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Low density lipoprotein receptor (LDLR) mutations cause familial hypercholesterolemia and early atherosclerosis. ABCA1 facilitates free cholesterol efflux from peripheral tissues. We investigated the effects of LDLR deletion (LDLR-/-) on ABCA1 expression. LDLR-/- macrophages had reduced basal levels of ABCA1, ABCG1, and cholesterol efflux. A high fat diet increased cholesterol in LDLR-/- macrophages but not wild type cells. A liver X receptor (LXR) agonist induced expression of ABCA1, ABCG1, and cholesterol efflux in both LDLR-/- and wild type macrophages, whereas expression of LXRα or LXRβ was similar. Interestingly, oxidized LDL induced more ABCA1 in wild type macrophages than LDLR-/- cells. LDL induced ABCA1 expression in wild type cells but inhibited it in LDLR-/- macrophages in a concentration-dependent manner. However, lipoproteins regulated ABCG1 expression similarly in LDLR-/- and wild type macrophages. Cholesterol or oxysterols induced ABCA1 expression in wild type macrophages but had little or inhibitory effects on ABCA1 expression in LDLR-/- macrophages. Active sterol regulatory element-binding protein 1a (SREBP1a) inhibited ABCA1 promoter activity in an LXRE-dependent manner and decreased both macrophage ABCA1 expression and cholesterol efflux. Expression of ABCA1 in animal tissues was inversely correlated to active SREBP1. Oxysterols inactivated SREBP1 in wild type macrophages but not in LDLR-/- cells. Oxysterol synergized with nonsteroid LXR ligand induced ABCA1 expression in wild type macrophages but blocked induction in LDLR-/- cells. Taken together, our studies suggest that LDLR is critical in the regulation of cholesterol efflux and ABCA1 expression in macrophage. Lack of the LDLR impairs sterol-induced macrophage ABCA1 expression by a sterol regulatory element-binding protein 1-dependent mechanism that can result in reduced cholesterol efflux and lipid accumulation in macrophages under hypercholesterolemic conditions.

Cellular cholesterol content reflects a balance of lipid influx by lipoprotein receptors and endogenous synthesis and efflux to cholesterol acceptor particles. The beneficial effect of high density lipoprotein (HDL) in protecting against the development of cardiovascular disease is thought to be mediated predominately through its induction of cellular cholesterol efflux and “reverse cholesterol transport” from peripheral tissues to the liver. We tested the hypothesis that HDL could inhibit cellular lipid accumulation by modulating expression of peroxisome proliferator-activated receptor-γ (PPARγ)-responsive genes. To this end, we evaluated expression of two PPARγ-responsive genes, CD36, a receptor for oxidized low density lipoprotein, and aP2, a fatty acid-binding protein. HDL decreased expression of macrophage CD36 and aP2 in a dose-dependent manner. HDL also decreased aP2 expression in fibroblasts, reduced accumulation of lipid, and slowed differentiation of fibroblasts into adipocytes. HDL stimulated mitogen-activated protein (MAP) kinase activity, and inhibition of CD36 expression was blocked by co-incubation with a MAP kinase inhibitor. HDL increased expression of PPARγ mRNA and protein, induced translocation of PPARγ from the cytoplasm to the nucleus, and increased PPARγ phosphorylation. Our data demonstrate that despite induction and translocation of PPARγ in response to HDL, MAP kinase-mediated phosphorylation of PPARγ inhibited expression of PPARγ-responsive genes and suggest mechanisms by which HDL may inhibit cellular lipid accumulation.

A nontransferable peptide analog of a plasma apolipoprotein diacyl lipid-associating peptide (diLAP) incorporates into model reassembled high density lipoproteins (R-HDL). In whole plasma in vitro, diLAP irreversibly transfers to native rat HDL2 and human HDL3, but not to rat HDL1 or human HDL2. The rate of transfer is dependent on the physical state of the lipid in the R-HDL. Exogenous cholesterol promotes the formation of larger HDL. When diLAP-labeled R-HDL were injected into rats, the diLAP that initially associated with HDL2 transferred to HDL1 over a period of 48 h. The rate of clearance of diLAP-labeled HDL was slower than that of apoA-I. The liver was the preferred site for diLAP-labeled HDL1 uptake. In contrast, diLAP-labeled HDL2 were associated with liver, ovaries, and adrenal glands, with the adrenal grands exhibiting the highest specific association. DiLAP was not found in the kidneys. These data show that 1) rat HDL is cleared more slowly than rat apoA-I; 2) HDL is removed from the plasma compartment as a particle; 3) there are tissue-specific differences in the removal of rat HDL1 and HDL2; 4) HDL2 is a precursor to HDL1; and 5) cholesterol and the activity of lecithin:cholesterol acyltransferase are essential to HDL remodeling.

Apolipoprotein (apo) B-100 mRNA is edited in the small intestine (in all mammals examined) and the liver (in mice and rats only) to produce apoB-48 mRNA. ApoB mRNA editing involves a C–>U conversion of the first base of the codon CAA for Gln-2153 in apoB-100, changing it to an in-frame stop codon (UAA). The edited mRNA encodes apoB-48, which is colinear with the N-terminal 48% of apoB-100. ApoB mRNA editing can be reproduced in vitro using cellular extracts from one species to edit synthetic apoB mRNA sequences from a different species. Editing of transcripts from transfected genes also appears not to be species-specific. We have produced transgenic mice that express full-length human apoB-100 mRNA at high levels in the liver and small intestine. Human apoB-100 (a 550-kDa protein) but not apoB-48 (a 260-kDa protein) is detected in total plasma (at approximately 22 mg/dl) and in very low density and low density lipoproteins. The endogenous mouse plasma apoB concentration is reduced by approximately 45% in the transgenic animals. Thus, the transgenic mice form an animal model for familial hyperapolipoprotein B, an inherited form of hyperlipidemia. To our surprise, we found that the full-length human apoB mRNA consists of > 99% apoB-100 mRNA in both the liver and small intestine

Through its interaction with the low density lipoprotein (LDL) receptor, apolipoprotein (apo) B-100 is a major determinant of LDL metabolism and plasma cholesterol. Its receptor binding ability is conformation-dependent and requires its expression on the right lipoprotein particles. The structural signal that targets apoB-100 to LDL is unknown. We have microinjected a human apoB-100 minigene construct comprising less than 25% of the apoB-100 sequence driven by the natural apoB promoter to produce transgenic mice. The transgene product was expressed at a high level and was present exclusively in the LDL of these animals. Analysis of the responsible sequence (residues 2878-3925 of apoB-100) reveals unique structural features that may be important in its role as an LDL-targeting domain.

