Inhibition of the phosphatase activity of the red cell membrane Ca2+ pump by acidic phospholipids.

The effect of phospholipids was tested on the p-nitrophenylphosphatase activity of the Ca2+ pump. Acidic phospholipids like phosphatidylserine and phosphatidylinositol inhibited the phosphatase activity, while neutral phospholipids like phosphatidylcholine did not. This result contrasts sharply with the known activating effect of acidic phospholipids on the Ca2(+)-ATPase activity of the pump. It is known that the phosphatase activity of the Ca2+ pump can be elicited either by calmodulin and Ca2+ or by ATP and Ca2+. Unlike calmodulin, acidic phospholipids failed to stimulate the phosphatase activity. Furthermore, calmodulin-activated phosphatase was completely inhibited by acidic phospholipids. Maximal inhibition of the ATP-activated phosphatase was only 70%. Inhibition by acidic phospholipids was non-competitive regarding to calmodulin, suggesting that acidic phospholipids and calmodulin do not bind to the same domain of the pump. The presence of Ca2+ was essential for the inhibition, and the apparent affinity for Ca2+ for this effect was increased by acidic phospholipids. Results are consistent with the idea that acidic phospholipids stabilize an enzyme-Ca complex lacking phosphatase activity.     

Radioiodination of naturally occurring phospholipids

A simple and rapid nonenzymatic method for radioiodination of phospholipids is described. It involves oxidation of Na125I with TlCl3 (or chloramine-T) in an aqueous medium, with subsequent exposure of the phospholipids, dissolved in chloroform/methanol, to the action of the oxidizing mixture. Purification of the radiolabelled phospholipids was effected by washing with sodium thiosulphate followed by thin-layer chromatography on silica gel. Specific radioactivity of 125I-labelled phosphatidylcholine was estimated to be about 10 muCi/mg phospholipid. The method is designed for radioiodination of various naturally occurring phospholipids.     

Effect of phospholipids on UDP-glucose: ceramide glucosyltransferase from Golgi membranes

1. The removal of phospholipids completely abolished the activity of the enzyme UDP-glucose:ceramide glucosyltransferase from Golgi membranes. 2. Modulation of enzyme activity by phospholipids was undertaken on the solubilized form of the enzyme. 3. Well-defined fatty acyl chains and polar head groups were necessary for maximal stimulation by phospholipids. 4. A specific requirement for phosphatidylcholine is suggested by preliminary experiments of reconstitution of enzyme activity with phosphatidylcholine vesicles.     

Effect of phospholipids from Saccharomyces cerevisiae at different stages of development on restoration of succinooxidase activity in lipid-depleted mitochondria

Phospholipids extracted fromSaccharomyces cerevisiae at different stages of development after glucose repression contain three major fatty acids: palmitic, palmitoleic and oleic. The ratio palmitic: palmitoleic strongly decreases beginning at the 6th hour of growth.To test the effect of fatty acid composition and in particular of unsaturation on succinoxidase activity, all these phospholipids, phospholipids from commercial yeast, and Asolectin were incubated with lipid-depleted yeast mitochondria. The amount of P bound was not much different for the various phospholipids; succinoxidase activity was restored best by Asolectin; the least effective reactivation was given by phospholipids from yeast at the middle stages of growth. There are not great differences between the various phospholipids and there is no correlation with unsaturation. If we compare the pattern of appearance of respiration during morphogenesis of yeast mitochondria with the pattern of the capability of the phospholipids from cells at different stages of mitochondrial morphogenesis to restore activity of lipid-depleted yeast mitochondria, we find no correlation. The results of this investigation are consistent with the idea that changes in phospholipids and changes in enzyme activities are not linked by a causal relation.     

Interaction with phospholipids of a membrane thiol peptidase that is essential for the signal transduction of mating pheromone in Rhodosporidium toruloides

