Biomedical subjects

M C Phillips

Publications and source records attributed to M C Phillips.

At least 73 records · Page 4Linked to original sources

PubMed1994

The molecular basis for the difference in charge between pre-beta- and alpha-migrating high density lipoproteins.

Several subspecies of human high density lipoprotein (HDL) exist, and particle surface charge is one parameter that can be used to distinguish them. For instance, pre-beta HDL has a lower negative surface charge than the bulk of plasma HDL resulting in slower migration when subjected to electrophoresis in an agarose gel at pH 8.6. The molecular basis for the variations in HDL particle surface charge have been established in this work. Quantitative analysis of electrophoretic mobilities in agarose gels indicates that the surface potentials of reconstituted pre-beta HDL and alpha-HDL3 are -7.6 and -11.4 mV, respectively. The difference of 3.8 mV corresponds to an apparent 1e more net negative charge on alpha-migrating HDL3. The contributions of the lipid and protein components to the charge of HDL3 were defined using reconstituted particles (rHDL) containing various combinations of HDL3 protein and lipid. The surface potentials of spherical and discoidal rHDL are not affected by the particle apoprotein composition. Thin layer chromatography of the HDL3 phospholipid fraction from five normolipidemic subjects revealed that it consists of approximately 84% phosphatidylcholine, 12% sphingomyelin, and 4% phosphatidylinositol (PI) by weight. PI significantly affects the charge of discoidal rHDL and is responsible for approximately 0.7 mV of the surface potential difference between pre-beta- and alpha-HDL. Reconstitution of spherical complexes containing apolipoprotein AI, palmitoyl-oleoyl phosphatidylcholine, and HDL3 neutral lipid esters showed that particles containing a neutral lipid core migrate to an alpha position in an agarose gel. The change in particle shape accounts for approximately 3 mV of the difference in surface potential between pre-beta and alpha-HDL. Overall, the charge of HDL particles is determined by 1) a direct contribution of negative charge from PI molecules in the surface of the lipoprotein and 2) the conformation of the resident apolipoprotein(s) which is influenced by the presence of neutral lipids and the particle shape. This quantitative understanding of surface charge provides a basis for understanding variations in function of HDL subspecies.

Apolipoproteins A↗

PubMed1993

Conformation of apolipoprotein E in lipoproteins.

Lysine (Lys) residues in apolipoprotein (apo) E are known to be involved in binding of apoE-containing lipoproteins to the low density lipoprotein (LDL) receptor. To examine the microenvironments of the Lys residues of apoE-3 in a variety of lipid-associated states, we have used a high resolution 13C-NMR method in which Lys were reductively methylated with [13C] formaldehyde. Over a wide pH range, the spectrum of apoE in canine HDLc, a spherical lipoprotein particle, exhibited two peaks from Lys epsilon-amino groups. The two pools of Lys in HDLc titrate with pK alpha values of 10.4 ("normal") and 9.3 ("active"). In contrast, eight epsilon-[13CH3]2Lys peaks (delta = 42.5-44.5 parts/million) with pK alpha values ranging from 8.2 to 10.1 and 7.8 to 10.5 were observed at pH 9.5 for human and canine apoE, respectively, in discoidal complexes with dimyristoyl glycerophosphocholine. A single Lys microenvironment was observed for apoE present in a disordered, lipid-free, state in 8 M urea, confirming the fact that the lipid environment is modulating the conformation of apoE. The above data demonstrate that the conformation of apoE, as reflected by the Lys microenvironments, on spherical HDLc particles is different from that on discoidal complexes.

Animals↗

PubMed1993

Effect of cholesterol on the charge and structure of apolipoprotein A-I in recombinant high density lipoprotein particles.