A series of apolipopeptides, in which single proline substitutions were made at various sites in the 20-residue sequence, have been synthesized and tested. These peptides have nearly the same hydrophobic content, but very different helical contents, in a structure-making solvent. The affinity of these peptides for phospholipids was evaluated on the basis of their intrinsic tryptophan fluorescence and equilibrium dialysis against model high density lipoproteins. Proline substitutions at one end of the peptide had little or no effect on the fluorescence, circular dichroism, affinity for model high density lipoproteins, or activation of human plasma lecithin:cholesterol acyltransferase. By contrast, there was a dramatic change in all of these variables as the site of substitution was moved progressively closer to the middle of the peptide. All of these data suggested that a helix breaker that is substituted at the midpoint of a helical surface-associating peptide will greatly reduce its affinity for phospholipid surfaces. These results demonstrate that helicity and hydrophobicity are independent determinants of the affinity of an apolipopeptide for a phospholipid surface.

Proteins conjugated with lactose residues exhibit enhanced hepatic uptake mediated by the galactose receptor. In this study, we demonstrate that lactosaminated Fab fragments (lac-Fab) of IgG can induce hepatic catabolism of specific antigens, especially low density lipoproteins (LDL). lac-Fab and human LDL-lac-Fab complex exhibited specific uptake in isolated rat hepatocytes. In vivo in the rat, lactosamination enhanced plasma clearance of Fab fragments 2-fold and hepatic localization 20-fold. Fab fragments retained their affinity after lactosamination. Hepatic uptake of rat 125I-IgG complexed in vitro with anti-rat lac-Fab was increased almost 5-fold, compared to rat 125I-IgG alone. Injection of rats with anti-LDL lac-Fab induced plasma clearance and hepatic uptake of tracer amounts of previously injected human 125I-LDL, which decreased 50% 10 min after injection of lac-Fab, with 30% present in the liver. Asialofetuin completely inhibited these processes. After a bolus of 6 mg of human LDL, administration of anti-LDL lac-Fab reduced the serum cholesterol of rats to basal values within 2.5 h. These findings suggest that lactosaminated Fab fragments of specific IgGs are effective reagents for inducing hepatic uptake of macromolecules through the galactose receptor. lac-Fab specific for LDL may be an effective hypocholesterolemic agent in vivo.

The origin and the functional significance of apo-A-I polymorphism in man has been investigated. Together with proapo-A-I (identified as A-I1 of the polymorphic series), four other isoforms are found in human plasma, namely A-I2, A-I3, A-I4, and A-I5. A-I3 is the "mature" product of proapo-A-I conversion in plasma. In this study we provide evidence that the other, more acidic, mature apo-A-I isoproteins are derived from A-I3 by a stepwise deamidation process. This conclusion is based on the following observations. 1) Incubation of A-I3 or A-I4, either free or associated with high density lipoprotein, produces a series of more acidic isoproteins corresponding to the sequence found in plasma. The conversion process fits in well with a first order reaction, and A-I3 to A-I4 conversion occurs virtually at the same rate as A-I4 to A-I5 conversion. 2) A-I3 and A-I4 have the same NH2- and C-terminal residues. 3) Formation of apo-A-I acidic isoproteins is accompanied by liberation of ammonia. In order to investigate whether deamidation of apo-A-I results in the production of forms which have different catabolism, a series of turnover studies was carried out in normal volunteers. A-I3 and A-I4 residence times in plasma were, respectively, 3.50 +/- 0.16 and 3.00 +/- 0.10 days (mean +/- S.E.; n = 3). Degradation rate of A-I3 was 8.81 +/- 0.69 mg/kg/day and that of A-I4 was 1.66 +/- 0.15 mg/kg/day (mean +/- S.E.; n = 3). Conversion of A-I3 to A-I4 and A-I4 to A-I5 occurred at the same rate in vivo as that observed in vitro. These results are consistent with the concept that A-I3 is the precursor to the other mature apo-A-I isoforms in plasma. A-I3 is the major isoform through which apo-A-I is eliminated from plasma.

Apolipoprotein-serine (apoLP-Ser or apoC-I) is one of the apoprotein constituents of human plasma very low density lipoprotein. The protein has 57 amino acid residues, including one residue of methionine and is lacking histidine, cysteine, cystine, and tyrosine. The NH2 terminus of apoLP-Ser is threonine and the COOH terminus is serine. Cleavage of apoLP-Ser with cyanogen bromide, followed by chromatography of the digest on Bio-Gel P-30 in 25% formic acid, yielded two fragments corresponding to the NH2-terminal (CNBr I) and the COOH-terminal (CNBr II) fragments and accounting for the 57 residues of the intact protein. The amino acid sequences of the tryptic peptides from CNBr I and chymotryptic peptides from CNBr II were determined by conventional methods. The amino acid sequence of apoLP-Ser is as follows: Thr-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys-Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Arg-Glu-Leu-Ile-Ser-Arg-Ile-Lys-Gln-Ser-Glu-Leu-Ser-Ala-Lys-Met-Arg-Glu-Trp-Phe-Ser-Glu-Thr-Phe-Gln-Lys-Val-Lys-Glu-Lys-Leu-Lys-Ile-Asp-Ser.

Ten murine monoclonal antibodies have been produced that are specific for bovine milk lipoprotein lipase. One monoclonal antibody, bLPL-mAb-7, inhibited completely the apolipoprotein C-II (apo-C-II)-dependent enzymic hydrolysis of trioleoylglycerol in a phospholipid-stabilized emulsion, but had no effect on the hydrolysis of the water-soluble substrate p-nitro-phenylacetate. Four times more bLPL-mAb-7 was required to achieve 50% inactivation of lipoprotein lipase activity when the enzyme was preincubated with excess apo-C-II. Disruption of the binding of a dansyl-labeled apo-C-II peptide to lipoprotein lipase by bLPL-mAb-7 was demonstrated by resonance energy transfer, both in the presence and absence of lipid. This antibody thus appears to recognize the apo-C-II binding site of lipoprotein lipase. In addition, bLPL-mAb-7 also inhibited the lipoprotein lipase activity of human post-heparin plasma.

