Elaine L. Gong

Characterization of human high-density lipoproteins by gradient gel electrophoresis

Gradient gel electrophoresis in conjunction with automated densitometry was applied to the identification and estimation of subpopulations of high-density lipoproteins (HDL) in the ultracentrifugal d less than or equal to 1.200 fraction from human plasma. The frequency distribution of relative migration distances (RF values) of subpopulation peaks in HDL patterns of a group (n = 194) of human subjects showed five apparent maxima: two in the RF range associated with the HDL2 subclass, and three in the RF range of the HDL3 subclass. HDL within RF intervals bounding these maxima were designated (HDL2b)gge, (HDL2a)gge, (HDL3a)gge, (HDL3b)gge and (HDL3c)gge and were shown to correspond approximately to material determined by analytic ultracentrifugation within the HDL2b, HDL2a and HDL3 components. Material represented by the HDL2a component, as resolved by three-component analysis of the ultracentrifugal Schlieren pattern, was found by gradient gel electrophoresis to be polydisperse in particle size. Mean hydrated densities and particle sizes of HDL corresponding to those with RF values of the frequency maxima were: 1.085 g/ml and 10.57 nm in the (HDL2b)gge; 1.115 g/ml and 9.16 nm in the (HDL2a)gge; 1.136 g/ml and 8.44 nm in the (HDL3a)gge; 1.154 g/ml and 7.97 nm in the (HDL3b)gge; and 1.171 g/ml and 7.62 nm in the (HDL3c)gge. The mean hydrated density values of the subpopulations within the (HDL3a)gge and (HDL3b)gge were comparable to those of the HDL3L and HDL3D components recently characterized by zonal ultracentrifugation. High order and statistically significant correlations between densitometric scans of the (HDL2b)gge, (HDL2a)gge and (HDL3)gge material, as obtained from gradient gels, and plasma concentrations of the HDL2b, HDL2a and HDL3 components, as obtained from analytic ultracentrifugation, were demonstrated.

Electron microscopic study on reassembly of plasma high density apoprotein with various lipids.

Products resulting from the sonification of mixtures of plasma high density lipoprotein apoprotein and specific lipids were studied by electron microscopy using negative staining. Sonicates of apoprotein plus lecithin produced disc-shaped structures which stacked in aggregates with a 50–55-A repeat; the discs were 100–200 A in diameter. Incorporation of unesterified cholesterol into the mixture produced structures morphologically similar to those observed in sonicates of apoprotein plus lecithin. Disc-shaped particles from sonified mixtures of apoprotein, lecithin and unesterified cholesterol were ultracentrifugally isolated in the d 1.063–1.21 g/ml fraction and were incubated with a plasma d > 1.21 g/ml fraction containing lecithin: cholesterol acyltransferase activity. Electron microscopy following the incubation procedure showed a transformation of the disc-like structures into approximately spherical particles (50–100 A diameter). Similar spherical particles were also obtained after sonification of apoprotein-lecithin-unesterified cholesterol-cholesteryl ester mixtures. Results indicate a requirement for the presence of cholesteryl esters to maintain normal morphology of plasma high density lipoproteins.

Plasma Lipoproteins in Familial Lecithin:Cholesterol Acyltransferase Deficiency: Effects of Incubation with Lecithin: Cholesterol Acyltransferase in vitro

To study the effect of lecithin: cholesterol acyltransferase (LCAT) on the plasma lipoproteins of patients with familial LCAT deficiency, whole plasma or the lipoprotein fraction of d smaller than 1.006 g/ml (VLDL) was incubated in the presence of LCAT and subsequently examined by chemical, physical, and immunological techniques. The following occured upon incubating either hyperlipemic or nonlipemic plasma: The concentrations of polar lipids decreased, particulary in the large molecular weight lipoprotein subfraction of d 1.019-1.063 g/ml (LDL2) and in the lipoprotein fraction of 1.06301.25 g/ml (HDL). The concentration of cholesteryl ester (CE) increased, particularly in the VLDL and in the lipoprotein fractions of d 1.006-1.019 g/ml (LDL1) and LDL2. The concentration of arginine-rich apolipoprotein decreased in the HDL and increased in the VLDL and LDL1. The concentrations of the C-apoliproteins appeared to change in the opposite direction. The concentration of apolipoprotein B in the LDL increased concomitantly with an increase in the concentration and flotation rsate of the small LDL2. The concentration apolipoprotein A-I in the HDL increased; and a major component in the HDL fraction became identical in apperance to normal HDL. Upon incubating a patients isolated VLDL in the presence of LCAT, lipoproteins with properties similar to normal LDL2 were formed. These experiments show that the LCAT reaction can alter the apolipoprotein content and physical properties as well as the lipid content of the patients lipoproteins.

