The interaction of prostaglandins with human serum lipoproteins

Using high density and low density lipoproteins (HDL and LDL) labeled with fluorescent analogues of phosphatidylcholine or sphingomyelin it was found that low amounts (10^–12 M) of prostaglandins E₁ and F₂ induced different structural rearrangements of the lipoprotein surface, whereas prostaglandins E₂ and F₁ had no effect. The effects of prostaglandin E₁ on HDL were largely paralled by those of this prostaglandin on synthetic recombinants prepared from pure apolipoprotein A₁, phospholipids and cholesterol and were demonstrated to be caused by prostaglandin-apolipoprotein interaction. The interaction resembled that of a ligand with a specific receptor protein because it was specific, reversible, concentration and temperature dependent and saturable. However the retaining capacity of HDL or LDL for prostaglandin E₁ as determined by equilibrium dialysis was very low and a single prostaglandin E₁ molecule was able to induce structural changes in large numbers of discrete lipoprotein particles. To explain this remarkable fact a non-equilibrium model of ligand-receptor interaction is proposed. According to that model in open systems characterized by weak ligand-receptor binding, high diffusion rate of the ligand and long relaxation times which exceed the interval between two successive receptor occupations, the ligand-induced changes will accumulate, resulting in transformation of the system into a new state which may be far away from equilibrium. It is emphasized that the low mobility of lipids constituting the environment of the receptor protein plays a critcal role in this type of signal amplification.It was further demonstrated that the PGE₁-induced changes of the lipoprotein surface resulted in an enhancement of LDL-to-HDL transfer of cholesterol esters and phosphatidylcholine especially in the presence of serum lipid transfer proteins. The acceleration of the interlipoprotein transfer caused by prostaglandin E₁ in turn increases the rate of cholesterol esterification in serum. It is suggested that in such a way prostaglandin E₁ may influence the homeostasis of cholesterol.Abbreviations LDL low density lioproteins - HDL high density lipoproteins - PG prostaglandin - ASM anthrylvinyl-labeled sphingomyelin (N-12-(9-anthryl)-11-trans-dodecanoylsphingosin-1-phosphocholine - APC anthrylvinylphosphatidylcholine (1-radyl-2-[(9-anthryl)-11-transdodecanoyl)-sn-glycerophosphocholine - NAP-SM nitroazidophenyl labeled sphingomyelin (N-[N-(2-nitro-4azidophenyl)-12-aminododecanoyl]-sphingosin-1-phosphocholine) - NAP-PC adizophenyl labeled phosphatidylcholine (1-radyl-2-[N-(2-nitro-4azidophenyl)-12-aminododecanoyl]-sn-glycero-3-phosphocholine - DPPC dipalmitoylphosphatidylcholine - P fluorescence polarization - E parameter of tryptophanyl to ASM resonance energy transfer - LEP lipid-exchange protein     

Interaction of prostaglandins with low-density lipoproteins in human blood

Interaction of prostaglandins (PG) with human plasma low density lipoproteins (LDL) was studied, using fluorescent spectroscopy and photoreactive labeling. It was demonstrated that PGE1 at low concentrations (less than 10(-9) M) induces specific lipid rearrangements on the surface of LDL globules. It was assumed that these rearrangements are brought about by the interaction of PG with apolipoprotein B to form short-living complexes. A possible mechanism and biological significance of the observed phenomenon are discussed.     

Effect of the proteolysis of low-density serum lipoproteins on their interaction with macrophages

125I-labelled human serum low density lipoproteins (LDL) were incubated with cultured mouse peritoneal macrophages at 37 degrees C, with the following study of cellular uptake and 125I-LDL degradation by measuring the content of TCA-soluble products of LDL hydrolysis in the cultural medium. It was shown that limited pepsin proteolysis of LDL (10%) led to a more effective LDL uptake and degradation by macrophages. The data suggest that enzyme-induced modification of LDL may increase their atherogenicity.     