The effects of cholesterol on the conformation and net charge of apoA-I have been investigated in homogeneous recombinant high density lipoprotein (HDL) particles. ApoA-I charge and structure in discoidal recombinant HDL complexes containing palmitoyloleoylphosphatidylcholine and cholesterol have been quantitated by guanidine HCl denaturation, circular dichroism, electrokinetic analysis, and NMR spectroscopy of [13C]lysine-labeled apoA-I. In a discoidal particle containing 2 molecules of apoA-I and 160 molecules of palmitoyloleoylphosphatidylcholine, apoA-I exhibits an alpha-helix content of 75%, and the particle has a net negative surface charge of -5.2e/mol of apoA-I at pH 8.6. Addition of 2 molecules of cholesterol to this complex has no significant effect upon particle size, but slightly decreases the net charge (-5.0e) and alpha-helix content (68%) of apoA-I and enhances the stability of the helical segments, as reflected by an increase in the free energy of unfolding from 2.9 to 3.5 kcal/mol. In contrast, increasing the cholesterol content to 20 molecules/particle progressively increases particle size and apoA-I net negative charge (-6.1e), and there is a concomitant reduction in the free energy of stabilization of the alpha-helical structure in apoA-I to 2.2 kcal/mol. (13CH3)2-Lys resonances from apoA-I in discoidal recombinant HDL exhibit six chemical shifts at pH 10; these peaks originate from dimethyl-Lys residues that have pKa values ranging from 8.4 to 10.3. The titration behavior of apoA-I Lys residues is generally similar in the presence and absence of cholesterol, except that 4 Lys residues titrate at a significantly higher pH in the presence of cholesterol. These data are consistent with cholesterol having a direct effect on apoA-I conformation and charge in HDL. Structural changes of this magnitude can affect the interactions between HDL and various plasma proteins and cell surfaces. It is therefore likely that the cholesterol content of HDL plays an important role in regulating the metabolism of this lipoprotein.

Apolipoprotein A-I↗

PubMed1993

Effect of end group blockage on the properties of a class A amphipathic helical peptide.

In a recent classification of biologically active amphipathic alpha-helixes, the lipid-associating domains in exchangeable plasma apolipoproteins have been classified as class A amphipathic helixes (Segrest, J.P., De Loof, H., Dohlman, J.G., Brouillette, C.G., Anantharamaiah, G.M. Proteins 8:103-117, 1990). A model peptide analog with the sequence, Asp Trp Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu Ala Phe (18A), possesses the characteristics of a class A amphipathic helix. The addition of an acetyl group at the alpha-amino terminus and an amide at the alpha-carboxyl terminus, to obtain Ac-18A-NH2, produces large increases in helicity for the peptide both in solution and when associated with lipid (for 18A vs Ac-18A-NH2, from 6 to 38% helix in buffer and from 49 to 92% helix when bound to dimyristoyl phosphatidylcholine in discoidal complexes). Blocking of the end-groups of 18A stabilizes the alpha-helix in the presence of lipid by approximately 1.3 kcal/mol. There is also an increase in the self-association of the blocked peptide in aqueous solution. The free energy of binding to the PC-water interface is increased only by about 3% (from -8.0 kcal/mol for 18A to -8.3 kcal/mol for Ac-18A-NH2). The Ac-18A-NH2 has a much greater potency in raising the bilayer to hexagonal phase transition temperature of dipalmitoleoyl phosphatidylethanolamine than does 18A. In this regard Ac-18A-NH2 more closely resembles the behavior of the apolipoprotein A-I, which is the major protein component of high-density lipoprotein and a potent inhibitor of lipid hexagonal phase formation. The activation of the plasma enzyme lecithin: cholesterol acyltransferase by the Ac-18A-NH2 peptide is greater than the 18A analog and comparable to that observed with the apo A-I. In the case of Ac-18A-NH2, the higher activating potency may be due, at least in part, to the ability of the peptide to micellize egg PC vesicles.

Apolipoprotein A-I↗

PubMed1993

The immunologic role of the gastrointestinal tract.

