Phosphatidylcholine Complexes Research Articles

Reaction of lecithin:cholesterol acyltransferase with micellar complexes of apolipoprotein A-I and phosphatidylcholine, containing variable amounts of cholesterol.

In a continued investigation of lecithin:cholesterol acyltransferase reaction with micellar, discoidal complexes of phosphatidylcholine (PC) . cholesterol . apolipoprotein A-I (apo-A-I), we prepared well defined complexes with variable free cholesterol contents and examined their reactivity with purified enzyme. The complexes, prepared by the sodium cholate dialysis method, were fractionated into "small" and "large" classes by gel filtration of the reaction mixtures through a Bio-Gel A-5m column. The small complexes had egg-PC/cholesterol/apo-A-I molar ratios from 68:14:1 to 80:1:1, discoidal shapes with diameters around 114 (+/- 13) A and widths of 42 A by electron microscopy, and Stokes radii from 47 to 49 A corresponding to molecular weights near 2 X 10(5). The corresponding properties of the large complexes, isolated from samples with higher cholesterol contents, were egg-PC/cholesterol/apo-A-I molar ratios from 84:26:1 to 96:17:1, diameters of 161 (+/- 20) A, widths of 43 A, Stokes radii around 80 A, and estimated molecular weights in the vicinity of 5 X 10(5). Both types of complexes, when adjusted to equal apo-A-I concentrations, gave essentially identical initial reaction velocities with purified lecithin:cholesterol acyltransferase over a wide range of cholesterol concentrations (from 2 X 10(-7) to 4 X 10(-4) M), PC/cholesterol molar ratios (from 3:1 to 12:1), and quite different lipid fluidity conditions as detected by diphenylhexatriene fluorescence polarization. When complexes were adjusted to a constant cholesterol concentration, the initial velocities of the lecithin:cholesterol acyltransferase reaction followed Michaelis-Menten kinetics relative to the apo-A-I concentrations. Arrhenius plots of initial reaction rates for various complexes with variable cholesterol content and fluidity, measured at constant apo-A-I concentrations, gave identical temperature dependences with an average activation energy of 18.0 kcal/mol. These results strongly suggest that the cholesterol esterification on high density lipoprotein particles does not depend on their unesterified-cholesterol contents, PC/unesterified-cholesterol molar ratios, nor on the fluidity of their lipid domains.

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.

Modulation of phospholipid transfer protein activity. Inhibition by local anesthetics

The transfer of phospholipid molecules between biological and synthetic membranes is facilitated by the presence of soluble catalytic proteins, such as those isolated from bovine brain which interacts with phosphatidylinositol and phosphatidylcholine and from bovine liver which is specific for phosphatidylcholine. A series of tertiary amine local anesthetics decreases the rates of protein-catalyzed phospholipid transfer. The potency of inhibition is dibucaine>tetracaine>lidocaine>procaine, an order which is compared with and identical to those for a wide variety of anesthetic-dependent membrane phenomena. Half-maximal inhibition of phosphatidylinositol transfer by dibucaine occurs at a concentration of 0.18 mM, significantly lower than the concentration of 1.9 mM required for half-maximal inhibition of phosphatidylcholine transfer activity of the brain protein. Comparable inhibition of liver protein phosphatidylcholine transfer activity is observed at 1.6 mM dibucaine. For activity measurements performed at different pH, dibucaine is more potent at the lower pH values which favor the equilibrium toward the charged molecular species. With membranes containing increasing molar proportions of phosphatidate, dibucaine is increasingly more potent. No effect of Ca 2+ on the control transfer activity or the inhibitory action of dibucaine is noted. These results are discussed in terms of the formation of specific phosphatidylinositol or phosphatidylcholine complexes with the amphiphilic anesthetics in the membrane bilayer.

Human high density lipoprotein (HDL 3) binding to rat liver plasma membranes

The binding of human 125I-labeled HDL 3 to purified rat liver plasma membranes was studied. 125I-labeled HDL 3 bound to the membranes with a dissociation constant of 10.5 μg protein/ml and a maximum binding of 3.45 μ g protein/mg membrane protein. The 125I-labeled HDL 3-binding activity was primarily associated with the plasma membrane fraction of the rat liver membranes. The amount of 125I-Iabeled HDL 3 bound to the membranes was dependent on the temperature of incubation. The binding of 125I-labeled HDL 3 to the rat liver plasma membranes was competitively inhibited by unlabeled human HDL 3, rat HDL, HDL from nephrotic rats enriched in apolipoprotein A-I and phosphatidylcholine complexes of human apolipoprotein A-I, but not by human or rat LDL, free human apolipoprotein A-I or phosphatidylcholine vesicles. Human 125I-labeled apolipoprotein A-I complexed with egg phosphatidylcholine bound to rat liver plasma membranes with high affinity and saturability, and the binding constants were similar to those of human 125I-labeled HDL 3. The 125I-labeled HDL 3-binding activity of the membranes was not sensitive to pronase or phospholipase A 2 however, prior treatment of the membranes with phospholipase A 2 followed by pronase digestion resulted in loss of the binding activity. Heating the membranes at 100° C for 30 min also resulted in an almost complete loss of the 125I-labeled HDL 3-binding activity.

