Modulation of cardiac glycoside inhibition of (Na+ + K+)-ATPase by membrane lipids. Difference between species

The role of lipids in the modulation of the ouabain-sensitivity of membrane (Na+ + K+)-ATPase from different species has been studied using a reconstitution procedure which promotes lipid exchange during detergent depletion by Sephadex chromatography. Hybrid reconstitution of delipidated (Na+ + K+)-ATPase preparations from bovine brain into the lipids obtained from crab nerve enzyme preparations significantly reduces the sensitivity of the brain enzyme to inhibition by ouabain. Conversely, reconstitution of crab nerve enzyme into the lipids from bovine brain enzyme preparations increases the sensitivity of the crab enzyme to ouabain inhibition. These opposing effects demonstrate the role of membrane lipids in modulating the enzyme-inhibition relationship in preparations from these different species.     

S-adenosyl-L-methionine modulates Na+ + K+-ATPase activity in rat colonic basolateral membranes.

Rat colonic basolateral membranes were incubated with S-adenosyl-L-[methyl-3H]methionine (0.3 mM) at 37 degrees C for 2 h at pH 9.0. This resulted in an increase in the specific activity of Na+ + K+-ATPase by 60%. Kinetic parameter analysis revealed a 2-fold increase in the Vmax. of this enzymatic activity, whereas the Km for ATP was unchanged. The methylation inhibitor S-adenosyl-L-homocysteine (2 mM) significantly reduced these S-adenosyl-L-methionine-stimulated increases in specific activity and the Vmax. of Na+ + K+-ATPase. S-Adenosyl-L-methionine treatment of basolateral membranes was also found to significantly increase the fluidity of these preparations, as assessed by steady-state fluorescence polarization techniques using the fluorophore 1,6-diphenyl-1,3,5-hexatriene; S-adenosyl-L-homocysteine (2 mM) again markedly reduced this S-adenosyl-L-methionine-induced increase in fluidity. While transmethylation reactions involving phospholipids, non-polar lipids and proteins were all found to exist in rat colonic basolateral membranes, based on a number of observations, the results of the present studies suggest that transmethylation of membrane phospholipids, but not membrane non-polar lipids or proteins, influenced the fluidity of basolateral membranes which, in turn, modified Na+ + K+-ATPase activity in these membranes.     

Dissimilar effects of Na+ and K+ on the promotion of glucosyltransferase secretion in Streptococcus salivarius

A defined growth medium (designated AP11), in which the concentrations of Na+ and K+ could be altered independently of one another, was developed for Streptococcus salivarius ATCC 25975. The addition of 100 mM-Na+ to AP11-medium containing 25 mM-K+ initially reduced the rate of expression of extracellular glucosyltransferase (GTFe). However, once S. salivarius had adaptated to grow in the presence of 100 mM-Na+, the rate of GTFe expression was stimulated. In fact once adapted to the presence of Na+ in the environment the same increase in the rate of enzyme expression was observed in all batch cultures irrespective of the K+ concentration (2-50 mM). At 2 mM-K+ there was no change in the level of saturation of the membrane lipids when the Na+ concentration was increased from 0 mM to 100 mM. Na+-stimulation of GTFe expression was confirmed in non-proliferating cell suspensions at different K+ concentrations. In non-proliferating cell suspensions, GTFe expression outlined a rectangular hyperbola with respect to K+ concentration when the K+ concentration was stepped up from 2 mM. The increase in GTFe synthesis and secretion was transient and was similar to that previously reported by us in Na+-rich medium, though it did not reach the same high levels. The reduced transient stimulation of GTFe expression correlated both with an enrichment in the saturated fatty acids of the membrane lipids of S. salivarius, and with the fact that the degree of saturation was only slightly reduced when the K+ concentration was stepped up from 2 mM to 50 mM. Needless to say, the final octadecenoic to octadecanoic (C18:1/C18:0) fatty acid ratio retained its direct correlation with the transient increase in GTFe production following the step up in K+ concentration, giving rise to an apparent biphasic plot when combined with that previously reported.     