Interaction with phospholipids of a membrane thiol peptidase [referred to as trigger peptidase (TPase), T. Miyakawa et al. (1987) J. Bacteriol. 169, 1626-1631] that plays a key role in the signalling of a lipopeptidyl mating pheromone at the cell surface of pheromone-target cell (mating type a) of Rhodosporidium toruloides was studied. The activity of highly purified TPase which requires phospholipids was restored by reconstitution of the enzyme into liposomes prepared with phospholipids extracted from the yeast cell. The presence of Ca2+ was essential for both the reconstitution process and the catalytic reaction of TPase. Triton X-100 mixed micelles containing phospholipids also activated the enzyme. The specificity and stoichiometry of activation by phospholipids was investigated by determination of TPase in the presence of mixed micelles that contained defined classes and numbers of phospholipid molecules in the Triton X-100 micelles. It was demonstrated that TPase is activated by mixed micelles containing 2-6 molecules of phosphatidylserine or phosphatidylethanolamine. Other phospholipids of the membranes of this organism, such as phosphatidylcholine and phosphatidylglycerol, had little effect on activation, indicating that the amino group of the phospholipids may be required for the function of TPase. Direct evidence for the interaction of TPase and Triton X-100/phosphatidylserine mixed micelles was obtained by molecular sieve chromatography on Sephacryl S-200. These data established that a phospholipid bilayer is not a requirement for TPase activation, and that the purified enzyme can be activated by a relatively small number of phospholipid molecules of specific classes.     

Compositional and molecular species analysis of phospholipids by high performance liquid chromatography coupled with chemical ionization mass spectrometry

High performance liquid chromatography (HPLC) was combined with chemical ionization mass spectrometry (CIMS) by the use of a moving-belt interface. The technique was employed for the analysis of naturally occurring phospholipids. Positive and negative ion mass spectra of various phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and sphingomyelin were obtained in the chemical ionization mode with ammonia or methane as the reagent gas. Specific ions for individual phospholipid "bases" were identified. These ions were used in specific ion monitoring of the phospholipids during HPLC-CIMS. CIMS of each phospholipid also provided extensive information on the molecular species of the individual class of phospholipids. Relative abundance of different molecular species of each phospholipid as determined by CIMS agreed well with the results obtained by gas-liquid chromatography. Rat brain phospholipids were analyzed by HPLC-CIMS in about 15 minutes. Routinely, about 5 micrograms of individual phospholipid was analyzed by HPLC-CIMS, however, with specific ion monitoring the method provides a detection capability at the subnanogram level.     

Corticosteroid effect on Caco-2 cell lipids depends on cell differentiation

Previous studies from our laboratory have indicated that secondary hyperaldosteronism affects phospholipids of rat colonic enterocytes. To assess whether this represents a direct effect of mineralocorticoids on enterocytes, the role of aldosterone and dexamethasone in the regulation of lipid metabolism was examined in Caco-2 cells during development of their enterocyte phenotype. Differentiation of Caco-2 cells was associated with increased levels of triglycerides (TG) and cholesteryl esters (CE), a decreased content of cholesterol and phospholipids and changes in individual phospholipid classes. The phospholipids of differentiated cells had a higher content of n-6 polyunsaturated fatty acids (PUFA) and lower amounts of monounsaturated (MUFA) and saturated fatty acids than subconfluent undifferentiated cells. Differentiated cells exhibited a higher ability to incorporate [3H]arachidonic acid (AA) into cellular phospholipids and a lower ability for incorporation into TG and CE. Incubation of subconfluent undifferentiated cells with aldosterone or dexamethasone was without effect on the content of lipids, their fatty acids and [3H]AA incorporation. In contrast, aldosterone treatment of differentiated cells diminished the content of TG, increased the content of phospholipids and modulated their fatty acid composition. The percentage of n-6 and n-3 PUFA in phospholipids was increased and that of MUFA decreased, whereas no changes in TG were observed. The incorporation of [3H]AA into phospholipids was increased and into TG decreased and these changes were blocked by spironolactone. Treatment of differentiated cells with dexamethasone increased their CE content but no effect was identified upon other lipids, their fatty acid composition and on the incorporation of [3H]AA. As expected for the involvement of corticosteroid hormones the mineralocorticoid and glucocorticoid receptors were identified in Caco-2 cells by RT-PCR. The results suggest that aldosterone had a profound influence on lipid metabolism in enterocytes and that its effect depends on the stage of differentiation. The aldosterone-dependent changes occurring in phospholipids and their fatty acid composition may reflect a physiologically important phenomenon with long-term consequences for membrane structure and function.     

Phospholipid-enzyme interactions of chitin synthase of Coprinus cinereus

Abstract The effect of phospholipids on chitin synthase activity has been studied with digitonin-solubilized and partially purified preparations from Coprinus cinereus . When cholate was used as detergent, it inhibited enzyme activity, but this inhibition was reversed by increasing concentrations of phospholipids. Preincubation with cholate and phospholipid caused irreversible loss of activity. When sonicated with solubilized enzyme preparation, dimyristoyl phosphatidyl choline strongly stimulated activity, while dioleoyl phosphatidyl choline was inhibitory. The Arrhenius plot of the effect of temperature on enzyme activity contained breaks, characteristic of a membrane-bound enzyme. It is suggested that chitin synthase requires an annulus of phospholipids for activity.     