Three cloned apolipoprotein A-II genes were isolated from a human genomic cosmid library constructed in our laboratory. An approximately 3-kilobase HindIII insert containing the entire gene was analyzed by RNA:DNA hybridization and electron microscopy. The apo-A-II gene was found to consist of 4 exons and 3 intervening sequences (IVS), and the lengths of each exon and IVS were estimated by direct observation of the hybrids. The entire approximately 3-kilobase HindIII insert was sequenced. The 5' end of the gene was determined by primer extension. The DNA sequence confirms the presence of 4 exons and 3 IVS: exon 1, 34 nucleotides; exon 2, 76 nucleotides; exon 3, 133 nucleotides; exon 4, 230 nucleotides; IVS-I, 169 nucleotides; IVS-II, 299 nucleotides; and IVS-III, 396 nucleotides. A "TATA box" is located at position -29 from the CAP site. A "CAT box" is present at position -78. A "TG" element consisting of (TG)19 is identified at the 3' end of IVS-III. Furthermore, an enhancer core sequence, CTTTCCA, is identified at position -355 in the 5' flanking sequence. At positions -497 to -471 upstream from the CAP site is a stretch of 27 nucleotides that show high homology to stretches of 5' flanking sequences in the apo-C-II, apo-A-I, apo-E, and apo-C-III genes. An Alu dimer sequence is located approximately 300 nucleotides from the 3' end of the gene. Within this Alu sequence, we have identified a polymorphic MspI site. Restriction fragment length polymorphism involving this site has been previously shown to correlate with apo-A-II levels and high density lipoprotein structure. Analysis of conformation by Chou-Fasman analysis and by the helical hydrophobic moment of Eisenberg et al. (Eisenberg, D., Weiss, R. M., and Tergwillager, T. C. (1982). Nature (Lond.) 299, 371-374) indicates that in all of the 5 apolipoproteins characterized at the nucleotide level to date, i.e. apo-C-II, apo-A-II, apo-E, apo-A-I, and apo-C-III, the 2 IVS within the peptide coding regions of the gene tend to occur at regions corresponding to the surface of the polypeptide chain and divide the protein into distinct functional domains.

Apolipoprotein-serine (apoLP-Ser) is one of the protein constituents of human plasma very low density lipoproteins (VLDL). The protein has 57 amino acids with 1 residue each of methionine and tryptophan. Cleavage of apoLP-Ser with cyanogen bromide yields the NH2-terminal fragment (CNBr I) and COOH-terminal fragment (CNBr II) having 38 and 19 residues, respectively. CNBr II contains the single residue of tryptophan. We have studied the interaction of egg phosphatidylcholine with apoLP-Ser and its cyanogen bromide fragments. The binding of phospholipid was studied in the following ways: (a) by inhibition of the reactivation of defatted β-hydroxybutyrate dehydrogenase from beef heart mitochondria; (b) by changes in circular dichroic and fluorescence spectra; (c) by isolation of complexes of the protein and peptide fragments with phospholipid, using density gradients of KBr; and (d) by rouleaux formation by phospholipid vesicles as determined by electron microscopy after negative staining with phosphotungstic acid. ApoLP-Ser strongly inhibited the reactivation of defatted β-hydroxybutyrate dehydrogenase; 10 µg of the apoprotein inhibited reactivation by 85%. CNBr I and CNBr II inhibited reactivation to a lesser extent than did the intact apoprotein. The α helical content of apoLP-Ser was about 56%, as determined by circular dichroism. Mixing the apoprotein with bilamellar vesicles of phosphatidylcholine resulted in an estimated increase in the α helical content to about 73%. The apparent helicity of CNBr I was increased from 34 to 65% and of CNBr II from 15 to 25% in the presence of phosphatidylcholine. The addition of phospholipid vesicles to apoLP-Ser produced a blue shift (6.0 nm) in the fluorescence maximum, indicating a shift of the tryptophan to a more hydrophobic environment. A similar finding was made with CNBr II in the presence of phospholipid. In ultracentrifugal flotation experiments, complexes of the phospholipid with apoLP-Ser, CNBr I, and CNBr II, each floated at a density less than 1.08 g per ml, whereas apoLP-Ser in the absence of phospholipid sedimented to a density greater than 1.18 g per ml. Finally, when the phosphatidylcholine vesicles were titrated with increasing amounts of the protein or cyanogen bromide fragments and negatively stained with phosphotungstic acid, the vesicles exhibited a series of morphological changes from free vesicles to linear arrays of "stacked discs" as determined by electron microscopy. These findings are interpreted in light of a theory of phospholipid-binding involving amphipathic helical regions that contain positively and negatively charged amino acids and are consistent with the localization of such regions in the primary chemical structure of the molecule.

Apolipoprotein glutamine I (apoLP-Gln-I or apoA-I) is one of the major protein constituents of human plasma high density lipoproteins. The protein has 245 amino acid residues, including 3 residues of methionine, and is lacking isoleucine, cystine, and cysteine. Cleavage of apoLP-Gln-I with cyanogen bromide yields four fragments, designated in their order of elution from Bio-Gel P-30 as CNBr I, II, III, and IV. In the present study, we report the complete amino acid sequence of the NH2-terminal fragment, CNBr II, a peptide that contains 90 amino acid residues.

The amino acid sequence of human lecithin:cholesterol acyltransferase has been determined by degradation and alignment of peptides obtained from tryptic and staphylococcal digestions and the cleavage with cyanogen bromide and consisted of 416 amino acid residues. All of the tryptic peptides of lecithin:cholesterol acyltransferase were isolated and sequenced. Peptides resulting from digestion by staphylococcal protease, cyanogen bromide cleavage, or the combination of the two methods were employed to find overlapping segments. The N terminus of human lecithin:cholesterol acyltransferase was determined to be phenylalanine by sequencing the whole protein up to 40 residues while the C terminus was identified as glutamic acid through carboxypeptidase Y cleavage. Cys50 and Cys74 and Cys313 and Cys356 were identified as the two disulfide bridges while the free sulfhydryl groups were located at positions 31 and 184. The N-glycosylated sites of the protein were assigned to asparagines at positions 20, 84, 272, and 384. The active site of lecithin:cholesterol acyltransferase was identified as serine on position 181 according to its homology with other serine-type esterases which have a common structure of glycine-variable amino acid-active serine-variable amino acid-glycine (Gly-X-Ser-X-Gly) with the variable amino acids disrupting the homology. No long internal repeats or homologies with apolipoproteins were found. The secondary structure is consistent with the results of predictive algorithms. A simple model of the enzyme is proposed on the basis of available chemical data and predictive methods.

