Apolipoproteins A-I,A-II and E are independently distributed among intracellular and newly secreted HDL of human hepatoma cells

Whereas hepatocytes secrete the major human plasma high density lipoproteins (HDL)-protein, apo A-I, as lipid-free and lipidated species, the biogenic itineraries of apo A-II and apo E are unknown. Human plasma and HepG2 cell-derived apo A-II and apo E occur as monomers, homodimers and heterodimers. Dimerization of apo A-II, which is more lipophilic than apo A-I, is catalyzed by lipid surfaces. Thus, we hypothesized that lipidation of intracellular and secreted apo A-II exceeds that of apo A-I, and once lipidated, apo A-II dimerizes. Fractionation of HepG2 cell lysate and media by size exclusion chromatography showed that intracellular apo A-II and apo E are fully lipidated and occur on nascent HDL and VLDL respectively, while only 45% of intracellular apo A-I is lipidated. Secreted apo A-II and apo E occur on small HDL and on LDL and large HDL respectively. HDL particles containing both apo A-II and apo A-I form only after secretion from both HepG2 and Huh7 hepatoma cells. Apo A-II dimerizes intracellularly while intracellular apo E is monomeric but after secretion associates with HDL and subsequently dimerizes. Thus, HDL apolipoproteins A-I, A-II and E have distinct intracellular and post-secretory pathways of hepatic lipidation and dimerization in the process of HDL formation. These early forms of HDL are expected to follow different apolipoprotein-specific pathways through plasma remodeling and reverse cholesterol transport.     

Apo A-II participates in HDL–liposome interaction by the formation of new pre-β mobility particles and the modification of liposomes

Interaction between high density lipoproteins (HDL) and liposomes results in both a structural modification of HDL and the generation of new pre-β HDL-like particles. Here, phosphatidylcholine liposomes and human HDL were incubated at liposomal phospholipid/HDL phospholipid (L-PL/HDL-PL) ratios of 1:1, 3:1 and 5:1 with a subsequent assessment of the distribution of apolipoprotein (apo) A-I, apo A-II, free cholesterol (FC) and PL between newly generated pre-β mobility lipoproteins and non-disrupted liposomes. Both at L-PL/HDL-PL ratios of 3:1 and 5:1 the fraction of liposomal-derived PL associated with pre-β fraction was significantly higher than those accepted by α-HDL. We found that 78% of apo A-I released from HDL was incorporated into pre-β mobility fraction. The relative contents of PL and apo A-I in pre-β fraction were constant irrespective of the initial L-PL/HDL-PL ratio in the incubation mixture and accounted for approximately 83 and 11%, respectively. Apo A-II was detached from HDL to a similar extent as apo A-I and distributed evenly between pre-β fraction and non-disrupted liposomes. Apo A-II constituted approximately 1%, by weight, in these fractions at all L-PL/HDL-PL ratios investigated. It corresponded approximately to 10% of pre-β fraction protein mass. Both liposomes and pre-β fraction accepted comparable amounts of FC released from HDL. This data indicated that during the interaction between human HDL and phosphatidylcholine liposome apo A-II participates both in structural modification of liposomes and in the generation of pre-β mobility fraction of constant content of PL, apo A-I and apo A-II. Involvement of apo A-II in HDL–liposome interaction may influence the anti-atherogenic properties of liposomes.     

Apolipoprotein A-II/A-I ratio is a key determinant in vivo of HDL concentration and formation of pre-beta HDL containing apolipoprotein A-II