Protection of chlorophyll by phospholipids from photooxidation

Rapid photooxidation of chlorophyll in chloroform was shown to be partly inhibited by various biologically significant compounds including β-carotene, xanthophyll and several synthetic and natural phospholipids. Protection from bleaching by phospholipids was most evident for the phosphatidyl cholines and was less for the phosphatidyl ethanolamines. Protection was independent of the chain length and unsaturation of the esterified fatty acids and depended primarily on the nature of the polar head group of the phospholipids.     

Phospholipid and Ca++ dependency of phorbol ester receptors

The phospholipid and Ca++ dependency of a partially purified phorbol ester apo-receptor from the soluble fraction of mouse brain homogenates was studied. This apo-receptor is believed to be identical with the Ca++ and phospholipid-dependent protein kinase C. Binding of phorbol esters to the receptor/kinase C was shown to be entirely dependent on phospholipids. The negatively charged phospholipids phosphatidylserine, phosphatidylinositol, and phosphatidic acid all fully reconstituted binding. The neutral phospholipids were inactive. Among active phospholipids and mixtures of phospholipids, substantial differences (greater than 100-fold) were observed in the amounts required to achieve reconstitution. Although Ca++ was not required for reconstitution of binding activity, it dramatically (up to 100-fold) increased the potency of phospholipids for reconstitution. The phospholipids not only permitted reconstitution of the apo-receptor but also played a major role in determining the binding characteristics of the complex. The KD values of [3H]phorbol 12,13-dibutyrate were in the range of 0.8 nM for the complex with phosphatidylserine to 30 nM for the complex with dioleoyl-phosphatidic acid. Like the binding affinity, the stimulation of protein kinase C activity by phorbol esters was dependent on the phospholipid into which the receptor/kinase C was reconstituted. The importance of the lipid domain for controlling the receptor/kinase C activity and for modulation of cellular sensitivity to phorbol esters is discussed.     

Comparison of the effects of carbisocaine and other local anesthetics on 32P incorporation into individual and total phospholipids in synaptosomes

The aim of the paper was to study the effect of carbisocaine, a new local anesthetic with high liposolubility on incorporation of 32P into individual and total phospholipids and to compare its effect with that of other local anesthetics (procaine, lidocaine, cinchocaine, heptacaine). Carbisocaine decreased 32P incorporation into neutral phospholipids and increased the incorporation into acid phospholipids, presumably by inhibiting phosphatidate phosphohydrolase, similarly as reported for other anesthetics (Brindley and Bowley 1975). The increased incorporation of 32P into phosphatidylserine induced by carbisocaine suggests that this phospholipid is also synthetised from phosphatidic acid. At low concentrations, the local anesthetics studies were found to increase 32P incorporation into total phospholipids, whereas at high concentrations they reduced 32P incorporation. This biphasic effect is in agreement with the incorporation of 14C from glucose into lipids (Lassánová et al. 1984) and with the effect of cinchocaine on glycerol incorporation into phospholipids (Allan and Michell 1975), suggesting that local anesthetics affect de novo synthesis of phosphatidic acid. Carbisocaine increased 32P incorporation into phospholipids, in concentrations lower by several orders of magnitude as compared to the other local anesthetics studied. A rough correlation was observed between the concentrations at which the local anesthetics showed stimulatory effect on 32P incorporation, and the average effective concentrations of the respective anesthetics. No such correlation could be found for carbisocaine.     

Phospholipid barrier to fibrinolysis: role for the anionic polar head charge and the gel phase crystalline structure

The massive presence of phospholipids is demonstrated in frozen sections of human arterial thrombi. Purified platelet phospholipids and synthetic phospholipids retard in vitro tissue-type plasminogen activator (tPA)-induced fibrinolysis through effects on plasminogen activation and plasmin function. The inhibition of plasminogen activation on the surface of fibrin correlates with the fraction of anionic phospholipid. The phospholipids decrease the amount of tPA penetrating into the clot by 75% and the depth of the reactive surface layer occupied by the activator by up to 30%, whereas for plasmin both of these parameters decrease by approximately 50%. The phospholipids are not only a diffusion barrier, they also bind the components of the fibrinolytic system. Isothermal titration calorimetry shows binding characterized with dissociation constants in the range 0.35-7.64 microm for plasmin and tPA (lower values with more negative phospholipids). The interactions are endothermic and thermodynamically driven by an increase in entropy, probably caused by the rearrangements in the ordered gel structure of the phospholipids (in line with the stronger inhibition at gel phase temperatures compared with liquid crystalline phase temperatures). These findings show a phospholipid barrier, which should be overcome during lysis of arterial thrombi.     