When lipoprotein [a] was isolated in the presence of the proteolytic inhibitor Trasylol, its apoprotein exhibited one dominant band corresponding to a molecular weight of about 1.2 million when analyzed by electrophoresis on 3.25% sodium dodecyl sulfate-polyacrylamide gels. After chemical reduction, this band was missing but was replaced by two bands, one corresponding to a molecular weight of about 490,000 and the other to a molecular weight of about 645,000. Before treatment with reducing agents, the apolipoprotein [a] and apolipoprotein B immunoreactivities were detectable in the same electrophoretic band, but after reduction the apolipoprotein [a] was demonstrated to be separate from the apolipoprotein B. These results suggest that the apoprotein of lipoprotein [a] is composed of two subunits which are similar in molecular weight and are held together by one or more disulfide bonds. One subunit possesses apolipoprotein [a] and the other apolipoprotein B immunoreactivity. The secondary structure of the apoprotein components within lipoprotein [a] has been studied by circular dichroism and found to differ significantly from the secondary structure of the apoproteins in low density lipoproteins and high density lipoproteins. About 30% alpha-helical structure was measured in lipoprotein [a] compared to 48% in low density lipoproteins and 70% in high density lipoproteins. Lipoprotein [a] exhibited a much higher percentage of disordered structure than either of the other two lipoproteins.

Using thrombin and trypsin as probes, we determined: first, that low-density lipoprotein (LDL) receptor binding determinants switch from apolipoprotein (apo) E to apo-B within the very-low-density lipoprotein (VLDL) Sf 20-60 region of the metabolic cascade from VLDL1 (Sf 100-400) of hypertriglyceridemic (HTG) human subjects to LDL. Second, two different conformations of apo-E exist in HTG-VLDL Sf greater than 60, one accessible (greater than or equal to 1 mol/mol of particle) and one inaccessible (1-2 mol/mol) to both thrombin and the LDL receptor; normal VLDL (Sf greater than 60) have only the inaccessible conformation and therefore do not bind to the LDL receptor. Third, thrombin degrades apo-B into large fragments, three of which have electrophoretic mobilities similar to B-48, B-74, and B-26; this, however, has no effect on apo-B-mediated receptor binding. Fibroblast studies showed that thrombin could abolish receptor uptake of HTG-VLDL1 and HTG-VLDL2 (Sf 60-100), had little or no effect on HTG-VLDL3 (Sf 20-60), and no effect on uptake of intermediate-density lipoprotein (IDL) or LDL. Trypsin abolished the binding of HTG-VLDL1 and HTG-VLDL2, reduced that of HTG-VLDL3, but had little to no effect on IDL or LDL binding. Immunochemical techniques revealed that thrombin cleaved some apo-E into the E-22 and E-12 fragments; after trypsin treatment no apo-E was detected in any HTG-lipoprotein. Normal VLDL subclasses contained less apo-E than the corresponding HTG-VLDL subclasses and it was not cleaved by thrombin. Apo-B immunoreactivities of VLDL subclasses were not significantly changed after treatment with thrombin, although thrombin cleaved some of the B-100 of each VLDL subclass, and all apo-B in IDL and LDL, into 4-6 major large fragments. Trypsin converted all of the apo-B of each lipoprotein into smaller fragments (Mr less than 100,000). We conclude that apo-E of the thrombin-accessible conformation mediates uptake of HTG-VLDL1 and HTG-VLDL2 but that apo-B alone is sufficient to mediate receptor binding of IDL and LDL; the switch from apo-E to apo-B as the primary or sufficient binding determinant occurs within the VLDL3 (Sf 20-60) region of the metabolic cascade, where receptor binding first appears in VLDL subclasses from normal subjects.

Cloned human apo-C-II cDNA was used as a hybridization probe to identify the human apo-C-II gene in a genomic library constructed in our laboratory. The isolated apo-C-II DNA was studied both by electron microscopy and by direct sequence analysis. Ultrastructural morphological analysis of RNA-DNA hybrids revealed that the apo-C-II gene had complex structures because of regions of inverted complementary sequences in and around the gene forming stem-and-loop structures which interfere with the formation of stable RNA:DNA hybrids. Extensive morphological analysis revealed a minimum of 3 intervening sequences (IVS), and their lengths were measured. Direct sequence analysis of the cloned gene confirmed the presence of 3 IVS. There are 4 Alu type sequences in IVS-I. We sequenced 4340 nucleotides which include 545 nucleotides in the 5' flanking region, the entire gene which spans 3320 nucleotides, and 475 nucleotides in the 3' flanking region which also encompasses an additional Alu sequence. The 5' end of the gene was identified by primer extension and sequencing of the primer extended cDNA. Apo-C-II mRNA structure was deduced from the cDNA sequence, the primer extension experiments, and the genomic sequence. It is 494 nucleotides in length. Its sequence differs from previously published sequences in that there are 7 additional nucleotides before the polyadenylate tail. In the 5' flanking region, nucleotides -234 to -213 encompass a GC-rich region which exhibits high homology (greater than 70%) to the 5' flanking regions of the genes of all the apolipoproteins published to date, namely, apo-A-II (-497 to -471), apo-A-I (approximately -196 to -179), apo-E (-409 to -391), and apo-C-III (approximately -116 to -103). This highly conserved region might represent some evolutionarily conserved sequences from these related genes and/or might represent a region with regulatory function.

In this study we have investigated the effects of very low density lipoprotein (VLDL) lipolysis on the removal of radiolabeled apolipoprotein C-II and apolipoprotein C-III-1 from in vitro lipolyzed lipoproteins. Lipolysis was carried out in vitro using lipoprotein lipase purified from bovine milk, and mixtures with or without plasma. Lipoproteins were isolated by ultracentrifugation and by gel filtration. Labeled apo-C-II and apo-C-III-1 distributed among plasma lipoproteins, predominantly VLDL and high density lipoprotein (HDL). Lipolysis induced transfer of apo-C-II and apo-C-III-1 from VLDL to HDL. The transfer was proportional to the extent of triglyceride hydrolysis, and similar for the two apoproteins. The apo-C-II/apo-C-III-1 radioactivity ratio did not change in either VLDL or the fraction of d greater than 1.006 g/ml during the progression of the lipolytic process. Similar observations were recorded while using plasma-devoid lipolytic systems. Gel filtration of incubation mixtures, on 6% agarose, revealed that the removal of labeled apo-C molecules from VLDL is not a consequence of either centrifugation or high salt concentration. These results suggest that there is no preferential removal of apo-C-II or apo-C-III-1 from lipolyzed VLDL particles. They further indicate that the ratio of apo-C-II to apo-C-III-1 does not regulate the extent of lipolysis of different VLDL particles, at least in VLDL isolated from normolipidemic humans.

