The effect of ferriprotoporphyrin IX and chloroquine on phospholipid monolayers and the possible implications to antimalarial activity

Ferriprotoporphyrin IX intercalates into phospholipid membranes, as evidenced from its effect on the surface pressure of monolayers composed of different phospholipids. Ferriprotoporphyrin intercalation is enhanced by membrane hydrophobicity and decreased by negative surface potential. Chloroquine enhances the effect of ferriprotoporphyrin in relatively hydrophobic membranes but reduces it in monolayers composed of highly unsaturated phospholipids. These results are consistent with the differential effect of chloroquine on ferriprotoporphyrin-induced lysis of erythrocytes and of malarial parasites, thus supporting the membrane-lesion hypothesis of antimalarial action.     

Interaction of antimalarial drugs with hemin

Hemin (ferriprotoporphyrin IX) is shown to form complexes with the chloroquine class of antimalarial drugs. The Soret band of hemin becomes optically active upon the addition of chiral drugs. Results on the hemin-induced quenching of the fluorescence of chloroquine are consistent with the formation of a 2:1 hemin:drug complex with a formation constant of 1.4 x 10(7) at 298 K. Also a direct comparison of the drug-treated and drug-free parasites themselves, by the noninvasive photoacoustic spectroscopic method, reveals an in vivo interaction between endogenous hemin and the added drug.     

Chloroquine-induced phospholipid fatty liver. Measurement of drug and lipid concentrations in rat liver lysosomes

A method has been developed to measure the concentration of chloroquine in lysosomes isolated from the liver of rats. It employs 3H2O and [U-14C]sucrose to determine the intralysosomal water volume of purified lysosomes obtained by free flow electrophoresis. Twelve h after a single dose, the concentration of chloroquine in lysosomes was 6.3 mM and at 24 h it rose to 16.5 mM. With continued treatment, lysosomal chloroquine concentrations were 61 and 74 mM at 48 and 72 h. The lysosomal concentrations of chloroquine attained were sufficient to block intralysosomal phospholipase A1 activity. The lysosomal content of phospholipid rises 1.7-fold and 2.6-fold over that of control at 12 and 24 h, respectively. At 72 h, lysosomal phospholipid was 3.7-fold greater than that of control. Lysosomes show an increased negative surface charge with chloroquine administration which is due in part to an increased ratio of acidic to neutral phospholipids in the lysosomal membrane. The phosphatidylinositol content of lysosomes rose rapidly with chloroquine treatment and accounted for the early increase in the ratio. Bis(monoacylglycero)phosphate, an acidic phospholipid synthesized only in lysosomes, increased later in the course of chloroquine treatment and accounted for the continued increase in acidic phospholipids.     

Cholesterol displacement from membrane phospholipids by hexadecanol

Adding cholesterol to monolayers of certain phospholipids drives the separation of liquid-ordered from liquid-disordered domains. The ordered phases appear to contain stoichiometric complexes of cholesterol and phospholipid. Furthermore, it has been suggested that the cholesterol in these complexes has a low chemical activity compared to that of the free sterol; i.e., that in excess of the phospholipid binding capacity. We have now tested the hypothesis that the membrane intercalator 1-hexadecanol (HD) similarly associates with phospholipids and thereby displaces the complexed cholesterol. HD introduced into monolayers of pure dimyristoylphosphatidylcholine generated highly condensed (stable and solid) domains. In contrast, the phase behavior of mixed monolayers of the phospholipid, sterol, and alcohol suggested that HD could substitute for cholesterol mole for mole in promoting liquid-ordered domains. We also found that the transfer of cholesterol from mixed monolayers to aqueous cyclodextrin was greatly stimulated by the presence of HD, but only at levels sufficient to competitively displace the sterol from the phospholipid. This enhanced efflux was interpreted to reflect an increase in uncomplexed cholesterol. We conclude that HD forms complexes with dimyristoylphosphatidylcholine that are surprisingly similar to those of cholesterol. HD competitively displaces cholesterol from the phospholipid and thereby increases its chemical activity.     