The interaction of prostaglandins with high-density lipoproteins: a non-equilibrium model of ligand-receptor interaction

Using high-density lipoproteins (HDL) labeled with a fluorescent phospholipid probe (an anthrylvinyl-labeled analogue of sphingomyelin) it was found that low amounts (10(-12) M) of the prostaglandins E1 and F2 alpha induced different structural changes of the HDL surface, whereas prostaglandin E2 had no effect. The effects of prostaglandin E1 on HDL were largely paralleled by those of this prostaglandin on synthetic recombinants prepared from apolipoprotein A1, phospholipids and cholesterol. The prostaglandin E1-HDL interaction resembled that of a ligand with a receptor site because it was specific, reversible, concentration- and temperature-dependent and saturable. However, the maximal HDL retaining capacity for prostaglandin E1 as determined by equilibrium dialysis was very low, and a single prostaglandin E1 molecule was able to induce structural changes in a large number of discrete lipoprotein particles. To explain this remarkable fact, a non-equilibrium model of ligand-receptor interaction is proposed. According to this model in open systems characterized by a short life-time of the ligand-receptor complex, high diffusion rates of the ligand and long relaxation times which exceed the interval between two successive ligand-receptor occupations, the ligand-induced changes will accumulate, resulting in amplification of the primary biological signal. It is emphasized that the low mobility of lipids constituting the environment of the receptor protein plays a critical role in this type of signal amplification.     

Action of lipases on low-density serum lipoproteins]

It was shown that the action of phospholipase A2 on low density serum lipoproteins (LDL) in the presence of serum albumin led to decrease in the floatation coefficient. Lipase hydrolyzed LDL triglycerides after pretreatment of the latter with phospholipase A2. Due to the action of lipases the LDL residue loses its solubility and the cholesterol-rich precipitate forms. The loss of solubility of lipoproteins treated with lipases and proteinases may occur in vivo, underlying athermao formation, i.e. can thus serve as one of the factors in the pathogenesis of atherosclerosis.     

Preparative isoelectric focusing of human serum very low-density and low-density lipoproteins.

Ten percent glycerol prevented the usual precipitation of human serum very low-density lipoproteins (VLDL) and low-density lipoproteins (LDL) at their isoelectric points during their preparative isoelectric focusing (IEF), IEF separated VLDL and LDL into two major fractions. The observed optical density peaks are not artifacts caused by binding of Ampholines to VLDL or LDL since no radioactivity accumulated in the fractions containing VLDL or LDL during IEF in the presence of [¹⁴C]Ampholine, and gel filtration completely separated the lipoproteins from [¹⁴C]Ampholine. These results suggest that IEF may separate subspecies of VLDL and LDL under suitable experimental conditions.     

Physical-chemical interaction of heparin and human plasma low-density lipoproteins

This study characterizes the physical-chemical interactions of heparin with human plasma low-density lipoproteins (LDL). A high reactive heparin (HRH) specific for the surface determinants of LDL was isolated by chromatography of commercial bovine lung heparin on LDL immobilized to AffiGel-10. HRH was derivatized with fluoresceinamine and repurified by affinity chromatography, and its interaction with LDL in solution was monitored by steady-state fluorescence polarization. Binding of LDL to fluoresceinamine-labeled HRH (FL . HRH) was saturable, reversible, and specific; HRH stoichiometrically displaced FL . HRH from the soluble complex, and acetylation of lysine residues on LDL blocked heparin binding. Titration of FL.HRH with excess LDL yielded soluble complexes with two LDL molecules per heparin chain (Mr 13,000) characterized by an apparent Kd of 1 microM. Titration of LDL with excess HRH resulted in two classes of heparin binding with two and five heparin molecules bound per LDL and apparent Kd values of 1 and 10 microM, respectively. At physiological pH and ionic strength, the soluble HRH-LDL complexes were maximally precipitated with 20-50 mM Ca2+. Insoluble complexes contained 2-10 HRH molecules per LDL with the final product stoichiometry dependent on the ratio of the reactants. The affinity of HRH for LDL in the insoluble complexes was estimated between 1 and 10 microM. Insoluble LDL-heparin complexes were readily dissociated with 1.0 M NaCl, and their formation was prevented by acetylation of the lysine residues on LDL.     