During the past few decades, researchers have shed new light on the role of nutritional support in the immunocompromised critically ill patient. "Nutritional pharmacology" has become the catch phrase of the 1990s due to the impact of selected nutrients on host immune defenses. When feasible, enteral nutrition is preferred to parenteral nutrition to preserve the integrity of the gut mucosal barrier, especially when administered jejunally in the early stages of hypermetabolism. Glutamine and fiber provide necessary fuels for the gastrointestinal tract, whereas arginine and omega-3 fatty acids exert beneficial effects on certain cells of the immune system. Daly et al demonstrated that "enteral nutrition with supplemental arginine, RNA nucleotides and omega-3 fatty acids (Impact, Sandoz Nutrition, Minneapolis, MN) compared with a standard enteral diet, significantly improved immune, metabolic and clinical outcomes (22% mean reduction in length of stay) in UGI cancer patients undergoing surgery." Although more research is needed in the area of nutritional pharmacology, newly available nutrient-specific enteral products (Table 1) provide promise for altering the clinical outcome of immunocompromised patients.

Critical Illness↗

PubMed1992

The conformation of apolipoprotein A-I in discoidal and spherical recombinant high density lipoprotein particles. 13C NMR studies of lysine ionization behavior.

To elucidate the molecular details of how high density lipoprotein (HDL) microstructure affects the conformation of apolipoprotein (apo) A-I in various classes of HDL particles, apoA-I structure in homogeneous recombinant HDL (rHDL) complexes containing palmitoyl-oleoyl phosphatidylcholine (POPC) and cholesteryl oleate has been investigated by NMR spectroscopy of [13C]lysine-labeled apoA-I. All Lys residues in rHDL apoA-I were labeled with 13C by reductive methylation, and then their ionization behavior was characterized by 13C NMR spectroscopy. Four discoidal particles were prepared to contain from 64 to 256 molecules of POPC and 2 molecules of apoA-I; their major diameters ranged from 9.3 to 12.1 nm. (13CH3)2-Lys resonances from apoA-I in discoidal complexes exhibit six distinct chemical shifts at pH 10. The various Lys have pKa values ranging from 8.3 to 10.5, indicating that they exist in different microenvironments. More than 80% of the Lys residues in small (9.3 nm) discoidal particles titrate at a significantly lower pH than in the large (12.1 nm) discoidal particles. This indicates that apoA-I has a different conformation on the differently size discs. Two spherical particles were prepared with POPC:cholesteryl oleate:apoA-I molar stoichiometries of 56:16:2 and 232:84:4 and diameters of 7.4 and 12.6 nm, respectively. On spherical rHDL, apoA-I (13CH3)2-Lys resonances exhibit five distinct chemical shifts at pH 10. The titration behavior of apoA-I Lys residues is the same in small and large spherical particles, indicating that apoA-I conformation is similar on the two particles. The Lys microenvironments indicate that the conformation of apoA-I in discoidal complexes is dependent on particle size and that these conformations are substantially different from that of apoA-I on spherical complexes. Lys microenvironments in discoidal complexes differ from that of spherical complexes by 4 to 5 ysines which titrate with relatively low pKa values on discs. This reflects apparent differences in conformation in the NH2-terminal one-third of apoA-I on discs and spheres.

Amino Acid Sequence↗

PubMed1992

The charge and structural stability of apolipoprotein A-I in discoidal and spherical recombinant high density lipoprotein particles.