Binding of Ca2+ to phosphoinositols, phosphatidylserines and gangliosides

The effect of K+, Mg2+ and serotonin on the interaction between Ca2+ and different phospholipids as well as glycosphingolipids (gangliosides) was studied by equilibrium dialysis using 45Ca as tracer. The highly polar phosphatidylinositol-4,5-bisphosphate (TPI) was found to bind more Ca2+ per lipid molecule than all other lipids tested and Ca2+ could not be released as easily as in the other lipids by K+, Mg2+ and serotonin. Ca2+ is released from all lipid-Ca2+ complexes most effectively by Mg2+, serotonin is less effective but enhances K+ in its capacity to displace Ca2+ from the respective binding sites. A remarkable dissociating influence of serotonin on ganglioside-Ca2+ and phosphatidylserine-Ca2+ complexes is observed. This effect is less pronounced with phosphatidylinositol-Ca2+ complexes under comparable comparable conditions. The possible functional role of phospholipids and gangliosides in vivo is discussed with regard to the specific Ca2+-binding properties of these lipids.     

Electrostatic control by lipids upon the membrane-bound (Na+ + K+)-ATPase

In this paper, the membrane-bound (Na+ + K+)-ATPase from bovine brain is shown to be controlled by electrostatic alterations of the charged lipids surrounding the enzyme. The properties under investigation are the enzymatic activity, activation energy and the response of the enzymatic system to temperature. Arrhenius plots of the ATPase activity are biphasic with a break at temperature Ti. The temperature Ti, the activation energies at temperatures above and below Ti, and the enzymatic activity at any constant temperature have been shown to depend upon the concentrations of alkali and alkaline-earth metal ions in the solution. These electrolyte dependencies are ascribed to changes of electrostatic conditions at the lipids surrounding the ATPase. If the higher electrostatic screening ability of divalent ions is taken into account, the results in the presence of mono- and divalent ions become virtually the same. As a result of this work, it is concluded that electrostatic alterations are transmitted to the ATPase from the lipids of the membrane in which the enzyme is embedded. Inhibition and activation of the enzyme by mono-and divalent metal ions may thus be explained without any auxiliary hypothesis, particularly without postulating specific binding sites for the different ionic species at the protein. In addition, the specific lipid requirement of the ATPase may be understood better in the light of this interpretation.     

Studies on the Ca2+-induced lysis of platelet alpha-granules

Platelet alpha-granules have been reported to lyse upon addition of submillimolar Ca2+ (J. Van der Meulen and S. Grinstein, J. Biol. Chem. 257, 5190). Similar observations in parotid granules have been attributed to extensive lipid hydrolysis. Experiments were performed to assess the role of lipases and proteases in Ca2+-induced lysis of alpha-granules. No differences were detected between lipids of Ca2+-treated and control granules by two-dimensional thin-layer chromatography. Moreover, several phospholipase inhibitors were without effect on Ca2+-induced lysis. Similarly, the polypeptide patterns of control and treated granules were identical and protease inhibitors failed to prevent lysis. In contrast, lysis could be suppressed by increasing the osmolarity of the medium or by substitution with nonpermeating ions. Lysis was unaffected by quinine, amiloride, furosemide, or tetraethylammonium but was inhibited by 4,4'-diisothiocyano-2,2'-stilbene disulfonate (DIDS), a powerful inhibitor of anion transport. The data suggest that Ca2+-induced lysis of alpha-granules does not result from wholesale hydrolysis of either lipids or proteins. Instead, the results are consistent with a Ca2+-mediated change in membrane permeability. In the presence of permeating ions, this leads to entry of salt and osmotically obliged water with consequent swelling and eventual lysis.     

Evidence that lipid lateral phase separation induces functionally significant structural changes in the Ca+2ATPase of the sarcoplasmic reticulum.

