Characterization of apolipoprotein A-IV complexes and A-IV isoforms in human lymph and plasma lipoproteins

We have isolated and characterised A-IV apolipoprotein (apo-A-IV) from human lymph and plasma by immunoabsorbance chromatography and two-dimensional electrophoresis. Two different apo-A-IV-containing lipoproteins were isolated from four different sources, human lymph triglyceride-rich fraction (TRL), lymph lipoprotein-deficient fraction (LDF), plasma high-density lipoprotein (HDL), and plasma lipoprotein-deficient fraction (LDF). The lipoprotein complexes obtained from lymph TRL and plasma HDL were similar and contained apo-A-IV, apo-A-I, and small molecular weight peptides (apo-C or -A-II). The second lipoprotein complex was isolated from lymph LDF and plasma LDF, and contained apo-A-IV, apo-A-I, and a peptide of Mr = 59,000. The lipid composition of the lipoprotein complexes varied according to the source: triglyceride predominating in lymph TRL and phospholipid and cholesteryl ester from the other sources. Free cholesterol was conspicuously present in very small amounts. Using two-dimensional electrophoresis and immunoblotting techniques, eleven isoproteins of apo-A-IV were identified (pI-4.98, 5.06, 5.10, 5.15, 5.20, 5.22, 5.25, 5.30, 5.34, 5.42, and 5.48). The isoprotein pattern of lymph TRL and plasma HDL was similar, but that of lymph and plasma LDF were different patterns. These results suggest that apo-A-IV associated with d less than 1.21 lipoproteins and apo-A-IV present in LDF may be in metabolically separate lipoproteins and may have different physiological roles.     

Evidence for two sites on rat liver plasma membranes which interact with high density lipoprotein.

There is little dispute that high density lipoprotein (HDL) binds to cells, however, the nature of the interaction is not fully understood. We now present evidence for a new binding site of higher affinity but lower capacity than the sites previously described in the literature. This new site is characterized by high affinity/low capacity for HDL binding (Kd = 0.94 microgram/ml, Bmax = 36 ng/mg), while the low affinity site (Kd = 36 micrograms/ml, Bmax approximately 700 ng/mg) appears to be consistent with the literature values for the interaction of HDL with cells and isolated membranes. Proteolysis of HDL with trypsin abolished its interaction with the high affinity site, suggesting an apolipoprotein requirement, while having no effect on binding to the lower affinity site. Kinetic rates of association/dissociation were determined in order to further characterize the high affinity site. At a concentration which favored the binding of HDL with the high affinity site (1 microgram/ml, 37 degrees C), the time course of association of HDL with rat liver plasma membranes, displayed a biphasic pattern, requiring 6-8 h to reach the level of binding predicted from the saturation studies. The second phase was highly sensitive to temperature, being considerably slower at 24 degrees C and totally abolished at 0 degrees C. A kinetic Kd, derived from the measured association and dissociation rate constants (Kd = 0.31 microgram/ml), was found to be of a similar magnitude to the Kd calculated for the high affinity site by Scatchard analysis (Kd = 0.94 microgram/ml). In summary, the high affinity site on rat liver plasma membranes displays an apoprotein requirement and kinetic parameters, consistent with a ligand-receptor interaction.     

Characterization of the discoidal complexes formed between apoA-I-CNBr fragments and phosphatidylcholine.

The structure, composition, and physico-chemical properties of lipid-protein complexes generated between dimyristoylphosphatidylcholine (DPMC) and the CNBr fragments of human apoA-I were studied. The fragments were separated by high performance liquid chromatography and purified on a reversed-phase column. The complexes with DMPC were isolated on a Superose column; their dimensions were obtained by gradient gel electrophoresis and by electron microscopy. The secondary structure of the protein in the complexes was studied both by circular dichroism and by attenuated total reflection infrared spectroscopy. The fragments 1 and 4 of apoA-I, containing, respectively, two and three amphipathic helices, recombined with the phospholipid to generate discoidal particles with sizes similar to that of apoA-I- and apoA-II-DMPC complexes. The infrared measurements indicated that in all complexes the apolipoprotein helical segments were oriented parallel to the phospholipid acyl chains and that the protein was located around the edges of the discs. Computer modelling of the complexes based on energy minimization techniques proposed a model for these particles in agreement with the dimensions measured experimentally. In conclusion, we propose that apoA-I and its longest CNBr fragments are able to generate discoidal particles with DMPC, with apolipoprotein helical segments oriented parallel to the acyl chains of the phospholipids.     