The possible role of enantiodiscrimination in bilirubin toxicity

The possibility that enantiodiscrimination of bilirubin might be involved in neuronal membrane destabilization, and therefore in bilirubin toxicity, was investigated, by circular dichroism, on model membranes composed of phospholipids. The equilibrium between bilirubin enantiomers is displaced at some extent by the interaction with certain phospholipids. The extent of equilibrium displacement depends on the molecular structure of phospholipids and on the state of charge of bilirubin. The results obtained validate the hypothesis of a possible involvement of chirality in bilirubin toxicity and support a previously suggested model for the molecular mechanism of the interaction of bilirubin with the synaptic membrane.     

The influence of polyunsaturated fatty acids on spermine inhibition of lipoperoxidation. Studies on liposomes prepared with microsomal and mitochondrial phospholipids of sea bass (Dicentrarchus labrax L.) and rat liver

1. Composition of phospholipids extracted from different organelles of European sea bass liver was determined and compared with that of phospholipids extracted from the same organelles of rat liver. 2. Spermine binding to the vesicles prepared from microsomal and mitochondrial phospholipids and their aggregation was studied: these parameters indicate that only the presence of acidic phospholipids and not their unsaturation was essential for polyamine action. 3. No correlation exists between polyunsaturated fatty acid and spermine inhibition of lipid peroxidation. In fact microsomal phospholipids, which have a low content of acidic phospholipids, and a prevalent presence of phosphatidylinositol, are not protected by spermine. 4. Mitochondrial phospholipids, which have high content of cardiolipin, elicit the capability of spermine to inhibit lipid peroxidation.     

Evidence for a lipid dependence of mitochondrial nicotinamide nucleotide transhydrogenase

1. The lipid dependence of mitochondrial nicotinamide nucleotide transhydrogenase from beef heart was investigated. With submitochondrial particles digestion of phospholipids by phospholipases A and C led to a partial inhibition that could not be readily reversed by phospholipids.

2. Extraction of neutral lipids including ubiquinone from lyophilized submitochondrial particles with pentane did not inhibit the transhydrogenase, whereas further extraction with water/acetone led to a complete and apparently irreversible inhibition.

3. A partially purified preparation of transhydrogenase, depleted of lipids (and inactivated) by treatment with cholate and ammonium sulphate, was reactivated by various purified phospholipids but not by detergents or triacylglycerols.

4. It is concluded that mitochondrial transhydrogenase, catalyzing the non-energy-linked transhydrogenase reaction, requires phospholipids specifically for its catalytic activity and not as dispersing agents. A mixture of phospholipids appears to fulfill this requirement better than the individual phospholipids.     

Inhibition of peroxyl radical-mediated lipid oxidation by plasmalogen phospholipids and alpha-tocopherol

The recently discovered peroxyl radical scavenging properties of plasmalogen phospholipids led us to evaluate their potential interactions with alpha-tocopherol. The oxidative decay of plasmalogen phospholipids and of polyunsaturated fatty acids as induced by peroxyl radicals (generated from 2,2'-azobis-2-amidinopropane hydrochloride; AAPH) was studied in micelles using 1H-NMR and chemical analyses. In comparison with alpha-tocopherol, a 20- to 25-fold higher concentration of plasmalogen phospholipids was needed to induce a similar inhibition of peroxyl radical-mediated oxidation of polyunsaturated fatty acids. Plasmalogen phospholipids and alpha-tocopherol protected each other from oxidative degradation. In low-density lipoproteins (LDL) and micelles supplemented with plasmalogen phospholipids plus alpha-tocopherol, the peroxyl radical-promoted oxidation was additively diminished. The differences in the capacities to inhibit oxidation processes induced by peroxyl radicals between the plasmalogen phospholipids and alpha-tocopherol were less pronounced in the LDL particles than in the micelles. In conclusion, plasmalogen phospholipids and alpha-tocopherol apparently compete for the interaction with the peroxyl radicals. Oxidation processes induced by peroxyl radicals are inhibited in an additive manner in the presence of the two radical scavengers. The contribution of the plasmalogen phospholipids to the protection against peroxyl radical promoted oxidation in vivo is expected to be at least as important as that of alpha-tocopherol.     