Thiolation of low-density lipoproteins and their interaction with L2C leukemic lymphocytes

We present here, a new method for coupling sulfhydryl groups (SH) to low-density lipoprotein (LDL) surface. This method uses homocysteine thiolactone (HCTL) which reacts with lysine residues in a very mild manner, and permits the selection of the number of SH bound per LDL. Under our experimental conditions (8 SH/LDL), the affinity of thiolated LDL for the specific receptors and their further internalization by L2C lymphocytes are preserved.     

Effect of prostaglandins on lipid transfer between high- and low-density lipoproteins in human plasma

Prostaglandin (PG) E1 was shown to stimulate the transfer of phosphatidylcholine and cholesterol esters from human high density lipoproteins to low density lipoproteins. The enhancement of the interlipoprotein lipid transfer by PGE1 was observed both at low prostaglandin concentrations under conditions of spontaneous exchange as well as in the presence of the lipoprotein-depleted plasma and the partly purified lipid transfer plasma protein. At the same time PGE2 showed no significant influence on the interlipoprotein lipid transfer. It is supposed that the effect of PGE1 is due to the PGE1-induced reorganization of the high density lipoprotein surface and that the PG-lipoprotein interaction is a factor which regulates cholesterol homeostasis.     

A fluorescent sterol probe study of human serum low-density lipoproteins

The fluorescent sterol probe, ergosta-5,7,9,(11),22-tetraen-3 beta-ol (dehydroergosterol), was utilized as a cholesterol analog to label human serum low-density lipoproteins (LDL). Quenching of dehydroergosterol fluorescence by KI indicated that most of the fluorophore was either buried within the outer phospholipid monolayer of LDL or within the neutral lipid core of LDL. The steady-state anisotropy of dehydroergosterol in LDL detected the cholesteric core phase transition near 30 degrees C. Fluorescence lifetime decays for dehydroergosterol contained two components, both below and above the cholesteric phase transition, with the major lifetime component near 1 ns. Neither lifetime component underwent a detectable change in duration at the core phase transition temperature. Time-correlated fluorescence anisotropy decays of dehydroergosterol indicated a single rotational correlation time near 1.7 ns, which was unaffected by the core phase transition. Time-correlated anisotropy decays also suggested hindered rotation of dehydroergosterol in LDL. These results indicate that unesterified cholesterol is primarily located in the outer phospholipid monolayer of LDL, with the majority of cholesterol not in direct contact with the aqueous phase.     

Acetaldehyde binding increases the catabolism of rat serum low-density lipoproteins

Acetaldehyde was found to form adducts with rat serum lipoproteins. The binding of [14C]acetaldehyde to lipoproteins was studied at low concentrations which are known to exist during ethanol oxidation. The amount of lipoprotein adducts was a linear function of acetaldehyde concentration up to 250 microM. Incubation of rat plasma low-density lipoproteins (LDL) with 200 microM acetaldehyde increased the disappearance rate of the 3H-label from the cholesterol ester moiety of LDL injected into normal rats. The data show that even low concentrations of acetaldehyde are capable of affecting LDL metabolism. These findings may provide an explanation for the low concentrations of serum LDL in alcoholics.     

Studies on pig serum lipoproteins. I. Separation and properties of low-density lipoproteins.

The low-density lipoproteins in pig serum were separated into two subclasses (LDL1 and LDL2) by 2 to 7% pore size gradient gel electrophoresis. Preparative gel electrophoresis in 2 to 4% gradient gel made it possible to isolate these components as distinct entities. After delipidation by chromatography on Sepharose 4B in the presence of SDS, both apo-LDL1 and apo-LDL2 were found to have a molecular weight of 2.6X10(5). However, when these apoproteins were incubated in 10% sodium dodecyl sulfate, fragmentation occurred and the minimum fragment molecular weight was estimated to be 2.4X10(4). No essential difference was found in the amino acid compositions or fragmentation patterns of the apoproteins. However, the amounts of carbohydrates in the two apoproteins were different (7.09% in apo-LDL1 and 5.08% in apo-LDL2). The carbohydrate composition was 0.8% sialic acid, 2.38% N-acetyl-glucosamine, and 4.01% neutral sugars in apo-LDL1 and 0.5, 1.75, and 2.83% in apo-LDL2, respectively. In both apoproteins, mannose, galactose, and fucose were present in almost the same molar ratio of 4-5 : 2-3 : 1.     

