Estrogen treatment increases phospholipid transfer activities in chicken liver.

The effect of subcutaneous beta-estradiol injection on liver phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol transfer activity of immature chicken has been determined. The estrogen administration significantly enhanced the transfer activity of both phosphatidylcholine (100%), phosphatidylethanolamine (160%), and phosphatidylinositol (150%). In vivo experiments revealed that the hormone-induced changes in liver lipid transfer activity were sensitive to a protein synthesis inhibitor, cycloheximide. A partial characterization of liver protein transfer on Sephacryl S-200 showed that multiple transfer proteins are involved in the beta-estradiol effect. This is the first time that hormonal modulation of phospholipid transfer activities is described, and the results suggest that the hepatic phospholipid transfer activities might be involved in the biosynthesis of plasma lipoproteins in vivo.

Effect of cholesterol and phosphatidylcholine of different chain length on acrosome breakdown and fertilizing capacity of amphibian spermatozoa.

The effect of different lipids on the fertilizing capacity of Bufo arenarum spermatozoa and on acrosome breakdown of Leptodactylus chaquensis spermatozoa was studied. Sonicated vesicles of egg yolk phosphatidylcholine (1 mM) were as effective as vesicles of egg yolk phosphatidylcholine:cholesterol (molar ratio 1:0.9) in inhibiting the fertilizing capacity of Bufo arenarum spermatozoa. This suggests that cholesterol depletion from the spermatozoa was not the cause of the fertility loss. Bufo arenarum spermatozoa were incubated with phosphatidylcholines with even chain length from 6 to 18 carbons. At a concentration of 0.01 mM, didecanoyl-phosphatidylcholine reduced fertilizing capacity to 10% in a few minutes and to 0% within 60 minutes. Didodecanoyl-phosphatidylcholine required 2 hours to reduce fertility to 10% and 4 hours to cause a 100% loss of fertilizing capacity. A concentration of didecanoyl-phosphatidylcholine as low as 5 x 10(-4) mM caused a more than 95% fertility loss in less than five minutes. At a concentration of 0.1 mM, didecanoyl-phosphatidylcholine induced complete acrosome breakdown in Leptodactylus chaquensis spermatozoa in 15 minutes, whereas didodecyl-phospatidylcholine required 2 hours. At a concentration 100-fold lower didecanoyl-phosphatidylcholine induced complete acrosome breakdown in 2 hours. Electron microscopic observations in both species showed loss of acrosome caused by the action of the didecanoyl-phosphatidylcholine. Longer chain phosphatidylcholines exerted an inhibitory effect on Bufo arenarum spermatozoa fertilizing capacity at a higher concentration when in a vesicular form.

Phospholipid transfer activities in toad oocytes and developing embryos.

The role of lipid transfer proteins during plasma membrane biogenesis was explored. Developing amphibia embryos were used because during their growth an active plasma membrane biosynthesis occurs together with negligible mitochondrial and endoplasmic reticulum proliferation. Sonicated vesicles, containing 14C-labeled phospholipids and 3H-labeled triolein, as donor particles and cross-linked erythrocyte ghosts as acceptor particles were used to measure phospholipid transfer activities in unfertilized oocytes and in developing embryos of the toad Bufo arenarum. Phosphatidylcholine transfer activity in pH 5.1 supernatant of unfertilized oocytes was 8-fold higher than the activity found in female toad liver supernatant, but dropped steadily after fertilization. After 20 hr of development, at the stage of late blastula, the phosphatidylcholine transfer activity had dropped 4-fold. Unfertilized oocyte supernatant exhibited phosphatidylinositol and phosphatidylethanolamine transfer activity also, but at the late blastula stage the former had dropped 18-fold and the latter was no longer detectable under our assay conditions. Our results show that fertilization does not trigger a phospholipid transport process catalyzed by lipid transfer proteins. Moreover, they imply that 75% of the phosphatidylcholine transfer activity and more than 95% of the phosphatidylinositol and phosphatidylethanolamine transfer activities present in pH 5.1 supernatants of unfertilized oocytes may not be essential for toad embryo development. Our findings do not rule out, however, that a phosphatidylcholine-specific lipid transfer protein could be required for embryo early growth.

Fusion of phospholipid vesicles induced by muscle glyceraldehyde-3-phosphate dehydrogenase in the absence of calcium.

