the incubation mixture and in a volatile fraction containing substances of low molecular weight derived from the side chain of cholesterol. The butanol extract was separated by paper chromatography into three radioactive fractions, one of which contained the steroids more polar than cholesterol. 2. The butanol extract from incubations with [4-(14)C]cholesterol contained a radioactive substance moving with the same R(F) as cholic acid on thin-layer chromatography. 3. After incubation with [26-(14)C]-cholesterol, 60-80% of the radioactivity extracted by steam-distillation of the incubation mixture at acid pH was recovered as [(14)C]propionic acid. 4. In the presence of [26-(14)C]cholesterol, mitochondria from thyroxine-treated rats produced more radioactivity in carbon dioxide and in the volatile fraction, and less radioactivity in the fraction containing the polar steroids, than did mitochondria from normal rats. In the presence of [4-(14)C]cholesterol, mitochondria from thyroxine-treated rats produced the same amount of radioactivity in the polar steroids as did normal mitochondria. 5. Thyroxine treatment had no effect on the capacity of the mitochondria to oxidize propionate to carbon dioxide. 6. These results are best explained by supposing that thyroxine stimulates a rate-limiting reaction leading to the cleavage of the side chain of cholesterol, but has little or no influence on the hydroxylations of the ring system or on the oxidation of the C(3) fragment removed from the side chain.     

Professor David Shapiro, on his seventieth birthday

The deuteron quadrupole splitting of lamellar mesophase samples containing lecithin and heavy water depends strongly on sample composition and temperature. Broadening effects due to cholesterol may arise from deuteron exchange between water and cholesterol. In samples composed of lecithin, cholesterol, alkali chloride and heavy water, or of lecithin, alkali cholate and heavy water, the degree of water orientation is lower with K⁺ ions than with the other alkali ions. ²³Na NMR experiments show K⁺ ions to interact more strongly with the amphiphilic molecules than other alkali ions. A decrease in ²³Na line width on cholesterol addition is ascribed to a partial release of sodium ions from the lamellae. The ²³Na quadrupole splitting increases with increasing cholesterol content and this may be due to a reduced motional freedom of the polar end of the lecithin molecule.     

Metabolic relationships of the isolated fractions of the pectic substances of actively growing sycamore cells

1. d-Glucose and l-arabinose serve as precursors of the pectic polysaccharides of sycamore suspension-callus tissue. 2. The rates and characteristics of the incorporation of radioactive sucrose, glucose and mesoinositol by sycamore callus tissue have been compared and shown to be different. 3. The time-course of the incorporation of radioactive glucose into the major fractions within the cells has been determined. Approx. 7-10% of the radioactivity incorporated is present in the whole pectin of the cells. 4. A study of the continuous incorporation of radioactive glucose showed that the neutral arabinan-galactan fraction of the pectin quickly became saturated with the radioactive label. During the incorporation of radioactivity from a pulse of radioactive glucose the neutral fraction became progressively less labelled, with a corresponding increase in the radioactivity of the weakly acidic pectinic acid, which is known to contain neutral sugars. 5. When the cells were exposed to a pulse of radioactive l-arabinose, the label accumulated first in the neutral fraction and then after 4hr. it passed to the weakly acidic pectinic acid with a corresponding decrease in the radioactivity of the neutral fraction. 6. The product that was initially labelled during the first hour of exposure of the cells in the stationary phase to radioactive glucose was identified as an incompletely methylated galacturonan in which the radioactivity was present in the anhydrogalacturonide residues. This polysaccharide probably acts as the precursor of the polyuronide portions of both the strongly acidic and weakly acidic pectinic acids. 7. The observations are discussed in relation to the structure of the pectic substances and their function in cell growth and development. A tentative model for their metabolic relationship is put forward.     