Apolipoprotein glutamine I (apoLP-Gln-I or apoA-I) is the major protein constituent of the human plasma high density lipoproteins. Cleavage of this protein with cyanogen bromide yields four fragments, designated in the order of elution from Bio-Gel P-30 as CNBr I, II, III, and IV. In the first paper in this series, the amino acid sequence of the NH2-terminal fragment, CNBr II, is reported. In the present study, CNBr IV, III, and I, containing, respectively, 25, 36, and 94 amino acids were sequenced by conventional means. To establish the alignment of the cyanogen bromide fragments, apoLP-Gln-I was digested with trypsin and two of the three methionine-containing tryptic peptides were isolated. The amino acid sequence of apoLP-Gln-I is as follows: (see article). With the complete amino acid sequence available, a CPK space-filling model of apoLP-Gln-I was constructed. The protein was placed into an alpha helical conformation wherever the primary structure permitted. Thirteen helical regions were identified. These regions account for 70% of the amino acid residues of the protein. Each helix is characterized as having two faces (amphipathic). One is a polar face that occupies approximately 180 degrees of the surface of the helix and the other is a hydrophobic face that occupies the other 180 degrees of the helical surface. Similar amphipathic helices have been identified previously in the other lipoprotein-proteins that have known sequences. It is suggested that the amphipathic helical regions of apoLP-Gln-I are important in the binding of phospholipids in high density lipoproteins.

ApoA-II and dimyristoylphosphatidylcholine (DMPC) spontaneously associate to give three different complexes whose structures are determined by the initial reactant concentration and by the reaction temperature with respect to Tc (23.9 degrees C), the gel to liquid crystalline transition temperature of DMPC. At an initial lipid to protein ratio of 45/1, a single complex (2.29 x 10(5) daltons) is quantitatively formed at all temperatures between Tc - 4 degrees C and Tc + 6 degrees C. When the 45/1 complex is mixed with DMPC liposomes there is lipid exchange but no net transfer of lipid, so that the structure of the complex remains unaltered. At an initial molar ratio of 100 to 300:1, the reaction scheme is more complex. At 24 degrees C a 240/1 complex (1.5 x 10(6) daltons) is formed from a precursor 75/1 complex (3.43 x 10(5) daltons) if excess (approximately 300 mol/mol) lipid is present. The 75/1 complex exhibits lipid exchange in the presence of added DMPC liposomes at 24 degrees C, and both the 75/1 and the 240/1 complex can be converted to smaller protein-rich complexes in the presence of added apoA-II. These results suggest that the initial lipid/protein ratio and the physical state of a lipid or lipid . protein complex determines the composition and structure of the resulting complex and support the view that lipid-protein interactions are stronger than protein-protein or lipid-lipid interactions.

The primary structure of bovine milk lipoprotein lipase (bLPL) was determined by alignment of peptides produced by tryptic digestion, Staphylococcus aureus V8 protease digestion, and cyanogen bromide cleavage. bLPL consists of 450 amino acid residues. Most tryptic peptides were isolated and analyzed, except for the dipeptide, Glu-Lys (position 423-424), and the 2 Lys at positions 416 and 488. Peptides resulting from digestion by S. aureus V8 protease and cyanogen bromide cleavage filled the missing part and completed the primary sequence of bLPL. The NH2 terminus of bLPL was determined to be Asp by sequencing the intact protein with a gas phase sequencer for up to 30 residues, whereas the COOH terminus was identified as Gly through, carboxyl peptidase Y cleavage. The enzyme contains 10 cysteine residues, all of which exist in disulfide linkages. They are formed between Cys29 and Cys42, Cys218 and Cys241, Cys266 and Cys285, Cys277, and Cys280, and Cys420 and Cys440. The sites of N-glycosylation were identified at Asn44 and Asn361. In accordance with a common structural homology of serine-type esterases, -G-X-S-X-G- (Yang, C. Y., Manoogian, D., Pao, Q., Lee, F., Knapp, R. D., Gotto, A. M., Jr., and Pownall, H. J. (1987) J. Biol. Chem., 262, 3086-3191), the active site serine of bLPL was assigned to the serine at position 134. The chymotrypsin nick of bLPL was determined to be between residues 390 and 391. A model of the enzyme is proposed on the basis of our data and available chemical data.

Three different but related abnormal lipoprotein species, LP-X1, LP-X2, and LP-X3, have been isolated from cholestatic plasma by ethanol precipitation and zonal ultracentrifugation. All three populations are rich in phospholipids (64.9 to 67.5%) and cholesterol (23.0 to 26.8%) but poor in cholesteryl esters (0.4 to 1.9%), triglycerides (1.8 to 3.2%), and protein (3.2 to 6.7%) with differences in chemical composition which result in buoyant densities (1.038, 1.049, and 1.058, respectively) to allow their separation. LP-X1, LP-X2, and LP-X3 exhibited apparent flotation rates of 17.3, 9.7, and 3.2 Svedbergs and Stokes radii of 339, 343, and 294 A, respectively. As determined from circular dichroic measurements, the protein constituents of all three particles possessed a high degree of alpha helical structure (41 to 65%). Each LP-X particle exhibited abnormally low fluidity as evaluated by electron paramagnetic resonance. All of the particles contained human serum albumin and the C-proteins as major protein constituents, but only LP-X2 and LP-X3 contained apolipoprotein A-I and apolipoprotein E.

Cholesteryl ester transfer to low density lipoproteins (LDL) was achieved either by incubation of a cholesteryl ester-rich lipid microemulsion with whole plasma or by incubation of the cholesteryl ester-rich microemulsion with lipoprotein-free plasma and LDL. Sequential ultracentrifugal isolation gave LDL that contained between 5 and 37% of the cholesteryl ester derived from the artificial cholesteryl ester donor. The labeling was essentially complete within 2 h of incubation. Resonance energy transfer of LDL, labeled with fluorescent cholesteryl esters and with the surface probe 5-(N-hexadecanoylamino) fluorescein, indicated that the added cholesteryl ester was located in the inner core of the lipoprotein. In addition, biological screening of the labeled LDL in cultured cells in vitro and in the rat in vivo showed that this labeling procedure had no detectable deleterious effect on LDL. This method of labeling lipoproteins allows a rapid and specific introduction of cholesteryl esters into lipoproteins.