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.     

Quantitation of apolipoprotein A-IV in human plasma using a competitive enzyme-linked immunosorbent assay

A method for measuring human apolipoprotein A-IV has been developed using the competitive enzyme-linked immunosorbent assay (ELISA) system. The assay described is relatively easy, rapid, and inexpensive to perform, uses convenient dilutions of plasma (1/8-1/32) but is sensitive enough to quantitate the apoA-IV content of lipoproteins following gel filtration of small (0.3-0.5 ml) volumes of plasma. The working range is 100-600 ng of apoA-IV per 50-microliters sample and the intra- and interassay coefficients of variations are 7.5 and 10.2% (means), respectively. The mean apoA-IV concentration of 100 subjects was found to be 16.4 +/- 5.4 mg/dl. The assay can be performed on untreated plasma samples which may be stored frozen (-20 degrees C) for up to 2 months.     

Changes in the concentration of plasma lipoproteins and apoproteins following the administration of Triton WR 1339 to rats.

Changes in whole plasma and lipoprotien apoprotein concentrations were determined after a single injection of Triton WR 1339 into rats. Concentrations of apoproteins A-I (an activator of lecithin:cholesterol acyl transferase), arginine-rich apoprotein (ARP), and B apoprotein were measured by electroimmunoassay. The content of C-II apoprotein (an activaor of lipoprotein lipase) was estimated by the ability of plasma and lipoprotein fractions to promote hydrolysis of triglyceride in the presence of cow's milk lipase and also by isoelectric focusing on polyacrylamide gels. Apoproteins C-II and A-I were rapidly removed from high density lipoprotein (HDL) after Triton treatment and were recovered in the d 1.21 g/ml infranate fraction. A-I was then totally cleared from the plasma within 10--20 hr after injection. Arginine-rich apoprotein was removed from HDL and also partially cleared from the plasma. The rise in very low density lipoprotein (vldl) apoprotein that followed the removal of apoproteins from HDL was mostly antributed to the B apoprotein, although corresponding smaller increases were observed in VLDL ARP and C apoproteins. The triglyceride:cholesterol, triglyceride:protein, and B:C apoprotein ratios of VLDL more closely resembled nascent rather than plasma VLDL 10 hr after Triton injection. These studies suggest that the detergent may achieve its hyperlipidemic effct by disrupting HDL and thus removing the A-I and C-II proteins from a normal activating environment compirsing VLDL, HDL, and the enzymes. The possible involvement of intact HDL in VLDL catabolism is discussed in relation to other recent reports which also suggest that abnormalities of the VLDL-LDL system may be due to the absence of normal HDL.     

Vitamin A and carotenoids. The enzymic conversion of β-carotene into retinal in hog intestinal mucosa

The conversion of beta-carotene into retinal was studied in vitro with enzyme preparations from homogenates of hog intestinal mucosa. The hog mucosal enzyme was purified about 27-fold by precipitation with ammonium sulphate, chromatography on DEAE-Sephadex and gel filtration on Sephadex G-200. The reaction displayed a narrow optimum pH range (approx. 7.8-8.2). The enzyme was stimulated strongly by the addition of thiols, and was inhibited by thiol inhibitors and by the chelating agents alphaalpha'-bipyridyl and o-phenanthroline. The reaction required the addition of an appropriate detergent (or bile salt); maximal activity was obtained by addition of an appropriate combination of detergents and lipid (specifically Tween 40, sodium glycocholate and sphingomyelin). The reaction displayed Michaelis kinetics with K(m)1.3x10(-6)m and V(max.)1.1nmole of retinal formed/hr. (for 0.7mg. of enzyme protein). The properties of the hog enzyme are similar to those previously reported for a less purified rat enzyme preparation.     