The details of how high density lipoprotein (HDL) microstructure affects the conformation and net charge of apolipoprotein (apo) A-I in various classes of HDL particles have been investigated in homogeneous recombinant HDL (rHDL) particles containing apoA-I, palmitoyl-oleoyl phosphatidylcholine (POPC) and cholesteryl oleate. Isothermal denaturation with guanidine HCl was used to monitor alpha-helix structural stability, whereas electrokinetic analyses and circular dichroism were used to determine particle charge and apoA-I secondary structure, respectively. Electrokinetic analyses show that at pH 8.6 apoA-I has a net negative charge on discoidal (POPC.apoA-I) particles (-5.2 electronic units/mol of apoA-I) which is significantly greater than that of apoA-I either free in solution or on spherical (POPC.cholesteryl oleate.apoA-I) rHDL (approximately -3.5 electronic units). Raising the POPC content (32-128 mol/ml of apoA-I) of discoidal particles 1) increases the particle major diameter from 9.3 to 12.1 nm, 2) increases the alpha-helix content from 62 to 77%, and 3) stabilizes the helical segments by increasing the free energy of unfolding (delta GD degree) from 1.4 to 3.0 kcal/mol of apoA-I. Raising the POPC content (28-58 mol/mol of apoA-I) of spherical particles 1) increases the particle diameter from 7.4 to 12.6 nm, 2) increases the percent alpha-helix from 62 to 69%, and 3) has no significant effect on delta GD degree (2.2 kcal/mol of apoA-I). This study shows that different HDL subspecies maintain particular apoA-I conformations that confer unique charge and structural characteristics on the particles. It is likely that the charge and conformation of apoA-I are critical molecular properties that modulate the metabolism of HDL particles and influence their role in cholesterol transport.

Apolipoprotein A-I↗

PubMed1992

Effects of membrane lipids and -proteins and cytoskeletal proteins on the kinetics of cholesterol exchange between high density lipoprotein and human red blood cells, ghosts and microvesicles.

To better understand the effects of plasma membrane lipids and proteins and the cytoskeleton on the kinetics of cellular cholesterol efflux, the effects of (1), selectively depleting either sphingomyelin (SM) or phosphatidylcholine (PC); (2), cross-linking the cytoskeleton, and (3), removing certain cytoskeletal and integral membrane proteins on radiolabelled cholesterol efflux from red blood cells (RBC) have been studied. When RBC were treated with either phospholipase A2 or sphingomyelinase C to hydrolyze either 30-40% of the PC or 40-50% of the SM, respectively, the halftimes (t1/2) for cholesterol efflux to excess HDL3 were not significantly altered, with the values being 4.4 +/- 0.8 h or 3.7 +/- 0.4 h, respectively, compared to 4.6 +/- 0.6 h for control RBC. To investigate the effects of the cytoskeleton on the rate of free cholesterol (FC) desorption from the plasma membrane, the cytoskeletal proteins were cross-linked by either heat-treatment or exposure to diamide and cholesterol efflux from ghosts of these cells was measured. Cross-linking the cytoskeletal proteins by diamide treatment resulted in no significant change in t1/2 for treated (3.6 +/- 0.6 h) compared to control (4.2 +/- 0.4 h) ghosts: this suggests that the cytoskeleton does not play a large role in modulating cholesterol efflux. To investigate the effects of membrane proteins on cholesterol efflux, RBC microvesicles, containing mainly band 3 and 4 proteins and little of the cytoskeletal proteins, such as spectrin (bands 1,2) or actin (band 5), were obtained by incubation with the ionophore A23187. With excess HDL3 present, microvesicles exhibited a t1/2 of 4.2 +/- 1.9 h (compared to the t1/2 of 4.2 +/- 0.4 h for control ghosts). The results described in this paper suggest that neither changing the SM/PC ratio in the membrane nor cross-linking the cytoskeletal proteins nor removing the cytoskeleton changes the t1/2 for cholesterol efflux to excess HDL3. Presumably, the cholesterol-phospholipid interactions are insensitive to these perturbations in membrane structure.

Cholesterol↗

PubMed1992

The phase behavior of cholesteryl esters in intracellular inclusions.