Insulin inhibits the secretion of lipoprotein components such as triglyceride, phospholipid, and apolipoproteins B and E in primary rat hepatocyte cultures. The aim of this study was to determine whether these hormonal effects are related to the interaction of insulin with its receptor on the surface of cultured hepatocytes. Half-maximal inhibition of secretion of apolipoprotein E and triglyceride occurred at 6 ng/ml porcine insulin, equivalent to a 20% receptor occupancy. When compared to porcine insulin, both guinea pig insulin and desoctapeptide insulin were 60 times less inhibitory on triglyceride and apolipoprotein secretion. These analogs were also 60 times less effective in competing with porcine 125I-insulin for receptor binding. Anti-insulin receptor IgG inhibited binding of porcine insulin to cells in a dose-dependent fashion. However, similar to the hormone itself, it reduced the secretion of triglyceride and apolipoproteins E and B. Preincubation of cells with 200 ng/ml porcine insulin for 15 h caused a 2.5-fold reduction of surface receptor number. These cells were less sensitive to the inhibitory effect of porcine insulin on secretion of triglyceride and apolipoproteins B and E. We conclude that the effects of insulin on lipoprotein processing by hepatocytes in culture are receptor-mediated, can be imitated by antibodies, to the insulin receptor, and are subject to control by receptor down-regulation.

Subcutaneous injection of murine macrophage cell line P388D1 into syngeneic DBA/2 produced tumors, which upon solubilization with 40 mM octyl glucoside contained acetylated low density lipoprotein binding activity. The tumor-derived receptor specifically bound acetylated low density lipoprotein with an affinity of approximately 3 X 10(-8) M but did not bind low density lipoprotein or high density lipoprotein. It was identical in binding specificity, affinity, and Pronase sensitivity to the receptor in intact cells or that obtained from solubilized cultured cell membranes. Partial purification of the receptor was achieved by solubilizing tumors with 1% Triton X-100 followed by chromatography on polyethyleneimine cellulose. After elution with a NaCl gradient in the presence of octyl glucoside and association with liposomes, a 287-fold purification of the receptor was achieved. The receptor was identified by specific ligand blotting as a 260,000-dalton protein having a pI of approximately 6.0. Binding to the receptor by acetylated low density lipoprotein, malondialdehyde-modified low density lipoprotein, and maleic anhydride-modified serum albumin was demonstrated by ligand blotting. A single receptor protein can, therefore, account for the binding of multiple types of charge-modified lipoprotein and nonlipoprotein ligands to the macrophage cell surface.

A sucrose-rich diet stimulates the biosynthesis of very low density lipoproteins in rat liver. This diet also increases the triglyceride content of hepatic very low density lipoproteins and changes their apolipoprotein composition. To study the changes of hepatic apolipoprotein biogenesis in response to such a diet, we measured secretory rates of apolipoproteins A-I, B, and E in cultured rat hepatocytes. In cultures from rats fed the sucrose-rich diet the production of apolipoprotein E was increased 2-fold as compared to controls, whereas the production of apolipoproteins A-I and B was unchanged. The enhanced production of apolipoprotein E could be accounted for by a 2-fold increase in hepatic apolipoprotein E mRNA, as measured by slot blot hybridization. To characterize the mechanisms leading to the increase of liver apolipoprotein E mRNA levels we measured the transcriptional activity of the apolipoprotein E gene in a cell-free transcription system using isolated liver cell nuclei. Transcriptional activity of the apolipoprotein E gene was 7% that of albumin gene transcription in control animals. In rats fed a sucrose-rich diet the transcription rate of the apolipoprotein E gene increased to 140 +/- 11% of controls. There was no change in albumin gene transcription. Thus, a sucrose-rich diet enhances apolipoprotein E biosynthesis in rat liver, at least in part, by stimulating transcription of the apolipoprotein E gene.

Fluorescent derivatives of a phosphatidylglycerol, phosphatidylserine, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, and diacylglycerol have been studied to establish the effect of different polar headgroups on the mechanism and kinetics of spontaneous phospholipid transfer between recombinants of human plasma apolipoprotein A-II and dimyristoylphosphatidylcholine. The fluorescent lipids are all 1-myristoyl-2-[9-(1-pyrenyl)nonanoyl] glycerides. The transfer of the lipids is a first order process where the rate is independent of the concentration over a 50 fold range of the acceptor recombinants. These results are consistent with the lipids transferring as monomers being a water-soluble intermediate. The rate of transfer of the different phospholipids are slightly slower than phosphatidylcholine, with that of phosphatidylethanolamine being about 4 times slower. The transfer of phospholipids with a titratable headgroup is pH-dependent. The difference in the rates and pH dependence may be a function of the interactions (hydrogen bonding) between polar headgroups. The rate of transfer of the diacylglycerol is 20 times slower than phosphatidylcholine, but its activation energy (21 kcal/mol) is only 2 to 3 kcal less than most of the phospholipids (23 kcal/mol). These results suggest that the rate and activation energy for the spontaneous transfer of phospholipids can be predicted to a first approximation on the basis of its hydrophobic content, irrespective of the pH or identity of the polar headgroup.

Synthetic fragments of apo-C-II, specifically labeled on their NH2-terminals with the 5-dimethylaminonaphthalene-1-sulfonyl (dansyl or DNS) fluorophore, have been prepared by solid phase peptide synthesis. When a complex is formed between bovine milk lipoprotein lipase and N-dansyl-apo-C-II peptides, resonance energy transfer occurs from the tryptophan residues of the enzyme to the dansyl-labeled peptides upon excitation at 280 nm. In the absence of lipid, the association constant increases 10-fold when the length of the DNS peptide is increased from apo-C-II-DNS(64-78) (0.04 X 10(6) M-1) to apo-C-II-DNS(60-78) (0.3 X 10(6) M-1). In the presence of lipid, the association constants are dependent on peptide chain length, and increase from 0.4 X 10(6) M-1 for apo-C-II-DNS(64-78) to 2.2 X 10(7) M-1 for apo-C-II-DNS(43-78). The interactions are specific for lipoprotein lipase, are disrupted by guanidinium chloride, are not affected by 1.0 M NaCl, and are competitive with the corresponding nondansylated peptide. Apolipoproteins C-III and A-I, at 5 to 1 molar ratios, had no effect on the interaction. These findings demonstrate the importance of the COOH-terminal region in the lipoprotein lipase-apo-C-II interaction and show that activation of the enzyme involves a specific protein-protein interaction.