We have studied lipid lateral phase separation (LPS) in the intact sarcoplasmic reticulum (SR) membrane and in bilayers of isolated SR membrane lipids as a function of temperature, [Mg+2], and degree of hydration. Lipid LPS was observed in both the intact membrane and in the bilayers of isolated SR lipids, and the LPS behavior of both systems was found to be qualitatively similar. Namely, lipid LPS occurs only at relatively low temperature and water content, independently of the [Mg+2], and the upper characteristic temperature (th) for lipid LPS for both the membrane and bilayers of its isolated lipids coincide to within a few degrees. However, at similar temperatures, isolated lipids show more LPS than the lipids in the intact membrane. Lipid LPS in the intact membrane and in bilayers of the isolated lipids is fully reversible, and more extensive for samples partially dehydrated at temperatures below th. Our previous x-ray diffraction studies established the existence of a temperature-induced transition in the profile structure of the sarcoplasmic reticulum Ca+2ATPase which occurs at a temperature corresponding to the [Mg+2]-dependent upper characteristic temperature for lipid LPS in the SR membrane. Furthermore, the functionality of the ATPase, and in particular the lifetime of the first phosphorylated enzyme conformation (E1 approximately P) in the Ca+2 transport cycle, were also found to be linked to the occurrence of this structural transition. The hysterisis observed in lipid LPS behavior as a function of temperature and water content provides a possible explanation for the more efficient transient trapping of the enzyme in the E1 approximately P conformation observed in SR membranes partially dehydrated at temperatures below th. The observation that LPS behavior for the intact SR membrane and bilayers of isolated SR lipids (no protein present) are qualitatively similar strongly suggests that the LPS behavior of the SR membrane lipids is responsible for the observed structural change in the Ca+2ATPase and the resulting significant increase in E1 approximately P lifetime for temperatures below th.     

Inhibition of erythrocyte membrane (Ca2+ + Mg2+)-ATPase by the organophosphorus insecticides parathion and methylparathion

Organophosphorus insecticides parathion and methylparathion non-competitively inhibited the activity of (Ca²⁺ + Mg²⁺)-ATPase bound to and solubilized from pig erythrocyte membrane. Both enzyme preparations exhibited biphasic substrate curves displaying the existence of two functional active sites with low and high affinity to ATP. Also, the relationship between the activity of bound enzyme and Ca²⁺ concentration was biphasic. The activity reached maximum at 20 μM then dropped progressively as the Ca²⁺ concentration was raised. The inhibition of the activity was more pronounced for parathion than for methylparathion and the solubilized enzyme preparation was more affected than the bound one. The inhibition constants (K_i) for parathion for bound enzyme were 55 and 158 μM for high- and low-affinity active sites, respectively; for methylparathion these values equalled 74 and 263 μM, respectively. K_i values for parathion were 36 and 118 μM for solubilized enzyme (high- and low-affinity sites, respectively), for methylparathion −62 and 166 μM, respectively. The magnitude of the effect was greater for a low Ca²⁺ concentration, which could arise from different conformational states of the enzyme at different calcium concentrations. The results of the experiment suggest that the insecticides inhibited the ATPase by binding to a site on the enzyme rather than by the interaction with associated lipids, although lipids could weaken the action of the compounds due to the strong affinity of organophosphorus insecticides to lipids.     

Influence of nitric oxide donors on the intrinsic fluorescence of Na+,K+-ATPase and effects on the membrane lipids.

Effects of the nitric oxide donors S-nitroso-glutathione (GSNO) and S-nitroso-N-acetylpenicillamine (SNAP) on Na+,K+-ATPase-rich membrane fragments purified from pig kidney outer medulla were studied using intrinsic fluorescence and ESR of spin-labeled membranes. These S-nitrosothiols differently affected the intrinsic fluorescence of Na+,K+-ATPase: GSNO induced a partial quenching, whereas SNAP produced no alteration. Quenching can be due to a direct modification of exposed tryptophan residues or to an indirect effect caused by reactions of nitrogen oxide reactive species with other residues or even with the membrane lipids. Pre-incubation of Na+,K+-ATPase with 0.4mM GSNO resulted in a modest inhibition of ATPase activity (about 24%) measured under optimal conditions. Stearic acid spin-labeled at the 14th carbon atom (14-SASL) was used to investigate membrane fluidity and the protein-lipid interface. SNAP slightly increased the mobility of bulk lipids from Na+,K+-ATPase-rich membranes, but did not change the fraction of bulk to protein-interacting lipids. Conversely, treatment with GSNO extinguished the ESR signals from 14-SASL, indicating generation of free radicals with high affinity for the lipid moiety. Our results demonstrated that membranes influence bioavailability of reactive nitrogen species and bias the activity of different S-nitrosothiols.     