Overexpression of human apolipoprotein A-II (apo A-II) in mice induced postprandial hypertriglyceridemia and marked reduction in plasma HDL concentration and particle size [Boisfer et al. (1999) J. Biol. Chem. 274, 11564-11572]. We presently compared lipoprotein metabolism in three transgenic lines displaying plasma concentrations of human apo A-II ranging from normal to 4 times higher, under ad libitum feeding and after an overnight fast. Fasting dramatically decreased VLDL and lowered circulating human apo A-II in transgenic mice; conversely, plasma HDL levels increased in all genotypes. The apo A-I content of HDL was inversely related to the expression of human apo A-II, probably reflecting displacement of apo A-I by an excess of apo A-II. Thus, the molar ratios of apo A-II/A-I in HDL were significantly higher in fed as compared with fasted animals of the same transgenic line, while endogenous LCAT activity concomitantly decreased. The number and size of HDL particles decreased in direct proportion to the level of human apo A-II expression. Apo A-II was abundantly present in all HDL particles, in contrast to apo A-I mainly present in large ones. Two novel findings were the presence of pre-beta migrating HDL transporting only human apo A-II in the higher-expressing mice and the increase of plasma HDL concentrations by fasting in control and transgenic mice. These findings highlight the reciprocal modifications of VLDL and HDL induced by the feeding-fasting transition and the key role of the molar ratio of apo A-II/A-I as a determinant of HDL particle metabolism and pre-beta HDL formation.     

Measurement of human high density lipoprotein apolipoprotein A-1 in serum by radioimmunoassay.

A sensitive and specific double antibody radioimmunoassay for the major apolipoprotein (apo A-I) of human serum high density lipoprotein (HDL) was developed. Initial studies indicated that direct measurements of apo A-I concentration in whole untreated sera or isolated high density lipoprotein fractions yielded variable results, which were lower than those obtained in the corresponding samples which had been subjected to delipidation. Subsequently, it was observed that heating diluted sera or HDL for 3 hr at 52 degrees C prior to assay resulted in maximal increases in apo A-I immunoreactivity to levels comparable to those found in the delipidated specimens. This simple procedure permitted multiple sera to be assayed efficiently with full recovery of apo A-I.     

Regulation of high density lipoprotein receptors in cultured macrophages: role of acyl-CoA:cholesterol acyltransferase

The interaction of human serum high density lipoproteins (HDL) with mouse peritoneal macrophages and human blood monocytes was studied. Saturation curves for binding of apolipoprotein E-free [125I]HDL3 showed at least two components: non-specific binding and specific binding that saturated at approximately 40 micrograms HDL protein/ml. Scatchard analysis of specific binding of apo E-free [125I]-HDL3 to cultured macrophages yielded linear plots indicative of a single class of specific binding sites. Pretreatment of [125I]HDL3 with various apolipoprotein antibodies (anti apo A-I, anti apo A-II, anti apo C-II, anti apo C-III and anti apo E) and preincubation of the cells with anti-idiotype antibodies against apo A-I and apo A-II prior to the HDL binding studies revealed apolipoprotein A-I as the ligand involved in specific binding of HDL. Cellular cholesterol accumulation via incubation with acetylated LDL led to an increase in HDL binding sites as well as an increase in the activity of the cytoplasmic cholesterol esterifying enzyme acyl-CoA:cholesterol acyltransferase (ACAT). Incubation of the cholesterol-loaded cells in the presence of various ACAT inhibitors (Sandoz 58.035, Octimibate-Nattermann, progesterone) revealed a time- and dose-dependent amplification in HDL binding and HDL-mediated cholesterol efflux. It is concluded that the homeostasis of cellular cholesterol in macrophages is regulated in part by the number of HDL binding sites and that ACAT inhibitors enhance HDL-mediated cholesterol efflux from peripheral cells.     

Interaction between high-density lipoprotein subpopulations in apo B-free and abetalipoproteinemic plasma.