Three spin-labeled derivatives of stearic acid and two derivatives of palmitic acid have been used to study the structure of the strong fatty acid binding site of bovine serum albumin. The steroid and indole binding sites have been studied using spin-labeled derivatives of androstol and indole, respectively. Paramagnetic resonance and fluorescence quenching data suggest that the fatty acid, steroid, and indole binding sites may be identical. The mobility of the nitroxyl group at C-8 of palmitic acid bound to albumin at a 1:1 molar ratio is unaffected when the carboxyl group is esterified. When the nitroxyl group is located at C-5 on this acid its motion is detectably increased by esterification of the carboxyl group but the magnitude of this change is small. This result suggests that the carboxyl group may play a minor role in the binding of fatty acids to the strongest fatty acid binding site of albumin. When stearic acid derivatives bearing the nitroxide at C-5, C-12, and C-16 are bound to albumin at a ligand to albumin ratio of 1, the order of mobility at 0-30 degrees is C-16 greater than C-12 congruent to C-5. Although motion at the methyl terminus is always greater than at the COOH terminus in the range 0-60 degrees, a simple monotonic increase in chain motion between the two termini is not observed. Arrhenius plots of the motion parameters for these bound fatty acids show two abrupt changes in slope. The temperature ranges for these changes are 15-23 degrees and 38-45 degrees. These results suggest that when one mole of spin-labeled fatty acid is bound to albumin, the protein undergoes a conformational change in each of these temperature ranges.

Dimyristoylphosphatidylcholine (DMPC) and apolipoprotein A-II (apoA-II) combine to form three isolatable complexes of molar stoichiometry of 240:1 75:1, and 45:1, respectively. Steady state fluorescence depolarization studies with diphenylhexatriene (DPH) and parinaric acid incorporated into the DMPC/apoA-II complexes reveal that increasing the protein content reduces fluidity and increases the apparent lipid phase transition (Tc). Time-resolved studies with DPH reveal hindered rotational motion and a loss of fluidity with increasing protein content, arising from an increase in the order of the lipid assay. The parinaric acid probes could detect no lateral phase separation of lipid ensembles in the DMPC/apoA-II complexes, suggesting that the DMPC adjacent to the protein is as mobile as bulk lipid. Measurements of the excimer-forming properties of a pyrene lecithin analog show that the increasing protein content in the DMPC/apoA-II complexes reduces lateral diffusion of the lipid.

High density lipoproteins (HDL) were isolated from pig plasma by ultracentrifugal flotation between densities 1.063 and 1.210 g per ml. After delipidation, the apolipoproteins were fractionated by chromatography on Sephadex G-150 and DEAE-cellulose in 5.4 m urea. One major apolipoprotein was isolated and characterized. From its chemical and physical properties, this major apolipoprotein appears very similar to apoLP-Gln-I from human HDL. Pig HDL appears to differ from human HDL in a marked decrease in the relative content of apoLP-Gln-II in the pig lipoprotein family. A distinct peak associated with apoLP-Gln-II was not identified by chromatography of pig apoHDL. A small quantity of a protein with the same mobility of human apoLPGln-II was detected in pig apoHDL after polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The major apolipoprotein from pig HDL had an approximate molecular weight of 26,000 as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Its calculated molecular weight from amino acid analysis was 29,800. Rabbit antisera prepared against the pig apolipoprotein formed precipitin lines of partial identity between this protein and apoLP-Gln-I from human HDL. Treatment of the pig apolipoprotein with carboxypeptidase established Leu-Gln as the COOH-terminal sequence. Pig and human apoLPGln-I had similar amino acid compositions, but the pig protein contained isoleucine and had 12 more residues of alanine than did human apoLP-Gln-I. Pig and human apoLP-Gln-I had indistinguishable absorption spectra and similar circular dichroism spectra indicating a relatively high content of α-helical conformation. Both proteins activated human lecithin : cholesterol acyltransferase. These studies demonstrate that the major apolipoprotein from human and pig HDL have similar chemical, immunological, physical, and physiological properties.

The interaction of human plasma high density apolipoproteins (apoHDL) with lipids was examined by circular dichroism (CD) of the peptide and aromatic chromophores. By CD criteria native HDL contained about 70% α helical, 5 to 15% β, and 15 to 20% disordered structure. Delipidation decreased the helical content by about 20% with a corresponding increase in disordered structure. The two major apoproteins of HDL (apoA-I and apoA-II), isolated by chromatography in urea, refolded to different extents after removal of urea; apoA-I had more ordered structure than apoA-II (approximately 55% and 35% α helix, respectively). Reconstitution of apoA-I, apoA-II, and apoHDL with both phosphatidylcholine and cholesteryl ester restored, respectively, 119%, 87%, and 100% of the helical structure of the parent HDL. Phosphatidylcholine alone restored 50 to 70% of the increase produced by both phosphatidylcholine and cholesteryl oleate. With each substrate, the increase in helical content on lipid-protein recombination was accompanied by a corresponding decrease in disordered structure. Spectra of HDL in the near-ultraviolet range showed peaks at 258, 264, 283.5, and 290.5 nm. Delipidation reduced and markedly altered the ellipticity bands of the near-ultraviolet CD spectrum. The bands at 283.5 and 290.5 nm, tentatively assigned to one or more of the tryptophan chromophores in apoA-I, were reversed in sign and shifted to 286 and 292 nm, respectively. The near-ultraviolet CD spectrum of the parent HDL was partially restored by recombination of apoHDL with phosphatidylcholine alone and almost completely restored by recombination with both phosphatidylcholine and cholesteryl oleate. Recombination with phosphatidylcholine alone was sufficient to restore the 286 and 292 nm peaks to their original sign and position. These studies indicate that about 20 to 30% of the amino acid residues in the two major HDL apoproteins are involved in a helix-disordered transition on lipid removal or restoration. The near-ultraviolet CD spectrum is especially sensitive to these lipid-induced structural changes. Both phospholipid and cholesteryl esters are required for complete reorganization of the secondary and tertiary structure of HDL.