Differential scanning calorimetry and polarizing light microscopy have been used to investigate kinetic and thermodynamic properties of the phase behavior of cholesteryl ester contained in Fu5AH rat hepatoma cells and J774 murine macrophages. These cultured cells store cholesteryl esters as cytoplasmic inclusions of approximately 1-micron diameter and thus are models of the foam cells characteristic of atherosclerotic plaque. Simple binary mixtures of cholesteryl palmitate and cholesteryl oleate, the predominant cholesteryl esters in cellular inclusions in both cell types serve as models to explain important aspects of the phase behavior of these inclusions. Although inclusions should exist as stable crystals at 37 degrees C under conditions of thermodynamic equilibrium, microscopic examination of cells indicates that inclusions exist as metastable liquid crystals at 37 degrees C for extended periods of time. Using an analytical model based on nucleation theory, we predict that the cholesteryl ester inclusions should be liquid-crystalline in the cytoplasm of living cells. This may not be true either for lysosomal cholesteryl ester or for extracellular cholesteryl ester present in advanced atherosclerotic plaque where fusion of droplets can enhance the possibility of crystallization. The enhanced metastability of the relatively fluid liquid-crystalline state in cellular inclusions should result in increased activity of the neutral cholesteryl ester hydrolase in living cells.

Animals↗

PubMed1992

Adsorption of apolipoprotein A-IV to phospholipid monolayers spread at the air/water interface. A model for its labile binding to high density lipoproteins.

The mechanisms that mediate the labile binding of apolipoprotein A-IV (apoA-IV) to high density lipoproteins (HDL) are not known. We therefore used a surface balance and surface radioactivity detector to investigate the adsorption of apoA-IV to egg phosphatidylcholine monolayers spread at the air/water interface. ApoA-IV bound rapidly and reversibly to phospholipid monolayers and generated a maximum increase in surface pressure of 19 millinewtons (mN)/m at a subphase concentration of 2 x 10(-5) g/dl. Binding decreased linearly with increasing initial surface pressure; at pressures greater than 28-29 mN/m, apoA-IV could no longer penetrate the lipid monolayer. The area occupied by the amino acid residues in apoA-IV reached an unusually low limiting molecular area of 10-12 A2/residue at surface saturation. The surface pressure of native HDL3 was calculated to be 33 mN/m, and it rapidly decreased with the action of lecithin:cholesterol acyltransferase on the particle surface. We conclude that the surface activity of apoA-IV is lower than that of any other human apolipoprotein; its binding and surface conformation are particularly sensitive to pressure; and at saturation, a significant portion of the molecule is excluded from the interface. The exclusion pressure of apoA-IV may be only slightly lower than the surface pressure of HDL; in vivo, the action of lecithin:cholesterol acyltransferase and lipid transfer proteins may cause the HDL3 surface pressure to oscillate about a narrow range that spans the exclusion pressure of apoA-IV. The resultant labile association of apoA-IV and HDL may be of central importance to its role in lipoprotein metabolism.

Adsorption↗

PubMed1992

Effect of dietary supplementation with n-3 polyunsaturated fatty acids on physical properties and metabolism of low density lipoprotein in humans.

The effects of marine n-3 polyunsaturated fatty acids were investigated in relation to the chemical and physical properties of low density lipoprotein (LDL) and how these changes affected LDL metabolism in humans. The subjects received supplements of six capsules daily, each capsule containing 1 g of either highly concentrated ethyl esters of n-3 fatty acids (85% eicosapentaenoic acid and docosahexaenoic acid) (n = 12) or corn oil (56% linoleic and 26% oleic acid) (n = 11). After 4 months of oil supplementation, the following changes were observed in the lipid moiety of the n-3-enriched LDL particles compared with LDL from the corn oil group: LDL cholesteryl ester, as well as the amount of total lipids of LDL, was significantly lower (0.97 +/- 0.12 versus 1.19 +/- 0.23 mg/mg protein and 1.88 +/- 0.40 versus 2.45 +/- 0.31 mg/mg, respectively; mean +/- SD, n = 6, p less than 0.05); the amount of eicosapentaenoic and docosahexaenoic acids and the unsaturation index increased (104.0 versus 29.4 micrograms/mg protein and 6.64 versus 5.49, respectively); and differential scanning calorimetry showed that LDL cholesteryl ester melting temperature was lowered by 2 degrees C (27.6 +/- 0.8 degrees versus 29.5 +/- 0.2 degrees C). The only effect observed on the protein moiety was an increase in the ratio of apolipoprotein (apo) B to cholesterol (0.66 +/- 0.17 versus 0.82 +/- 0.14 mg/mg cholesterol; p less than 0.05). Circular dichroism spectra of LDL indicated an alpha-helix content of 46 +/- 5% in apo B from both groups. No difference was observed by 13C nuclear magnetic resonance spectroscopy in the ratio of "active" to "normal" lysine residues of apo B. No detectable differences in the size of n-3 fatty acid-enriched LDL particles versus control LDL could be measured by either electron microscopy of negatively stained LDL (24.5 +/- 2.0 versus 25.0 +/- 1.5 nm) or dynamic light scattering (24.9 +/- 0.9 versus 24.9 +/- 0.4 nm). LDL from the fish oil and corn oil groups showed similar susceptibility to Cu(2+)-catalyzed lipid peroxidation, as indicated by the amount of lipid peroxides formed during the oxidation time, and degradation of oxidatively modified LDL in J774 macrophages as a function of Cu2+ oxidation time. No effect of n-3 fatty acids was observed on LDL metabolism. Specific uptake and degradation of n-3 fatty acid-enriched LDL were similar to those for control LDL in HepG2 cells as well as in human skin fibroblasts, and they showed the same ability to stimulate cholesteryl ester synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗

PubMed1992

Turnover of synthetic class A amphipathic peptide analogues of exchangeable apolipoproteins in rats. Correlation with physical properties.

Peptide analogues of the class A amphipathic helixes from exchangeable apolipoproteins mimic apolipoprotein (apo) A-I in a number of ways, including the ability to activate the enzyme lecithin:cholesterol acyltransferase, to associate with high density lipoproteins (HDLs), and to form HDL-like particles in the presence of lipids. This study investigated the metabolic properties of several of these peptide analogues in the rat. Peptide analogues studied were 18A (referred to as L-18A to differentiate it from D-18A, and which mimics apolipoprotein amphipathic helical domains in its charge distribution), 37pA (a dimer of two 18A monomers separated by a proline), 18R (with reversed charge distribution compared with 18A), and D-18A (identical in amino acid sequence to 18A but synthesized from D-amino acids). Peptides were radiolabeled with 125I. In addition, metabolism of rat and human 125I-apo A-I and human 14C-apo A-I was studied; no significant differences in clearance of these preparations were seen. Clearance data were fitted to multiexponential equations to give half-times of clearance; biexponential equations consistently provided the best nonlinear least-squares curve fit. The order of relative lipid affinity determined in vitro was 37pA greater than apo A-I greater than D-18A = L-18A greater than 18R. Half-times of clearance were in the same approximate rank order: 37pA, 6.9 +/- 3.3 hours (mean +/- SD); apo A-I, 6.9 +/- 1.8 hours; D-18A, 4.0 +/- 1.0 hours; L-18A, 4.6 +/- 1.6 hours; and 18R, 0.9 +/- 0.1 hour.(ABSTRACT TRUNCATED AT 250 WORDS)

Adsorption↗

PubMed1992

Apolipoproteins, membrane cholesterol domains, and the regulation of cholesterol efflux.

Published data related to both cell membrane biology and apolipoprotein structure are reviewed and used to formulate a new model describing the mechanisms of cholesterol efflux from cell plasma membrane to high density lipoprotein (HDL) particles. The central premise of this model is the existence of heterogenous domains of cholesterol within plasma membranes. We propose that cholesterol efflux from cell membranes is influenced by three factors: 1) the distribution of cholesterol between cholesterol-rich and cholesterol-poor membrane domains, 2) the diffusion of cholesterol molecules through the extracellular unstirred water layer, and 3) the transient interaction of segments of the amphipathic helix of the HDL apolipoprotein with cholesterol-poor membrane domains resulting in enhanced cholesterol efflux.

Animals↗

PubMed1992

Quantitative measurement of lipoprotein surface charge by agarose gel electrophoresis.