Ferriprotoporphyrin IX, phospholipids, and the antimalarial actions of quinoline drugs

Two subclasses of quinoline antimalarial drugs are used clinically. Both act on the endolysosomal system of malaria parasites, but in different ways. Treatment with 4-aminoquinoline drugs, such as chloroquine, causes morphologic changes and hemoglobin accumulation in endocytic vesicles. Treatment with quinoline-4-methanol drugs, such as quinine and mefloquine, also causes morphologic changes, but does not cause hemoglobin accumulation. In addition, chloroquine causes undimerized ferriprotoporphyrin IX (ferric heme) to accumulate whereas quinine and mefloquine do not. On the contrary, treatment with quinine or mefloquine prevents and reverses chloroquine-induced accumulation of hemoglobin and undimerized ferriprotoporphyrin IX. This difference is of particular interest since there is convincing evidence that undimerized ferriprotoporphyrin IX in malaria parasites would interact with and serve as a target for chloroquine. According to the ferriprotoporphyrin IX interaction hypothesis, chloroquine would bind to undimerized ferriprotoporphyrin IX, delay its detoxification, cause it to accumulate, and allow it to exert its intrinsic biological toxicities. The ferriprotoporphyrin IX interaction hypothesis appears to explain the antimalarial action of chloroquine, but a drug target in addition to ferriprotoporphyrin IX is suggested by the antimalarial actions of quinine and mefloquine. This article summarizes current knowledge of the role of ferriprotoporphyrin IX in the antimalarial actions of quinoline drugs and evaluates the currently available evidence in support of phospholipids as a second target for quinine, mefloquine and, possibly, the chloroquine-ferriprotoporphyrin IX complex.     

The condensing effect of cholesterol in lipid bilayers

The condensing effect of cholesterol on phospholipid bilayers was systematically investigated for saturated and unsaturated chains, as a function of cholesterol concentration. X-ray lamellar diffraction was used to measure the phosphate-to-phosphate distances, PtP, across the bilayers. The measured PtP increases nonlinearly with the cholesterol concentration until it reaches a maximum. With further increase of cholesterol concentration, the PtP remains at the maximum level until the cholesterol content reaches the solubility limit. The data in all cases can be quantitatively explained with a simple model that cholesterol forms complexes with phospholipids in the bilayers. The phospholipid molecules complexed with cholesterol are lengthened and this lengthening effect extends into the uncomplexed phospholipids surrounding the cholesterol complexes. This long-range thickening effect is similar to the effect of gramicidin on the thickness of lipid bilayers due to hydrophobic matching.     

Kinetics of interactions between apomyoglobin and phospholipid membrane

The interaction of apomyoglobin and its mutant forms with phospholipid membranes was studied using tryptophan fluorescence and circular dichroism in the far UV region. It is shown that a negatively charged phospholipid membrane can have a dual effect on the structure of protein molecule upon their interaction. On the one hand, the membrane induces denaturation of the protein native structure to its intermediate state, acting as a moderate denaturing agent. On the other hand, it can stabilize the structure of unfolded protein to the same intermediate state, acting as a moderate structuring agent. The kinetics of interaction between apomyoglobin and its mutant forms and the phospholipid membrane depends on the membrane surface charge. Here the interaction rate depends on the concentration of phospholipids vesicles and stability of protein molecule, which increase with a decrease in the latter. The roles of these factors in the folding of membrane proteins and the choice of the targeted delivery pathways for protein drugs are discussed.     

Effect of chloroquine and O,O'-bis(diethylaminoethyl)hexestrol on acidic phospholipid membranes

The amphiphilic drugs chloroquine and O,O'-bis(diethylaminoethyl)hexestrol are able to form complexes with the acidic-phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol. The dissociation constants of the complexes with chloroquine are independent of pH in the range investigated here (4--7) as well as of temperature (4 degrees C--40 degrees C). The phase transition temperature of phospholipid is markedly reduced by both drugs, the effect is reversible by addition of Ca2.     