Apolipoprotein glutamine II (apoLP-Gln-II) is one of the major protein constituents of human plasma high density lipoproteins. The protein is of known amino acid sequence and contains two identical polypeptide chains of 77 amino acids, linked by a single disulfide bridge at residue 6. We have investigated the effects of chemical modification of this disulfide linkage on the physical, immunological, and lipid-binding properties of the protein. ApoLP-Gln-II was reduced and alkylated with three reagents which are selective for the sulfhydryl linkage: (a) iodoacetic acid; (b) the spin label, N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)bromoacetamide (TBA); and (c) a fluorescent label, 1-dansyl-2-maleimido ethylenediamine (DME). The three alkylated derivatives were isolated and chemically characterized. It was determined that 0.9 mole of carboxymethylcysteine, 0.8 mole of TBA, and 0.8 mole of DME were present per mole of protein monomer. ApoLP-Gln-II, Cm-apoLP-Gln-II, and TBA-apoLP-Gln-II were reconstituted with phosphatidylcholine. The protein-phospholipid complexes were isolated by ultracentrifugation in sucrose density gradients. The complexes contained, respectively, 1.23, 1.18, and 0.95 mg of phosphatidylcholine per mg of protein, representing approximately 13 moles of phosphatidylcholine per mole of protein. When ribonuclease was reduced and alkylated with iodoacetic acid, incubated with phosphatidylcholine, and subjected to density gradient centrifugation in a way identical with apoLP-Gln-II, the isolated ribonuclease contained less than 0.05 mg of phosphatidylcholine per mg of protein. The helicity of apoLP-Gln-II was reduced from approximately 46 to 37% by reduction and alkylation. This reduction could be reversed by reconstitution with phosphatidylcholine. The helicity of apoLP-Gln-II was increased from 46 to 61% and Cm-apoLP-Gln-II from 37 to 56% by reconstitution with phosphatidylcholine. Similar results were obtained with TBA-apoLP-Gln-II. By contrast, circular dichroism measurements indicated no increase in the α helical content of Cm-RNase when the reconstitution with phosphatidylcholine was performed as with apoLP-Gln-II. The electron paramagnetic resonance spectrum of TBA-apoLP-Gln-II indicated a rapidly tumbling spin label with a high degree of rotational freedom. The mobility of the spin label was not significantly altered by reconstitution with either phosphatidylcholine or with phosphatidylcholine plus cholesteryl oleate. A characteristic fluorescent spectrum of the dansyl chromophore was obtained with DME-apoLP-Gln-II. Reconstitution with phosphatidylcholine did not produce any significant change in the position or intensity of the dansyl fluorescence. ApoLP-Gln-II and each of the alkylated derivatives gave precipitin lines of complete identity with antisera prepared against apoLP-Gln-II. No immunological changes were detected after reconstitution of the apoprotein or its alkylated derivatives with phosphatidylcholine. Our findings show that, while reduction of the disulfide linkage of apoLP-Gln-II may affect the secondary structure of the protein, this effect is seen only in the absence of lipid. The integrity of the disulfide bond does not appear to be critical for binding of phosphatidylcholine. As assessed by EPR and by fluorescent labels selective for the sulfhydryl group, the environment of apoLP-Gln-II at or near residue 6 does not appear to be significantly altered by the interaction with and binding of phosphatidylcholine by the protein.

Apolipoprotein-glutamine-II (apoLP-Gln-II) is one of the two major protein constituents of human plasma high density lipoproteins (HDL). ApoLP-Gln-II contains two identical chains of 77 amino acids each, which are linked by a single disulfide bond at residue 6. Each chain contains a single methionine at position 26. In the present study we have examined the phospholipid binding properties of apoLP-Gln-II, of the NH2-terminal cyanogen bromide fragment both in the reduced form (CNBr IV, residues 1 to 26) and with the disulfide bond intact (CNBr IV)2, of the COOH-terminal cyanogen bromide fragment (CNBr III, residues 27 to 77) and of performic acid-oxidized apoLP-Gln-II. The binding of phosphatidylcholine was monitored by the inhibition of the reactivation of delipidated β-hydroxybutyrate dehydrogenase from beef heart mitochondria and by the formation of phospholipid-protein complexes which were isolated by density gradient ultracentrifugation in sucrose. The circular dichroism and immunochemical properties of these fragments and derivatives of apoLP-Gln-II were studied both in the presence and absence of phosphatidylcholine. The results may be summarized as follows. (a) ApoLP-Gln-II and its performic acid-oxidized derivative were potent inhibitors of the reactivation of β-hydroxybutyrate apodehydrogenase. One microgram of these preparations gave approximately 50% inhibition. The COOH-terminal CNBr fragment, CNBr III, also inhibited reactivation of the apodehydrogenase at low concentrations, but was less effective than the intact protein. Both CNBr IV and (CNBr IV)2 failed to inhibit reactivation of the dehydrogenase. (b) ApoLP-Gln-II, performic acid-oxidized apoLP-Gln-II and CNBr III formed complexes with phosphatidylcholine which were isolated by density gradient ultracentrifugation in sucrose (d 1.063 to 1.210). These isolated complexes contained, respectively, 1.16, 1.56, and 2.00 mg of phosphatidylcholine per mg of protein or peptide. When (CNBr IV)2 was reconstituted with phosphatidylcholine and subjected to ultracentrifugation under identical conditions, the isolated peptide contained only 0.08 mg of phospholipid per mg of peptide. (c) Reconstitution with phosphatidylcholine resulted in an apparent increase in α helical content (as judged by an increase in θ222 nm in the circular dichroism spectrum). The magnitude of the changes were as follows: 49% to 64% for apoLP-Gln-II, 32 to 57% for performic acid-oxidized apoLP-Gln-II, and 32 to 44% for CNBr III. There was no significant change in the CD spectra of (CNBr IV)2 detected in the presence of phosphatidylcholine. (d) Performic acid oxidation of apoLP-Gln-II did not alter the qualitative immunoprecipitin lines when this antigen was tested against rabbit antisera prepared against apoLP-Gln-II. The same antisera reacted with CNBr III and (CNBr IV)2, but formed lines of only partial identity between the fragments or between each fragment and apoLP-Gln-II. Rabbit antisera to CNBr III formed precipitin lines of complete identity between this fragment, apoLP-Gln-II, and performic acid-oxidized apoLP-Gln-II. No differences were noted after reconstitution with phosphatidylcholine. Anti-CNBr III did not form detectable precipitin lines with (CNBr IV)2. By the tests we have employed, we conclude that CNBr III or the COOH-terminal two-thirds of apoLP-Gln-II exhibits preferential interaction with phosphatidylcholine, although to a lesser extent than does the intact protein. The NH2-terminal fragment did not show a significant interaction with phosphatidylcholine whether the disulfide linkage was intact or not. Modification of the disulfide linkage or of the methionine at residue 26 did not alter the qualitative immunoprecipitin test with the antisera employed.