ESR spin-label studies of lipid-protein interactions in membranes

Lipid spin labels have been used to study lipid-protein interactions in bovine and frog rod outer segment disc membranes, in (Na+, K+)-ATPase membranes from shark rectal gland, and in yeast cytochrome oxidase-dimyristoyl phosphatidylcholine complexes. These systems all display a two component ESR spectrum from 14-doxyl lipid spin-labels. One component corresponds to the normal fluid bilayer lipids. The second component has a greater degree of motional restriction and arises from lipids interacting with the protein. For the phosphatidylcholine spin label there are effectively 55 +/- 5 lipids/200,000-dalton cytochrome oxidase, 58 +/- 4 mol lipid/265,000 dalton (Na+, K+)-ATPase, and 24 +/- 3 and 22 +/- 2 mol lipid/37,000 dalton rhodopsin for the bovine and frog preparations, respectively. These values correlate roughly with the intramembrane protein perimeter and scale with the square root of the molecular weight of the protein. For cytochrome oxidase the motionally restricted component bears a fixed stoichiometry to the protein at high lipid:protein ratios, and is reduced at low lipid:protein ratios to an extent which can be quantitatively accounted for by random protein-protein contacts. Experiments with spin labels of different headgroups indicate a marked selectivity of cytochrome oxidase and the (Na+, K+)-ATPase for stearic acid and for cardiolipin, relative to phosphatidylcholine. The motionally restricted component from the cardiolipin spin label is 80% greater than from the phosphatidylcholine spin label for cytochrome oxidase (at lipid:protein = 90.1), and 160% greater for the (Na+, K+)-ATPase. The corresponding increases for the stearic acid label are 20% for cytochrome oxidase and 40% for (Na+, K+)-ATPase. The effective association constant for cardiolipin is approximately 4.5 times greater than for phosphatidylcholine, and that for stearic acid is 1.5 times greater, in both systems. Almost no specificity is found in the interaction of spin-labeled lipids (including cardiolipin) with rhodopsin in the rod outer segment disc membrane. The linewidths of the fluid spin-label component in bovine rod outer segment membranes are consistently higher than those in bilayers of the extracted membrane lipids and provide valuable information on the rate of exchange between the two lipid components, which is suggested to be in the range of 10(6)-10(7) s-1.

Fluorescence Depolarization Studies of Fluidity and Phase Transition in Human Apoprotein · Phospholipid Complexes

The microviscosity of unilamellar vesicles of dimyristoyl-3-sn-phosphatidylcholine and that of phosphatidylcholine . apoprotein complexes was followed by fluorescence depolarization after labeling with 1,6-diphenyl-1,3,5-hexatriene. The transition temperature from gel-crystalline to liquid-crystalline phase in 24 degrees C for the dimyristoyl-phosphatidylcholine vesicles and is shifted to around 30 degrees C in the complexes between phosphatidylcholine and apoA-I, apoA-II, apoC-I, apoC-III proteins while the cooperativity of the transition is decreased. At temperatures below the transition of the phospholipid, the microviscosity of the complexes of phosphatidylcholine with apoA-I, apoA-II and apoC-I proteins is lower than that of the phosphatidylcholine, while the opposite effect is observed above 30 degrees C. The phosphatidylcholine . apoprotein complexes isolated on a Sepharose 6B column have a molecular weight around 100 000 and a phosphatidylcholine/apoprotein ratio of 2--2.6 (w/w). The microviscosity measurments at 35 degrees C performed after elution of the column enable the complex to be detected. The size and microviscosity of the apoprotein . phosphatidylcholine complex is compatible with a model where the vesicular structure has disappeared and the amino acid side chains present hydrophobic interaction with the phosphatidylcholine acyl chains.

Protein-immobilized lipid in dimyristoylphosphatidylcholine-substituted cytochrome oxidase: Evidence for both boundary and trapped-bilayer lipid

Cytochrome oxidase-dimyristoyl phosphatidylcholine complexes have been prepared at defined lipid:protein ratios to study the effects of protein packing density on the lipid fluidity. All the complexes reveal a two-component ESR spectrum from an incorporated phosphatidylcholine spin label, corresponding to both an immobilized lipid boundary layer and fluid bilayer regions. Difference spectra, obtained by subtracting the same immobilized spectrum from the spectra of the various complexes, demonstrate a strong perturbation of the lipid bilayer fluidity which is quite distinct from the immobilized boundary layer formation.

Structural studies of cholesteryl acetate - phosphatidylcholine complexes

The structural order of cholesteryl acetate-dimyristoylphosphatidylcholine (DMPC)-water mixtures was examined at room temperatures using Raman spectra in the CH and CC stretch regions. Intensity ratios I 2890/I 2850, I 2830/I 2930 and I 1064/I 1089 were used as structural parameters to measure the order of DMPC acyl chains. These three ratios pass through a well defined minimum at ca. 14 mol% cholestryl acetate. These data also show that cholesteryl acetate disrupts the packing order of the DMPC acyl chains from 0 to ca. 35 mol% cholesteryl acetate. As the concentration of cholesteryl acetate is increased from ca. 14 to ca. 35 mol% the DMPC acyl chains become more ordered and cholesteryl acetate appears to form a crystalline phase that remains inn suspension with the lecithin. As more cholesteryl acetate is added, these crystalline regions become larger and begin to form a precipitate which is structurally identical to those particles which remain in suspension.

Interaction of phosphatidylcholine and apolipoprotein-alanine: electron microscopic studies

Protein-lipid complexes of apolipoprotein-alanine and phosphatidylcholine were studied by electron microscopy using the technique of negative staining. An unfractionated sonicated dispersion of phosphatidylcholine vesicles of varying size was combined with apolipoprotein-Ala without exposure to further sonication. The resulting complexes appeared as linear arrays of stacked discs varying in length from 500 to 5000 Å. These stacks appeared to form by two processes: (1) by the transverse cleavage of segments of discs from multilamellar vesicles; and (2) by alignment of bilamellar vesicles into linear arrays. When fractionated bilamellar vesicles of uniform diameter (250 Å) were titrated with increasing amounts of apolipoprotein-Ala, the vesicles underwent a series of morphological changes from free vesicles to stacked discs to parallel groups of stacked discs to myelin figures.