Regulation of lipases by lipid-lipid interactions: implications for lipid-mediated signaling in cells

Lipases are extracellular peripheral proteins that act at the surface of lipid emulsions stabilized, typically, by phospholipids. At a critical composition lipase activity toward substrates in phospholipid monolayers is discontinuously switched on by a small increase in substrate mole fraction. This occurs in part because lipase binding is inhibited by phospholipids. Binding of the lipase cofactor, colipase, is also inhibited by phospholipids. The initial rate of colipase binding increases abruptly at a substrate mole fraction that is approximately half the critical composition for lipase activity and just above that in substrate-phospholipid complexes. Moreover, complex collapse areas show an approximately 1:1 correlation with phospholipid excluded areas determined from an analysis of colipase adsorption rates. Thus, complexes inhibit colipase binding rate. Additionally, the switching of lipase activity likely occurs when uncomplexed substrate becomes the majority species in the interface. Lipase substrates, e.g. diacylglycerols, are typically the same lipids generated in the cytoplasmic surface of the plasma membrane of stimulated cells. As colipase binding is nonspecific and complexes involving lipase substrates form on the basis of lipid-lipid interactions alone, complexes should form in the plasma membrane of stimulated cells and may regulate protein translocation to the membrane.     

The interaction of bacterial lipopolysaccharide with phospholipid bilayers and monolayers

The association of bacterial lipopolysaccharide with artificial membranes was studied in an attempt to understand the mechanism of binding of lipopolysaccharide to cell surfaces and to look for an effect on membrane stability. The membrane models used were phospholipid bilayers and monolayers. As measured by survival time, lipopolysaccharide was found to decrease the stability of bilayers at a concentration of 300 μg/ml. When assayed by dielectric breakdown, an effect of lipopolysaccharide was noticeable at concentrations of 50 μg/ml. In studies involving the penetration of monomolecular films of various phospholipids, native and alkali-treated lipopolysaccharide both caused increases in surface pressure, and therefore penetrated the films. However, alkali-treated lipopolysaccharide was at least ten times more efficient than the native product in penetration. Alkali-treated lipopolysaccharide had a greater degree of surface activity than native lipopolysaccharide, since alkali-treated lipopolysaccharide formed monomolecular films by itself, whereas native lipopolysaccharide did not. The changes in the surface pressure and surface potential of phospholipid films produced by lipopolysaccharide in the subsolution suggested that the interaction of lipopolysaccharide with phospholipid monolayers was by a combination of penetration and adsorption to the undersurface.     

Peripheral protein adsorption to lipid-water interfaces: the free area theory

In fluid monolayers approaching collapse, phospholipids and their complexes with diacylglycerols hinder adsorption to the monolayer of the amphipathic protein, colipase. Herein, a statistical, free-area model, analogous to that used to analyze two-dimensional lipid diffusion, is developed to describe regulation by lipids of the initial rate of protein adsorption from the bulk aqueous phase to the lipid-water interface. It is successfully applied to rate data for colipase adsorption to phospholipid alone and yields realistic values of the two model parameters; the phospholipid excluded area and the critical free surface area required to initiate adsorption. The model is further developed and applied to analyze colipase adsorption rates to mixed monolayers of phospholipid and phospholipid-diacylglycerol complexes. The results are consistent with complexes being stably associated over the physiologically relevant range of lipid packing densities and being randomly distributed with uncomplexed phospholipid molecules. Thus, complexes should form in fluid regions of cellular membranes at sites of diacylglycerol generation. If so, by analogy with the behavior of colipase, increasing diacylglycerol may not trigger translocation of some amphipathic peripheral proteins until its abundance locally exceeds its mole fraction in complexes with membrane phospholipids.     

Adaptive changes in Plasmodium transmission strategies following chloroquine chemotherapy.