The electrophoretic mobilities of low density lipoprotein (LDL) and six pure proteins in a 0.5% agarose gel have been compared to literature electrophoretic mobility values determined by the Tiselius moving boundary method. There is a strong correlation (r = 0.99) between the electrophoretic mobilities determined by the two techniques. The electrophoretic behavior of charged particles smaller than very low density lipoproteins (VLDL) is not markedly perturbed by a 0.5% agarose matrix, and variations in mobility primarily reflect differences in particle valence and density of surface charge. Application of electrokinetic theory to derive protein and lipoprotein net charges from the electrophoretic mobilities in agarose yields a quantitative delineation of lipoprotein electrophoretic migration patterns wherein the beta mobility region comprises a surface potential range of -4.5 to -7.0 mV; the pre-beta region a range of -7.0 to -10.5 mV; the alpha mobility region a range of -10.5 to -12.5 mV and the serum albumin region a range of -12.5 to -14.0 mV. Because protein conformation and charge are critical in metabolic regulation, the agarose gel electrophoresis technique provides a valuable analytical tool that should help to elucidate further details of the structure-function relationships of serum lipoprotein particles.

Electrochemistry↗

PubMed1991

Cholesterol transport between cells and high-density lipoproteins.

Various types of studies in humans and animals suggest strongly that HDL is anti-atherogenic. The anti-atherogenic potential of HDL is thought to be due to its participation in reverse cholesterol transport, the process by which cholesterol is removed from non-hepatic cells and transported to the liver for elimination from the body. Extensive studies in cell culture systems have demonstrated that HDL is an important mediator of sterol transport between cells and the plasma compartment. The topic of this review is the mechanisms that account for sterol movement between HDL and cells. The most prominent and easily measured aspect of sterol movement between HDL and cells is the rapid bidirectional transfer of cholesterol between the lipoprotein and the plasma membrane. This movement occurs by unmediated diffusion, and in most situations its rate in each direction is limited by the rate of desorption of sterol molecules from the donor surface into the adjacent water phase. The net transfer of sterol mass out of cells occurs when there is either a relative enrichment of sterol within the plasma membrane or a depletion of sterol in HDL. Recent studies suggest that certain minor subfractions of HDL (with pre-beta mobility on agarose gel electrophoresis and containing apoprotein A-I but no apo A-II) are unusually efficient at promoting efflux of cell sterol. To what extent efflux to these HDL fractions is balanced by influx from the lipoprotein has not yet been established clearly. The prevention and reversal of atherosclerosis require the mobilization of cholesterol from internal (non-plasma membrane) cellular locations. To some extent, this may involve the retroendocytosis of HDL. However, most mobilization probably involves the transport of internal sterol to the plasma membrane, followed by desorption to extracellular HDL. Several laboratories are investigating the transport of sterol from intracellular locations to the plasma membrane. Studies on biosynthetic sterol (probably originating mostly in the smooth endoplasmic reticulum) suggest that there is rapid transport to the plasma membrane in lipid-rich vesicles. Important features of this transport are that it bypasses the Golgi apparatus and may be positively regulated by the specific binding of HDL to the plasma membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1991

13C NMR evidence that substitution of glutamine for arginine 3500 in familial defective apolipoprotein B-100 disrupts the conformation of the receptor-binding domain.

Familial defective apoB-100 is a genetic mutation that is characterized by abnormal low density lipoprotein (LDL) and moderate hypercholesterolemia. Heterozygotes for this disorder possess two populations of LDL. One has normal receptor binding, and the other, which can be isolated by monoclonal antibody 19 immunoaffinity chromatography, has almost no binding activity. The mutation that disrupts binding is a Gln for Arg substitution of apoB-100 residue 3500. NMR spectra of LDL containing (13CH3)2Lys residues show that chemically modified Lys exist in two microenvironments. In normal human LDL, there are about 50 Lys with pK 8.9 and 170 Lys with pK 10.5; an upper limit of 10 pK 8.9 Lys may be particularly involved in binding to the LDL receptor. Examination of the mixture of normal LDL and mutant LDL from five patients shows that the latter have fewer pK 8.9 Lys. In purified defective LDL at least seven Lys are redistributed from the active to normal pool. The CD spectra of mutant and normal LDL are identical. Therefore, substitution of Gln for Arg at position 3500 induces a change in local conformation which disrupts the receptor-binding domain of apoB-100.