Characterization of discoidal complexes of phosphatidylcholine, apolipoprotein A-I and cholesterol by gradient gel electrophoresis

Complexes of egg yolk phosphatidylcholine and apolipoprotein A-I were prepared by a detergent (sodium cholate)-dialysis method and characterized by gradient gel electrophoresis, gel filtration, electron microscopy and chemical analysis. Multicomponent electrophoretic patterns were obtained indicating formation of at least eight classes of discoidal complexes. The relative contribution of the different classes to the electrophoretic pattern was a function of the molar ratio of phosphatidylcholine:apolipoprotein A-I in the interaction mixture. Molar ratios of phosphatidylcholine:apolipoprotein A-I in isolated complexes were strongly and positively correlated with disc diameter obtained by electron microscopy. Incorporation of unesterified cholesterol into phosphatidylcholine/apolipoprotein A-I interaction mixtures also resulted in formation of unique complexes but with considerably different particle size distributions relative to those observed in the absence of cholesterol. One common consequence of cholesterol incorporation into interaction mixtures of 87.5:1 and 150:1 molar ratio of phosphatidylcholine:apolipoprotein A-I was the disappearance of a major complex class with diameter of 10.8 nm and the appearance of a major component with diameter of approximately 8.8 nm. Electrophoretic patterns of cholesterol-containing complexes showed a strong similarity to patterns recently published for high density lipoproteins from plasma of lecithin:cholesterol acyltransferase-deficient subjects, suggesting that the complexes formed in vitro by the detergent-dialysis method may serve as appropriate models for investigation of the origins of the HDL particle size distribution.

Molecular pathways in the transformation of model discoidal lipoprotein complexes induced by lecithin: Cholesterol acyltransferase

Incubation (24 h, 37 degrees C) of discoidal complexes of phosphatidylcholine and apolipoprotein A-I (molar ratio 95 +/- 10 egg yolk phosphatidylcholine-apolipoprotein A-I; 10.5 X 4.0 nm, long X short dimension; designated, class 3 complexes) with the ultracentrifugal d greater than 1.21 g/ml fraction transformed the discoidal complexes to a small product with apparent mean hydrated and nonhydrated diameter of 7.8 and 6.6 nm, respectively. Formation of the small product was associated with marked reduction in phosphatidylcholine-apolipoprotein AI molar ratio of the complexes (on average from 95:1 to 45:1). Phospholipase A2 activity of lecithin:cholesterol acyltransferase participated in the depletion process, as evidenced by production of unesterified fatty acids. In the presence of the d greater than 1.21 g/ml fraction or partially purified lecithin:cholesterol acyltransferase and a source of unesterified cholesterol, the small product could be transformed to a core-containing (cholesteryl ester) round product with a hydrated and nonhydrated diameter of 8.6 and 7.5 nm, respectively. By means of cross-linking with dimethylsuberimidate, the protein moiety of the small product was shown to contain primarily two apolipoprotein A-I molecules per particle, while the large product contained three apolipoprotein A-I molecules per particle. The increase in number of apolipoprotein A-I molecules per particle during transformation of the small to the large product appeared to result from fusion of the small particles during core build-up and release of excess apolipoprotein A-I from the fusion product. The results obtained with the model complexes were consistent for the most part with recent observations (Chen, C., Applegate, K., King, W.C., Glomset, J.A., Norum, K.R. and Gjone, E. (1984) J. Lipid Res. 25, 269-282) on the transformation, by lecithin:cholesterol acyltransferase, of the small spherical high-density lipoproteins of patients with familial lecithin:cholesterol acyltransferase deficiency.