Both theory and data suggest that malaria parasites divert resources from within-host replication to the production of transmission stages (gametocytes) when conditions deteriorate. Increased investment into transmission stages should therefore follow subcurative treatment with antimalarial drugs, but relevant clinical studies necessarily lack adequate control groups. We therefore carried out controlled experiments to test this hypothesis, using a rodent malaria (Plasmodium chabaudi) model. Infections treated with a subcurative dose of the antimalarial chloroquine showed an earlier peak and a greater rate of gametocyte production relative to untreated controls. These alterations led to correlated changes in infectivity to mosquitoes, with the consequence that chloroquine treatment had no effect on the proportion of mosquitoes infected. Treatment of human malaria commonly does not result in complete parasite clearance. If surviving parasites produce compensatory increases in their rate of gametocyte production similar to those reported here, such treatment may have minimal effect on decreasing, and may actually increase, transmission. Importantly, if increased investment in transmission is a generalized stress response, the effect might be observed following a variety of antimalarial treatments, including other drugs and potential vaccines. Similar parasite life history counter-adaptations to intervention strategies are likely to occur in many disease-causing organisms.     

Cholesterol homeostasis and the escape tendency (activity) of plasma membrane cholesterol

We review evidence that sterols can form stoichiometric complexes with certain bilayer phospholipids, and sphingomyelin in particular. These complexes appear to be the basis for the formation of condensed and ordered liquid phases, (micro)domains and/or rafts in both artificial and biological membranes. The sterol content of a membrane can exceed the complexing capacity of its phospholipids. The excess, uncomplexed membrane sterol molecules have a relatively high escape tendency, also referred to as fugacity or chemical activity (and, here, simply activity). Cholesterol is also activated when certain membrane intercalating amphipaths displace it from the phospholipid complexes. Active cholesterol projects from the bilayer and is therefore highly susceptible to attack by cholesterol oxidase. Similarly, active cholesterol rapidly exits the plasma membrane to extracellular acceptors such as cyclodextrin and high-density lipoproteins. For the same reason, the pool of cholesterol in the ER (endoplasmic reticulum) increases sharply when cell surface cholesterol is incremented above the physiological set-point; i.e., equivalence with the complexing phospholipids. As a result, the escape tendency of the excess cholesterol not only returns the plasma membrane bilayer to its set-point but also serves as a feedback signal to intracellular homeostatic elements to down-regulate cholesterol accretion.     

Brain spectrin exerts much stronger effect on anionic phospholipid monolayers than erythroid spectrin

Red blood cell spectrin and its nonerythroid analogues are linked to integral proteins of the membrane by several skeletal protein receptors, such as ankyrin and protein 4.1 together with p55. However, there are also many reasons for believing that they are insufficient to engender all the properties that characterise the native membrane. Therefore, we are concerned with the mechanism by which brain spectrin interacts with phospholipids of the membrane bilayer. Brain and erythrocyte spectrin were shown previously to bind phospholipid vesicles as well as monolayers prepared from aminophospholipids: phosphatidylethanolamine and phosphatidylserine and their mixtures with phosphatidylcholine (PC).In the present study, it is shown that brain spectrin binds to monolayers prepared from anionic phospholipids, such as phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidyl glycerol, diphosphatidylglycerol, and their mixtures with PC. Brain spectrin injected into the subphase to reach nanomolar concentration induced a substantial increase in the surface pressure of monolayers prepared from the phospholipids and their mixtures mentioned above, possibly by penetrating them. This effect is stronger in the case of monolayers prepared from anionic phospholipids alone and weaker when monolayers were prepared from mixtures with PC. The weakest effect was observed in the case of phosphatidylinositol-4,5-bisphosphate monolayers. An interaction of brain spectrin with monolayers prepared from anionic phospholipids (PI/PC 7:3 and PA/PC 7:3) was inhibited (PI/PC much stronger than PA/PC) by purified erythrocyte ankyrin, which indicates that the binding site for those lipids is located in the β-subunit, possibly in, or in close proximity of, the ankyrin-binding site.In contrast, erythrocyte spectrin injected into the subphase induced a change in the surface pressure of monolayers prepared from anionic phospholipids, which was equal or smaller than the value of surface pressure change induced by protein without a monolayer. This effect was different from what had been observed previously for monolayers prepared from aminophospholipids and their mixtures with PC, and from the data for nonerythroid spectrin presented here.     