Apolipoprotein B-100↗

PubMed1991

Effects of apolipoprotein structure on the kinetics of apolipoprotein transfer between phospholipid vesicles.

The kinetics and mechanism of transfer of 14C-labeled human apolipoproteins A-I, A-II and C-III1 between small unilamellar vesicles (SUV) have been investigated. Ion exchange chromatography was used for rapid separation of negatively charged egg phosphatidylcholine (PC)/dicetyl phosphate donor SUV containing bound 14C-labeled apoprotein from neutral egg PC acceptor SUV present in 10-fold molar excess. The transfer kinetics of these apolipoproteins at 37 degrees C are consistent with the existence of fast, slow and apparently 'nontransferrable' pools of SUV-associated lipoprotein: the transfers from these pools occur on timescales of seconds (or less), minutes/hours and days/weeks, respectively. For donor SUV containing about 15 apoprotein molecules per vesicle and at a donor SUV concentration of 0.15 mg phospholipid/ml incubation mixture, the sizes of the fast kinetic pools for apolipoproteins A-I, A-II and C-III1 associated with donor SUV are 2, 10 and 11%, respectively. The sizes of the slow kinetic pools for these apolipoproteins are 16, 71 and 50%, respectively. The transfer of the various apolipoproteins from the slow kinetic pool follows first order kinetics and the half-time (t1/2) values are in the order: apo C-III1 less than apo A-I. Increasing the number of apoprotein molecules per donor SUV enlarges the size of the fast pool and increases the t1/2 of slow transfer. The differences in the kinetics of apolipoprotein transfer between SUV are consequences of the variations in the primary and secondary structures of the apolipoprotein molecules. The slow transfer of apoprotein molecules is mediated by collisions between donor and acceptor SUV; the rate is dependent on the apoprotein molecular weight with larger molecules transferring more slowly from donor SUV containing the same lipid/protein molar ratio. The hydrophobicity of the apoprotein molecule is also significant with less hydrophobic molecules transferring more rapidly. Further understanding of the differences in the kinetics of transfer of these apolipoproteins will require more knowledge of their secondary and tertiary structures.

Apolipoproteins↗

PubMed1991

Effects of apolipoproteins on the kinetics of cholesterol exchange.

The effects of apolipoproteins on the kinetics of cholesterol exchange have been investigated by monitoring the transfer of [14C]cholesterol from donor phospholipid/cholesterol complexes containing human apolipoproteins A, B, or C. Negatively charged discoidal and vesicular particles containing purified apolipoproteins complexed with lipid (75 mol % egg PC, 15 mol % dicetyl phosphate, and 10 mol % cholesterol) and a trace of [14C]cholesterol were incubated with a 10-fold excess of neural, acceptor, small unilamellar vesicles (SUV; 90 mol % egg PC and 10 mol % cholesterol). The donor and acceptor particles were separated by chromatography on DEAE-Sepharose, and the rate of movement of labeled cholesterol was analyzed as a first-order exchange process. The kinetics of exchange of cholesterol from both vesicular and discoidal complexes that contain apoproteins are consistent with an aqueous diffusion mechanism, as has been established previously for PC/cholesterol SUV. The addition of 2-3 molecules of apo A-I to a donor SUV does not significantly alter the half-time (t1/2), which is 80 +/- 9 min at 37 degrees C. However, addition of 5-12 apo A-I molecules progressively decreases t1/2 from 65 +/- 2 to 45 +/- 4 min. This enhancement in the rate of desorption of cholesterol molecules is presumed to arise from the creation of packing defects at boundaries around the apoprotein molecules, which are intercalated among the phospholipid and cholesterol molecules in the surface of the donor SUV.(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoprotein A-I↗