Two populations of high-density lipoprotein (HDL) particles exist in human plasma. Both contain apolipoprotein (apo) A-I, but only one contains apo A-II: Lp(AI w AII) and Lp(AI w/o AII). To study the extent of interaction between these particles, apo B-free plasma prepared by the selective removal of apo B-containing lipoproteins (LpB) from the plasma of three normolipidemic (NL) subjects and whole plasma from two patients with abetalipoproteinemia (ABL) were incubated at 37 degrees C for 24 h. Apo B-free plasma samples were used to avoid lipid-exchange between HDL and LpB. Lp(AI w AII) and Lp(AI w/o AII) were isolated from each apo B-free plasma sample before and after incubation and their protein and lipid contents quantified. Before incubation, ABL plasma had reduced levels of Lp(AI w AII) and Lp(AI w/o AII), (40% and 70% of normals, respectively). Compared to the HDL of apo B-free NL plasma, ABL HDL had higher relative contents of free cholesterol, phospholipid and total lipid, and contained more particles with apparent hydrated Stokes diameter in the 9.2-17.0 nm region. These differences were particularly pronounced in particles without apo A-II. Despite their differences, the total cholesterol contents of Lp(AI w AII) increased, while that of Lp(AI w/o AII) decreased in all five plasma samples and the amount of apo A-I in Lp(AI w AII) increased by 6-8 mg/dl in four during the incubation. These compositional changes were accompanied by a relative reduction of particles in the 7.0-8.2 nm Stokes diameter size region and an increase of particles in the 9.2-11.2 nm region. These data are consistent with intravascular modulation between HDL particles with and without apo A-II. The observed increase in apo A-II-associated cholesterol and apo A-I, could involve either the transfer of cholesterol and apo A-I from particles without apo A-II to those with A-II, or the transfer of apo A-II from Lp(AI w AII) to Lp(AI w/o AII). The exact mechanism and direction of the transfer remain to be determined.     

Lipoproteomics II: mapping of proteins in high-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry

High-density lipoprotein (HDL) is the most abundant lipoprotein particle in the plasma and a negative risk factor of atherosclerosis. By using a proteomic approach it is possible to obtain detailed information about its protein content and protein modifications that may give new information about the physiological roles of HDL. In this study the two subfractions; HDL(2) and HDL(3), were isolated by two-step discontinuous density-gradient ultracentrifugation and the proteins were separated with two-dimensional gel electrophoresis and identified with peptide mass fingerprinting, using matrix-assisted laser desorption/ionisation time of flight mass spectrometry. Identified proteins in HDL were: the dominating apo A-I as six isoforms, four of them with a glycosylation pattern and one of them with retained propeptide, apolipoprotein (apo) A-II, apo A-IV, apo C-I, apo C-II, apo C-III (two isoforms), apo E (five isoforms), the recently discovered apo M (two isoforms), serum amyloid A (two isoforms) and serum amyloid A-IV (six isoforms). Furthermore, alpha-1-antitrypsin was identified in HDL for the first time. Additionally, salivary alpha-amylase was identified as two isoforms in HDL(2), and apo L and a glycosylated apo A-II were identified in HDL(3). Besides confirming the presence of different apolipoproteins, this study indicates new patterns of glycosylated apo A-I and apo A-II. Furthermore, the study reveals new proteins in HDL; alpha-1-antitrypsin and salivary alpha-amylase. Further investigations about these proteins may give new insight into the functional role of HDL in coronary artery diseases.     

Apolipoprotein E genotypes and metabolic risk factors for coronary heart disease in middle-aged women