The effect of lipid composition and physical state of phospholipid monolayer on the binding and incorporation of a basic amphipathic peptide from the C-terminal region of the HIV envelope protein gp41

The interaction of a peptide identical to the carboxy terminal region of the envelope glycoprotein gp41(828) of HIV with negatively charged phospholipids in a monolayer was studied by a Wilhelmy film balance. No significant interaction of the peptide with a monolayer composed of pure neutral but a strong affinity to negatively charged phospholipids could be observed. In mixed phospholipid monolayers the binding of the gp41(828) is primarily limited by the amount of acidic phospholipids. The physical state of the monolayer is another important parameter for binding. Clustering of negatively charged phospholipids and the surface pressure are crucial. Ca(2+) ions strongly interfere with the peptide-lipid interaction up to complete abolishment. The effects observed are dependent on the nature of the acidic lipid. Phosphatidylglycerol was found to be more sensitive than phosphatidylserine. The significance of the results for processes like virus assembly and budding will be discussed.     

Loading of lipophorin particles with phospholipids at the midgut of Rhodnius prolixus

32P-Labelled midguts (32P-midguts) of Rhodnius prolixus females were incubated in the presence of nonradioactive purified lipophorin and the release of radioactivity to the medium was analysed. The radioactivity found in the medium was associated with lipophorin phospholipids. When the 32P-midguts were incubated in the absence of lipophorin, no 32P-phospholipids were found in the medium. Comparative analysis by thin-layer chromatography of 32P-phospholipids derived from metabolically labelled 32P-midgut or lipophorin particles after incubation with 32P-midgut showed some differences, revealing a possible selectivity in the process of phospholipids transfer. The transfer of phospholipids to lipophorin was linear with time up to 45 min, was saturable with respect to the concentration of lipophorin, and was half-maximal at about 5 mg/ml. The binding of 32P-lipophorin to the midgut at 0 degrees C reached the equilibrium at about 1 h of incubation. The binding of 32P-lipophorin was inhibited by an excess of nonradioactive lipophorin, which suggests a specific receptor for lipophorin. The capacity of midguts and fat bodies to transfer phospholipids to lipophorin varied during the days following the meal. When lipophorin enzymatically depleted of phospholipids by treatment with phospholipase A2 was incubated with 32P-midguts, the same amount of phospholipids was transferred, indicating a net gain of phospholipids by the particle.     

Phospholipid-dependence of oestrone UDP-glucuronyltransferase and p-nitrophenol UDP-glucuronyltransferase.

Hepatic UDP-glucuronyltransferase activity was resolved into two fractions, one exhibiting oestrone glucuronyltransferase activity and the other exhibiting p-nitrophenol glucuronyltransferase activity. Hydroxyapatite-column chromatography removed greater than 95% of the phospholipids from both preparations. The partially purified delipidated enzymes were essentially devoid of catalytic activity, but activities were restored by the addition of phospholipids or phosphatidylcholine mixtures containing various saturated and unsaturated fatty acids. Both oestrone and p-nitrophenol glucuronyl-transferase activities were reconstituted to similar degrees with the phosphatidylcholine mixtures. When purified phospholipids were tested, phosphatidylcholine and lysophosphatidylcholine were most effective in restoring activity, whereas phosphatidylethanolamine was the least effective. These results further suggest that oestrone and p-nitrophenol UDP-glucuronyltransferases are dependent on phospholipids for their activity.     

Role of phospholipids in the lipophorin particles of Rhodnius prolixus.

The lipophorin of Rhodnius prolixus metabolically labelled with 32P exclusively in the phospholipid moiety was purified on a potassium bromide gradient and treated with phospholipase A2 in the presence of an excess of fatty acid-free albumin. The treatment completely removed the phospholipids from the particles and generated [32P]-lysophosphatidylcholine, [32P]-lysophosphatidylethanolamine, and free fatty acids that remained bound to albumin. The phospholipid-depleted lipophorin particles remained soluble, indicating that phospholipids are not essential in maintaining the stability of the particles in aqueous solution. Complete removal of phospholipids did not affect the association of apolipophorin III with lipophorin particles. Lipophorin density increased slightly from 1.120 to 1.134 g/ml after treatment. The phospholipid-depleted particles also retained their ability to be recognized and loaded in vitro with phospholipids delivered by the fat body, thus supporting the concept of lipophorin's role as a reusable lipid shuttle for phospholipids.