Brain spectrin exerts much stronger effect on anionic phospholipid monolayers than erythroid spectrin

Red blood cell spectrin and its nonerythroid analogues are linked to integral proteins of the membrane by several skeletal protein receptors, such as ankyrin and protein 4.1 together with p55. However, there are also many reasons for believing that they are insufficient to engender all the properties that characterise the native membrane. Therefore, we are concerned with the mechanism by which brain spectrin interacts with phospholipids of the membrane bilayer. Brain and erythrocyte spectrin were shown previously to bind phospholipid vesicles as well as monolayers prepared from aminophospholipids: phosphatidylethanolamine and phosphatidylserine and their mixtures with phosphatidylcholine (PC).In the present study, it is shown that brain spectrin binds to monolayers prepared from anionic phospholipids, such as phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidyl glycerol, diphosphatidylglycerol, and their mixtures with PC. Brain spectrin injected into the subphase to reach nanomolar concentration induced a substantial increase in the surface pressure of monolayers prepared from the phospholipids and their mixtures mentioned above, possibly by penetrating them. This effect is stronger in the case of monolayers prepared from anionic phospholipids alone and weaker when monolayers were prepared from mixtures with PC. The weakest effect was observed in the case of phosphatidylinositol-4,5-bisphosphate monolayers. An interaction of brain spectrin with monolayers prepared from anionic phospholipids (PI/PC 7:3 and PA/PC 7:3) was inhibited (PI/PC much stronger than PA/PC) by purified erythrocyte ankyrin, which indicates that the binding site for those lipids is located in the beta-subunit, possibly in, or in close proximity of, the ankyrin-binding site.In contrast, erythrocyte spectrin injected into the subphase induced a change in the surface pressure of monolayers prepared from anionic phospholipids, which was equal or smaller than the value of surface pressure change induced by protein without a monolayer. This effect was different from what had been observed previously for monolayers prepared from aminophospholipids and their mixtures with PC, and from the data for nonerythroid spectrin presented here.     

Electrogenesis from an ATPase-ATP-sodium pseudo pump.

The short circuit current and the open circuit voltage responses of membranes to ATP, which have been attributed to membrane ATPase acting as a sodium pump, have been reproduced not only in a lipid membrane containing solubilized ATPase but also in membranes formed of the phospholipids contained in ATPase. The response is greatest with cardiolipin, but occurs with other acidic phospholipids. This observation of electrogenesis without hydrolysis is a surface phenomenon probably due to the alignment of ATP on the phospholipid by ion association at its interface with the water phase. The finding constitutes a precaution for interpreting studies of membrane Na-K-ATPase or for its incorporation into an artificial membrane. The substances necessary for electrogenesis are present at the mitochondrial membrane, and the particular orientation of the ATP on the phospholipids in vitro suggests a role for this ion association in the function of Na-K-ATPase.     

Electrogenesis from an ATPase-ATP-sodium pseudo pump

Summary The short circuit current and the open circuit voltage responses of membranes to ATP, which have been attributed to membrane ATPase acting as a sodium pump, have been reproduced not only in a lipid membrane containing solubilized ATPase but also in membranes formed of the phospholipids contained in ATPase. The response is greatest with cardiolipin, but occurs with other acidic phospholipids. This observation of electrogenesis without hydrolysis is a surface phenomenon probably due to the alignment of ATP on the phospholipid by ion association at its interface with the water phase. The finding constitutes a precaution for interpreting studies of membrane Na–K-ATPase or for its incorporation into an artificial membrane. The substances necessary for electrogenesis are present at the mitochondrial membrane, and the particular orientation of the ATP on the phospholipids in vitro suggests a role for this ion association in the function of Na–K-ATPase.     