Apo E genotypes and plasma metabolic risk factors (total cholesterol, triglycerides, HDL and LDL cholesterol, total/HDL cholesterol ratio, lipoprotein Lp (a), apolipoprotein A-I, A-II, apo B, and apo E) were determined in 134 healthy middle-aged (X +/- SD 49.62 +/- 4.83) women. The aim of this study was to investigate metabolic risk markers according to various apo E genotypes, and to evaluate a possible risk for coronary heart disease. The results revealed that the frequencies of apo E3/3 are the most frequent (46%), followed by E4/4 (2%), E3/4 (14%), E2/3 (14%), and E2/4 (2%) in the middle-aged women. Higher mean triglycerides, LDL-C and apo B levels were found with apo E3/4, and lower mean levels of HDL-C i.e. apo A-I than in other analyzed genotypes. Greater mean of total/HDL ratio and lower levels of apo A-II were seen with E2/4. Serum lipoprotein Lp (a) concentration was higher in women with genotypes E3/3. Apo E concentration was the lowest with genotypes E4/4, i.e. the highest with E2/3. Serum total cholesterol tended to be higher in women with genotypes E4/4. Genotype E3/4 is connected with the highest concentrations of (X +/- SD) triglycerides (1.74 +/- 0.78), LDL (4.28 +/- 1.88), apo B (1.03 +/- 0.32) and with the lowest concentrations of HDL cholesterol (1.11 +/- 0.21) in the relation to the other analyzed genotypes. This group of women could possibly represent high risk women for CHD. Genotype E3/3 is associated with the highest concentration of independent genetic risk marker for CHD, lipoprotein Lp (a) (0.19 +/- 0.27). The genotype E4/4 has the highest concentration of total cholesterol (5.93 +/- 1.01), and has to be taken in account for risk evaluation in women. High level of apo E (0.11 +/- 0.05) and low level of apo A-I (1.80 +/- 0.44) were associated with E2/3 genotypes. The significance of E3/4 with the high total/HDL ratio (5.52 +/- 2.21) and low apo A-II (0.53 +/- 0.09) is important indicator, because total/HDL cholesterol ratio represents independent Established Risk Factor (ERF) for CHD. Apolipoprotein E genotypes as genetic markers and investigation of serum metabolic risk markers appear to be important in view for further evaluation of high risk women for CHD in our population.     

Plasma factors required for human apolipoprotein A-II dimerization

Although plasma high-density lipoproteins (HDL) have been implicated in several cardioprotective pathways, the physiologic role of apolipoprotein (apo) A-II, the second most abundant of the HDL proteins, remains ambiguous. Human apo A-II is distinguished from most other species by a single cysteine (Cys6), which forms a disulfide bond with other cysteine-containing apos. In human plasma, nearly all apo A-II occurs as disulfide-linked homodimers of 17.4 kDa. Although dimerization is an important determinant of human apo A-II metabolism, its mechanism and the plasma and/or cellular sites of its dimerization are not known. Using SDS-PAGE and densitometry we investigated the kinetics of apo A-II dimerization and observed a slow (t(1/2) = approximately 10 days), second-order process in Tris-buffered saline. In 3 M guanidine hydrochloride, which disrupts apo A-II secondary structure and self-association, the rate was 3-fold slower. In contrast, lipid surfaces that promote apo A-II alpha-helix formation and lipophilic interaction profoundly enhanced the rate. Reassembled HDL increased the second-order rate constant k(2) by 7500-fold, unilamellar 1-palmitoyl-2-oleoylphosphatidylcholine vesicles increased k(2) 850-fold, and physiological concentrations of human serum albumin increased k(2) 220-fold. Thus, while dimerization of apo A-II in aqueous buffer is too slow to account for the high fraction of dimer found in plasma, lipids and proteins "catalyze" dimer formation, a process that could occur either intracellularly prior to secretion or in the plasma compartment following secretion. These data suggest that formation of disulfide links within or between polypeptide chains can be controlled, in part, by coexisting lipids and proteins.     

Characterization of human high-density lipoprotein subclasses LP A-I and LP A-I/A-II and binding to HepG2 cells

Plasma HDL can be classified according to their apolipoprotein content into at least two types of lipoprotein particles: lipoproteins containing both apo A-I and apo A-II (LP A-I/A-II) and lipoproteins with apo A-I but without apo A-II (LP A-I). LP A-I and LP A-I/A-II were isolated by immuno-affinity chromatography. LP A-I has a higher cholesterol content and less protein compared to LP A-I/A-II. The average particle mass of LP A-I is higher (379 kDa) than the average particle weight of LP A-I/A-II (269 kDa). The binding of 125I-LP A-I to HepG2 cells at 4 degrees C, as well as the uptake of [3H]cholesteryl ether-labelled LP A-I by HepG2 cells at 37 degrees C, was significantly higher than the binding and uptake of LP A-I/A-II. It is likely that both binding and uptake are mediated by apo A-I. Our results do not provide evidence in favor of a specific role for apo A-II in the binding and uptake of HDL by HepG2 cells.     