Ca2+-induced fusion of phospholipid vesicles (phosphatidylcholine/phosphatidic acid, 9:1 mol/mol) prepared by ethanolic injection was followed by five different procedures: resonance energy transfer, light scattering, electron microscopy, intermixing of aqueous content, and gel filtration through Sepharose 4-B. The five methods gave concordant results, showing that vesicles containing only 10% phosphatidic acid can be induced to fuse by millimolar concentrations of Ca2+. When the fusing capability of several soluble proteins was assayed, it was found that concanavalin A, bovine serum albumin, ribonuclease, and protease were inactive. On the other hand, lysozyme, L-lactic dehydrogenase, and muscle and yeast glyceraldehyde-3-phosphate dehydrogenase were capable of inducing vesicle fusion. Glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle, the most extensively studied protein, proved to be very effective: 0.1 microM was enough to induce complete intermixing of bilayer phospholipid vesicles. Under conditions used in this work, fusion was accompanied by leakage of internal contents. The fusing capability of glyceraldehyde-3-phosphate dehydrogenase was not affected by 5 mM ethylenediaminetetraacetic acid. The Ca2+ concentration in the medium, as determined by atomic absorption spectroscopy, was 5 ppm. Heat-denatured enzyme was incapable of inducing fusion. We conclude that glyceraldehyde-3-phosphate dehydrogenase is a soluble protein inherently endowed with the capability of fusing phospholipid vesicles.

Heterogeneity of rabbit intestine brush border plasma membrane cholesterol.

Nonspecific lipid transfer protein accelerated cholesterol exchange from brush border vesicles according to a biphasic time course, but sonicated vesicles made from brush border phospholipids and glycosphingolipids showed a single phase exchange. Removal of surface protein with papain or opening brush border vesicles with deoxycholate did not abolish the biphasic exchange pattern. In brush border vesicles treated with cholesterol oxidase, 21 +/- 10% of the free cholesterol was oxidized rapidly, and the remaining cholesterol was oxidized at a slower rate. Opening vesicles with sodium deoxycholate or treatment with phospholipase C, which degraded 55% of the phospholipids, did not increase the size of the rapidly oxidizable cholesterol pool. The rapidly exchangeable and the rapidly oxidizable cholesterol pools appear to represent the same fraction. In double-labeled brush border vesicles 27 +/- 9% of the cholesterol is present in a readily accessible pool, which slowly equilibrates with the remaining membrane cholesterol. The fractional turnover rate of cholesterol in the readily accessible pool equals 0.07 +/- 0.04 h-1 and is increased to 3.35 h-2 by 12 micrograms/ml of nonspecific lipid transfer protein. The heterogeneous distribution of cholesterol in the intact brush border vesicles may not reflect an inside-outside distribution or interaction of cholesterol with membrane lipids but rather an association of more than two-thirds of the membrane cholesterol with a membrane protein fraction.

Lipid transfer proteins in the study of artificial and natural membranes.

Lipid transfer proteins, differing in their specificity for the transfer of lipids and for the surfaces on which they act, have been purified from various mammalian tissues and subsequently characterized. Several of their properties make them useful research tools. They have been used alone or with other techniques to study the distribution and mobility of phospholipids in artificial vesicles and in natural membranes, and have been used to create asymmetric phospholipid vesicles. Lipid transfer proteins are capable of altering the lipid composition of membranes by introducing new lipids or by depletion of existing lipids. Some of the transfer proteins can effect a net transfer of phospholipids, glycosphingolipids and cholesterol from one structure to another, whereas others appear to act primarily in promoting exchange. Some lipid transfer proteins are capable of introducing spin labeled and fluorescent lipid analogs into the outer surface of membranes. Because of lipid transfer proteins do not seem to alter membrane lipid asymmetry or permeability of membranes, they are useful tools for studying the effect of lipid substitution on membrane-mediated transport processes and on various membrane-bound enzyme systems.

Accelerated transfer of neutral glycosphingolipids and ganglioside GM1 by a purified lipid transfer protein.

The transfer of labeled neutral glycosphingolipids from sonicated phosphatidylcholine vesicles to erythrocyte ghosts is greatly stimulated by a nonspecific lipid transfer protein purified from beef liver. Globo-tetraglycosylceramide is transferred at a rate 40% of that for dipalmitoylphosphatidylcholine. II3-alpha-N-Acetylneuraminosyl-gangliotetraglycosylceramide is also transferred by the transfer protein, either from sonicated phosphatidylcholine vesicles or from ganglioside micelles to erythrocyte ghosts. The nonspecific lipid transfer protein catalyzes the net transfer of glycosphingolipids from brush border membrane vesicles (from rabbit intestine) to sonicated phosphatidylcholine/cholesterol vesicles.

Rat liver proteins capable of transferring phosphatidylethanolamine. Purification and transfer activity for other phospholipids and cholesterol.