The identification of specific high density lipoprotein3 binding sites on human blood monocytes using fluorescence-labeled ligand.

We previously reported the identity and purification of two HDL3-binding proteins in rat liver plasma membranes. As these proteins are candidate high density lipoprotein (HDL) receptors and probably multifunctional, including a role in HDL metabolism, we have considerable interest in identifying corresponding proteins that are present in human tissue. This report describes the identification of HDL3-binding sites on human monocytes with the use of fluorescence microscopy and flow cytometry assay. After the incubation of mononuclear cells from human blood with fluorescein isothiocyanate (FITC)-labeled human HDL3, fluorescence micrographs showed dense signals of fluorescent grains on monocytes, but not lymphocytes. A significant increase in FITC intensity on monocytes, but not lymphocytes, was observed by flow cytometry analysis, and the interaction between FITC-HDL3 and human monocytes was concentration-dependent. Although very low density (VLDL) and low density lipoprotein (LDL) were ineffective competitors and HDL2 only partially competed for binding, a 50-fold concentration of HDL3 did compete effectively for binding of FITC-HDL3 to human monocytes. Trypsin treatment reduced the FITC intensity of monocytes, showing that a portion of cell-associated FITC-HDL3 remained bound to the cell surface. Two major HDL-binding proteins were identified in CHAPS-solubilized human mononuclear cells by ligand blotting, using HDL3 as the ligand. Both showed similar binding parameters, specificity, and molecular weight identical to HB1 and HB2 from rat liver plasma membrane. We conclude that corresponding candidate HDL receptors or a similar receptor complex also exist on human blood monocytes.     

Identification and characterization of mixed disulphide complexes of E apoprotein in high density lipoprotein of subjects with acute alcoholic hepatitis.

Two E apoprotein complexes have been isolated from the plasma high density lipoprotein fraction of patients with alcoholic hepatitis. Both were mixed disulphide complexes and could be dissociated into subunits with β-mercaptoethanol but not with 1% sodium dodecyl sulphate or 8 M urea. One of the complexes (molecular weight 46,000) was identified as an E-AII unit and the other, of molecular weight 106,000 was consistent in properties with an E trimer. The latter has not been described before and the E-AII complex has been reported once previously in plasma of patients with Type III hyperlipo-proteinaemia. It is proposed that the presence of E complexes in abnormal disease states may affect the normal recognition of E monomer by cells thereby altering the subsequent fate of their host lipoproteins and their lipid constituents.     

Rotational diffusion of human lipoproteins and their receptors as determined by time-resolved phosphorescence anisotropy

Time-resolved phosphorescence anisotropy has been used to assess the rotational dynamics of human serum lipoproteins labeled with phosphorescent probes of high triplet yield. Labeling the lipid phase of low density, very low density, and high density lipoproteins with an eosinyl fatty acid revealed the existence of two motions. The shorter time constant was attributed to motion of the chromophore within the lipoprotein particle, while the longer time constant represented the global tumbling of the particles in solution. The measured correlation times for this global motion were about twice those predicted from the Stokes-Einstein relationship. Covalent labeling of the apolipoproteins of the low and high density lipoproteins with erythrosin revealed the existence of segmental motion of labeled domains of the apolipoprotein within their respective particles. The correlation times for this motion were within the range 10-50 microseconds. The binding of low density lipoproteins to receptors on membranes isolated from the adrenal cortex resulted in a freezing of the global motion, but maintenance of the faster segmental motion of the labeled domains of the apolipoprotein. The experiments imply that in these membranes there is no global motion of the low density lipoprotein-receptor complex on the phosphorescence time scale. Similar results were found for the binding of high density lipoproteins to liver plasma membranes. The contributions of nonspecific binding of the labeled lipoproteins to the measured phosphorescence anisotropy were carefully assessed.