Consistent relationship between selenium and apolipoprotein A-II concentrations in the sera of fasting middle-aged male abstainers and regular consumers of alcohol

Several studies have suggested that selenium serum levels may be associated with serum lipids and apolipoproteins. In the present study, 99 clerical workers aged 40–49 yr were selected based on their drinking and smoking habits. The serum concentration of selenium was not affected by these lifestyle factors. The regular drinkers had raised serum high-density lipoprotein cholesterol, apo A-I, and apo A-II concentrations. Correlation analysis showed that serum selenium was positively and consistently associated with apo A-II regardless of alcohol consumption. Factor analysis revealed that serum selenium had no association with factors that represented each lipoprotein fraction (LDL, HDL, and VLDL). The present study indicates that serum selenium is positively correlated only with apo A-II levels.     

Comparison of the metabolic behavior of rat apolipoproteins A-I and A-IV, isolated from both lymph chylomicrons and serum high density lipoproteins

Rat apolipoprotein (apo) A-I and A-IV, isolated from both lymph chylomicrons and serum high density lipoproteins (HDL) were analyzed by isoelectric focusing. Lymph chylomicron apo A-I consisted for 81 +/- 2% of the pro form and for 19 +/- 2% of the mature form, while apo A-I isolated from serum HDL was present for 36 +/- 4% in the pro form and for 64 +/- 4% in the mature form. Apo A-IV also showed two major protein bands after analysis by isoelectric focusing. The most prominent component is the more basic protein that amounts to 80 +/- 2% in apo A-IV isolated from lymph chylomicrons and to 60 +/- 3% in apo A-IV isolated from serum HDL. Apo A-I (or apo A-IV), isolated from both sources (lymph chylomicrons or serum HDL), was iodinated and the radioactive apolipoproteins were incorporated into rat serum lipoproteins. The resulting labeled HDL was isolated from serum by molecular sieve chromatography on 6% agarose columns and injected intravenously into rats. No difference in the fractional turnover rate or the tissue uptake of the two labeled HDL preparations was observed, neither for apo A-I nor for apo A-IV. It is concluded that the physiological significance of the extracellular pro apo A-I conversion or the post-translational modification of apo A-IV is not related to the fractional turnover rate in serum or to the rate of catabolism in liver and kidneys.     

Studies on the interaction between apolipoprotein A-II-enriched HDL3 and cultured bovine aortic endothelial (BAE) cells

The specific binding of high-density lipoproteins (HDL) to a number of cell and membrane types has been reported. The aim of this study was to investigate the ligand specificity of HDL binding sites on bovine aortic endothelial (BAE) cells and in particular to investigate the role of apo A-II in the interaction. In order to do this we prepared AII-HDL3 particles by incubating HDL3 with apo HDL. These particles were enriched in apo A-II, contained virtually no apo A-I, and were similar to HDL3 in terms of size and lipid composition. As these particles resemble the native HDL3 structure we believe they are probably a more suitable model for investigation of ligand specificity than artificial recombinants. AII-HDL3 particles were shown to bind to cells with similar affinity and capacity as HDL3. Further experiments indicated that HDL3 and AII-HDL3 competed with each other for binding and displayed similar affinities for a common binding site(s). The results suggest that apo A-II, as well as apo A-I, play an important role in the process of HDL recognition by putative HDL receptors on endothelial cells.     

A rapid and simple quantification of human apolipoprotein E-rich high-density lipoproteins in serum.

A rapid and simple method for the quantitative determination of human serum apo E-rich high-density lipoproteins is described. A sample was divided into two parts; one part was mixed with an equal volume of 13% polyethylene glycol 6000, and the other part was mixed with a solution containing dextran sulfate, sodium phosphotungstate, and Mg2+, respectively. The mixed solutions were centrifuged (2000 g; 15 min). The supernate obtained by the former procedure contained both apo E-rich HDL and apo E-poor HDL, but that obtained by the latter procedure contained solely apo E-poor HDL. The serum apo E-rich HDL concentration in terms of apo E (E) and cholesterol (C), was given by the following equations: E = EP x 2, and C = (CP - CD) x 2, where EP and CP were the concentrations of apo E and cholesterol, respectively, in the supernate obtained with 13% polyethylene glycol, and CD was the concentration of cholesterol in the supernate obtained with the mixture solution of dextran sulfate, sodium phosphotungstate, and Mg2+. Normal serum apo E-rich HDL concentrations were 2.6 +/- 1.5 and 6.7 +/- 2.3 mg/dl (means +/- SD, n = 38) in terms of apo E and cholesterol, respectively. Apo E-rich HDL was increased strikingly in the sera from three patients with hepatobiliary diseases.     