Two proteins, one in a highly purified form, have been isolated from the soluble fraction of rat liver homogenate. These proteins accelerate the transfer of labeled phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, sphingomyelin, and cholesterol from liposomes to mitochondria or erythrocyte ghosts. The fraction obtained after ammonium sulfate precipitation, gel filtration on Sephadex G-75, ion-exchange chromatography on CM-cellulose, ampholyte displacement chromatography, and heat treatment exhibited an 876-fold increase in its phosphatidylethanolamine transfer activity as compared with the postmitochondrial supernatant adjusted to pH 5.1. Isoelectric focusing on polyacrylamide gels shows a single band between pH 8.6 and 9.0. The transfer activity is abolished by trypsin, but withstands 5-min heating at 90 degrees. After heat treatment, a single major band is seen on polyacrylamide gel electrophoresis followed by two minor ones. The molecular weight of the major band is 12,500, as determined by electrophoresis on 15% polyacrylamide gels in the presence of sodium dodecyl sulfate. A molecular weight of 13,500 was calculated from molecular filtration through Sephadex G-50. The relative transfer activities toward the different phospholipids remain constant throughout the last three steps of the purification procedure in spite of the extensive change in the electrophoretic profile of the protein mixture. The cholesterol transfer activity remains unchanged after the final heat treatment as well. This indicates that all of the transfer activities are present in a single protein.

Kinetic modifications of the acetylcholinesterase and (Ca2+ + Mg2+)-ATPase in rat erythrocytes by cholesterol feeding.

The influence of cholesterol on the membrane-bound acetylcholinesterase and (Ca2+ + Mg2+)-ATPase was studied in erythrocytes of five groups of male rats fed different fat-supplemented diets. Two groups of rats were fed essential fatty acid (EFA) sufficient diets with 5% lard or corn oil as the dietary fat, and two groups were fed EFA-deficient diets: a basic, fat-free diet and the same diet supplemented with 5% hydrogenated beef fat. One additional group of rats was fed a stock diet. The kinetic changes recorded were in the degree of the cooperativity of the inhibition by F- of the acetylcholinesterase and the activation by Ca2+, and by Mg2+ of the (Ca2+ + Mg2+)-ATPase. The kinetic behavior of the enzymes was only modified by cholesterol feeding when they were bound to a membrane with a high fatty acid fluidity (e.g. derived from rats fed the corn oil-supplemented diet). The enzymes from a membrane with a low fatty acid fluidity (e.g. derived from rats fed a lard-supplemented diet) were not altered by cholesterol feeding. The changes were noticeable after 24 hours of cholesterol feeding. It is suggested that the in vivo cholesterol sites are involved in a regulatory mechanism for mammalian membrane-bound enzymes.

Asymmetry and transposition rates of phosphatidylcholine in rat erythrocyte ghosts.

Purified phospholipid exchange protein from beef heart cytosol is used to accelerate the exchange of phospholipids between labeled sealed ghosts and phosphatidylcholine/cholesterol liposomes. The purified protein accelerates the transfer of phosphatidylcholine and, to a lesser degree, that of sphingomyelin, phosphatidylinositol, and lysophosphatidylcholine. The presence of exchange protein does not accelerate the exchange of phospholipids between intact red blood cells and liposomes, but 75% of the phosphatidylcholine of sealed ghosts is readily available for exchange. The remaining 25% is also exchangeable but at a slower rate. When the exchange is assayed between inside-out vesicles and liposomes, 37% of the phosphatidylcholine is readily available, and 63% is exchanged at a slower rate. These results are consistent with an asymmetric distribution of phosphatidylcholine in isolated erythrocyte membrane fractions. The sum of the forward and backward transposition of phosphatidylcholine between the inside and outside layers of sealed ghost membranes amounts to 11% per hour, and the half-time for equilibration is 2.3 h. Significatnly lower values are obtained for the inside-out vesicles (half-time for equilibration: 5.3 h). These results suggest that, during the formation of the vesicles, the asymmetry of phosphatidylcholine is partially preserved, but structural changes occur in the membrane that affect the rate of membrane transposition of phosphatidylcholine.

Kinetic changes of the erythrocyte (Mg2+ + Ca2+)-adenosine triphosphatase of rats fed different fat-supplemented diets.

The activation by Mg2+, in the presence of 0.2 mM Ca2+, of the erythrocyte ATPase from rats fed with six different fat-supplemented diets has been studied. A sigmoid kinetic curve was found. The values of the Hill coefficient showed a positive correlation with the membrane fatty acid fluidity, which is expressed as the ratio between double bond index and saturated fatty acid content. The values of the Hill coefficient ranged from 1.0, in animals fed with lard-supplemented diet, to 2.0, in animals fed with corn oil-supplemented diet. When the effect of increasing Ca2+ concentration in these two groups was studied at pH 8.1, an activation with the latter group and an inhibition with the former one were found. The activation by Ca2+ found in corn oil-fed animals was lost after treatment with phospholipase C and restored after the addition of homologous phospholipids. The activation could not be restored by addition of phospholipids from lard-fed animals. In this group, treatment with phospholipase C left the kinetic behavior unmodified, but an activation by Ca2+ could be detected after adding phospholipids from corn oil-fed animals. It is suggested that membrane fatty acid fluidity is involved in the cooperative transitions and cryptic activity of the (Mg2+ + Ca2+)-ATPase.