Influence of emulsion type and atmospheric oxygen on the fading of latent image in electron microscopic radioautography

Summary The intensity of radioautographic reactions in Ilford L4, Sakura NR-H2 and Kodak NTE emulsions was compared after exposure in either dry air or dry helium gas at 4°C to test the stability of latent images in the presence or absence of oxygen. A light proof container is described in which slides bearing radioactive sections coated with the three emulsions were exposed in dry helium at a constant pressure of approximately 0.5 atm. The comparison of air and helium atmospheres during exposure of radioautographs was estimated qualitatively for ¹²⁵I-labeled thyroid sections stored for several years and, in addition, quantitative data was derived from ³H-labeled methacrylate sections stored from 21 days to 1 year.With the three emulsions under study, the background fog remains low under both exposure conditions at 4°C for as long as several years duration. Using L4 emulsion, similar high grain densities are obtained in air and helium and, therefore, the latent images in L4 emulsion remain stable in the presence of oxygen. In the case of NTE and NR-H2 emulsions, as the exposure time increases, substantially lower reaction intensities are observed in air than in helium. This difference in reaction intensity is evident by 3 weeks with NTE and after 4 weeks with NR-H2. Hence, there is fading of the latent images in the latter emulsions in the presence of oxygen.It is concluded that reliable results may be obtained with the L4 emulsion by exposure of radioautographs in dry air, whereas with the NR-H2 and NTE emulsions, exposure should be in an oxygen-free medium, such as is provided by a dry helium atmosphere.     

Synthesis of nuclear lipids in L2C leukemic lymphocytes

We previously reported that propiconazole strongly inhibits cholesterol synthesis, but not cell division in a stimulated cell, the human lymphocyte cultured with phytohemagglutinin, showing that newly synthesized cholesterol is not necessary for cell division. In this study we labeled the L2C leukemic guinea pig lymphocyte, a naturally stimulated cell, with [2-14C]acetate, and compared the composition of newly synthesized lipids isolated from nuclei and whole cells (or microsomes). We observed that the proportion of cholesterol in labeled non-saponifiable lipids extracted from nuclei was lower than in non-saponifiable lipids isolated from whole cells, whereas the proportion of squalene and polar lipids was higher. By analyzing total lipid extracts, the polar lipids were identified as alkylglycerols, and the above mentioned distribution of constituents was confirmed. The identification of alkylglycerols was also supported by the comparison of radioactive lipid composition after labeling cells with three different lipid precursors: [2-14C]mevalonate, [2-14C]acetate and [2-14C]stearate. When cells were labeled in the presence of dodecylimidazole, the percentage of squalene and alkylglycerols decreased in nuclear lipids, but was not altered when cells were cultured in the presence of propiconazole, a cholesterol synthesis inhibitor which does not affect cell division of human stimulated lymphocytes. We have shown that dodecylimidazole inhibited alkylglycerol biosynthesis and squalene uptake by the nucleus, suggesting that these compounds could play a role in the regulation of cell division.     

13C-NMR studies of the membrane structure of enveloped virions (vesicular stomatitis virus).

The mobility of the lipids in the bilayer of the envelope of vesicular stomatitis virus has been probed over its complete space by the biosynthetic incorporation of [N-13CH3]- choline as a probe for the polar head groups and [3-13C]- and [11-13C] oleic acid and [16-13C]- palmitic acid for the hydrophobic region of the bilayer. These precursors were effectively incorporated as established by the concomitant administration of the same precursors in radioactive form. Spin lattice relaxation time measurements (T1) of the 13C enriched segments in complete virus envelope allowed estimation of their mobility. The mobility of the polar head groups is restricted, probably due to ionic interactions with neighbouring acidic phospholipids (phosphatidylserine) and/or acidic side chains of the glycoprotein (G-protein). The rigidity of the hydrophobic part of the bilayer is due to the high cholesterol content and interaction with the immersing polypeptide chains of the G- and possibly M-protein. The rigidity is limited to a depth of about 15 A ranging from the inner and outer surface, whereas the inner core of the bilayer is fluid. Tryptic cleavage of the hydrophilic part of the G-protein allows the lipophilic immersing polypeptide fragment to enter further the bilayer which then reduces the fluidity of the hydrocarbon chains in the core region by lipid-protein interactions.     