A sensitive sandwich enzyme-linked immunosorbent assay of rat apolipoprotein A-I: effect of various sample treatments on apolipoprotein A-I immunoreactivity and an application to young and aged rat sera.

A highly sensitive sandwich enzyme-linked immunosorbent assay for rat apo A-I was developed. Samples and standards were added to each well of microtiter plates precoated with immunoaffinity-purified IgG. Bound apo A-I was detected with immunoaffinity-purified Fab'-horseradish peroxidase conjugate by a colorimetric method. The sensitivity reached 2.5 pg/well, and the working range for the measurement of serum apo A-I concentration was 0.1 to 1.0 ng/well. The mean intra- and interassay coefficients of variation were 2.8 and 4.1%, respectively. The epitopes of apo A-I in serum were effectively exposed by the use of 6 mol/liter guanidine.HCl. Serum apo A-I concentrations in 36- to 40-week-old rats (62.3 +/- 8.6 mg/dl, mean +/- SD, n = 16) were significantly higher (P less than 0.05) than those in 8- to 12-week-old rats (55.1 +/- 4.3 mg/dl, n = 9). But the age-related change of serum apo A-I was much smaller than that of serum apo E. Apo A-I was contained in smaller HDL particles (or HDL2) in normal rat serum.     

Stimulation of lecithin:cholesterol acyltransferase activity by apolipoprotein A-II in the presence of apolipoprotein A-I

Various combinations of incorporation and addition of apolipoprotein A-I (apo A-I) and apolipoprotein A-II (apo A-II) individually or together to a defined lecithin-cholesterol (250/12.5 molar ratio) liposome prepared by the cholate dialysis procedure were used to study the effect of apo A-II on lecithin:cholesterol acyltransferase (LCAT, EC 2.3.1.43) activity of both purified enzyme preparations and plasma. When apo A-I (0.1-3.0 nmol/assay) alone was incorporated or added to the liposome, apo A-I effectively activated the enzyme. By contrast, when apo A-II (0.1-3.0 nmol/assay) alone was incorporated into or added to the liposome, apo A-II exhibited minimal activation of LCAT activity, approximately 1% of the activity obtained by an equal amount of apo A-I. Addition of apo A-II (0.1-3.0 nmol/assay) together with apo A-I (0.8 nmol/assay) to the liposome reduced the LCAT activity to approximately 30% of the level obtained with addition of apo A-I alone. On the other hand, addition of apo A-II (0.1-3.0 nmol/assay) or addition of lecithin-cholesterol liposome containing apo A-II (0.1-3.0 nmol/assay) to lecithin-cholesterol liposome containing apo A-I (0.8 nmol/assay) did not significantly alter apo A-I activation of LCAT activity. However, when the same amounts (0.1-3.0 nmol/assay) of apo A-II were incorporated together with apo A-I (0.8 nmol/assay) into the liposome, apo A-II significantly stimulated LCAT activity as compared to activity obtained with incorporation of apo A-I alone. The maximal stimulation was obtained with 0.4 nmol apo A-II/assay for both purified and plasma enzyme. At this apo A-II concentration, approximately 4-fold and 1.8-fold stimulation was observed for purified enzyme and plasma enzyme, respectively. These results indicated that apo A-II must be incorporated together with apo A-I into lecithin-cholesterol liposomes to exert its stimulatory effect on LCAT activity and that apo A-II in high-density lipoprotein may play an important role in the regulation of LCAT activity.     