Hg2+ and Cd2+ interact differently with biomimetic erythrocyte membranes

In order to characterize the potentially deleterious effects of toxic Hg²⁺ and Cd²⁺ on lipid membranes, we have studied their binding to liposomes whose composition mimicked erythrocyte membranes. Fluorescence spectroscopy utilizing the concentration dependent quenching of Phen Green™ SK by Hg²⁺ and Cd²⁺ was found to be a sensitive tool to probe these interactions at metal concentrations ≤1 μM. We have systematically developed a metal binding affinity assay to screen for the interactions of Hg²⁺ or Cd²⁺ with certain lipid classes. A biomimetic liposome system was developed that contained four major lipid classes of erythrocyte membranes (zwitterionic lipids: phosphatidylcholine and phosphatidylethanolamine; negatively charged: phosphatidylserine and neutral: cholesterol). In contrast to Hg²⁺, which preferentially bound to the negatively charged phosphatidylserine compared to the zwitterionic components, Cd²⁺ bound stronger to the two zwitterionic lipids. Thus, the observed distinct differences in the binding affinity of Hg²⁺ and Cd²⁺ for certain lipid classes together with their known effects on membrane properties represent an important first step toward a better understanding the role of these interactions in the chronic toxicity of these metals.     

Dynamic lipid-protein stoichiometry on E1 and E2 conformations of the Na+/K+ -ATPase

Annular lipid-protein stoichiometry in native pig kidney Na+/K+ -ATPase preparation was studied by [125I]TID-PC/16 labeling. Our data indicate that the transmembrane domain of the Na+/K+ -ATPase in the E1 state is less exposed to the lipids than in E2, i.e., the conformational transitions are accompanied by changes in the number of annular lipids but not in the affinity of these lipids for the protein. The lipid-protein stoichiometry was 23 ± 2 (α subunit) and 5.0 ± 0.4 (β subunit) in the E1 conformation and 32 ± 2 (α subunit) and 7 ± 1 (β subunit) in the E2 conformation.     

Temperature-induced change in the Ca2+-dependent ATPase activity and in the state of the ATPase protein of sarcoplasmic reticulum membrane.

The temperature dependence of the Ca2+-dependent ATPase activity and of the conformational fluctuation of the ATPase molecule has been measured for four kinds of preparations: fragmented sarcoplasmic reticulum, MacLennan's enzyme (purified ATPase preparation), and DOL and egg PC-ATPase (purified ATPase preparations in which lipids are replaced with dioleoyllecithin and egg yolk lecithin, respectively). It has been found that Arrhenius plots of the Ca2+-dependent ATPase activity show a break at about 18 degrees C for all the preparations. Hydrogen--deuterium exchange kinetics of the peptide NH protons were used to measure the conformational fluctuation of the protein molecules. Van't Hoff plots of the conformational fluctuation amplitude of a region near the surface of the ATPase molecule also show a break at about 18 degrees C for all the preparations. It is concluded that the break at around 18 degrees C is not related to a gel-liquid crystalline transition of lipids but to a change in the conformation of the ATPase molecule existing in fluid lipids.     

Study of catalytically active center of the Ca2+-Mg2+-dependent ATPase of sarcoplasmic reticulum by triplet probe method

It was shown that eosine and erythrosine are competing inhibitors of the Ca2+-Mg2+- dependent ATPase active center of sarcoplasmic reticulum. The eosine and erythrosine inhibition constants are equal to 1.4 x 10(-6) M and 1.1 x 10(-6) M, respectively. Nitroxide radicals of various hydrophobicity and K3Fe(CN)6 were used to compare the constants of triplet states exchange quenching of erythrosine in aqueous solution, in lecithine liposomes and in ATPase active center of sarcoplasmic reticulum. It was established that ATPase binding center was immersed into a liquid phase and was not connected with lipids. Mn2+ and Gd3+-ions, which are competing with Mg2+ and Ca2+ for binding sites in the enzyme active center, diminished the phosphorescence quenching time of eosine at 77K. This means that the ion binding sites are less than 12 A apart from ATP-binding center.     