Cholesterol catabolism in the rabbit in fasted and fed states.

Urinary and fecal endogenous steroid excretion of fed or fasted New Zealand white rabbits was determined by the isotopic steady state method after subcutaneous implantation of radioactive cholesterol. While plasma cholesterol was increasing during a 9-day fast, fecal steroid excretion decreased to 10% of the excretion rates in the fed state. Refeeding the fasted rabbits led to a decrease in plasma cholesterol and an increase in fecal endogenous steroid excretion. Urinary steroid excretion, which represented 18% of total endogenous steroid excretion for fed animals, decreased during fasting and increased during refeeding, but these changes were relatively small. The small intestine, cecum, and colon of fed or fasted rabbits had similar endogenous steroid was acidic steroid. During attempts to alter the circulating bile acid concentration by supplying deoxycholate (200 mg/day) to fed rabbits or cholestyramine (2 g/day) to fasted rabbits, plasma cholesterol concentration did not change to the same extent as during fasting or refeeding, respectively. The decreased cholesterol catabolism and the hypercholesterolemia that are seen in the fasting rabbit may result from decreased clearance of plasma cholesterol.     

Attenuated total reflection (ATR) Fourier transform infrared spectroscopy of dimyristoyl phosphatidylserine-cholesterol mixtures

Mixtures of cholesterol with dimyristoyl phosphatidylserine or deuterated dimyristoyl phosphatidylserine were investigated by polarized and non polarized attenuated total reflection (ATR) Fourier transform infrared (FTIR) Spectroscopy. From polarized spectra the dichroic ratios of various vibrations as a function of cholesterol were calculated. Dichroic ratios of methylene vibration (CH(2)) 2934 cm(-1) of cholesterol decreases with increase of cholesterol concentration leveling off in the region where cholesterol phase separation takes place. The orientation of deuterated methylene (CD(2)) symmetric and asymmetric bands of the deuterated dimyristoyl phosphatidylserine is influenced little by cholesterol. In the polar region of dimyristoyl phosphatidylserine no effect of cholesterol on the dichroic ratios of carbonyl (C==O) and asymmetric phosphate (PO(2)(-)) vibrations were detected. For nonpolarized spectra the broad bands in the polar region of the phospholipid were deconvoluted. The carbonyl band (C==O) in pure dimyristoyl phosphatidylserine is composed of five bands; in the presence of increasing concentrations of cholesterol conformational change of these vibrations takes place evolving into one predominant band. Similar conformational change takes place in the presence of 75 molecules water/molecule DMPS. For the asymmetric phosphate band very small shifts due to interaction with cholesterol were detected.     

A STAINING TECHNIQUE FOR AUTORADIOGRAPHS OF NONDIFFUSIBLE ISOTOPES

Nondeparaffinized radioactive tissue sections are stained with hematoxylin and eosin by being floated on aqueous solutions for 1 hr each. The sections are then thoroughly washed, dried and exposed to autoradiographic plates or emulsions for predetermined periods of time. When desirable, both stained and unstained adjacent tissue sections can be mounted on a single slide of autoradiographic plate for exposure. Kodak DK-19 and 30% Na₂S₂O₃.5H₂O solutions are used for subsequent developing and fixing. The finished autoradiographs show excellent resolution and cytologic detail, since the gelatin remains unstained while the tissue retains its stain. Stains other than hematoxylin and eosin can be applied to this technique, provided they withstand the developmental and fixation processes.     