Determination of human apolipoprotein C-II by electroimmunoassay. Studies on standardization and determination before and after physical training

1. Human VLDL and HDL were fractionated by sequential ultracentrifugation until free of contaminant plasma proteins. 2. Column chromatofocusing method was used to isolate apolipoprotein C-II from apoVLDL and apo HDL. C-apoprotein peak was rechromatofocused and the second peak was the apo C-II (pI 4.7, homogeneous band on SDS slab gel). 3. New Zealand white rabbits were immunized with apo C-II. Antiserum gave a single precipitate are of identity between whole serum, apoVLDL, apoHDL and apo C-II. 4. Apo C-II concentration was measured by electroimmunoassay method. During standardization 1% Triton X-100 improved the rocket shapes and contours. Total delipidation did not affect the assay system and so the antigenic determinants of apo C-II are all available to antiserum. The lowest concentration of apo C-II possible to determine with this method was 70 ng/sample well. 5. There was no difference between the apo C-II values before (39.8 +/- 7.1 mg/l, n = 19) and after (41.6 +/- 6.4 mg/l, n = 19) moderate physical training among normolipemic subjects. 6. Specific immunoprecipitation technique was also used to determine apo C-II content in standard pool serum.     

Serum levels of apolipoprotein A-I, A-II and HDL-cholesterol in second half of normal pregnancy and in pregnancy complicated by pre-eclampsia

Serum concentrations of Apolipoprotein A-I and A-II, (Apo A-1 and Apo A-II) HDL-cholesterol (HDL-C), Total Cholesterol (TC), triglycerides (TG) and lipoprotein electrophoresis were assayed serially in the second half of normal pregnancy (21 women), in pre-eclampsia (26 women) and in both groups one and six weeks after delivery. In the normal group we found increased concentrations of Apo A-I and HDL-C, which remained unaltered during pregnancy. Apo A-II was unchanged. Correlation coefficients for Apo A-II vs HDL-C and Apo A-I vs Apo A-II decreased gradually towards delivery while it remained at an elevated and unaltered level for Apo A-I vs HDL-C. The Apo A-I/HDL-C ratio was unaltered during the whole study while the Apo A-I/A-II ratio was elevated during pregnancy and the Apo A-II/HDL-C ratio was reduced. These results may indicate a gradual change in the surface structure of the HDL particle or its subfractions. In pre-eclampsia Apo A-I and HDL-C concentrations were reduced, TG was increased and Apo A-II and TC were unchanged when compared with the normal pregnancy group. A more pronounced correlation coefficient was recorded for Apo A-I vs HDL-C than for Apo A-II vs HDL-C and Apo A-I vs Apo II. The results indicate that from an atherogenic point of view normal pregnancy seems more beneficial than pre-eclampsia.     

The single proline-glutamine substitution at position 5 enhances the potency of amyloid fibril formation of murine apo A-II

The primary structure of murine apolipoprotein A-II (apo A-II) has been determined. Apo A-II consists of a single polypeptide chain of 78 amino acid residues, of which the amino-terminus is pyrrolidone carboxylic acid. Except for residues 5 and 38, the amino acid sequence is identical to that of murine senile amyloid protein (ASSAM), which has a common antigenicity with apo A-II. Substitution of glutamine (ASSAM) for proline (apo A-II) at position 5 is distinct and may possibly be related to murine senile amyloid-ogenesis.     

Cholesterol-induced alteration of apolipoprotein A-I conformation in reassembled high density lipoprotein.

Recent reports have shown that apolipoprotein A-I (apo A-I), the major protein of high density lipoprotein (HDL) may exist in different conformational states. We studied the effects of apolipoprotein A-II and/or cholesterol on the conformation of apo A-I in reassembled HDL. Analysis of tryptophan fluorescence quenching in the presence of iodine suggested that cholesterol increased the number of apo A-I tryptophan residues accessible to the aqueous phase, but decreased their mean degree of hydration. These observations cannot be totally explained on the basis of the effect of cholesterol on phospholipid viscosity as determined by fluorescence anisotropy of diphenyl hexatriene. We did not observe any effect of apo A-II on the conformation of apo A-I.