Phase behavior of membranes reconstituted from dipentadecanoylphosphatidylcholine and the Mg2+-dependent, Ca2+-stimulated adenosinetriphosphatase of sarcoplasmic reticulum: evidence for a disrupted lipid domain surrounding protein

A new method was used for reconstituting active sodium deoxycholate solubilized Ca2+-ATPase of rabbit skeletal muscle sarcoplasmic reticulum. Removal of the detergent by dialysis at the pretransition temperature of the pure lipid (22 degrees C) favored the formation of sheet-like structures with a lipid and protein content close to that of the detergent-solubilized sample. Freeze-fracture electron micrographs revealed the Ca2+-ATPase to be organized in rows corresponding to the typical banded pattern seen in low-temperature freeze-fracture micrographs of pure lipid bilayers. Incubation of the sheetlike structures at a temperature (38 degrees C) above the pure lipid main phase transition (33.5 degrees C) caused closure of the sheets into vesicles displaying homogeneous intramembranous particle distributions, at least for membranes containing less than 150 lipids per Ca2+-ATPase. However, in membranes of higher lipid content, free lipid patches were seen both above and below the lipid phase transition. By use of high-sensitivity differential scanning calorimetry, three classes of excess heat capacity peaks were observed in the vesiculated samples. A broadened "free lipid" peak occurred for samples containing between 550 and 200 lipids per protein (Tm = 33.5 degrees C, as for the order-disorder transition in pure lipid vesicles). Between 200 and 150 lipids per Ca2+-ATPase, a broad shoulder became apparent in the range of 29-32 degrees C. Below 150 lipids per Ca2+-ATPase, a peak at 26-28 degrees C became increasingly prominent with lower lipid content. At a lipid to protein ratio of about 30, no peaks in heat capacity were observed. The temperature dependence of diphenylhexatriene fluorescence anisotropy revealed a similar pattern of membrane phase behavior, except that a phase transition was detected at 33.5 degrees C in all membranes studied. On the basis of these observations, we propose that the Ca2+-ATPase is surrounded by a "lipid annulus" of motionally inhibited lipid molecules that do not contribute to a calorimetrically detectable phase transition. Beyond the annulus, "secondary domains" of disrupted lipid packing account for the peak at 26-28 degrees C and the 29-32 degrees C shoulders. At high lipid to protein ratios, the secondary domains coexist with protein-free, lipid-bilayer patches, which account for the peak at 33.5 degrees C.     

Effects of salinity and replacement of K+ by Na+ on lipid composition in two sugar beet inbred lines

The effects of NaCl and replacement of K⁺ by Na⁺ on the lipid composition of the two sugar beet inbred lines FIA and ADA were studied (a) with increasing additions of NaCl to the basal medium, and (b) with increasing replacement of K⁺ by Na⁺ at the same total concentration as in the basal medium. Direct relations were noted between NaCl concentration of the nutrient solution and the phospholipid concentration in the roots of FIA, the genotype characterized by a low K⁺/Na⁺ ratio, as well as between NaCl in the medium and the phospholipid concentration in the shoots of ADA, the genotype with a high K ⁺/Na ⁺ ratio. The sulfolipid level in the roots of FIA was maintained at higher NaCl concentrations, while it was decreased in ADA. The glycolipid concentration in the shoots of ADA and the degree of unsaturation of the fatty acids of the total lipid fraction were decreased by salinity, indicating reduced biosynthesis of chloroplast glycolipids and/or accelerated oxidation of these lipids in the presence of NaCl.
In the Na⁺ for K⁺ replacement experiment a low content of K⁺ in the medium resulted in decreased levels of total lipids, phospholipids and sulfolipid in the roots of both genotypes, which did not relate to root growth. K⁺-leakage from the roots at low K⁺-level in the medium may be reduced by the increase in saturation of the lipids. In the shoots of ADA increased levels of total lipids, phospholipids and Sulfolipid were noted at a low K⁺-concentration of the nutrient solution.     

Modulation of rat distal colonic brush-border membrane Na+-H+ exchange by dexamethasone: role of lipid fluidity