Cholesterol-dependent pore formation of Clostridium difficile toxin A

The large clostridial cytotoxins toxin A and toxin B from Clostridium difficile are major virulence factors known to cause antibiotic-associated diarrhea and pseudomembranous colitis. Both toxins mono-glucosylate and thereby inactivate small GTPases of the Rho family. Recently, it was reported that toxin B, but not toxin A, induces pore formation in membranes of target cells under acidic conditions. Here, we reassessed data on pore formation of toxin A in cells derived from human colon carcinoma. Treatment of 86Rb+-loaded cells with native or recombinant toxin A resulted in an increased efflux of radioactive cations induced by an acidic pulse. The efficacy of pore formation was dependent on membrane cholesterol, since cholesterol depletion of membranes with methyl-beta-cyclodextrin inhibited 86Rb+ efflux, and cholesterol repletion reconstituted pore-forming activity of toxin A. Similar results were obtained with toxin B. Consistently, methyl-beta-cyclodextrin treatment delayed intoxication of cells in a concentration-dependent manner. In black lipid membranes, toxin A induced ion-permeable pores only in cholesterol containing bilayers and at low pH. In contrast, release of glycosylphosphatidylinositol-anchored structures by phosphatidylinositol specific phospholipase C treatment did not reduce cell sensitivity toward toxins A and B. These data indicate that in colonic cells toxin A induces pore formation in an acidic environment (e.g. endosomes) similar to that reported for toxin B and suggest that pore formation by clostridial glucosylating toxins depends on the presence of cholesterol.     

An improved method for the isolation and assay of the acid lipase from human liver.

An improved method for the isolation and assay of the lysosomal acid lipase from human liver has been developed. Over 90% of the enzymatic activity was extracted in soluble form by brief homogenization of frozen tissue with the nonionic surfactant, Triton X-100. With cholesterol, [1-14C]oleate and 4-methylumbelliferyl plamitate as substrate in emulsions with the amphoteric surfactant, N-tetradecyl-N,N,-dimethyl-3-ammonio-1-propanesulfonate, and ethanol, an apparent V of 1.9 nmol . min-1 . mg-1 protein was obtained with the radioactive substrate and 29 nmol . min-1 . mg-1 protein with the fluorogenic substrate analog, respectively. The released radioactivity-labelled oleic acid was quantitated by selective extraction with a new biphasic solvent system containing carbon tetrachloride and hexane. This assay procedure offers the advantages over other procedures that subcellular fractionation of the tissue is not required for the isolation of the cellular fractionation of the tissue is not required for the isolation of the enzyme; the enzymatic activity toward these emulsions is much greater than previously reported for other methods of substrate solubilization and cholesterol esters with saturated and unsaturated fatty acids can be employed as substrate since both types of fatty acids can be efficiently partitioned and quantitated with this solvent system.     

Regulation of the metabolism of lipid emulsion model lipoproteins by a saturated acyl chain at the 2-position of triacylglycerol

Lipid emulsion particles were prepared by sonicating four different lipid mixtures (triacylglycerol (TAG), 70%; phospholipid, 25%; cholesteryl oleate (CO), 3%; and free cholesterol, 2%), then purified by density gradient ultracentrifugation. For three test mixtures, the TAG contained 50, 75, or 100% 1,3-dioleyl-2-stearylglycerol (OSO) with the remainder being triolein (OOO); 100% triolein in the lipid mixture was used as the control. After intravenous injection of the lipid particles into unanesthetized rats, removal of radioactive TAG fatty acid and CO from plasma was measured for 30 min, then liver and spleen uptakes were measured. When emulsions contained 75% or 100% OSO as TAG, the plasma removal rates of CO were, respectively, 60% or 30% of the rate when the TAG was 100% triolein; smaller recoveries of CO were found in the liver. The clearances of TAG fatty acid did not differ significantly and the recoveries of TAG fatty acid in the organs were not affected by the type of emulsion injected. Remnant particles were derived from donor rats in which uptake was blocked by exclusion of liver and other viscera from the circulation before injection of 100% OOO and 100% OSO emulsions. When injected into recipient intact rats, the removal of remnants from plasma was slower for remnants derived 15 min after injection of 100% OSO emulsions than from 100% OOO emulsions, showing that the slower removal of emulsion CO was due to slower remnant uptake from the plasma with OSO emulsions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Behaviour of bovine phosphatidylethanolamine-binding protein with model membranes. Evidence of affinity for negatively charged membranes.