Polypeptides from serum low-density lipoproteins of pigs (Sus domesticus).

Low-density lipoproteins floating between densities 1-006 and 1-063 g cm-3 were isolated by centrifugation of blood serum obtained from 24-h fasted pigs (Sus domesticus). This lipoprotein fraction contained two components with Sf 1-063 values of 3-4 and 2-3 at 20 degrees C when examined by analytical ultracentrifugation. Delipidation of the lipoprotein yielded 15% recovery of soluble protein. Chromatography on Sephadex G100 in 8 M urea of these delipidation products yielded three fractions of different sizes which were present in both native and succinylated apoproteins. These fractions from the succinylated apolipoproteins were further characterized. A polypeptide fraction comprising 70% of the total protein had an apparent molecular weight of 34000 and contained greater amounts of amino acids with hydrophobic side chains than did the second fraction of apparent molecular weight 22000 which contained 15% of the protein. The third fraction of apparent molecular weight 12500 contained 15% of the protein.     

Modeling the interaction of peroxynitrite with low-density lipoproteins. III: the role of antioxidants

Peroxynitrite is a candidate for the substance responsible for the initiation of peroxidation of low-density lipoproteins (LDL) in blood. This is believed to be the initial step in the formation of atherosclerotic plaque. Using kinetic arguments, this paper examines possible routes in both LDL particles and in the surrounding plasma for antioxidants to block peroxidation. The antioxidants considered include ascorbate ion, glutathione and human serum albumin in plasma as well as alpha -tocopherol, ubiquinone-10 and carotenoids in the LDL particles. The results suggest that in the plasma compartment the most efficacious blocking mechanism is the reaction of ascorbate ion with the peroxynitrite precursor, superoxide ion. The situation in LDL particles is much less clear cut because of the paucity of kinetic data in this medium. However, some constraints are suggested on the requirements for an effective antioxidant.     

Modeling the interaction of peroxynitrite with low-density lipoproteins. I. Plasma levels of peroxynitrite

Peroxynitrite is a strong candidate for the material responsible for the initiation of peroxidation of low-density lipoproteins (LDLs) which is considered the first step in the formation of atherosclerotic plaque. Recent advances in the understanding of peroxynitrite chemistry allow the construction of a kinetic model that can be used to understand the factors controlling levels in plasma. These results indicate that the carbon dioxide catalysed decomposition of peroxynitrite produces large quantities of reactive species, but the rapid decomposition of this intermediate, ONOOCO(-)(2), may limit its availability to attack LDLs at points distant from the site of production. In this case, peroxynitrite itself may be of greater quantitative importance in LDL peroxidation.     

Modeling the interaction of peroxynitrite with low-density lipoproteins. II: reaction/diffusion model of peroxynitrite in low-density lipoprotein particles

Peroxynitrite is a possible initiator for the free radical chain reaction that results in peroxidation of low-density lipoproteins (LDL) which is the first step in atherogenisis. This paper reports on the use of a diffusion/reaction model to examine the processes involved in peroxynitrite attack on LDL particles. Results indicate that because of the short distance involved, diffusion is much more rapid than chemical decomposition. Because of this decoupling the free radicals generated by peroxynitrite decomposition may be found at any point in the LDL particle. At the concentrations expected in physiological systems only a small proportion of LDL particles may contain peroxynitrite molecules. However, these particles may still be profoundly effected because of the long reaction chain length expected after initiation.     

In vitro binding of human serum low-density lipoproteins by sulfated pectin derivatives

It was shown that sulfated pectin derivatives bind human serum low-density lipoproteins (LDLs) in vitro to a greater extent than native pectins. At the same time, the number of sulfate groups and the molecular weight of sulfated derivatives were crucial factors. The sulfated pectin derivatives with molecular weights more than 200 kDa containing 45 wt % sulfate groups had the greatest ability to bind LDLs, while the sulfated derivatives with molecular weights lower than 50 kDa containing 5% sulfate groups exhibited the lowest activity.