Earlier studies by our laboratory have suggested a relationship between an amiloride-sensitive Na+-H+ exchange process and the physical state of the lipids of rat colonic brush-border membrane vesicles. To further assess this possible relationship, a series of experiments were performed to examine the effect of dexamethasone administration (100 micrograms/100 g body wt. per day) subcutaneously for 4 days on Na+-H+ exchange, lipid composition and lipid fluidity of rat distal colonic brush-border membrane vesicles. The results of these studies demonstrate that dexamethasone treatment significantly: (1) increased the Vmax of the Na+-H+ exchange without altering the Km for sodium of this exchange process, utilizing the fluorescent pH-sensitive dye, acridine orange. 22Na flux experiments also demonstrated an increase in amiloride-sensitive proton-stimulated sodium influx across dexamethasone-treated brush-border membrane vesicles; (2) increased the lipid fluidity of treated-membrane vesicles compared to their control counterparts, as assessed by steady-state fluorescence polarization techniques using three different lipid-soluble fluorophores; and (3) increased the phospholipid content of treated-membrane vesicles thereby, decreasing the cholesterol/phospholipid molar ratio of treated compared to control preparations. This data, therefore, demonstrates that dexamethasone administration can modulate amiloride-sensitive Na+-H+ exchange in rat colonic distal brush-border membrane vesicles. Moreover, it adds support to the contention that a direct relationship exists between Na+-H+ exchange activity and the physical state of the lipids of rat colonic apical plasma membranes.     

The influence of Ca2+ on the lateral lipid distribution and phase transition in phosphatidylethanolamine/phosphatidylserine vesicles

The lateral lipid distribution within dipalmitoylphosphatidylethanolamine (DPPE)/dipalmitoylphosphatidylserine (DPPS) vesicle membranes was investigated under the influence of Ca²⁺ using a lipid cross-linking method. To characterize the phase transition in DPPE/DPPS vesicles and to correlate the different phase states of the membrane lipids with the obtained lipid distribution ESR measurements using a fatty acid spin label were carried out. It is shown that Ca²⁺ has a significant influence on the lateral lipid distribution within the fluid phase of the membrane lipids; instead of a slight alternating lipid arrangement in absence of Ca²⁺ due to the electrostatic interaction between the DPPS headgroups after addition of Ca²⁺ a lateral cluster structure is characteristic of the fluid phase.     

Reconstitution of a bacterial Na+/H+ antiporter

Membrane proteins from alkalophilic Bacillus firmus RAB were extracted with octylglucoside, reconstituted into liposomes made from alkalophile lipids. The proteoliposomes were loaded with 22Na+. Imposition of a valinomycin-mediated potassium diffusion potential, positive out, resulted in very rapid efflux of radioactive Na+ against its electrochemical gradient. That the Na+ efflux was mediated by the electrogenic Na+/H+ antiporter is indicated by the following characteristics that had been established for the porter in previous studies: dependence upon an electrical potential; pH sensitivity, with activity dependent upon an alkaline pH; inhibition by Li+; and an apparent concentration dependence upon Na+ that correlated well with measurements in cells and membrane vesicles.     

The effects of Ca2+ on lipid diffusion

The effects of Ca2+ on rotational and translational diffusion of lipids in multilamellar dimyristoylphosphatidylcholine (DMPC)-water systems were investigated by time-resolved phosphorescence anisotropy steady-state fluorescence polarization and fluorescence recovery after photobleaching (FRAP) experiments. Above the phase transition temperature (Tm), addition of Ca2+ caused a steady increase in the segmental motion of the phosphorescent probe, but resulted in slower diffusion of the fluorescent and lateral diffusion probes. The former result is attributed to changes in the structure of the lipid/water interface that affects the chromophore mobility on the phosphorescence time scale but does not reflect lipid motion. Below the phase transition temperature, slower diffusion of all probes were observed with increasing concentrations of Ca2+, with sudden large changes occurring at [Ca2+] approximately 500 mM. This behaviour is attributed to association of Ca2+ with the lipid phosphate groups and the exclusion of water molecules which results in tighter packing of lipids and smaller segmental motion, leading eventually to the immobilization of lipid molecules.     

Distinct structural and adhesive roles of Ca2+ in membrane binding of blood coagulation factors

The GLA domain, a common membrane-anchoring domain of several serine protease coagulation factors, is a key element in membrane association and activation of these factors in a highly Ca2+-dependent manner. However, the critical role of Ca2+ ions in binding is only poorly understood. Here, we present the atomic model of a membrane-bound GLA domain by using MD simulations of the GLA domain of human factor VIIa and an anionic lipid bilayer. The binding is furnished through a complete insertion of the omega-loop into the membrane and through direct interactions of structurally bound Ca2+ ions and protein side chains with negative lipids. The model suggests that Ca2+ ions play two distinct roles in the process: the four inner Ca2+ ions are primarily responsible for optimal folding of the GLA domain for membrane insertion, whereas the outer Ca2+ ions anchor the protein to the membrane through direct contacts with lipids.