Effects of guanidine hydrochloride on human plasma high density lipoproteins

Denaturation of human plasma high density lipoproteins during ultracentrifugation in guanidine-HCl is characterized by: dissociation of apoA-I, in the range of 2-3 M guanidine-HCl, and dissociation of apoA-I and apoA-II in 5-6 M guanidine-HCl. Denaturation of high density lipoprotein species, during a sequence of timed exposure to guanidine-HCl followed first by removal of the denaturant by dialysis and then by ultracentrifugation, is characterized by:dissociation of lipid-poor apoA-I, which follows a time course similar to denaturation-related changes in reported spectroscopic parameters; and apparent formation of lipoprotein aggregation products depleted in apoA-I and relatively enriched in apoA-II. These studies indicate differential properties of the major apoproteins in stabilizing high density lipoprotein structure and characterize a mode of lipoprotein transformation and degradation which apparently results from apoprotein dissociation coupled with aggregation of denatured lipoprote species.

Use of sonicated dispersions of mixtures of cholesterol with lecithin as substrates for lecithin:Cholesterol acyltransferase☆

1. 1. Sonicated dispersions of mixtures of unesteriued cholesterol with lecithin can serve as substrates for radioassay of lecithin:cholesterol acyltransferase activity in the ultracentrifugal d > 1.21 g/ml fraction. 2. 2. Net esterification yields (24-h incubation) and initial reaction rates decrease with increasing molar proportion of unesterified cholesterol in the sonicated substrate. 3. 3. Negligible lecithin:cholesterol acyltransferase activity is observed when lecithin in the sonicated substrates is replaced by phosphatidyl ethanolamine, phosphatidyl serine or sphingomyelin. 4. 4. Ultracentrifugal fractionation of incubated assay mixtures shows product cholesteryl esters distributed primarily between the d < 1.063 and the d = 1.063–1.21 g/ml fractions. 5. 5. The present findings support the hypothesis that the significant differences in reactivity of the different classes of serum lipoproteins with lecithin: cholesterol acyltransferase may result from differences in the relative proportions of unesterified cholesterol and lecithin on the lipoprotein surface accessible to the enzyme.

Interaction by sonication of C-apolipoproteins with lipid: an electron microscopic study.

1. 1. Individual C-apolipoproteins (apoC-I, apoC-II and apoC-III) isolated from human plasma very low density lipoproteins (VLDL) were sonically reassembled with lecithin or lecithin plus nonpolar lipids (cholesteryl ester or triglyceride). Protein-lipid complexes were isolated at densities <1.006, 1.006–1.063 and 1.063–1.21 g/ml and were examined in the electron microscope. 2. 2. The bulk of the protein recovered was in the d 1.063–1.21 g/ml fraction. Each of the C-apolipoproteins formed discoidal complexes (minor axis 40 A; major axis 100–200 A) after reassembly with lecithin. Extensive rouleaux with 50–55 A periodicity were formed by the discs. ApoC-I sonicated with lecithin plus cholesteryl oleate or triolein produced spherical complexes (approximately 100 a diameter) similar to native high density lipoprotein HDL2 in the d 1.063–1.21 g/ml fraction. Both apoC-II and apoC-III formed mixed structures consisting of discs in rouleaux and HDL-like particles when nonpolar lipids were added. Rouleaux predominated in mixtures of apoC-II or apoC-III plus lecithin and cholesteryl oleate. Results were more variable with apoC-II or apoC-III plus lecithin and triolein; in some instances small spherical particles predominated while in others rouleaux predominated. This probably relates to variable incorporation of triglyceride into the protein-lipid complex. 3. 3. Structures in the d 1.006–1.063 g/ml fraction included myelin figures, vesicles with diameters greater than 200 A and small spherical particles 100–200 A in size. The latter were observed in protein-lipid mixtures containing apoC-I lecithin and nonpolar lipids. In most instances less than 10% of the protein was recovered in this density fraction. 4. 4. Particles similar to VLDL and ranging from 150–1200 A in diameter Were noted in the d < 1.006 g/ml fraction when apoC-proteins were sonicated with lecithin plus triolein. Only negligible amounts of protein were recovered in this density range.