The ability of phosphatidylethanolamine-binding protein (PEBP) to bind membranes was tested by using small and large unilamellar vesicles and monolayers composed of l-alpha-1,2-dimyristoylphosphatidylcholine, l-alpha-1,2-dimyristoylphosphatidylglycerol and l-alpha-1,2-dimyristoylphosphatidylethanolamine. PEBP only bound to model membranes containing l-alpha-1,2-dimyristoylphosphatidylglycerol; the interaction was primarily due to electrostatic forces between the basic protein and the acidic phospholipids. Further experiments indicated that the interaction was not dependent on the length and unsaturation of the phospholipid acyl chains and was not modified by the presence of cholesterol in the membrane. PEBP affinity for negatively charged membranes is puzzling considering the previous identification of the protein as a phosphatidylethanolamine-binding protein, and suggests that the association of PEBP with phospholipid membranes is driven by a mechanism other than its binding to solubilized phosphatidylethanolamine. An explanation was suggested by its three-dimensional structure: a small cavity at the protein surface has been reported to be the binding site of the polar head of phosphatidylethanolamine, while the N-terminal and C-terminal parts of PEBP, exposed at the protein surface, appear to be involved in the interaction with membranes. To test this hypothesis, we synthesized the two PEBP terminal regions and tested them with model membranes in parallel with the whole protein. Both peptides displayed the same behaviour as whole PEBP, indicating that they could participate in the binding of PEBP to membranes. Our results strongly suggest that PEBP directly interacts with negatively charged membrane microdomains in living cells.     

Cholesterol-rich intracellular membranes: a precursor to the plasma membrane

The disposition of newly synthesized sterols in cultured human fibroblasts has been examined in this study. We began by demonstrating that cholesterol mass and exogenously added [3H]cholesterol both are markers for the plasma membrane, perhaps better than 5'-nucleotidase. Cells were incubated with radioactive acetate to label their endogenous sterols biosynthetically, treated with cholesterol oxidase to convert plasma membrane cholesterol to cholestenone, and then homogenized and spun to equilibrium on sucrose gradients. The density gradient profiles of the various organelles were monitored using these markers: plasma membrane, radioactive cholestenone; smooth endoplasmic reticulum, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase); and Golgi apparatus, galactosyltransferase. The buoyant density profiles of radioactive intracellular cholesterol and lanosterol both had a peak at 1.12 g/cm3, similar to 5'-nucleotidase and galactosyltransferase but not to HMG-CoA reductase. This result suggests that cholesterol biosynthesis is not taken to completion in the endoplasmic reticulum. Digitonin treatment shifted the profiles of both plasma membrane and intracellular cholesterol to higher densities. Pretreatment of intact cells with cholesterol oxidase abolished the digitonin shift of plasma membranes but not the intracellular cholesterol, indicating that these two membrane pools are not entirely physically associated. Because intracellular cholesterol was shifted more than any of the organelle markers, it must reside in a separate membrane. Since digitonin selectively shifts the density of membranes rich in cholesterol, we infer that newly synthesized cholesterol accumulates in such membranes prior to its delivery to the plasma membrane. Taken together, these results suggest that cholesterol may be concentrated for delivery to the plasma membrane by being synthesized from a sterol precursor such as lanosterol in a discrete but undefined intracellular membrane.     