Lipid-protein interactions of the human erythrocyte concanavalin a receptor in phospholipid bilayers

The interaction of the human erythrocyte concanavalin A receptor (a subpopulation of Band 3) with phospholipids has been investigated using differential scanning microcalorimetry of reconstituted vesicles prepared by detergent dialysis. The mean diameter of dialyzed phospholipid vesicles jumps dramatically on inclusion of the concanavalin A receptor and then increases linearly with the fraction of protein in the bilayer. The glycoprotein has a dramatic effect on the phospholipid gel to liquid-crystalline phase transition, and ΔH decreases linearly with increasing mole fraction of protein up to a protein/lipid mole ratio of around 1:1160. Extrapolation of this data indicates that each concanavalin A receptor is able to perturb about 685 molecules of dimyristoylphosphatidylcholine, withdrawing them from the main phase transition. The cooperativity of phospholipid melting is profoundly disrupted by small amounts of glycoprotein, with the cooperative unit dropping to less than half its initial values at a protein/lipid mole ratio of 1:3800. A break occurs in the ΔH curve as the protein/lipid mole ratio is increased above 1:1160, and ΔH then increases linearly with increasing amounts of concanavalin A receptor in the bilayer. This phenomenon may be interpreted in terms of protein-protein aggregation which occurs in the phospholipid bilayer above a certain critical mole fraction of concanavalin A receptor, resulting in perturbed phospholipids being returned to the phase transition. In addition, the hydrophilic domains of the glycoprotein may exist in two different conformations depending on the protein concentration in the bilayer, and these may differ in their ability to interact with phospholipid headgroups at the membrane surface.     

Effect of phospholipids and a transmembrane peptide on the stability of the cubic phase of monoolein: implication for protein crystallization from a cubic phase

The cubic phase of monoolein has successfully been used for crystallization of a number of membrane proteins. However, the mechanism of protein crystallization in the cubic phase is still unknown. It was hypothesized, that crystallization occurs at locally formed patches of bilayers. To get insight into the stability of the cubic phase, we investigated the effect of different phospholipids and a model transmembrane peptide on the lipid organization in mixed monoolein systems. Deuterium-labeled 1-oleoyl-rac-[(2)H(5)]-glycerol was used as a selective probe for (2)H NMR. The phase behavior of the phospholipids was followed by (31)P NMR. Upon incorporation of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, or phosphatidic acid, the cubic phase of monoolein transformed into the L(alpha) or H(II) phase depending on the phase preference of the phospholipid and its concentration. The ability of phospholipids to destabilize the cubic phase was found to be dependent on the phospholipid packing properties. Electrostatic repulsion facilitated the cubic-to-L(alpha) transition. Incorporation of the transmembrane peptide KALP31 induced formation of the L(alpha) phase with tightly packed lipid molecules. In all cases when phase separation occurs, monoolein and phospholipid participate in both phases. The implications of these findings for protein crystallization are discussed.     

Inhibition of T lymphocyte activation by cyclosporin A: interference with the early activation of plasma membrane phospholipid metabolism

Rabbit lymph node and thymus lymphocytes were stimulated with concanavalin A (Con A). Cyclosporin A (CSA) inhibited in a dose-dependent way the induction of RNA and DNA synthesis; nearly complete inhibition was observed at a concentration of 200 ng/ml. Results of kinetic studies suggested that the immunosuppressive drug interfered with an early event occurring in activated lymphocytes. Among the earliest changes detectable in activated lymphocytes, the turnover of plasma membrane phospholipids is increased, predominantly of their fatty acid moieties, catalyzed by the membrane-bound lysophosphatide acyltransferase. CSA, at concentrations identical with those inhibiting macromolecular synthesis, also inhibited the Con A-stimulated specific increase in the incorporation of labeled fatty acids into plasma membrane phospholipids. When lymphocytes were stimulated with Con A for 1 hr, incorporation of labeled oleic acid and arachidonic acid approximately doubled in plasma membrane phospholipids. CSA at a concentration of 200 ng/ml prevented the elevated incorporation of labeled fatty acids into plasma membrane phospholipids of Con A-stimulated thymocytes. Concomitantly, the activation of lysolecithin acyltransferase, the key enzyme for the incorporation of long-chain fatty acids into phospholipids, was strongly inhibited. Up to high concentrations, CSA had no effect on the phospholipid metabolism of unstimulated lymphocytes. The results suggest that CSA inhibits the activation of T lymphocytes by interfering with the early activation of plasma membrane phospholipid metabolism.