Interaction of plasma high density lipoprotein HDL2b (d 1.063-1.100 g/ml) with single-bilayer liposomes of dimyristoylphosphatidylcholine.

Incubation of a major subfraction, HDL2b (d 1.063–1.100 g/ml), of human plasma high density lipoproteins, HDL (d 1.063–1.21 g/ml), with single-bilayer liposomes of dimyristoylphosphatidylcholine (DMPC) resulted in uptake of DMPC by the HDL2b and dissociation of lipid-free apolipoprotein A-I (apoA-I). In the presence of excess DMPC, the dissociated apoA-I was also incorporated with DMPC into discoidal complexes. Preliminary studies with model apoA-I-DMPC complexes indicated that they also can interact with native HDL2b with the resultant transfer of their DMPC to HDL2b and the concomitant release of their apoA-I. After interaction of HDL2b with DMPC liposomes, the DMPC-enriched HDL2b product showed a lower hydrated density and a larger particle size than the control HDL2b. The molecular properties of the lipoprotein product suggest that stabilization of the apoA-I-depleted HDL2b probably occurred via substitution of DMPC for the apoA-I at the HDL2b surface rather than by fusion of the apoA-I-depleted HDL2b. The above interactions of HDL2b with single-bilayer liposomes and discoidal complexes indicate pathways of phospholipid transfer relevant to the possible role of HDL in the metabolism of lipoprotein surface components in vivo.

Characterization of A-I-containing lipoproteins in subjects with A-I milano variant

The A-I Milano variant of apolipoprotein A-I (A-IM), by virtue of its Arg-173----Cys substitution, is capable of forming a disulfide bond with the 77-amino-acid apolipoprotein A-II polypeptide (A-IIS) as well as with itself to produce dimers, A-IM/A-IIS and A-IM/A-IM, respectively. A-I-containing lipoproteins (Lp): particles with A-II (Lp(A-I with A-11)) and particles without A-II (Lp(A-I without A-II)) in the plasma of two nonhyperlipidemic A-IM carriers were investigated to determine the effect of A-IM on these lipoproteins. Despite the existence of abnormal apolipoprotein dimers and the unusually low HDL cholesterol (17 and 14 mg/dl), A-I (67 and 75 mg/dl), and A-II (18 and 18 mg/dl) levels in the two carriers, the plasma A-I of the carriers was distributed between Lp(A-I with A-II) and Lp(A-I without A-II) in a proportion comparable to that observed in normals. As expected, A-IM/A-IIS mixed dimer was found in carrier Lp(A-I with A-II). However, A-IM/A-IM dimer was located almost exclusively in carrier Lp(A-I without A-II). Chemical (dimethylsuberimidate) crosslinking of the protein moieties of the major subpopulations of Lp(A-I with A-II) and Lp(A-I without A-II) of normal and A-IM carriers showed that Lp(A-I with A-II), which is located predominantly in the 7.8-9.7 nm interval ((HDL2a + 3a + 3b)gge), had an apparent protein molecular weight equivalent to two molecules of A-I and one to two molecules of A-II per particle. Most of the Lp(A-I without A-II) particles, located predominantly in the size intervals of 9.7-12.9 nm (designated (HDL2b)gge) and 8.2-8.8 nm (HDL3a)gge) had protein moieties exhibiting a molecular weight equivalence predominantly of four and three molecules of A-I, respectively. A small quantity of particles with apparent protein content of two molecules of A-I in the 7.2-8.2 nm interval ((HDL3b + 3c)gge) was also detected. These studies showed that in nonhyperlipidemic A-IM carriers, the occurrence of apolipoprotein dimers had not markedly affected the protein stoichiometry of Lp(A-I with A-II) and Lp(A-I without A-II).