Fate of intravenously administered particulate and lipoprotein cholesterol in the rat

Unesterified radioactive cholesterol, both bound to serum lipoproteins and dispersed in ethanol-saline, was injected into bile fistula and intact rats. Due to phagocytosis, mainly by the liver macrophages, intravenously injected cholesterol in ethanol-saline disappears from the bloodstream significantly faster than lipoprotein-bound cholesterol. Soon after the initial phagocytosis, the particulate isotopic cholesterol started to reappear in blood, reaching a maximal radioactivity in blood 10-24 hr after injection. Although the radioactive cholesterol reappears in serum in both esterified and unesterified form, it is likely that cholesterol is released from the phagocytic cells as unesterified cholesterol which is then esterified intravascularly or at other sites. In the bile fistula rats, somewhat more of the lipoprotein cholesterol than of the particulate cholesterol appeared in bile early after injection. However, cholesterol turnover calculated from a twopool model was the same for rats injected with lipoproteinbound or particulate cholesterol.     

The formation of lithocholic acid, chenodeoxycholic acid and α- and β-muricholic acids from cholesterol incubated with rat-liver mitochondria

1. When rat-liver mitochondria were incubated with [4-(14)C]cholesterol in the presence of a soluble supernatant fraction, various steroids more polar than cholesterol were formed. These included 3beta-hydroxycholest-5-en-26-oic acid, 3beta-hydroxychol-5-enoic acid, lithocholic acid, chenodeoxycholic acid and alpha- and beta-muricholic acids. 2. All the radioactive C(24) bile acids recovered were in conjugated form, probably as taurine conjugates. 3. The formation of 3beta-hydroxychol-5-enoic acid from cholesterol shows that liver mitochondria are capable of carrying out the oxidative removal of the isopropyl unit of the side chain before any modification has occurred in the ring system.     

The N-terminal 17% of apoB binds tightly and irreversibly to emulsions modeling nascent very low density lipoproteins

The N-terminal 17% of apolipoprotein B (apoB-17) readily associates with dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles (MLV) to form large (240-A diameter) discoidal particles. Because apoB is normally secreted with triacylglycerol (TAG)-rich lipoproteins, we studied the binding of apoB-17 to triolein-rich emulsions modeling nascent TAG-rich very low density-like lipoproteins. Emulsions with the following composition (by weight) were prepared: 85--89% triolein, 1.1--1.4% cholesterol, and 10--14% phosphatidylcholines (PC) including either egg yolk (EY)-, dimyristoyl (DM)-, or dipalmitoyl (DP)-PC representing (at 25 degrees C), respectively, a fluid surface, a surface at transition, and a mainly solid surface. The respective sizes were 1,260 +/- 500, 1,070 +/- 450, and 830 +/- 300 A mean diameter. The emulsions were incubated with conditioned medium containing apoB-17, and then reisolated by ultracentrifugation. Analysis of the emulsion-bound proteins by gel electrophoresis showed that all three emulsions bound primarily apoB-17. The DPPC emulsions bound more apoB-17 than EYPC or DMPC emulsions. Immunoaffinity-purified apoB-17 exhibited saturable, high affinity binding to EYPC and DPPC emulsions. The respective K(d) values were 32 +/- 23 and 85 +/- 27 nM and capacities (N) were 10 and 58 molecules of apoB-17 per particle. When apoB-17 bound to emulsions was incubated with DMPC MLV at 26 degrees C for 18 h, it remained bound to the emulsions, indicating that once bound to these emulsions it is unable to exchange off and solubilize DMPC into discs. In contrast, apoE-3 bound to emulsions dissociated from the emulsions when incubated with DMPC MLV and formed discs.Thus, apoB-17 binds strongly and irreversibly to emulsions modeling nascent lipoproteins. It therefore may play an important role in the stabilization of nascent VLDL and chylomicrons.- Herscovitz, H., A. Derksen, M. T. Walsh, C. J. McKnight, D. L. Gantz, M. Hadzopoulou-Cladaras, V. Zannis, C. Curry, and D. M. Small. The N-terminal 17% of apoB binds tightly and irreversibly to emulsions modeling nascent very low density lipoproteins. J. Lipid Res. 2001. 42: 51;-59.